Livro de Resumos - Abstracts Book Final 13jul16 PDF

Poster
Abstracts

SBBC-Sociedade Brasileira de Biologia Celular
Av. Prof Lineu Prestes, 1524
05508-000 So Paulo, SP
Email: sbbc@sbbc.org.br
Meeting Presidents
Prof. Dr. Hernandes Faustino de Carvalho

Departamento de Biologia Estrutural e Funcional
Instituto de Biocincias
UNICAMP
Profa. Dra. Patrcia Gama

Departamento de Biologia Celular e do Desenvolvimento
Instituto de Cincias Biomdicas
Universidade de So Paulo
Committees
Organizing Committee
Chao Yun Irene Yan (USP)
Deborah Schechtman (USP)
Flavia Alcntara de Carvalho Gomes (UFRJ)
Giselle Zencker (UNIFESP)
Marimlia Porcionatto (UNIFESP)
Marinilce Fagundes dos Santos (USP)
Marisa Ionta (UNIFAL)
Tiago Goss dos Santos (CIPE Hospital A.C. Camargo)
Vanessa Morais Freitas (USP)

Scientific Committee
Alexandre Bruni Cardoso (USP)
Chao Yun Irene Yan (USP)
Cludia Mermelstein (UFRJ)
Deborah Schechtman (USP)
Eder Schmidt (UFSC)
Edvaldo F Trindade (UFPR)
Elizabeth Bilsland (UNICAMP)
Enilza Maria Espreafico (USP)
Fbio Luis Forti (USP)
Glaucia Maria Machado Santelli (USP)
Guilherme Oliveira Barbosa (UNICAMP)
Luis Lamberti da Silva (USP)
Luiz Eurico Nasciutti (UFRJ)
Manoel Luis Pereira Costa (UFRJ)
Marcelo L Lamers (UFRGS)

Maria Clia Jamur (USP)
Marisa Ionta (UNIFAL)
Moacyr Jesus Barreto (UFPE)
Nathalie Cella (USP)
Patrcia Gama (USP)
Rodrigo Martins (UFRJ)
Silvana Allodi (UFRJ)
Silvya Stuchi Maria-Engler (USP)
Tiago Goss dos Santos (CIPE Hospital A.C. Camargo)
Vanessa Morais Freitas (USP)
Vilma R Martins (CIPE Hospital A.C. Camargo)
Wilson Savino (FIOCRUZ)

Abstract Evaluation Committe

Coordination
Giselle Zenker
Irene Yan
Marimlia Porcionatto
Marisa Ionta
Tiago Goss dos Santos
Vanessa Freitas

Abstract Reviewers
Adriana Hemerly
Adriana Miti Nakahata
Alexandre Bruni-Cardoso
Aline Mara dos Santos
Alison Colquhoun
Andrea Goncalves Trentin
Andrea Paffaro
Arielle Arena
Bettina Malnic
Carmem Gottfried
Carolina del Debbio
Cezar Fuziwara
Cilene Rebouas
Danilo Candido
Dawidson Gomes
Eduardo Moraes Rego Reis
Elen Haruka Miyabara
Fabio Luis Forti
Flavio Beraldo
Giordano Calloni
Glaucia Hajj
Ingo Reiderer
Julio Cesar Batista Ferreira
Luciana Capello
Luciana Romo
Luiz Anastcio Alves
Manoel Luis Costa
Marcelo Lamers
Marcia Wink
Marco Augusto Stimamiglio
Maria Ins Borella
Marilene Hohmuth Lopes
Martin Roffe
Michele Landemberger
Milena Botelho
Murilo Geraldo
Nathalie Cella
Ricardo Castilho Garcez
Ricardo J Giordano
Rodrigo Martins
Srgio Felisbino
Simone C. Motta
Simone Molz
Tatiana Takiishi
Valdemar Paffaro Junior
XVIII Meeting of Brazilian Society for Cell Biology 3
POSTER SESSIONS
July 14 (Thursday)
Poster ID
CANCER AND CELL BIOLOGY
A1-A170
CELL BIOLOGY OF INFLAMMATION
B1-B38
CELL DEATH
D1-D9
CYTOSKELETON AND MIGRATION
H1-H6
DEVELOPMENTAL BIOLOGY
I1-I18
EDUCATION IN CELL BIOLOGY
J1-J8
EXTRACELLULAR MATRIX
L1-L42
HOST-PATHOGEN INTERACTION
O1-O18
METHODS IN CELL BIOLOGY
P1-P8

July 15 (Friday)

Poster ID
CELL CYCLE AND PROLIFERATION
C1-C10
CELL DIFFERENTIATION
E1-E10
CELL SIGNALING
F1-F18
CELL TRAFFICKING AND ORGANELLES
G1-G6
EPIGENETICS
K1-K3
GENE AND CELL THERAPY
M1-M8
GENERAL CELL BIOLOGY
N1-N56
MORPHOLOGY AND CELL BIOLOGY
Q1-Q63
NEUROBIOLOGY
R1-R37
PLANT CELL BIOLOGY
S1-S3
REPRODUCTIVE BIOLOGY
T1-T68
STEM CELL BIOLOGY
U1-U23
TISSUE ENGINEERING
V1-V11
Poster presentation guidelines

Poster Sessions will be held at Rooms Minas Gerais + Rio de Janeiro and will be
organized according to the different reas (check table in the previous page. Please
check your presentation date accorging to the Board ID list above.
Set up: 8h00 (8 am) of each day
Tear down: before 18h00 (6 pm) of each day
Poster Session I: Thursday, July 14th
Poster Session II: Friday, July 15th
Author presentation time:
13h30- 14h15 even numbers

14h15- 15h00 odd numbers

PLEASE BE AWARE:
Poster numbers will identify the boards.
Presenters should bring their own tapes and hangers, as needed. The
Organizing Committee will not provide these items.
The Organing Committee will not collect and keep posters that are left on the
boards.


A CANCER AND CELL BIOLOGY
A1
A2
PROTECTIVE EFFECTS OF CAPSAICIN ON DMH-INDUCED

LEUKOCYTE GENOTOXICITY AND COLONIC CELL PROLIFERATION
TRANSLATIONAL CONTROL IN INVASIVE BREAST CANCER: A

TRANSLATOMICS APPROACH
Brunno Felipe Ramos Caetano* (1), Mariana Baptista Tablas (2), Natlia Elias
Ferreira Pereira (2) Maria Aparecida Marchesan Rodrigues (1) and Luis Fernando
Barbisan (2).
Bellato, H., M.1; Sanz, L., M.2; Suo, L. 2; Lupinacci, F., C., S1.; Roff, M.1 ; Martins, V.,
R. 1; de Andrade, V., P. 1; Larsson, O. 2; Hajj, G., N., M. 1
(1) Department of Pathology, School of Medicine, So Paulo State University,

Brazil.
(2) Department of Morphology, Institute of Biosciences, So Paulo State University,
Brazil.
*Presenting author - email: brnncaetano@gmail.com phone: +55 14 3880-0501
mobile: +55 14 98162-6224
Capsaicin (8-Methyl-N-vanillyl-(trans)-6-nonenamide), a lipophilic alkaloid
compound, is the major pungent ingredient found in red peppers and consumed
worldwide. Most reports on capsaicin potential carcinogenicity have yielded
inconsistent findings. Thus, we evaluated the modifying effects of capsaicin on 1,2dimethylhydrazine (DMH)-induced leukocyte genotoxicity and colonic cell
proliferation in rats. Male Wistar rats were randomly assigned into six experimental
groups (n=6). During the first four weeks, corn oil was given to groups 1 and 6,
while intragastric capsaicin was administered at 5mg/kg to groups 2 and 4, and at
50mg/kg to groups 3 and 5 three times/week. On weeks 3 and 4, the animals received
a subcutaneous injection of either DMH (groups 1-3, 40mg/kg) or EDTA (groups 46, vehicle), twice a week. Genotoxicity testing was performed by the comet assay in
peripheral blood samples, 24 hours after the last DMH injection. Ki-67
immunohistochemical expression was assessed in colon paraffin-embedded sections.
DNA damage levels as well as Ki-67 expression were significantly higher
(p<0.0001) in the DMH-treated groups, G1 and G2, than in groups G3-G6. In
conclusion, our results suggest that capsaicin at 50mg/kg had antigenotoxic and
cytostastic effects, significantly reducing DMH-induced leukocyte DNA damage and
colonic cytotoxicity in rats.
Ethical Approval: 1153/2015-CEUA. Funding support: FAPESP 2014/21951-6 and
2014/24762-0.
1.
2.
A.C.Camargo Cancer Center

Karolinska Institutet
BACKGROUND: Today, genome-wide projects attempt to define patterns of gene

expression by analyzing total mRNA populations. However, this approach provides
little information about protein levels, since transcription and translation are not
coupled. Therefore, the study of mRNAs subjected to post-transcriptional regulation in
breast cancer may lead to new gene expression profiles that better reflect protein levels
and provide new information on the nature of this disease. AIM: Analyze
preferentially translated mRNAs in breast cancer tissue. METHODS: A cohort was
selected from breast cancer patients at the AC Camargo Cancer Center. Clinical data
was evaluated and tumor samples were collected. mRNAs being actively translated
were isolated by polysome profiles and identification was performed by deep
sequencing using Smart-Seq2 for the construction of libraries with low amount of
material. RESULTS: We were able to include in our study 249 patients. The molecular
classification of tumors consisted of 70% Luminal, 10% HER2 and 20% Triple
Negative cases. Polysome profiles were successfully done in all samples and
demonstrated that triple negative had increased polysome-associated RNA when
compared with luminal, indicating a relationship between translational status and
tumor type. Representative libraries of cytosolic and polysome-associated mRNAs
were built using Smart-Seq2. 161 samples had their total and polysomal RNA
successfully converted into high quality libraries using only 2 ng of
RNA. CONCLUSIONS: Differentially translated mRNAs were isolated from a
representative cohort of breast cancer patients. Sequencing libraries were performed
and deep sequencing will reveal gene expression patterns that better reflect the
population of proteins.
A3
A4
TUMOR HETEROGENEITY EVALUATION IN GLIOBLASTOMAS USING

MICROARRAY OF POLYSOMAL MRNAS
DIETARY INDOLE-3 CARBINOL AND SYNBIOTICS PROMOTE TUMOR

DEVELOPMENT IN HEMIN-FED RATS
Fernanda Cristina Sulla Lupinacci (1*); Martin Roff (1); Hermano Bellato (1);
Hellen Kuasne (2), Tiago Goss dos Santos (1), Victor Piana de Andrade (3), Paulo
Sanematsu (4),Vilma Regina Martins(1),Silvia Regina Rogatto (2) and Glaucia
Noeli Maroso Hajj(1)
Nelci Antunes de Moura (1), *Brunno Felipe Ramos Caetano (2), Leonardo Nazario
de Moraes (1), Robson Francisco De Carvalho (1), Lus Fernando Barbisan (1)
(1) Department of Molecular and cell biology, A C Camargo Cancer Center, So

Paulo, Brasil
(2) Department of cytogenetic and molecular biology, A C Camargo Cancer Center,
So Paulo, Brasil
(3) Department of investigative pathology, A C Camargo Cancer Center So Paulo,
Brasil
(4) Department of neurosurgery, A C Camargo Cancer Center So Paulo, Brasil
*
Rua
Tagu,
440,
Liberdade,
flupinacci@cipe.accamargo.org.br
Institutional phone: (11) 2189-5000 r2977
So
Paulo-SP
01508-010.
Mobile: (11) 98206-3513
Glioblastoma is among the most aggressive tumor type and less responsive to
chemotherapeutic agents, thus a better understanding of the behavior of these tumors
may help to develop new treatments for this disease. Currently, many genome-wide
projects attempt to define general patterns of gene expression based on deep
sequencing or microarray data from total mRNA populations. However, this
approach provides little information about the molecular mediators of tumor biology,
because the expression levels of mRNAs do not necessarily reflect the levels of
proteins. The identification of mRNAs target of translational alterations in tumors
can show gene expression profiles that better reflect the population of proteins. In
this project we intend to identify mRNAs differentially translated in a single
glioblastoma by microarray of mRNAs associated with polysomes (actively engaged
in translation). The sample was divided in 8 pieces that were classified as high or
low grade based on histological characteristics. Polysomes were isolated from the
glioblastoma by continuous gradient of sucrose 7-47% ultracentrifugation. RNA was
extracted with TRIzol . We were able to identified mRNAs differentially translated
in a human glioblastoma that presented histologically different parts. By comparing
high vs low grade tumor pieces, we were able to identify more differentially
translated genes than differentially expressed genes. Among the differentially
translated mRNAs, there are many related to proliferation, development and cancer.
Results were validated with qPCR. The technic of isolating mRNA engaged in
translation can be used to identify biomarkers of tumor progression, leading to new
therapeutic approaches.
Ethical approval: 1775/13. Funding sup
port: FAPESP 2013/03315-2, 2014/15550-9
(1) Department of Pathology, School of Medicine, So Paulo State University, Brazil.

(2) Department of Morphology, Institute of Biosciences, So Paulo State University,
Brazil.
*Presenting author - email: brnncaetano@gmail.com phone: +55 14 3880-0501
mobile: +55 14 98162-6224
Hemin, a genotoxic compound found in red meat, may lead to colorectal cancer
whereas Indole-3-carbinol (I3C) and synbiotics are suggestive inhibitors of
carcinogenesis. Therefore, we assessed the effects of a synbiotic (inulin+
Bifidobacterium lactis) and/or I3C diet on dimethylhidrazine (DMH)-induced colon
carcinogenesis in hemin-fed male rats. Five groups of animals were given four
subcutaneous DMH injections. Two weeks after DMH-administration, G1 was fed
basal diet (BD) while G2, G3, G4 and G5 groups received BD+hemin,
BD+hemin+I3C, BD+hemin+synbiotic and BD+hemin+I3C+synbiotic, respectively,
for 23 weeks. At the sacrifice, colon tumors were removed and each tumor volume
was recorded. Colon tumors were processed and evaluated histopathologically and
tumor incidence and multiplicity were analyzed. Colonic tumor tissue samples were
collected for gene expression analysis. Tumor volume was higher in the group treated
with hemin+I3C+ synbiotic (G5), (p<0.02) than in the group fed BD+hemin (G2).
Invasive adenocarcinoma was more frequent in the group treated with
BD+hemin+I3C+synbiotic (G5), (p<0.001) than in the group fed BD+hemin (G2). The
gene expression analysis showed that in comparison with group fed BD+hemin (G2),
Cdh1, Tgfb1 and Appl1 were downregulated while Raf1 was upregulated in the group
treated with hemin+ I3C+ synbiotic (G5), (0.001<p<0.02). Cdh1 was downregulated
in the group treated with BD+hemin+I3C (G3) while Tnf was upregulated (p=0.004).
Our results suggest a interaction among hemin, I3C and synbiotic that promotes tumor
development.
Ethical Approval: CEEA 921/2012. Funding support: FAPESP 2011/23699-4 and
2012/12631-2.

A5
A6
HEAT SHOCK ANTAGONIZES UVA-INDUCED RESPONSES IN

MELANOCYTES AND MELANOMA: AN UNEXPECTED INTERACTION
EVALUATION OF CIRCULATING TUMOR MICROEMBOLI

PROGNOSTIC MARKER IN PATIENTS WITH LUNG CANCER
Leonardo Vinicius Monteiro de Assis, Maria Nathlia Moraes, Ana Maria de

Lauro Castrucci
Mnica T. de M. Diaz* (1), Emne A. Abdallah (1) Alexcia C. Braun (1), Celso A. L
Mello (2), Milena S. Tariki (2), Aldo L.A. Dettino (2), Marcello F. Fanelli (2),
Ludmilla T. D. Chinen (1).
Laboratory of Comparative Physiology of Pigmentation, Department of

Physiology, Institute of Biosciences, University of So Paulo, R. do Mato, trav.
14, no. 101, So Paulo, 05508-900, Brazil; email: deassis.leonardo@usp.br , + 55
11 3091 7523.
Background: The skin is under the influence of oscillatory factors such as light and
temperature. This organ possesses a local system that controls several aspects in a
time-dependent manner; moreover, the skin has a well-known set of opsins whose
function is still unknown. Aims: To evaluate the responses of normal (Melan-a)
and malignant (B16-F10) murine melanocytes to UVA radiation, heat shock, or a
combination of both stimuli, seeking to analyze opsins and clock gene expression
as well as melanin content. Methods: Melan-a and B16-F10 cells were kept under
constant darkness at 37 C. On the 4th day the cells were exposed to heat shock
(40 C), UVA radiation (4.4 KJ/m2) combined with heat shock (40 C), or UVA
radiation only (4.4 KJ/m2). Results: The mentioned-above stimuli did not lead to
apoptosis or cell cycle arrest in either cell line. The expression of Opn2 decreased
in response to heat shock in normal melanocytes; the opposite effect was found in
malignant ones. Opn4 expression increased in response to UVA radiation in both
cell lines. The clock gene machinery of malignant melanocytes is more responsive
to UVA radiation when compared to normal ones. In both cell lines, melanin
content increased in response to UVA. Most UVA-induced effects were
antagonized by heat shock. Conclusion: The responses displayed by melanoma
cells may represent an important step in cancer development/progression. The
knowledge of this new interaction UVA/temperature may be useful to enhance the
efficiency of current therapy for depigmentary diseases. Funding Support:
FAPESP and CNPQ.
Conflict of interest:
None to declare
(1)
(2)
AS
International Research Center, A.C. Camargo Cancer Center, So Paulo, Brazil.

Department of Clinical Oncology, A. C. Camargo Cancer Center, So Paulo,
Brazil.
* taiane_diaz@hotmail.com, +55 11 98539 0929 / +55 11 2189 2993, Rua Tagu, 440,
So Paulo, 01508-010, Brazil.
Background: Circulating Tumor Cells (CTCs) has been proved to be a good marker
for tumor
progression, particularly in
the
form
of Circulating Tumor
Microemboli (CTM), which is defined as a clusters of 3 CTCs or more, and may
be found in the peripheral blood from lung cancer patients. Aims: To evaluate if the
presence of CTMs is associated with tumor progression. Methods: CTCs were
isolated by ISET Technology. The samples of metastatic lung cancer patients were
collected and analyzed before the beginning of systemic treatment and after 2
months.
CTCs and CTMs
were identified
according
to its morphological
features. Patients were followed and evaluated according to RECIST
criteria. Progression-free survival (PFS) analysis was made by Kaplan-Meier method
and
the
difference
between
curves
was
calculated
by
Log-rank
test. Results: Nine patients were analyzed. Patients who had CTMs previously at the
beginning of systemic therapy had poor PFS compared to those with absence of
CTMs (2.6 months X
5.1 months, p= 0.07).
On
the
contrary, after
systemic chemotherapy, patients who had CTMs had better PFS than those with
absence of CTMs (7.4 months X 2.6 months, p= 0.08). Conclusion: elevated baseline
levels of CTMs were associated with poor prognosis for these patients. In our limited
number of patients, post chemotherapy CTM were inversely correlated to
PFS. We suppose that CTMs may suffer constitutive changes after chemotherapy and
lost its invasion capacity. This project has been approved by ethical committee (n
1367/10) and received funding support by CNPq.
A7
A8
KAISO KNOCKDOWN INHIBITS THE EXPRESSION OF CELLSURFACE DIFFERENTIATION MARKERS IN K562 CELLS
METASTATIC COLORECTAL CANCER FOLLOW-UP: EVALUATION BY

CTC KINETICS
Jaime Cofre (1), Eliana Abdelhay (2).
Emne Ali Abdallah (1)*, Virgilio Souza e Silva (2), Marcello Ferretti Fanelli (2),
Marcelo Calil Machado Netto (2), Vanessa da Silva Alves (1), Ludmilla Thom
Domingos Chinen (1).
(1) Universidade Federal de Santa Catarina, Laboratrio de Embriologia Molecular

e Cncer. Florianpolis, SC, Brazil
(2) Diviso de Laboratrios do CEMO, Instituto Nacional do Cncer, Rio de
Janeiro, Brazil
Presenting and Corresponding author: jaimecofre@yahoo.com.br; Laboratrio de
Embriologia Molecular e Cncer, UFSC, Sala 313b, CEP 88040-900, Brazil; +55
48 37219754
Background: There is consistent evidence of Kaisos role and the involvement in
human tumorigenesis, but there is no indication about its role in hematopoietic
differentiation or establishment of chronic myeloid leukemia (CML). Aims: We
used K562 cell line, established from a CML patient in blast crisis to investigate
Kaisos contribution to cell differentiation status, in the blast crisis of CML (CMLBP). Methods: For FACS analysis, it was possible to use antibodies that recognize
cell surface myeloid-specific antigens GPA-phycoerythrin (PE), CD33-fluorescein
isothiocyanate (FITC), CD11b-PE, CD15-FITC, CD117-PE, CD71-FITC (Becton
Dickinson). Appropriated isotype-matched controls (Becton Dickinson) were also
used. It is worth mentioning the use of siRNA to knock-down either Kaiso or
p120ctn alone or in combination. Results: We investigated whether knockdown of
either Kaiso or p120ctn alone or in mixture affects the global cell differentiation.
We quantified the amounts of hematopoietic differentiation markers CD15,
CD11b, CD33 and CD117 expressed on the plasma membrane of K562 cells by
FACS analysis. CD15 and CD11b were utilized widely as indicators of maturation
of the hematopoietic cells as well as granulocytic markers. We found that knockdown of Kaiso or p120 alone or Kaiso/p120ctn double knock-down decreased
CD15, CD33 and CD117 by 25-35%, 8% and 13%, respectively. Conclusion:
These findings indicate that knock-down of Kaiso and p120ctn are blocking the
differentiation program of CML-BP.
(1) International Center of Research, A. C. Camargo Cancer Center, So Paulo, Brazil.

(2) Department of Clinical Oncology, A. C. Camargo Cancer Center, So Paulo,
Brazil.
* emne22@gmail.com, (+55011) 98230-1537 / (+55011) 2189-2993.
Background: Circulating tumor cells (CTCs) are considered as a compartment of liquid
biopsy and have been demonstrated as a possible biomarker of tumor progression,
including metastatic colorectal cancer (mCRC). Aims: to verify if CTC kinetics could
reflect the tumor response to treatment. Methods: CTCs were isolated by ISET
Technology (Isolation by Size of Epithelial Tumor Cells), and identified by containing
malignant morphology and non-expressing CD45. Patients were followed and
evaluated according to RECIST criteria. Progression-free survival (PFS) analysis was
made by Kaplan-Meier method and the difference between curves was calculated by
Log-rank test. Results: 54 mCRC patients were enrolled in this study. It was possible
to evaluate CTCs at baseline (t1, before the beginning of systemic chemotherapy) and
follow-up (t2, 2 months after baseline, according imaging) in 42 patients. Patients with
lower CTCs levels who became with higher levels had worse PFS than patients with
higher levels and that became with lower levels (6.9 months vs. 14.7 months, P= 0.06).
In the group of patients that did not have change in CTC kinetics (always with higher
CTC levels or always with lower CTCs levels), there was no difference in PFS (P=
0.6). Conclusion: Our results showed a viable tool to monitor tumor progression and
response to treatment before their evidence by imaging. However, further studies are
needed to support this information. This project has been approved by ethical
committee (n 1367/10) and received funding support by FAPESP and Capes.
Conflict of interest statement: None declared.

Information on Ethical approval: This work was approved by the Ethics
Committee, CAAE/02878012.6.0000.0121/UFSC.
Funding support: INCa-RJ

A9
A10
ANALYSIS OF LAMININ-111 CLEAVAGE IN BREAST CANCER

1
ADIPOSE DERIVED MESENCHYMAL STEM CELL CONDITIONED MEDIA

MODULATE MCF-7 CELL PROLIFERATION AND DIFFERENTIATION
STATE
Basilio Smuczek Ruy G. Jaeger

1
Department of Cell and Developmental Biology, Institute of Biomedical Sciences,

University of So Paulo, So Paulo, Brazil.
Breast cancer is a public health problem. Cancer microenvironment plays a major
role in tumorigenesis and tumor progression. The extracellular matrix (ECM),
mainly formed by collagen, proteoglycans and glycoproteins, is an important
component of the tumor microenvironment. Our laboratory studies the effect of
laminin, key ECM glycoprotein, in tumor biology. Laminin peptides are involved
in central steps in tumor biology, such as migration, invasion, invadopodia
formation, protease secretion, and generation of reactive oxygen species (ROS).
However, a fundamental point to be elucidated is to determine the presence of
laminin-derived peptides at tumor sites in vivo and in vitro. This aspect so far
remains speculative. This prompted us to investigate proteolytic processing of
laminin in vivo and in vitro in breast cancer (invasive ductal carcinoma).
Quantitative immunohistochemistry in tissue microarrays (TMAs) addressed
laminin chains present in these tumors, showing 1 and 1 chains in breast tumors
compared to normal breast. Cells derived from breast cancer (MDA-MB-231 and
MCF-7) were grown on Matrigel, a reconstituted basement membrane with
prominent laminin expression. Normal-like MCF-10A breast cells grown on the
same substrate served as controls. Cells were grown on Matrigel for 24 hours.
Electrophoresis and immunoblot showed that MDA-MB-231 cells exhibited
cleavage of laminin alpha1, beta1 and gamma1 chains, generating fragments with
different molecular weights (200 kDa, 80 kDa, 70 kDa and 50 kDa). Proteomic
analysis is under way to identify putative bioactive peptides derived from laminin
cleavage. We conclude that 1 and 1 chains of laminin-111 are present in breast
tumors in vivo. Furthermore, laminin chains are cleaved in vitro, probably
generating relevant fragments and peptides.
Work approved by the Ethics Committee in Research Involving Human (CESPH1.233.185) and approved by the Ethics Committee on Animal Use (CEUA-n001,
pages 028, book 03). Financial supported: CNPq 471411/2013-2, FAPESP
2015/03393-9 and CAPES.
Marcelo Coutinho de Miranda (1); Andrea da Fonseca Ferreira (1); Mariane Izabella
Abreu de Melo (1); Marianna Kunrath Lima (1); Alfredo de Miranda Goes (1); Jerusa
Arajo Quinto Arantes Faria (1); Dawidson Assis Gomes (1).
(1)- Departament of Biochemistry and Immunology, Universidade Federal de Minas
Gerais, Minas Gerais, Brazil.
Correspondence: marcelocdempos@gmail.com. Instituto de Ciencias Biologicas.
Antonio Carlos Av. 6627, Pampulha. Cep:31270-901. Institutional number:
+5531334092631
Cancer initiation and progression are dependent on the acquisition of several
modifications that activate oncogenes, and inhibit tumor suppressors pathways.
Differents soluble factors can modulate tumor behavior. These changes allow the cells
disruption involved in physiological processes promoting the maintenance of the
affected cells. With the progression of cancer, the crosstalk between different cell types
present in and around the tumor affects tumor behavior. The mesenchymal stem cells
are present in the tumor stroma and have a different secretory profile with the ability to
modulate various cellular processes. To understand the secretome interference in cells
proliferation, death, and phenotypic alteration in breast tumor cells, mesenchymal stem
cells derived from adipose tissue conditioned medium (mcASC) was used for cultivate
MCF-7 cells. These cells when grown in mcASC showed shorter population doubling
time and increased frequency of phosphorylated histone 3 and EdU-positives cells.
Augmented expression of cyclin B1 was observed, suggesting the induction of
proliferation by mcASC. The mcASC was also able to increase apoptosis, confirmed
by capase-7 activation. The frequency of tumor initiating cells CD44+CD24-/low were
increased when in mcASC. Collectively, these data suggest that stem cells are able to
induce cell proliferation, likely by paracrine stimulation, also selection and phenotypic
changes in MCF-7 cells, resulting in a more aggressive tumor cells.
This work was supported by CAPES, CNPq, and had the UFMG ethical committee
appreciation (CAAE: 22912213.3.0000.5149).
The authors have not had any financial or personal relationships with individuals or
organizations that could be perceived to bias this work.
A11
A12
EXPRESSION AND RELEVANCE OF THE IMPACT PROTEIN IN

PANCREATIC CANCER
ANALYSIS OF KIN17 PROTEIN DETECTION ASSOCIATED WITH

NUCLEAR MATRIX IN THE MURINE MELANOMA CELLS
Vitria Bianchi Alves, Camila Braggion, Maria Dirlei Begnami, Helano Carioca
Freitas, Vilma Regina Martins, Martin Roff, Glaucia Noeli Maroso Hajj.
Sabrina Marques Godinho Kelmer, Lorena Gomes Polizelli, Anelise Cardoso Ramos,
Fabiana dos Santos Rando,Quirino Alves de Lima Neto, Vanessa Pinatto Gaspar e
Maria Aparecida Fernandez*
Departamento de Biotecnologia, Gentica e Biologia Celular, Universidade Estadual
de Maring,87020-900 Maring, Paran, Brasil.
(1)
(2)
(3)
International Research Center, A.C. Camargo Cancer Center, So

Paulo, Brazil.
Pathology Department, A.C. Camargo Cancer Center, So Paulo,
Brazil.
Clinical Oncology Department, A.C. Camargo Cancer Center, So
Paulo, Brazil.
New protein synthesis is a highly controlled process whose deregulation may

affect cellular processes such as proliferation or apoptosis. One of the main points
of mRNA translation control occurs through the phosphorylation of the alpha
subunit of the eukaryotic translation initiation factor 2 (eIF2), which leads to
inhibition of general protein synthesis. GCN2 is an eIF2 kinases activated upon of
amino acid starvation and its activity is dependent upon binding to GCN1.
IMPACT is a protein that binds to GCN1 thus competing with the GCN1-GCN2
interaction. Consequently, the deregulation in the expression or activity of
IMPACT, GCN1 or GCN2 could lead to an imbalance in the translational process.
Research in databases using the cBioPortal demonstrated genetical alterations in
the genes encoding IMPACT, GCN1 and GCN2 in a significant number of
pancreatic adenocarcinomas. In Brazil, this tumor it is responsible for 2% of all
cancers diagnosed and 4% of deaths. Therefore, to unravel the molecular
mechanisms associated with this tumor is of utmost importance for the
development of new therapeutic approaches. In this project, we intend to observe
the expression and function of IMPACT in pancreatic tumor cells lines and human
tumors.
Supported by CNPq (800434/2014-5). Ethical approval: 2186/16.
*Contact: Sabrina Marques Godinho Kelmer, Maria Aparecida Fernandez

E-mail: sabrinamg12@hotmail.com ; mafernandez@uem.br ;
aparecidafernandez@gmail.com
Tel.: +55 44 3011 5398
Among cancers that affect the skin, melanoma is the third most common type
worldwide and in most cases, it leads to mortality, because of its aggressiveness and
metastatic potential. Genetic, epigenetic and proteomic studies have reclassified
molecularly the findings of pathological melanoma basis, indicating biomarkers in the
progression of melanoma and the development of effective therapies against tumor
resistance. Previously published by our laboratory, there was the analysis of the
detection and localization of the kin17 protein at the nuclear and chromatin-associated
fractions in the murine melanoma cell line B16F10-Nex2 and in two derived subclones
with different metastatic potential, B16-8HR and B16-10CR. It revealed detection in
both fractions, with an elevated concentration in the chromatin-associated fraction in
B16-10CR, suggesting that the kin17 expression level could be a potential marker for
melanoma. This protein has been previously classified as a promising cancer
biomarker, by different groups, as it has been described involved in DNA metabolism
like replication initiation, recombination and repair. In this study we verified the
association of protein kin17 to the nuclear matrix in the same cell lines indicated
above. The preparation and extraction of nuclear matrix using lithium 3,5diiodosalicylate. The nucleus and nuclear matrix fractions were separated by
electrophoresis using polyacrylamide gels by SDS-PAGE and Western blotting. The
antibodies used were anti-kin17 (sc-32769, Santa Cruz Biotechnology Inc.), anti-CTCF
(07-729, Millipore) and nuclear matrix marker anti-p84 (ab487, Abcam). The results
confirm the co-localization of the kin17 protein to the nuclear matrix in all cell lines
analyzed and revealed a significant expression in lineage B16-10CR, compared to the
other ones. In conclusion, the kin17 protein is more associated to nuclear matrix in
lineage B16-10CR, in accordance with its proliferative potential and aggressiveness.
Funding support: CAPES, CNPQ, Fundao Araucria, Secretria de Estado da
Cincia, Tecnologia e Ensino Superior Fundo Paran and COMCAP facilities from
the Universidade Estadual de Maring-PR.

A13
A14
EVALUATION OF THE RASSF9 ROLE IN MELANOMA BY USING THE

REVERSE GENETIC CRISPR/CAS9 SYSTEM
IN VITRO ANTI-TUMOR POTENTIAL OF ABYSSINONE V-4 METHYL

ETHER
ISOLATED
FROM
ERYTHRINA
DROOGMANSIANA
(LEGUMINOSAE)
Elizabeth Cristina Rodrigues1, Roger Chammas2, Gustavo Pessini AmaranteMendes1
Stephane Zingue1, Abel Jol Gbaweng Yaya2, Julia Cisilotto3, Evelyn Winter3, Jelver
Alexander Sierra3, Emmanuel Talla4, Dieudonn Njamen5, Tnia Beatriz CreczynskiPasa3
1 - ICB-USP
2 - FMUSP
1.
The Ras-association family (RASSF) comprises ten proteins (RASSF1-10), which
2.
several of them are related to tumor suppression. RASSF9 is a member of 3.
Nterminal RASSF proteins and was first described as P-CIP1, a protein that4.is
involved in vesicle trafficking, and has been shown to bind Ras proteins.
5.
Experiments using RASSF9 knockout mice have shown this protein is
predominately expressed in the stratified epithelium, where it is crucial to
epidermal homeostasis. However, its function and importance in melanoma is still
obscure. Aiming to understand the role of RASSF9 in melanoma growth and
metastasis, we have developed RASSF9 knockout TM1-luciferase cells by using
the CRISPR/Cas9 system. The TM1 is an aggressive and non-metastatic murine
melanoma cell line. Our research group has genetically modified TM1 to express
the luciferase enzyme, allowing its use in non-invasive in vivo assays. For the
CRISPR/Cas9 system, we have designed specific sgRNAs for the RASSF9 and for
the nme-1 genes. As the nme-1 gene was the first characterized metastasis
suppressor, we will use TM1.nme-1 KO cells as a positive control. These sgRNA
sequences were cloned in lentiCRISPRv2 plasmid and transfected in HEK293T
cells to obtain the viral particles. TM1 cells were transduced with the obtained
lentivirus and the down regulation of RASSF9 or nme-1 was confirmed through
Western Blot and PCR analysis. In vivo assays are now being performed to
evaluate growth rate and metastatic behavior, whereas in vitro assays will be used
to check migration capacity and drug resistance.
(1) University of Maroua, Maroua, Cameroon

(2) Institute of Medical Research and Medicinal Plants Studies, Yaounde, Cameroon
(3) Federal University of Santa Catarina, Florianpolis, Santa Caterina, Brazil
(4) University of Ngaoundere, Ngaoundere, Cameroon
(5) University of Yaound 1, Yaounde, Cameroon
The Erythrina genus is known as a source of various secondary metabolites, but
less is known on their biological activities. Among them is abysonne V-4 methyl ether
(AVME) which are prenylated flavonoids isolated from Erythrina droogmansiana.
AVME has been recently reported to exhibit estrogenic/antiestrogenic properties in our
previous work. However the antitumor activity of AVME has not yet been evaluated.
The present work was therefore designed to assess the in vitro antitumor potential of
AVME. To achieve our goal, AVME was evaluated for its cytotoxicity using resazurin
reduction assay on four cancer and two non-tumoral cell lines. Cell cycle and apoptosis
were assessed using flow cytometry; cell migration/invasion and cell morphology were
performed using optic and electronic microscopes, while the ability of AVME to
inhibit metalloproteinase was assessed using Zymography assay. AVME induced
cytotoxic in all tested cell lines with a CC50 around of 20 M and seems to be weakly
selective for cancer cells (selectivity index = 1.5). AVME induced apoptosis (42%) in
MCF-7 cells, anti-migration, anti-invasion activities in MDA-MB-231 cells. However,
it did not induced change in cell cycle. Taken all together these results suggest that
AVME endow antitumor properties and could be a source of traditional medicine.
Further works are underway to elucidate the underlying mechanism by which AVME
induced its antitumor effects.
Support funding: The present work has been done with the support from CNPq-TWAS.
A15
A16
ANKHD1 SILENCING LEADS TO ACCUMULATION OF DNA DAMAGE

AND MODULATION OF DNA REPAIR GENES
TARGETED
PHOTORESPONSIVE
CHLOROALUMINUM
PHTHALOCYANINE OF NANO-DRUG DELIVERY SYSTEM FOR
EFFICIENT COMBINATION THERAPY IN BREAST CANCER CELLS
Anamika Dhyani(1,2), Patricia Favaro(3), Sara T Olalla Saad(1,2).

(1)Hematology and Hemotherapy Center, University of Campinas/HemocentroUnicamp.
(2)Instituto Nacional de Cincia e Tecnologia do Sangue, Campinas, So Paulo,
Brazil.
(3)Department of Biological Sciences, Federal University of Sao Paulo, Diadema,
So Paulo, Brazil.
Presenting author contact details: ANAMIKA DHYANI
Rua Carlos Chagas 480,
Hemocentro, UNICAMP
Campinas, 13083-878
Sao Paulo, Brazil.
Cel: 19-982275734
email: anamikadhyani2009@gmail.com
Background: ANKHD1, Ankyrin repeat and KH domain-containing protein 1, is
required for progression through S phase and the inhibition of ANKHD1
downregulates core histones.
Objective: To understand the mechanism behind downregulation of histones in
ANKHD1 silencing.
Methods: Multiple Myeloma (MM) cell line U266 was transduced with lentiviral
mediated shRNA targeting ANKHD1 and Lac z (control shRNA), separately. Cells
were harvested and functional analysis was carried out.
Results: DNA replication is coupled with histone synthesis during S phase and
downregulation of histones is known to be associated with replication stress and
DNA damage. Hence, we checked for expression of PCNA (Proliferating Cell
Nuclear Antigen), a protein involved in DNA replication and repair. PCNA
expression was significantly decreased in ANKHD1 inhibited cells, suggesting
ANKHD1s role in DNA replication and repair. For confirmation, we studied the
effect of ANKHD1 silencing upon the expression of -H2AX, a marker for DNA
double stranded breaks (DSBs) and an early sign of DNA damage induced by
replication stress. We observed a significant increase in -H2AX suggesting
accumulation of DSBs. Therefore, the decrease of core histones might be linked to
DNA damage and replication stress occurring during the S phase of MM cells with
ANKHD1 silencing. We also observed modulation in the expression of several
genes implicated in DNA damage and repair such as CHK2, ATM, PTEN, BRCA1
etc.
Conclusions: ANKHD1 silencing in MM cells leads to DNA damage and
modulates expression of several genes implicated in DNA damage and repair.
Thus, the accumulation of DNA damage explains the mechanism behind
downregulation of histones in ANKHD1 silenced cells.
Leonardo Barcelos de Paula (1), Maryanne Trafani de Melo (1), Antonio Claudio
Tedesco (1)
(1)
Department of Chemistry, University of Sao Paulo, Ribeirao Preto,

Brazil.
barcelos@usp.br
Background: Breast cancer is the most main cause of cancer deaths in women
worldwide, and it affects the physical and mental health of patients seriously.
Photodynamic therapy (PDT) is a well-established innovated treatment for tumor due
to its high cure rate and low recurrence rate. Aims: Evaluate the photosensitizer (PS)
activity combination with PDT in the development of breast cancer. Methods: We
developed the poly(lactic-coglycolic acid) nanocapsules (NC) loaded with
chloroaluminum phthalocyanine (ClAlPc) was obtained by a high thermodynamic
stability. Human breast cell lines (MCF-7 and MDA-MB-231) grown in culture were
treated with NC/ClAlPc defining the best drug concentration, and further irradiated
with monochromatic laser light. Cell uptake of the NC/ClAlPc was analyzed after
incubation for three hours at 37 C. Results: Our findings demonstrate excellent
physical and chemical stability of NC with a size less than 200 nm. The results
suggested that the cell uptake was kept at the cytoplasmic level. Evaluation of the PDT
light effects was performed using a diode-laser at doses from 100, 200, 700 and 1000
mJ/cm2. Mitochondrial activity describes a cellular viability of 58% (24 h), 43% (48 h)
and 25% (72 h) compared to the absence of activation of the PS, which is related to the
association of the effects of photodamage based on PDT mechanisms and cell injury.
Conclusion: PDT is an appropriate stand alone intervention, or as an adjunct to
surgery. This modality causes tissue destruction pathway the interaction between
oxygen, light and release of photosensitizing drug nanoentrapped.
Funding support: FAPESP.
Research was funded by FAPESP and INCT, Sao Paulo Brazil.


A17
A18
3D CO-CULTURE: SIMULATING THE TUMOR MICROENVIRONMENT
IMPACT OF FIBROBLASTS IN GENE AND MICRORNA EXPRESSION IN

PROSTATE TUMOR CELLS (LNCAP)
Najla Adel Saleh (1), Jelver Alexander Sierra Restrepo (2), Fabola Branco Filippin
Monteiro (3), Tnia Beatriz Creczynski-Pasa (1).
(1) Department of Pharmacy, Federal University of Santa Catarina, Florianpolis,
Brazil.
(2) Post-graduation course in Materials Science and Engineer, Federal University of
Santa Catarina, Florianpolis, Brazil.
(3) Department of Clinical Analysis, Federal University of Santa Catarina,
Florianpolis, Brazil.
Contact Details: Grupo de Estudos de Interaes entre Micro e Macromolculas. Rua
Roberto Sampaio Gonzaga Campus Universitrio, Trindade. CCS,.Bloco H, quarto
andar. E-mail: najladelsaleh@gmail.com.Telefone: +55 48 37212212, +55 48
99883956.
An in vitro way to mimic tumor microenvironment interactions including tumoral
and stromal cells is using a tridimensional (3D) co-culture model. Therefore, the aim
of this study was to characterize a 3D co-culture system of tumoral and stromal cells
using B16F10 (murine melanoma), J774 (murine macrophages) and NIH-3T3
(murine embryo fibroblasts) cell lines. Agarose gel 2% was used to block cell-plate
adhesion and the 3D co-culture was established with 4 103 cells of each cell line.
The spheroids developed after 4 days were treated for 3 additional days with
lipopolysaccharide (LPS) and dexamethasone (DEX) to induce M1 and M2
polarization, respectively. Spheroids area and morphology were analyzed by
photomicroscopy and scanning electron microscopy (SEM), respectively. The
membrane integrity and cellular viability were evaluated by flow cytometry using
propidium iodide (PI). According to the results obtained by photomicroscopy, the
spheroid area developed after 7 days was similar to the cells from the control and
treated group, which shows that the treatment with LPS and DEX does not affect the
cell growth. The interactions between tumoral and stromal cells, as well as cell
surface and sphere rigidity were visualized by SEM. Throughout flow cytometry
analysis was possible to confirm the treated 3D co-cultures and control cells
viability. In conclusion, 3D co-culture spheroid model using tumoral and stromal
cells was characterized and can be potentially used as a model to study the tumor
microenvironment.
Maira Smaniotto Cucielo*1, Brenda de Carvalho Minatel1, Bruno Martinucci1,

Gabriel Henrique Caxali1, Mariana Medeiros1, Flvia Karina Delella1.
1
Departament of Morphology; Institute of Biosciences; UNESP; Botucatu; So

Paulo; Brazil.
*mairacucielo@ibb.unesp.br; (14) 3880-0498
For many years, cancer research had focused primarily on the tumor cells. However,
it was found that the surrounding stroma was also essential for the carcinogenesis
regulation. For prostate gland, the stroma relevance is even clearer, since the normal
physiology of the organ is maintained due to a complex regulatory mechanism
between epithelial and stromal components; and when disrupted can promote cancer.
Thus, this study aim to evaluate the expression of genes and microRNAs in prostate
cancer cells (LNCaP) grown in co-culture with fibroblasts (HFF-1). After cultivation,
total RNA was extracted and relative expression of six mRNAs and the microRNAs:
-21 (oncomiR) and -29b (tumor suppressor) was quantified by RT-qPCR. Our results
showed that LNCaP cells co-cultivated with fibroblasts had significantly increased
BAK expression (2.50 fold), a pro-apoptosis gene, and increased miR-29b
expression (3.02 fold), with decreased expression of genes related with tumor
progression, as: BCL2 (0.31 fold), STAT3 (0.20 fold), MMP-2 (0.32 fold), AKT-1
(0.38 fold), AKT-2 (0.43 fold) and miR-21 (0.29 fold) (p<0.05). All genes and
microRNAs with differential expression are part of STAT3 pathway, a transcription
factor that regulates cell proliferation and survival, besides promote the
tumorigenesis. Therefore, it is apparent that fibroblasts have a fundamental role in
gene modulation of tumor cells, as they reduce the malignant phenotype of LNCaP
cells.
Financial support: CAPES, FAPESP 2013/26114-2.
Financial support: CNPq, FAPESC and CAPES.
A19
NINTEDANIB (BIBF 1120) RECOVERED THE
COMPLEX IN PROSTATE DORSOLATERAL
PROGRESSION FROM TRAMP MICE
A20
DYSTROGLYCAN
LOBE CANCER
HORMONE RECEPTOR AND NEOVASCULARIZATION FEATURES

IN CANCER PROGRESSION OF PROSTATE VENTRAL LOBE IN TRAMP
MICE AFTER NINTEDANIB TREATMENT
Ellen Nogueira Pangrazi (1), Raquel Frenedoso da Silva (1), Larissa Akemi Kido (1),
Fabio Montico (1), Valria Alves Cagnon (1)
Raquel Frenedoso da Silva, Ellen Nogueira Pangrazi, Letcia Ferreira Alves, Valria
Helena Alves Cagnon
(1)
Department of Structural and Functional Biology, State University of Campinas, So

Paulo, Brazil.
Department of Structural and Functional Biology, State University of

Campinas, So Paulo, Brazil.
Email: ellen_lima48@hotmail.com
Inst.: (19) 3521-6113
Mobile: (19) 98218-5112
The authors have nothing to disclose.
Nintedanib (BIBF 1120) has been reported as an inhibitor of FGF and VEGF
pathways, which are the main regulators of epithelial proliferation and invasive
ability in prostate cancer. The aim of this study was to evaluate the general
morphology, extracellular matrix ( and dystroglycans and -actin) and
angiogenesis molecules in the dorsolateral lobe of transgenic adenocarcinoma in the
mouse prostate (TRAMP), at different cancer progression grades, followed by
Nintedanib treatment. TRAMP mice (Treated groups) were treated with Nintedanib
(10mg/kg/day) from 8 to 12 or 12 to 16 weeks of age, and were euthanized at 12 or
16 weeks of age, respectively. The control groups received the vehicle (Tween 20,
10%) at the same age of the treated groups. The prostate dorsolateral lobe was
collected and processed for light microscopy, immunohistochemistry for dystroglycan and -actin, Western Blotting for -dystroglycan and microvessel
density analysis. The results showed progressive prostatic lesions according to aging.
We also observed a dystroglycan complex decrease and stromal hypertrophy in the
control groups. The Nintedanib treatment decreased adenocarcinoma severity,
delaying the angiogenesis process and increasing the dystroglycan complex
occurrence. The dystroglycan complex recovery was stronger in 16 week old mice.
The Nintedanib treatment did not interfere with the -actin reactivity. Thus, we can
conclude that Nintedanib was effective to restored the structural organization of the
prostate stroma and membrane, especially the dystroglycan complex, in prostate
cancer progression, helping to delay prostate cancer.
Ethics Committee: 3649-1
Email: raquelfrenedoso@yahoo.com.br
Inst.: (19) 3521-6113
Mobile: (19) 992417983
Nintedanib is a compound capable of inhibiting angiogenesis, preventing cell
proliferation and decreasing tumor volume. This study aimed to evaluate Nintedanib
effects on morphology, AR, ER, FGF-2, VEGF features in prostate cancer
development in transgenic adenocarcinoma of the mouse prostate (TRAMP). 72
animals were divided into: Control group treated with vehicle (Tween 20(10%),
orally) and Treated groups receiving the drug (10mg/Kg/day, orally) for 4 weeks.
The animals which had started treatment at 8 weeks of age were sacrificed at 12
weeks of age, and the other ones remained without treatment for 10 weeks and were
sacrificed at 22 weeks of age. Likewise, part of the animals which started treatment
at 12 weeks of age were sacrificed at 16 weeks of age, and another part remained
without treatment for six weeks and sacrificed at 22 weeks of age. Prostate ventral
lobes were collected for structural, immunohistochemical and protein level analyses
to AR, ER, FGF-2, VEGF, microvessel density and proliferative index.
Morphological evaluation showed that Nintedanib decreased prostate lesions and the
stromal hypertrophy. There was also significant AR reduction in the epithelial cells
and FGF-2 in both prostatic stromal and epithelial cells, besides led to decreased
microvessel density and VEGF immunolocalization. Furthermore, the proliferative
index was diminished in animals that received the drug. Thus, we can conclude that
Nintedanib led to a protective effect against prostate cancer in TRAMP mice due to
delaying the neoplastic development in the glandular microenvironment and
decreased different pathways involved in tumorigenesis.
FAPESP 2013/26677-7, Ethics Committee 3285-1

A21
A22
MAST CELLS ACTIVITY IN ANNEXIN A1 DEFICIENT MICE DURING

EARLY MNNG-INDUCED CARCINOGENESIS
TERMINAL CARBOHYDRATE RESIDUES ON THE SURFACE OF

LARYNGEAL
CARCINOMA
CELL
ARE
ALTERED
AFTER
PHOTODYNAMIC THERAPY
Lucas Ribeiro de Azevedo (1), Marina de Paula Silva (2), Sonia Maria Oliani (1,2)
Souza, R.K.F (1); Pacheco Soares, C.(1), da Silva,N.S.(2)
(1) Department of Biology, So Paulo State University (IBILCE/UNESP), So
Paulo, Brazil.
(2) Post-graduation Program of Structural and Functional Biology, So Paulo Federal
University (UNIFESP), So Paulo, Brazil.
Contact details:
e-mail: mpsilva.bio@gmail.com
Institutional telephone: +55 17 3221-2731
Mobile telephone: +55 17 99166-0902
Disclosure: The authors have no competing financial interests to disclose in relation
to this abstract.
Colorectal cancer (CRC) is a major worldwide health problem owing to its high
prevalence and mortality rates. Though advances in the diagnosis and treatment of
CRC have had a major impact on management of this malignancy, but new diagnosis
and treatment are therefore needed. Substantial evidences suggest that mast cells
modulate the adenoma to carcinoma sequence of CRC development and annexin 1
(AnxA1) is an anti-inflammatory protein that regulates intestinal mucosal injury,
inflammation and repair. The aim of our investigation was to evaluate the effect of
acute response on mast cells after intrarectal induction by MNNG on BALB/C (wildtype WT) and annexin 1 deficient (AnxA1-/-) mice after 8, 24 and 48h carcinogen
exposure. We observed differences in the colon length of AnxA1-/- and
histopathological changes in the colonic epithelium. In WT, AnxA1 expression
increased progressively with peak around 24h after carcinogen inoculation. In both
mice strains there was an increase in the number of mast cells, but more
desgranulated cells in AnxA1-/-. After MNNG administration, IL-1, IL-2, IL-6, IL10, IL-12, LIX, IL-17, IP-10, KC, MCP-1, VEGF, TNF- and IFN- expression were
significant in AnxA1-/-, whereas IP-10 increase in WT. Immunostaining was
observed for tryptase, and chymase after induction with MNNG, characterizing the
emergence of a different subpopulation of mast cells. Altogether, our finds suggest
that during early carcinogenesis AnxA1 promotes the protection against the MNNGinduced damage and an inhibitory action in the mast cell response that may result as
a potential target for prevention and treatment of CRC.
(1) Institute of Research and Development IP&D, Universidade do Vale do Paraba

UNIVAP, Laboratory Dynamics of Cellular Compartments, So Jose dos Campos,
So Paulo, Brazil. *55(12) 39471138, email: rkellyfarias@yahoo.com.br
(2) Institute of Research and Development IP&D, Universidade do Vale do Paraba
UNIVAP, Laboratory of Cell Biology and Tissue, Sao Jose dos Campos, Sao
Paulo, Brazil.
Background: Photodynamic therapy has been widely used as therapy for cancer, in
which a light source is used to activate a photosensitizer, causing a photo-oxidation
process in biological tissues and it is considered safe and minimally invasive,
reducing or destructing tumors. Cellular membranes have been identified as an
important therapeutic target, since glycoproteins present on the cell surface are
involved in many functions. This study analyzed the expression of glycoproteins
present on the surface of laryngeal carcinoma (HEp-2) cells after PDT with the
photosensitizer aluminum phthalocyanine tetrasulfonate (AlPcS4). Methods: HEp-2
cells were irradiated with a diode laser at 685 nm, energy density of 4.5 J/cm2 and
power density of 35 mW/cm2, during for 1min43s. Glycoproteins were evaluated by
fluorescence microscopy, using lectins ConA, PNA and DBA conjugated with
fluorescein. Results: After PDT, cells changed their surface glycoproteins and it was
observed the presence of terminal residues -D-glucose/Mannose (ConA). However,
after PDT, it was not observed the presence of galactose beta 1, 3, Nacetylgalactosamine (PNA) and N-acetyl--D-galactosaminyl terminal (DBA)
residues when compared to the control group. Conclusion: The effect of PDT using
AlPcS4 is able to change terminal carbohydrate residues on the surface of carcinoma
laryngeal cell.
Key-words: Photodynamic therapy; Lectins;
tetrasulfonate (AlPcS4); carcinoma laryngeal
Aluminum
phthalocyanine
Acknowledgements
The authors acknowledge support from FAPESP (Fundao de Amparo a Pesquisa
do Estado de So Paulo The State of So Paulo Research Foundation contract grant
number 2011/05404-7 and 2012/10643-3).
Ethics Committee approval: CIBIO/USP (01200.003570/1998-08) and

IBILCE/UNESP (n 118/2015).
Financial Support: CNPq (308144/2014-7) and CNPq/CAPES-MinCyT
(23038.001239/2014-43)
A23
A24
STAT3 REGULATES THE INTRACELLULAR ROS LEVELS AND

EXPRESSION OF APEX1 AND MBDA GENES, FROM BASE EXCISION
DNA REPAIR, IN BREAST CANCER MDA-MB-231 CELL LINE.
EVALUATION
OF
THE
TOXICITY
OF
EXPERIMENTAL
NANOPARTICLES ASSOCIATED WITH PACLITAXEL THROUGH THE
LIPID PROFILE OF NON-HUMAN PRIMATES CEBUS APELLA
TREATED AS A TOOL FOR CANCER THERAPY
Juliana Alves Rodrigues(1), Carol Panis(2), Isis Salviano(1), Layane Duarte e

Souza(1), Andre Luiz Mencalha(1).
(1) Department of Biophysics and Biometry, Rio de Janeiro State University, Rio de
Janeiro, Brazil.
(2)Department of Internal Medicine, Health Sciences Center, University of Londrina,
Paran, Brazil
Dejair da Silva Duarte (1), Nayara Cristina Lima de Oliveira (1), Danielle Cristinne
Azevedo Feio (1), Jos Augusto Pereira Carneiro Muniz (3), Patrcia Danielle Lima
de Lima (2), Rommel Mario Rodrguez Burbano (1)
(1)
(2)
Corresponding author: ju_federal@hotmail.com

(personal +55 021 969357216, institucional +55
021 2334-2058)
Background: The high incidence of death and metastasis in breast cancer has been
related to resistance to chemo/radiotherapy. STAT3 signaling and imbalance of
reactive oxygen species (ROS), which also induces DNA damage by nitrogen base
oxidation, have emerged as one of causes for resistance phenotype. However, the
crosstalk between STAT3, ROS and DNA damage repair in breast cancer is largely
unknown. Aim: This study intends to explore the role of STAT3 in ROS levels and
for expression of base excision repair (BER) genes in breast cancers cell line.
Methodology: To investigate the relationship between STAT3, ROS levels and BER
we used the cell line MDA-MB-231, inhibits STAT3 using Stattic, measured the
cellular ROS levels using DCFH-DA in FACScallibur (BD). The changes in BER
genes expression were carrying out by RT-qPCR. Lipid peroxidation was estimated
by tert-butyl hydroperoxide and the Nitric Oxide (NO) concentration was estimated
by chemilunicescense. Results: Our results showed a decrease in 50%, 80% and 98%
of ROS levels, using doses of 5 M, 10 M and 20 M of Sttatic, respectively, at
24h. The APEX1, the main player of BER, mRNA levels diminished 3 fold and
MBD4, a glycosilases, increased 4 fold in Stattic-treated cell compared with
untreated. Analyses of redox profile showed no change in NO and hydroperoxides
concentration after STAT3 inhibition. Conclusion: Our preliminary results suggest
that STAT3 inhibition decreases ROS levels and regulated the expression of BER
genes.
Funding: FAPERJ, CNPq, CAPES.
(3)
Human Cytogenetics Laboratory, Institute of Biological

Sciences, Federal University of Par, Belm, Par.
Laboratory of Molecular Biology, Center Biological and
Health Sciences, State University of Par, Belm, Par.
National Primate Center, Evandro Chagas Institute, Belm,
Par.
Presenting author: Dejair da Silva Duarte

Address: Lane So Pedro, N: 97, edifice Victor V, apartment 602
E-mail: dejair_duarte@outlook.com ; Telephone contact (mobile): (91) 98398-8099
(institutional): 3201-8188
Nayara Cristina Lima de Oliveira; Email: nayara.oliveira@icb.ufpa.br ; Telephone:
mobile: (91) 8018-5409 ; Institutional: 3201-8188
Danielle Cristinne Azevedo Feio; Email: daniellefeio@yahoo.com.br ; Telephone:
Institutional: 3201-8188
Patrcia Danielle Lima de Lima; Telephone: Institutional: (91) 3276-3387
Jos Augusto Pereira Carneiro Muniz; Telephone: Institutional: (91) 3213-0411
Rommel Mario Rodrguez Burbano; Email: rommel@ufpa.br ; Telephone: Mobile:
(91) 8836-4667 Institutional: 3201-8188
The chemotherapy placement system, called LED, has the ability to concentrate in
tumor tissue after injection into the bloodstream, thus being able to direct tumor
drugs incorporated into the nanoemulsion. The objective of this study is to evaluate
the chronic toxicity of nanoparticles associated with Paclitaxel chemotherapy (LDEPTX) analyzing the lipid profile of non-human primate species Cebus apella.The
groups were assigned to negative control (NC); Experimental (EXP 1 and EXP2)
where animals received the LDE-PTX for intravenously at two different doses of 175
mg/m2 and 250 mg/m2, respectively; and the positive control (CP1 and CP2) animals
received the drug intravenously in commercial form at the same doses used in the
experimental group, respectively. Data were submitted to analysis of variance
ANOVA with Bonferroni post-test with significance set at p <0.05, by BioEstat5.3.
The experimental treatment showed a concentration of lower triglycerides and VLDL
than CP 2, group treated with the highest concentration of drug commercial. The
concentration of HDL EXP group 1 and 2 were higher than in CP1 group. While the
concentrations of cholesterol and LDL levels were higher in EXP 2 group than in CN
and CP2 groups.Thus, the analysis of the experimental drug toxicity may contribute
to reduced side effects and knowledge of future patients for treating cancer and for
improving the use of LDE-PTX. The project was approved by the ethics committee
in research with experimental animals UFPA (BIO008-11) and funded by the
National Council for Scientific and Technological Development.

A25
A26
NINTEDANIB EFFECTS ON DELAYING CANCER PROGRESSION AND

DECREASING COX-2 IMUNOREACTIVITY IN THE PROSTATE
ANTERIOR LOBE
ASSESSMENT OF ALLELOPATHY BETWEEN DIFFERENT CELL LINES:

A MODEL FOR EARLY STAGES OF CANCER CELL PROLIFERATION
Letcia Ferreira Alves (1), Raquel Frenedoso da Silva (1), Ellen Nogueira Pangrazi
(1), Valria Helena Alves Cagnon (1).
(1) Department of Structural and Functional Biology, State University of Campinas,
So Paulo, Brazil.
Email: leferralves@gmail.com
Inst.: (19) 3521-6113
Mobile: (11) 99792-8631
Prostate cancer is the most prevalent type of cancer in men around the world. Due to
its high incidence, new therapies have been evaluated, including drugs capable of
inhibiting the FGF pathways, as Nintedanib. The aim of study herein is to evaluate
the Nintedanib therapy effects on the morphology and in COX-2 imunoreactivity in
the prostate anterior lobe in different grades of the tumor progression in TRAMP
mice. A total of 25 TRAMP mice was separated into experimental groups; control
groups (TC) and Nintedanib groups (TN) which were divided into TC0 (8 week old
mice), TC12 (12 week old mice), TC16 (16 week old mice), receiving vehicle and
TN12 (12 week old mice) - treated from 8 to 12 week of age with
10mg/Kg/day dose, and TN16 (16 week old mice) treated from 12 to 16 week of age
with 10mg/Kg/day dose. At the end of the treatment, animals were sacrificed and the
anterior lobe of the prostate collected and submitted to morphological and
immunohistochemistry analyses. The results showed that Nintedanib delayed the
prostate carcinogenesis progression, with reduction of over 20% at the frequency of
tissue injuries, considering particularly the TN16 group in relation to its control
group. Also, decreased COX-2 levels were verified in both groups treated with
Nintedanib in the prostate anterior lobe. Thus, we concluded that Nintedanib was
efficient in reducing the tumor progression and also, despite not directly being
related to inflammation, Nintedanib may adversely affect inflammatory pathways,
favoring prostate cancer delay.
CEUA:4020-1 FAPESP:16747-3
Mauro Csar Cafund de Morais (1,2,*), Alexandre Sarmento Queiroga (1,2), Alan
Sabino (2), Tharcsio Tortelli Jr. (1), Yuri Suhov (3), Izabella Stuhl (4), Roger
Chammas (1), Alexandre Ferreira Ramos (1,2)
(1) Department of Radiology and Oncology, Faculty of Medicine, University of Sao
Paulo, Sao Paulo, Brazil.
(2) School of Arts, Science and Humanities, University of Sao Paulo, Sao Paulo,
Brazil.
(3) DPMMS, University of Cambridge, UK.
(4) University of Debrecen, Hungary.
* Contact information:
mauro.morais@hc.fm.usp.br
Av. Dr. Arnaldo, 251, 8o andar
CEP 01246-000, Pacaembu, Sao Paulo, SP, Brazil
Ph.: +55(11)3893-3552
Mob.: +55(11)99724-1166
Background: Allelopathy is the process of growth inhibition of one species of plant
by another through the release of toxic molecules in its surroundings. This process is
analogous to the inhibition of cell proliferation by contact that controls tissue
density. Therefore, we propose to apply the theoretical machinery used for
description of allelopathy in the early stages of cancer cell proliferation, when the
tumor cell coexists with normal cells in its tissue of origin (in situ carcinoma, e.g.).
Aims: Here, we propose a strategy to approach contact inhibition (or allelopathy) in
cell culture and co-culture experiments and show the appropriateness of our
approach on description of cell proliferation. Methods: Cell density was estimated
daily for melanoma (SK-MEL-147) and keratinocyte (HaCaT) cell lines.
Fluorescence microscopy images from co-culture of the two cell lines (density ratio
1:10) were captured after fixing and labeling with anti-CDH1 antibody for
distinction. Nuclei was stained with Hoechst 33258 to count cells. Results: The
formation of melanoma clusters was observed in co-culture experiments.
Proliferation rate was measured using logistic growth model. Density ratio decreased
to 1:5 after 8 days in co-culture despite showing approximately the same
proliferation rates. Conclusion: While cell proliferation could be described using
logistic growth when there was no interaction between two or more cell lines, for a
more complex system, our results showed that loss of contact inhibition (interaction
by repulsion) needs to be taken into account, as an underlying mechanism for the
dominance of tumor cells in neoplasia growth.
This work is supported by CAPES (CAPES Project n. 88881.062174/2014-01)
A27
A28
EVALUATION OF THE ANTITUMOR ACTIVITY OF HER2 BINDING

PEPTIDES
RUTHENIUM COMPLEX [RU(CH3CO2)(DPPB)(BIPY)]PF6 INDUCES

APOPTOSIS IN MDA-MB-231 TRIPLE NEGATIVE BREAST CANCER
CELLS
Natalia Lima Coelho (1), Giuliana Bolognese Muniz (1), Carina Mucciolo Melo (1),
Maria Aparecida Pinhal (1,2)
(1)
(2)
Departament of Biochemistry / Molecular Biology, Federal University

of So Paulo, So Paulo, Brazil.
Department of Biochemistry, Faculdade de Medicina do ABC, Santo
Andr, So Paulo, Brazil.
E-mail: coelho.natalia@outlook.com
Address: Rua Trs de Maio, 100 4 andar, Disciplina de Biologia Molecular,
Departamento de Bioqumica, Vila Clementino, So Paulo, SP CEP 04044-020,
Brazil
Phone: (+55) (11) 55764442, ext. 1185
Mobile: (+55) (11) 973985155
Background: The receptor for epidermal growth factor HER2 has fundamental role
in cell proliferation and differentiation. In colorectal adenocarcinoma, HER2 is
overexpressed in approximately 85% of the cases. The overexpression of such
receptor is directly associated with more aggressive disease, resistance to chemo- and
hormone-therapy and poor prognosis. Previous work in our group, using Phage
Display technology selected two specific peptides that bind specific to HER2. Aims:
The objective of this study was to investigate the effect of the HER2-binding
peptides as potential anticancer compounds for colorectal cancer lineages. Methods:
The antitumor activity of these peptides was evaluated in cell proliferation and cell
viability assays. It was also examined the glycosaminoglycans profile, HER2,
heparanase and metalloprotease-9 expression of colorectal tumor cell lines. Results:
HER2, heparanase and metalloprotease-9 presented higher mRNA expression in the
Caco-2 compared to the HCT-116. Both lineages synthesized heparan sulfate and
chondroitim sulfate, whereas HCT-116 also has dermatan sulfate. HCT-116 line
secretes higher proportion of glycosaminoglycans compared to Caco-2 cells. The
anti-HER2 peptides have the ability to reduce cell viability of both cell lineages.
Furthermore, it was showed that such peptides have greater efficiency as
antineoplastic drug compared to the anti-angiogenic monoclonal antibody
bevacizumab, that binds specific to VEGF inhibiting the ligation with VEGF
receptor. Conclusion: The data obtained in this study indicate that possibly antiHER2 peptides evaluated in the present study may be used as potential anticancer
drug and present the prospect of being used as target therapy.
Funding: CAPES, CNPq. Ethics Committee: UNIFESP, #0645/10.
Keywords: colorectal carcinoma, HER2, peptides.
Ceclia Patrcia Popolin1, Joo Paulo Barolli Reis2, Amanda Blanque Becceneri1,
Anglica Ellen Graminha1, Angelina Maria Fuzer1, Mrcio Aurlio Pinheiro
Almeida3, Rodrigo de Souza Corra4, Alzir Azevedo Batista2 and Mrcia Regina
Cominetti1.
1
Universidade Federal de So Carlos, Departamento de Gerontologia,

ceciliapopolin@gmail.com
(16)
3306-6672,
amandabecc@gmail.com,
angelina_fuzer@yahoo.com.br,
mcominetti@ufscar.br
(16)
3306-6663;
2
Universidade Federal de So Carlos, Departamento de Qumica, daab@ufscar.br,
3
jpbarolli@yahoo.com.br; Universidade Federal do Maranho, Coordenao
de Cincia e Tecnologia, almeida.marcio@ufma.br; 4Universidade Federal de Ouro
Preto, Departamento de Qumica, , rodrigoquimic@yahoo.com.br
Background: Triple negative breast cancer (TNBC), in which cells do not have
estrogen, progesterone, or HER2 receptors, are responsible for 10-20% of breast
cancers. TNBC is typically treated with a combination of therapies such as surgery,
radiation therapy, and chemotherapy. Metal based drugs for chemotherapy, as
cisplatin, have been widely used. However, its side effects represent a limitation for
clinical use. Ruthenium has unique properties that justify its use as a candidate for
antitumor medication, such as possibility to occupy a high number of spatial
positions compared to cisplatin and the ability to mimic iron in binding several
biomolecules. These characteristics have made Ruthenium complexes promising
antitumor and antimetastatic candidates. Aim: The aim of this study was to
investigate the effects of [Ru(CH3CO2)(dppb)(bipy)]PF6 on the morphology and
apoptosis in TNBC MDA-MB-231 cells. Methods: Cells were treated with different
concentrations of [Ru(CH3CO2)(dppb)(bipy)]PF6 and adequately prepared for each
experiment. Experiments were as follows: morphological analysis, flow cytometry
with the PE-Annexin V Apoptosis Detection Kit (BD), nuclear fragmentation with
DAPI and Quantitative Real Time PCR (RT-qPCR). Results: The complex
[Ru(CH3CO2)(dppb)(bipy)]PF6 was able to change the morphology of MDA-MB231 cells and to induce apoptosis and nuclear fragmentation in these cells. The
complex was also able to upregulate the expression of Bax and caspase-3 genes
whereas did not affect the Bcl-2 gene expression. Conclusion: These results show
that [Ru(CH3CO2)(dppb)(bipy)]PF6 was able to induce apoptosis on TNBC MDAMB-231 cells. However, more studies should be performed in order to elucidate the
mechanisms of action of this complex.
Financial support: CAPES, FAPESP 2013/00798-2
As opinies, hipteses e concluses ou recomendaes expressas neste material so
de responsabilidade dos autores e no necessariamente refletem a viso da FAPESP.

A29
A30
METFORMIN
AND
RUXOLITINIB
INHIBITS
CELL
CYCLE
PROGRESSION AND RB PROTEIN ACTIVATION IN JAK2V617F CELLS
IGF1R SIGNALING MODULATES MYC EXPRESSION IN ACUTE

LYMPHOBLASTIC LEUKEMIA CELLS
Joo Agostinho Machado-Neto, Bruna Alves Fenerich, Renata Scopim-Ribeiro, Ana

Paula Nunes Rodrigues Alves, Jaqueline Cristina Fernandes, Priscila Santos
Scheucher, Eduardo Magalhes Rego, Fabiola Traina
Jaqueline Cristina Fernandes, Ana Paula Nunes Rodrigues Alves, Joo Agostinho
Machado-Neto, Renata Scopim-Ribeiro, Bruna Alves Fenerich, Fernanda Borges da
Silva, Belinda Pinto Simes, Eduardo Magalhes Rego, Fabiola Traina.
Department of Internal Medicine, University of So Paulo at Ribeiro Preto Medical

School, Ribeiro Preto, So Paulo, Brazil
Department of Internal Medicine, University of So Paulo at Ribeiro Preto Medical

School, Ribeiro Preto, So Paulo, Brazil.
The authors declare that they have no competing interests.
The authors declare that they have no competing interests.
Presenting author: jamachadoneto@gmail.com; phone: 55-16-3602-2888
Presenting author: jamachadoneto@gmail.com; phone: 55-16-3602-2888
Background: A recurrent gain-of-function mutation, V617F in JAK2, confers growth

factor-independent proliferation for hematopoietic cells in myeloproliferative
neoplasms (MPN). The current therapies for MPN are limited and do not lead to
elimination of the malignant clone. Aims: To investigate the effects of metformin (a
mitochondrial complex I inhibitor) and/or ruxolitinib (a selective JAK1/2 inhibitor)
on cell signaling and cell cycle progression. Methods: HEL and SET2 (JAK2V617F)
cell lines treated or not with metformin (10 mM) and/or ruxolitinib (300 nM) were
submitted to cell cycle analysis (flow cytometry) and PCR-array for PI3K/AKTrelated genes. Gene and protein expressions were evaluated by qPCR and Western
blot. Statistical analyzes were performed by ANOVA test. Results: In HEL cells,
PCR-array identified 11, 14 and 17 genes modulated by metformin, ruxolitinib and
combined treatment, respectively. The validation experiments confirmed that
metformin and/or ruxolitinib treatment reduced cyclin D1 (CCND1) and upregulated
p27 (CDKN1B) in HEL and SET2 cells (p<0.05). Notably, we observed a
downregulation of retinoblastoma (RB) phosphorylation, a cyclin D1 target and a
key cell cycle progression-related protein, in both JAK2V617F cell lines upon all
treatment conditions. In agreement, our functional analysis revealed an increase in
the percentage of cells in G1/S phase and a significant reduction in the percentage of
cells in G2/M phase (p<0.05) for metformin- and/or ruxolitinib-treated HEL and
SET2 cells. Conclusion: Our exploratory study establishes novel molecular
mechanisms of metformin and ruxolitinib action on JAK2V617F aberrant signaling
and provides insights for development of therapeutic strategies for MPN.
Background: Insulin-like growth factor 1 (IGF1) and its receptor regulate normal cell
growth and contribute to transformation of malignant cells. In fibroblasts, IGF1 was
found to induce nuclear IRS1 and beta-catenin translocation and MYC transcription.
Aims: to investigate the IRS1/beta-catenin axis in acute lymphoblastic leukemia
cells. Materials and Methods: Samples were obtained from 58 patients with ALL and
13 healthy donors. ALL cell lines (Jurkat, MOLT-4, Namalwa and Raji) were used.
Gene expression was measured by quantitative real-time PCR. Protein expression,
associations and cellular localization were evaluated by immunoprecipitation,
western blotting analysis, subcellular fractionation and confocal microscopy. Cells
were submitted to starvation in serum-free medium for 24 hours, followed by IGF1
stimulation (20 ng/mL) and/or IGF1R pharmacological inhibition (OSI-906; 20 M),
for 24 hours. Results: IRS1, beta-catenin and MYC mRNA expression were elevated
in ALL patients compared to normal controls. A positive correlation between betacatenin and MYC mRNA expression and between IRS1 and MYC was found. In ALL
cell lines, high expression of IGF1R, IRS1, beta-catenin and MYC protein were
observed. IRS1 and beta-catenin was found to be located in the nuclei and the
cytoplasm of ALL cell lines. Co-localization of IRS1/beta-catenin, in both nuclei and
cytoplasm of ALL cell lines, were observed. In Jurkat cells, a constitutive IRS1 and
beta-catenin protein interaction were observed; and IGF1 stimulation increased
nuclear translocation of IRS1 and beta-catenin. OSI-906 decreased IGF1R tyrosine
phosphorylation, nuclear beta-catenin translocation and MYC protein expression in
Jurkat cells. Conclusion: The IRS1/beta-catenin axis is activated in ALL cells.
Supported by CNPq and FAPESP.
A31
A32
ENGAGEMENT OF PRION PROTEIN WITH THE CO-CHAPERONE HOP

REGULATES THE MAINTENANCE OF GLIOBLASTOMA STEM-LIKE
CELLS
ANALYSIS OF ANTIOXIDANT AND MUTAGENIC PROPERTIES OF

QUERCETIN: AN EXAMPLE OF PARADOXICAL EFFECT
Rebeca Piatniczka Iglesia1, Mariana Brando Prado1, Lilian Cruz1, Vilma Regina
Martins2, Tiago Gss dos Santos2, Marilene Hohmuth Lopes1
(1)
(2)
1
Department of Cell and Developmental Biology; Institute of Biomedical Sciences;
University of Sao Paulo; Sao Paulo, Brazil. Zip code 05508-900.
2
International Center for Research and Education; A.C. Camargo Hospital; Sao
Paulo, Brazil. Postal code 02056-070.
Glioblastoma (GBM), a highly aggressive brain tumor, contains a subpopulation of
glioblastoma stem-like cells (GSCs) that play roles in tumor maintenance, invasion,
and therapeutic resistance. GSCs are therefore a promising target for GBM
treatment. Our group identified the prion protein (PrP) and its partner, the cochaperone HOP (heat shock organizing protein), as potential target candidates due to
their role in GBM tumorigenesis and in neural stem cell maintenance. Herein, we
show that, when GBM cells are cultured as neurospheres, they express specific
stemness markers such as CD133, CD15, Oct4 and SOX2, PrP is upregulated
compared to monolayer culture and co-localizes with CD133, suggesting that they
are appropriated models for GSCs. PrP silencing downregulates the expression of
molecules associated with cancer stem cells, upregulates markers of cell
differentiation and affects GSC self-renewal, pointing to a pivotal role for PrP in the
maintenance of GSCs. Exogenous HOP treatment increases proliferation and selfrenewal of GSCs in a PrP-dependent manner while HOP knockdown disturbs
proliferation process. Besides, disruption of the PrP-HOP complex by a HOP
peptide, which mimics PrP binding site, affects GSC self-renewal and proliferation,
indicating that HOP-PrP complex is required to GSCs stemness. Additionally, PrPdepleted GSCs downregulates cell-adhesion related proteins and impairs cell
migration indicating a putative role for PrP in cell surface stability of cell adhesion
molecules and GBM cell invasiveness, respectively. In conclusion, our results show
that HOP-PrP engagement may be a promising target for therapeutic intervention in
GBM.
Thaylene Alexandra (1,2), Rodrigo Pinheiro Araldi (1)

(1) Universidade Mogi das Cruzes, So Paulo, Brazil
(2) Genetics Laboratory, Butantan Institute, So Paulo, Brazil
Quercetin is a flavanoid found in bracken fern Peteridium aquilinum, which is
present in both human and bovine diet. Although studies had demonstrated the
antioxidant potential of quercetin, its consumption is considered a co-factor for
papillomavirus-associated cancer development, demonstrating a paradoxical effect.
However, due to antioxidant and cardiovascular protective properties, the dietary
supplementation with quercetin has been verified. Thus, this study evaluated the
antioxidant and mutagenic properties of quercetin to know the benefits and eventual
risks of its supplementation. All methods were approved by Ethic Committee
(process 747515/16). Antioxidant properties was evaluated through the
mitochondrial potential analysis (m), using the MitoTracker Deep Red probe and
DCFH-DA assay. These analyses were performed in primary culture of
papillomavirus-free bovine epithelial and VERO cell lines by flow cytometry.
Mutagenic analysis was performed by comet assay, using five samples of bovine
peripheral blood, which were divided in three groups: negative control, not treated
with any drug, positive control, treated with 50 g/mL of cyclophosphamide and
treated with 10 g/mL of quercetin. Results showed that the addition of quercetin
promotes an expressive reduction of reactive oxygen species (ROS) levels, but
without change the m. These results demonstrate that quercetin is able to bind to
free radical, conferring antioxidant properties, as described in literature. However,
cells treated with quercetin showed levels of clastogenicity similar to those verified
in cells treated with cyclophosphamide. This result demonstrates the quercetin,
although antioxidant can interact with DNA promoting breaks (clastogenesis). Thus,
the dietary supplementation with quercetin can lead to genomic instability,
increasing the cancer risk.

A33
A34
EVALUATION OF THE ANTITUMOR EFFECTS OF THE RUTHENIUM

COMPLEX [RU(AG)(DPPE)2]PF6 ON BREAST CANCER CELLS
CYTOTOXIC EFFECTS AND THE PROBABLE MECHANISM OF CELL

DEATH INDUCED BY BRAZILIAN STINGLESS BEES PROPOLIS
TOWARDS A HUMAN MELANOMA CELL LINE
*Marina Arajo Naves1, Liany Johanna Luna Dulcey1, Anglica Ellen Graminha1,
Alzir Azevedo Batista2, Marcia Regina Cominetti1
Jlia Cisilotto (1); Helosa Fernandes (1); Tnia Beatriz Creczynski-Pasa (1)
Department of Gerontology, 2 Department of Chemistry, Federal University of So

Carlos, CEP 13565-905, So Carlos SP, Brazil.
(1) Programa de Ps-Graduao em Farmcia, Universidade Federal de Santa

Catarina, Florianpolis, Santa Catarina, Brazil
*Contact Information:
e-mail: marinanaves_5@hotmail.com
Adress: Laboratory of Biology of Aging - LABEN, Department of Gerontology,
Federal University of So Carlos - UFSCar
Rod. Washington Luis, km 235 - So Carlos SP, Brazil - CEP:13565-905.
Phone: (16) 3306-6672
Mobile: (16) 99717-1991
juliacisilotto@hotmail.com/telephone number: (48) 3721-2212 and (48) 9648-4420
Background: Ruthenium complexes emerged as an alternative for less aggressive

treatment since cisplatin presents several side effects. Objectives: This study aims to
evaluate effects of [Ru(AG)(dppe)2]PF6 complex as a potential antitumor agent. This
compound was tested in MDA-MB-231 cells. Effects on cytotoxicity, induction of
apoptosis, migration and morphology of cells were investigated. Methodology: To
evaluate the effects of this complex on cell morphology, these were exposed to
different concentrations and photos were taken in different times of treatment.
Inhibition of cell proliferation was evaluated through a MTT colorimetric assay,
where cells were exposed to different concentrations, along with IC50 determination.
To evaluate the effects on migration, a wound healing assay was performed.
Apoptotic cells rate after treatment was determined using a PE Annexin V Apoptosis
Detection Kit from BD Pharmigen. Results: The sensibility of cells to
[Ru(AG)(dppe)2]PF6 can be seen in the morphology assay, in which cells structure
begins to alter in 8h at 12.5M, in 24h at 6.25M and in 48h at 0.195M, with cell
death at higher concentrations in the same times. The IC50 value for
[Ru(AG)(dppe)2]PF6 over MDA-MB-231 cell proliferation in 48h was 0.80M. The
complex inhibited the migration, proliferation of cells and induced apoptosis in a
concentration-response manner. Conclusion: These results suggest that
[Ru(AG)(dppe)2]PF6 complex affects the structure, proliferation and triggers cell
death of MDA-MB-231 cells in a concentration-response manner, which makes this
compound an interesting alternative in the study of future pharmaceuticals with
potential and less aggressive antitumor effects.
Melanoma is an aggressive type of cancer known as resistant against current

chemotherapeutic agents. Propolis has been showing several biological activities,
including cytotoxicity. Nevertheless, there are few works that report Brazilian
stingless bees propolis activity on cancer cells. Therefore, the present study aimed to
evaluate the cytotoxicity of propolis extracts from Scaptotrigona bipunctata
(Tubuna) and Melipona quadrifasciata (Mandaaia) bees in a melanoma cellular
model. The cytotoxicity was assessed by MTT assay in the SK-MEL-28 melanoma,
and the non-tumoral human melanocyte (NGM) cell lines. Cell cycle and apoptosis
were assessed by flow cytometry. Reactive oxygen species (ROS) and mitochondrial
membrane potential (m) were evaluated using the fluorescent dyes DCFH-DA
and JC-1, respectively. Finally, the expression of some key proteins was measured
by Western Blot. The results of cytotoxicity showed that CC50 for Mandaaia
propolis was 60 g/mL and for Tubuna propolis was 197 g/mL. Propolis extracts
seem to be selective towards melanoma cells (~two-fold), and when associated with
Vemurafenib, a drug used in melanoma treatment, a synergic effect was noted.
Furthermore, propolis extracts increased the ROS accumulation (~two-fold), reduced
the proteins Bcl-2 and AKT-3 levels and reduced cell migration and invasion. Both
extracts induced apoptosis by reducing the m, while only Mandaaia propolis
extract induced cell cycle arrest in G2/M. In conclusion, these results suggest that
stingless bees propolis induce cell death by apoptosis associated to the AKT survival
pathway, highlighting the potential of these raw materials in melanoma
chemotherapy.
Financial support: Capes
Funding Support: CNPq 131502/2015-8/ FAPESP 2013/00798-2.

de responsabilidade dos autores e no necessariamente refletem a viso da CNPq
e/ou FAPESP.

A35
A36
ROLE OF HEXOSAMINE BIOSYNTHETIC PATHWAY FOR GROWTH

AND PROGRESSION OF COLON CANCER
THE ROLE OF ECHINOCHROME A, DERIVED FROM L. VARIEGATUS,

IN TUMOR CELL LINE MCF-7
Andria Vasconcelos-dos-Santos1, Hector Loponte1, Natalia R. Mantuano1, Isadora

A. Oliveira1, Leonardo K. Teixeira2, Jlio Cesar Madureira de-Freitas-Junior2,
Fernanda Moll3, Ronaldo Mohana Borges1, Jos Andrs Morgado-Daz2, Wagner B.
Dias1 and Adriane R. Todeschini1*
Luciana Machado Dzik (1), Karina Fernandes Oliveira Rezende (1), Douglas Oscar
Ceolin Mariano (2), Daniel Carvalho Pimenta (2), Jos Roberto Machado Cunha Da
Silva (1), Vanessa Morais Freitas (1).
Instituto de Biofsica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro;

Instituto Nacional do Cncer; 3Instituto de Cincias Biomdicas, Universidade
Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
E-mail: andreia@biof.ufrj.br
Fone: Institutional: 55 (21)3938-654355
Mobile: (21)997725305
2
Background: Hyperglycemia per se has been considered a risk factor for cancer. Our
hypothesis is that high glucose increases colon cancer malignity by altering cell
glycosylation though hexosamines biosynthetic pathway (HBP). Aims: To test our
hypothesis we increased glucose availability of HBP and modulated its rate-limiting
enzyme, glutamine-fructose-6-phosphate amidotransferase (GFAT) in colon cancer.
Methods: Colon adenocarcinoma (MC38) cells were cultured in normal glucose (5
mM-LG) or high glucose (25 mM-HG). Hyperglycemia was induced by
streptozotocin (STZ) treatment in C57BL/6 mice. GFAT was modulated by
inhibition with 6-Diazo-5-oxo-L-norleucine or by gene silencing. Glycophenotype
was monitored by lectins/flow cytometry. Cell proliferation and invasion were also
evaluated. Cells were injected in euglycemic or STZ-treated animals followed by
tumor growth analysis. Tumor glycosylation was detected by lectins/histochemistry
and UDP-GlcNAc biosynthesis by MS imaging. The metastasis study included
fluorescence analysis of GFP-MC38 cells in lungs. We analyzed the GFAT protein
and mRNA levels of adenocarcinoma and normal tissues from patients. Results: We
showed altered glycosylation, increased cell proliferation and invasion in MC38-HG
cells. STZ-treated animals presented exacerbated tumor growth, metastasis and
aberrant glycophenotype. Modulation of GFAT by, both, pharmacological and gene
silencing lead to significant reduction of tumor progression. Agreeing with
experimental results are the findings showing that biopsies from patients colon
carcinoma express more GFAT and aberrant glycans, compared to normal tissue.
Conclusion: Our study suggests an important role of the HBP, in special its limiting
enzyme GFAT, for tumor progression of colon cancer and brings to light a new
target for cancer chemotherapy.
(1) Department of Cell and Tissue Biology, University of Sao Paulo, So Paulo,
Brazil
(2) Butantan Institute, So Paulo, Brazil
lu.dzik@usp.br
Institutional phone: +55(11)3091-7223/ mobile: +55(11)98235-6135
karinaforezende@yahoo.com.br
vfreitas@usp.br
jrmcs@usp.br
douglasceolin@gmail.com
dcpimenta@butantan.gov.br
Breast cancer is the world's leading cause of death among women, and the treatments
include hormone therapy, total mastectomy, and chemotherapy. However, tumors
that present worse prognosis dont respond to current therapies, imposing a need to
development of new therapeutic approaches. The use of marine animals as sources
for new bioactive molecules appears to be a quite promising method. Echinochrome
A (EA) is the most abundant pigment from L. variegatus, and was reported to have
anti-inflammatory and antioxidant activity in different cell lines. Moreover,
naphthoquinones are recognized as anti-tumor agents, acting through the inhibition
of cell proliferation. The aim of this project is to investigate possible EA anti-tumor
activity, evaluating the specific function of EA on human breast cancer cells (MCF7) viability and morphology. EA was isolated from celomatic fluid of L. variegatus,
using RP-HPLC. MCF-7 cells were then treated with different concentrations of EA
and growth inhibitory activity was tested using MTT assay. Cell morphology was
observed through staining of f-actin, and subsequently analysis by fluorescence
microscopy. Treatment starting from 25uM of EA induced decrease of cell viability
and cells exhibited morphological changes, including an increase in ring-like
formations of F-actin, condensed exclusively in the perinuclear zone, suggesting a
disruption of the microtubule network. Together with decreased cell viability in a
dose-and-time-dependent manner, this data suggest an antitumor activity of EA on
MCF-7 cell line, however, more studies are needed to confirm the role of EA on
tumor cell biology. Supported by CAPES, FAPESP and CNPQ.
The animal study was approved by the Committee for the Use of Experimental
Animals of our institution (IBCCF214-09/16). The human sample study was carried
out with approval of the Brazilian National Cancer Institutes Ethic Committee
(Registration number: 84/04).
Funding. CNPq, CAPES, FAPERJ.

A37
A38
SPATIAL STOCHASTIC MODEL FOR INVESTIGATION OF CELL

PROLIFERATION ON CARCINOMA IN SITU
A SPATIAL MODEL OF TUMOR PROGRESSION AND HETEROGENEITY
Alan Utsuni Sabino (2,*), Alexandre Queiroga (1,2), Mauro C. Cafund de Morais
(1,2), Yuri Suhov (3), Isabela Stuh (4), Roger Chammas (1), Alexandre Ferreira
Ramos (1,2)
(1) Department of Radiology and Oncology, Faculty of Medicine, University of So
Paulo, So Paulo, Brazil.
(2) School of Arts, Science and Humanities, University of So Paulo, So Paulo,
Brazil.
(3) Department of Pure Mathematics and Mathematical Statistics, University of
Cambridge, United Kingdom.
(4) University of Debrecen, Hungary.
alan.sabino@usp.br
Av. Arlindo Bttio, 1000
Background: Allelopathy is the inhibition of the growth of a species by another
through the release of chemicals on its surroundings. That phenomenon is analogous
to cell density control on tissues by contact inhibition. Hence, provided the
appropriate interpretation, those phenomena can be described by the same theoretical
machinery. The Widom-Rowlinson (WR) model describing a system of two (or
more) species interacting by repulsion (inhibition) is used for the description of
allelopathy. In that model, the repulsive degree of the species can be classified in
increasing order and, for a sufficiently high birth-death rates, the species having the
smallest repulsive degrees shall prevail on a spatial domain after a sufficiently large
time interval. Aims: We propose a modified version of the WR model for the
description of the progression of the cell population in a carcinoma in situ. Methods:
We simulate the dynamics of the WR model in terms of a markovian cell bith-deathmigration process in a 2D grid. The interaction among cells is given in terms of
exclusion diameters whose sizes depend on the degree of inhibition between two
cells. Results: We show that the prevalence of the most tolerant species on the
spatial domain occurs for a range of the birth-death ratios of the interacting cell
types. Conclusion: Our simulation results demonstrated that our model has
dynamical properties observed on a carcinoma in situ and, hence, can be a useful tool
on the investigation of tumor progression.
This work is supported by PVE-CAPES (CAPES Project n. 88881.062174/2014-01)
and PET MEC.
Alexandre Sarmento Queiroga (1**), Mauro Cafund Morais (1,2), Alan Utsuni
Sabino (2), Roger Chammas (1), Alexandre Ferreira Ramos (1,2).
(1) Department of Radiology and Oncology, Medicine College, University of Sao
(2) School of Arts, Science and Humanities, University of Sao Paulo, Sao Paulo,
Brazil.
* *Contact information:
asq@usp.br
Av. Dr. Arnaldo, 251, 8o andar
CEP 01246-000, Pacaembu, Sao Paulo, SP, Brazil
Mob.: +55(11)94343-9252
Background: Somatic evolution, resulting from the combination of both genetic

instability and local environment selective pressure, is a key aspect of
carcinogenesis. It induces individual heritable variation among tumor cell
populations, as observed in terms of differential survival and duplication rates of
subpopulations. Such variability could be responsible for intrinsic mechanisms of
treatment resistance.
Aim: We are proposing a spatial stochastic model to investigate the evolutionary
dynamics of carcinogenesis.
Methods: In silico experiments are performed in a grid (2D) by considering a
minimal set of effective cellular processes: duplication, death, migration, inheritable
variation rate and quiescence.
Results: Our in silico experiments show that cell proliferation is well described in
terms of the above effective processes. The appearance of new variants has a
relationship with the ratio between cell duplication and death rates. Some values of
that ratio are more favorable for the increasing of the variant subpopulations and
display an inherent topographical bias.
Conclusion: The results of the in silico experiments show qualitative features
observed in cancer cell proliferation and show the potential application of our model
for the investigation of carcinogenesis. The discovery of specific parameters favoring
the increase of new variants are giving insights for the design of chemotherapeutic
strategies. Experimental investigation for estimating input parameters of our model
and enhance its predictive capacity are ongoing.
This work is supported by CAPES (CAPES Project n. 99999.003586/2014-06)
A39
A40
EXPRESSION AND INTERACTION OF CLAUDIN-3 AND OCCLUDIN

DURING THE COLORECTAL CANCER PROGRESSION
TACRINE DERIVATIVES: NEW APPROACH FOR GLIOBLASTOMA

TREATMENT
Jennifer Francisco de S (1,2), Maria Teresa dos Santos Guedes (3), Ivanir Martins
de Oliveira (4), Jos Andrs Morgado Daz (1), Waldemir Fernandes de Souza (1).
Fernanda Nunes (1), Evelyn Winter (1), Letcia Barros Silva (2), Sarah Coelho
Feitosa (2), Helio Gauze Bonacorso (2), Tnia Beatriz Creczynski Pasa (1)
(1) Cell Biology Program, Research Center, Brazilian National Cancer Institute, Rio
de Janeiro, Brazil.
(2) Bioscience Institute, Federal University of the State of Rio de Janeiro, Rio de
Janeiro, Brazil.
(3) National Tumor and DNA Bank, Research Center, Brazilian National Cancer
Institute, Rio de Janeiro, Brazil.
(4) Pathology Division, Brazilian National Cancer Institute, Rio de Janeiro, Brazil.
Rua Andr Cavalcanti, 37, 5 andar Centro - Rio De Janeiro - RJ - Brazil
CEP: 20231-050
Phone: +552132076537
E-mail: wfsouza@inca.gov.br; jennifer.de.sa@hotmail.com
BACKGROUND: During colorectal cancer (CCR) progression, epithelial cells
undergo cell-cell adhesion disassembly increasing their malignant potential. In this
context, the claudins and occludin (tight junctions proteins) play important role in
regulating events related with carcinoma progression. Previous studies have shown
altered expression of claudin proteins in human CCR samples. Nevertheless, the
molecular interactions that modulate the Tight Junction (TJ) functions and their role
regulating the malignant potential remain to be defined. AIMS: Evaluate the
importance of expression and interaction of the claudin-3 and occludin proteins
during the colorectal cancer progression. METHODS: Human colorectal specimens
were obtained from surgical resection of CCR patients treated in Brazilian National
Cancer Institute (INCA in portuguese). In all cases, we collected adenocarcinoma
specimens and their paired normal mucosa, which were classified by TNM staging.
Claudin-3 and occludin protein levels were analyzed by imunoblotting and the
interaction between these proteins were evaluated by immunoprecipitation. This
study is being carried out with approval of the INCA Research Ethics Committee
(Prot 84/04). RESULTS: Our results showed that samples in the earliest stage
presented decreasing of claudin-3 and occludin expression in tumors. Nevertheless,
samples in advanced stage presented a raised expression of these proteins in tumors.
Moreover, we observed decreasing of interaction between claudin-3 and occludin in
tumors during all stages of this disease. CONCLUSION: Together, our findings
indicate that during CCR progression there is increase of claudin-3 and occludin
expression in tumor tissue, which is accompanied by decrease of interaction between
these proteins.
(1)
(2)
Departamento de Cincias Farmacuticas, Universidade Federal de

Santa Catarina, SC, Brazil. Email: eve_winter@hotmail.com.
Telephone: (48) 3721-2200
Ncleo de Qumica de Heterociclos NUQUIMHE, Departamento de
Qumica, Universidade Federal de Santa Maria, RS, Brazil.
Glioblastoma is the more common and aggressive tumor of CNS. It is very resistant
to chemotherapies and present low survival rates. Temozolomide is the drug of
choice for glioblastoma but not effective enough. Therefore, new drugs for this kind
of cancer have to be developed. Thus, in this work, six molecules containing
spiroheterocycles,
namely,
7-amine-spiro[chromeno[4,3-b]quinoline-6,1cycloalkanes] and associated with tacrine drug were tested in a non-resistant (SF295)
and in a resistant (SF295-R) glioblastoma cell lines. Tacrine has been used for
Alzheimer treatment, being interesting because it might cross the blood brain barrier,
and spiroheterocycles are described as cytotoxic compounds. Six tacrine derivatives
were then synthesized and tested. For this, cells were maintained in standard culture
conditions and checked by their viability, cell death pathway, caspases activities,
ROS generating, mitochondrial membrane potential (MMP) disturbance. The
compounds induced cytotoxicity in SF295 and in SF295-R cells with CC50 of about
25 and 40 M, respectively, smaller than for temozolomide, mainly in resistant cell
line. The main cell death pathway induced was apoptosis in agreement with the
increase in caspase-3 activity. Compounds TR5 and TR6 decreased the MMP and
TR5 increased the caspase-9 activity, indicating a mitochondrial disruption and
action by intrinsic-apoptosis. Compounds TR6-3Me and TR6-4Me increased the
MMP and, as well as TR7 increased the caspase-8 activity, indicating action by
extrinsic apoptosis. Compound TR6-4TBu did not change caspases activities but
induced ROS generation. These results indicate these compounds as promising
because they were more active and selective then temozolomide, the drug of choice.
Funding support: CAPES, CNPq, UFSM and UFSC.
SUPPORT: FAPERJ, INCa/MS.

A41
A42
ANALYSIS OF SUSCEPTIBILITY/RESISTANCE AT CISPLATIN BY

TUMORSPHERES ISOLATES OF OVARIAN CANCER CELL LINES
AN ISOTHIOURONIUM SALT INDUCES MITOTIC ARREST AND

APOPTOSIS BY CASPASE CASCADE ACTIVATION IN LEUKEMIA CELL
LINES
Matheus Henrique Souza Santos(1,2), Heloisa Helena Marques Oliveira(1) , Letcia da

Conceio Braga(1,2) Luciana Maria Silva(1)
*Email: matheusrique@hotmail.com
(1)
Laboratrio de Biologia Celular, Fundao Ezequiel Dias - Rua Conde Pereira

Carneiro, 80, Gameleira, Belo Horizonte/MG, Brasil - CEP: 30510-010.
(2)
Centro Universitrio UNA - Rua dos Guajajaras, 175 - Centro, Belo

Horizonte/MG, Brasil - 30180-100.
Ovarian cancer (OC) is the most fatal gynecological malignancy worldwide.
Residual tumor cells after cytotoxic therapy has shown cancer stem cells (CSC's)
enrichment, suggesting the importance of these cells in chemoresistance and relapse
of disease. Considering the great heterogeneity associated at renewal capacity
described for OC, this study aims to available the resistance profile of tumorspheres,
derived from OC cell lines after treatment with cisplatin used like chemoresistance
predictor model in OC. Cell lines TOV-21G and SKOV- 3 were used to obtain
tumorspheres enriched of CSC's. For isolation of tumorspheres was used culture
medium as described in literature. Flow cytometry was perfomed using DAPI for cell
cycle profile analysis. The cytotoxicity assay was performed by MTT colorimetric
method. The IC50 (concentration that reduce cell viability to 50%) of cisplatin,
determined for monolayer OC cells, was 112 g/mL for SKOV-3 and 75 g/mL for
TOV-21G. The IC50 were applied on tumorspheres-derived of respective tumor cells
and the IC50 obtained for monolayer was not enough to kill the cells present on
TOV-21G tumorspheres derived. This result is a indicative of importance of
tumorspheres (enriched of CSC's) in resistance tumor cell line TOV-21G. Cell cycle
analysis showed that cisplatin cause S-phase arrest in response to cellular stress in
SKOV3 tumorspheres-derived. However, some cells have ability to move to G2/M
phase, being an indicator of resistance to damage caused by cisplatin. More tests will
be realized but the preliminary information are important to understand the cisplatin
resistance mechanisms in OC.
Laura Sartori Assuno1; Iara Fabrcia Kretzer1; Jelver Alexander Sierra Restrepo1;
Misael Ferreira2; Marcus Mandolesi S2; Tnia Beatriz Creczynski Pasa1
(1) Departamento de Cincias Farmacuticas, Universidade Federal de Santa
Catarina, Florianpolis, Brazil.
(2) Departamento de Qumica, Universidade Federal
de Santa Catarina,
laura_sartori22@hotmail.com - phone number: (055) (48) 3721-2212/ (055) (48)
965240-70
Isothiouronium salts present important antimicrobial properties as antibacterial and
antifungal, which developed our interest if they could present antitumoral properties
also. Therefore, in a previous work we showed the cytotoxic effect of 28
isothiouronium salts against leukemia cell lines. Thus, the aim of this work was to
investigate the cell death pathway induced by the isothiouronium salt, namely MF08,
which were the most active. For that, its activity against leukemia cell lines was
evaluated mechanistically. Firstly, the cytotoxicity of MF08 was tested in acute
lymphocytic leukemia, chronic myelogenous leukemia, promyelocytic leukemia and
non-tumoral cell lines by a colorimetric assay. The CC50 values were calculated and
the selectivity index (SI) was determined as the ratio between the CC50 of nontumoral and leukemia cell lines. Morphological evaluation was performed by
fluorescence and transmission electron microscopy. Cell cycle and cell death were
performed by flow cytometry. The reactive oxygen species (ROS) production and
caspases -3, -8, -9 and -12 activities were observed fluorimetrically. Autophagic
process was observed by confocal and transmission electron microscopy. The CC50
of MF08 was approximately 3 M and the SI close to 7. Also, MF08 seems to cause
a mitotic block with an increase in G2/M population and internal imbalance, leading
to endoplasmic reticulum stress and increase in ROS level, autophagic lysosomes
formation and caspase -8 and -3 activation, culminating in apoptosis. In conclusion,
MF08 presents a selective cytotoxicity to leukemia cells by different pathways, being
an interesting strategy to cancer treatment to overcome drug resistance.
Supported by: CAPES/CNPQ/FAPESC/UFSC
A43
A44
FASN EXPRESSION REDUCTION AND ANTI-ADIPOGENIC ACTIVITY

INDUCED BY N-PHENYLMALEIMIDES
HEAT SHOCK PROTEIN 70 (HSP70) AND PROTEASOME INHIBITORS

AS A THERAPEUTIC TARGET IN MULTIPLE MYELOMA USING IN
VITRO AND IN VIVO ANALYSES
Daiane Rosolen (1), Iara Fabricia Kretzer (1), Evelyn Winter (1), Vnia Floriani
Noldin (3), caro Andrade Rodrigues do Carmo (1), Fabola Branco FilippinMonteiro (2), Valdir Cechinel-Filho (3), Tnia Beatriz Creczynski-Pasa (1)*.
(1) Departamento de Cincias Farmacuticas, Centro de Cincias da Sade,
Universidade Federal de Santa Catarina, Florianpolis, SC, Brasil.
(2) Departamento de Anlises Clnicas, Centro de Cincias da Sade, Universidade
Federal de Santa Catarina, Florianpolis, SC, Brasil.
(3) Ncleo de Investigaes Qumico-Farmacuticas (NIQFAR), Universidade do
Vale do Itaja (UNIVALI), Itaja - SC, Brasil.
Correspondence to: Tnia B. Creczynski-Pasa, Departamento de Cincias
Farmacuticas, Centro de Cincias da Sade, Universidade Federal de Santa
Catarina, 88040-900, CP 476 Florianpolis, SC, Brasil. Tel: + 55 48 3721-2212; email: tania.pasa@ufsc.br
Several neoplasms overexpress fatty acid synthase (FASN) upon their dependence on
increased lipogenesis; targeting this protein is being considered a strategy in
anticancer drug development. This can be relevant for aggressive tumors such as
melanoma in which FASN overexpression has been associated with increased depth
of invasion and worse prognosis. We have shown that N-phenylmaleimides,
presented antitumor activity against L1210 leukemia and B16F10 melanoma with
evidences of interference in the energetic metabolism. Here, we aimed to investigate
if N-phenylmaleimides (M1 and M5) interfere with fatty acids metabolism and its
relation with cancer. For that, a model of pre-adipocytes differentiation (3T3-L1
cells) and also human melanoma cells (SK-Mel-147) were used. As results, when
3T3-L1 cells were exposed to M1 and M5 in the presence of an adipogenic
cocktail, intracellular lipid content decreased by 26 to 36%, marking the inhibition of
adipocyte differentiation. High selectivity indexes were obtained with both
compounds for tumoral cells. Cell cycle phases analysis revealed a remarkable
proportion of cells with DNA fragmented, and this was correlated to apoptosis and
necrosis, showed by Annexin-V/PI assay. Furthermore, M1 and M5 reduced FASN
expression by 19 to 39%, respectively. In conclusion, the compounds presented
antiadipogenic and antitumoral activities. The antitumoral activity is a possible
consequence of the FASN reduction, which in turn, may result in a fuel decrease to
cell proliferation. Notably, reduction of fatty acid synthesis might be a potential
target for cancer treatment in a strategy of hunger-strike, which valorizes
these molecules as candidates for drug development.
Supported by: CAPES/CNPQ/FAPESC.
Angela Isabel Pereira Eugnio (1); Veruska Lia Fook Alves (1); Mariana Bleker de
Oliveira (1); Bryan Strauss (2); Daniela Zanatta (2); Marimlia Porcionatto (1);
Gisele Wally Braga Colleoni (1).
(1)
(2)
Department of Clinical and Experimental Oncology, UNIFESP, So Paulo,

Brazil.
Translational Research Centre of Oncology, Institute of Cancer of So Paulo,
USP, So Paulo, Brazil.
Contact Details:
Address: Rua Doutor Diogo de Faria, 824, Hemocentro UNIFESP, CEP 04037-003,
So Paulo, SP, Brazil.
Email: angelaipem@gmail.com/a.eugenio@unifesp.br
Institutional number: (11) 5576-4848 (extension 2659)
Disclosure of Conflicts of Interest:
The authors declare no conflicts of interest to disclosure.
Background: In multiple myeloma (MM), HSP70 helps to prevent proteotoxic stress
and cell death due to overload of unfolded/misfolded proteins produced by tumor
cells and it has integrative role in protein degradation due to the interaction with
many pathways, such as ubiquitin proteasome, unfolded protein response and
autophagy. Aims: To explore the role of HSP70 as a therapeutic target for MM
through in vitro and in vivo analysis. Methods: Bioluminescent cell lines
RPMI8226-LUC-PURO and U266-LUC-PURO were treated with HSP70 inhibitor
(VER155008) and proteasome inhibitor (bortezomib) for evaluation of apoptosis by
flow cytometry. Immunodeficient mice were used for subcutaneous xenograft model,
with intravenous VER155008 (40mg/kg) and bortezomib (1mg/kg), immediately
after transplant of the cell lines. Bioluminescence was measured once a day for seven
days. Results: The best result for RPMI8226-LUC-PURO was 65% of late apoptosis
after treatment with bortezomib and U266-LUC-PURO presented more than 60% of
late apoptosis after VER155008 (80M) combined with bortezomib (100nM). Mice
treated with VER155008, alone or in combination with bortezomib, showed
inhibition of tumor growth for both cell lines when compared with control group
after one week of treatment (p<0.001, Two-way ANOVA). Conclusion: Our study
shows that HSP70 and proteasome inhibitors combination induced apoptosis in
tumor cells in vivo for both MM cell lines. Since HSP70 connects several signaling
pathways that maintain cell survival, it can represent a key role to establish a new
approach for the treatment of MM. Financial support: FAPESP 2010/17668-6 and
CNPq (155272/2013-6). UNIFESP Ethics Committee (0219/12).

A45
A46
IN VITRO CYTOTOXIC EFFECTS OF QUINOLINES ANALOGS IN

OVARIAN CANCER
PARTICIPATION OF NON-CODING RNAS (NCRNAS) IN THE

MECHANISM EMT (EPITHELIAL-MESENCHYMAL TRANSITION) ON
HUMAN TUMOR CELL LINES
Aline Brito de Lima(1,3), Carolina Ferreira de Alencar(1,3), Agnaldo Lopes da Silva

Filho(2), Maria Raquel Santos Carvalho(3), Letcia da Conceio Braga(1), Sergio
Antnio Fernandes(4), Luciana Maria Silva(1).
*Email: lima.alinebrito@gmail.com
1.
2.
(1) Laboratrio de Biologia Celular, Fundao Ezequiel Dias - Rua Conde Pereira
Carneiro, 80, Gameleira, Belo Horizonte/MG, Brasil - CEP: 30510-010.
3.
4.
(2) Universidade Federal de Minas Gerais, Faculdade de Medicina, Departamento de
Ginecologia e Obstetrcia - Avenida Professor Alfredo Balena 190, Santa Efignia,
Belo Horizonte/MG, Brasil CEP: 30130-100.
(3) Universidade Federal de Minas Gerais, Instituto de Cincias Biolgicas,
Departamento de Biologia Geral - Avenida Antnio Carlos, 6627, Pampulha, Belo
Horizonte/MG, Brasil CEP: 31270-901
(4) Universidade Federal de Viosa, Departamento de Qumica - Avenida Peter
Henry Rolfs, s/n - Campus Universitrio, Viosa/MG, Brasil CEP: 36570-900
Ovarian cancer (OC) is the most lethal of gynecologic malignancies. This disease
was responsible for 239000 new cases and 152000 deaths in 2012, representing 4%
of all cancer cases and 4.2% of all cancer deaths in women around the world. Factors
that contribute to poor prognosis of OC is the lack of symptoms in early stages of
disease and chemotherapy resistance reinforcing the need for development of new
molecules with potential antitumor action. In this study, new five analogues of
quinolines, called Q1, Q2, Q15, Q22 and Q1d, were tested in human cell line of OC
(TOV-21G - ATCC). The cytotoxicity assay was performed by MTT colorimetric
method and reproductive cell death was available through clonogenic assay. The
same molecules were tested in an immortalized cell line of lung fibroblast (WI-26VA4 - ATCC) to analyze the selective action. Flow cytometry analysis with DAPI
was performed to available the cell cycle effects. MTT assay showed that IC50 of
quinolines varied between 92M and 435M for WI-26-VA4 and between 110M
and 877M for TOV-21G. Clonogenic assay demonstrated that Q22 significantly
inhibited the tumor cells growth TOV-21G and caused minor reproductive damage to
normal line WI-26-VA4. This result is interesting because recently researches have
focused to find compounds that act under tumor cells with minimal toxicity to
normal cells. Cell cycle analysis showed that Q22 cause G2/M arrest in response to
cellular stress. Based on these data, Q22 represent a promising template for
developing new molecules of anticancer agents. Financial support: Fapemig, CNPq.
Bryan rtero Perez Gonalves1, Silvia Ligrio Fialho2, Luciana Maria Silva1
(1) Laboratrio de Biologia Celular, Fundao Ezequiel Dias
(2) Diretoria de Desenvolvimento Tecnolgico Farmacutico, Fundao Ezequiel
Dias
(3) Laboratrio de Biologia Celular, Fundao Ezequiel Dias
Non-coding RNAs (microRNAs and long RNAs) have many biological functions,
including in cancer. This study aims to identify and isolate non-coding RNAs from
in vitro culture human tumor cells as model for future studies of crosstalk between
lineages, and the involvement of these molecules in carcinogenesis. The cell lines
BT-549 and RKO-AS45-1, are grown in RPMI 1640 with 10% FBS and TOV-21G
line in DMEM High Glucose with 15% FBS, incubated in both humid atmosphere at
37C with 5% CO2. Extraction of total RNA using TRIZOL. Evaluation of potential
TOV-21G membrane resistance before and after treatment with: total RNA, TGF-
and Everolimus using Millicell ERS-2. The cell morphology was analysed by
fluorescence microscopy and cell cycle by flow cytometry. The total RNA used in
cellular resistance test, it was shown intact. Our results show that tumor cell lines
BT-549 e TOV-21G compared the normal cell line Wi-26VA4 before treatment
present the following cell cycle profile: Wi-26VA4 40.16% in G1/G0, 13.7% in S
and 34.5% in G2/M; BT-549 51.5% in G1/G0, 29.6% in S and 10.2% in G2/M and
TOV-21G 84.86% in G1/G0, 13.43 in S and 23.37% in G2/M. Preliminary cell cycle
analysis of TOV post-everolimus treatment showed accentuated decrease of cells in
G2/M . The parameters of cell resistance to TOV-21G showed higher value after the
incubation period with TGF- (4.5cm/7.5cm) Everolimus (3cm/11.4cm)
and total RNA (4.5cm/8.4cm). Our results to date show that everolimus has the
ability to change the profile of the cell cycle TOV-21G decreasing cell progression.
A47
A48
DISCOVERY OF NOVEL HORMONE-DEPENDENT PROSTATE CANCER

INHIBITORS USING PHENOTYPIC CELL-BASED ASSAYS
Authors: Elisa Castaeda Santa Cruz, Marta Erica Saidel, Andrei Leito
INTRATUMORAL APOPTOSIS HAS PROGNOSTIC IMPACT IN LUNG

CANCER AND APOPTOTIC BODIES DERIVED FROM DIVERSE
INDUCERS MODULATES DIFFERENTLY THE POLARIZATION STATUS
OF MACROPHAGES
Medicinal Chemistry Group (NEQUIMED)

So Carlos Institute of Chemistry-University of So Paulo (IQSC-USP), Brazil
andleitao@iqsc.usp.br (16)3373-9943, (16)98210-1968
Matheus Becker1, Marco A. De Bastiani1, Carolina B. Muller1, Mariana M. Parisi2,

Melissa M. Markoski3, Lucinara D. Dias3, Mauro A. A. Castro4, Florencia M. BarbTuana2, Rosalva T. Meurer5, Marilda da C. Fernandes5, Fbio Klamt1
1
Prostate cancer is one most common diagnosed in the male population and it is
androgen-dependent in its initial phase. Despite many drugs have been developed for
the treatment of these pathologies over time, they lose efficacy in case of resistant
cancers bearing mutations on the target macromolecules. Here we performed in vitro
assays to evaluate the pharmacological and cytotoxic activities of new bioactive
substances using hormone-dependent (LNCaP) and independent (PC-3 DU145)
prostate cancer and fibroblast (Balb/C 3T3 clone A31) cell lines. MTT assays were
performed according to previously published work following incubation of the
chemicals for 72 h. Neq0502 was the most potent against LNCaP, with IC50 = 20
mol/L. In the cell cycle assay, Neq0502 had a similar profile to enzalutamide
(reference drug) without substantial disruption of the LNCaP cycle. Moreover,
Neq0502 was selective for LNCaP in relation to androgen-independent prostate
cancer and fibroblast cells. These data allow us to optimize new substances based on
the Neq0502 lead compound in future studies.
This work was supported by FAPESP projects 11/07025-3, 13/18009-4 and CNPq.
Laboratory of Cellular Biochemistry, ICBS/UFRGS, 90035-003 Porto Alegre (RS),

Brazil; 2Laboratory of Molecular Biology and Bioinformatics, ICBS/UFRGS, 90035003 Porto Alegre (RS), Brazil; 3Laboratory of Cellular and Molecular Cardiology,
IC/FUC, Porto Alegre (RS) 90620-000, Brazil; 4Laboratory of Bioinformatics,
Professional and Technological Education Sector, Polytechnic Center, UFPR, 81531970 Curitiba (PR), Brazil; 5Laboratory of Pathology Research, UFCSPA, Porto
Alegre (RS) 90050-170, Brazil.
Background Tumor-associated macrophages (TAM) are abundant components of
nonsmall cell lung cancer (NSCLC) and present at least two distinct phenotypes: the
pro-inflammatory (M1), and the alternatively-activated (M2), which is antiinflammatory and pro-tumoral. TAM polarization status depends on factors from the
tumor microenvironment such as the phagocytosis of apoptotic bodies derived from
tumoral cells.
Aims
We aimed to analyze the influence of macrophages and intratumoral
apoptosis in NSCLC patients survival, and evaluate whether the phagocytosis of
apoptotic cells obtained from different inducers are able to differently modulate
macrophages M1/M2 phenotypes.
Methods
Prognostic impact of intratumoral apoptosis (cleaved-caspase-3+ cells)
and macrophages (CD68+ cells) was evaluated in an NSCLC patient cohort (IA-IIB
stages) (n=40). The influence of apoptotic bodies in the modulation of macrophages
activation status was evaluated by inducing apoptosis in A549 human NSCLC cell
line with CDDP (chemotherapeutic drug), TnCl (physiological oxidant) and 3-BP
(metabolic inhibitor) (titrated with annexin V/PI flow cytometry stain). The apoptotic
bodies were collected and incubated with monocytes-derived macrophages (MDM).
rtPCR of M1/M2 markers and cytokines were analyzed.
Results
CD68+ cells were associated with good prognosis and high cleavedcaspase-3+ cells were correlated with worse prognosis. Phagocytosis efficiency of
apoptotic bodies induced by TnCl was lower than CDDP and 3-BP, and
macrophages exposed to apoptotic bodies obtained from TnCl treatment showed a
14-fold increase in IL-6 (pro-inflammatory cytokine) while CDDP-derived apoptotic
bodies raised 4-fold IL-10 levels (anti-inflammatory cytokine).
Conclusion Apoptosis and M have prognostic impact and different therapeutic
approach have dissimilar effect over antitumoral activity of macrophages.
Support
MCTI/CNPq Universal (476114/2008-0),
(465113/2014-1), MCTI/CNPq Universal (445457/2014-7).
MCTI/CNPq
CBAB

A49
A50
SSi6 INDUCES ALTERATIONS IN THE MORPHOLOGY AND INHIBITS

MIGRATION AND INVASION ON TRIPLE NEGATIVE BREAST TUMOR
CELLS
CYTOTOXIC AND CYTOSTATIC EFFECTS OF CYSTEINE PROTEASE

INHIBITORS ON PANCREATIC CANCER CELLS
Liany Johanna Luna Dulcey1, Marina Arajo Naves1, James Almada da Silva2,
Mrcia Regina Cominetti1.
1
Department of Gerontology, Federal University of So Carlos, CEP 13565-905, So

Carlos SP, Brazil;
2
Pharmacy Center, Federal University of Sergipe, CEP 49100-000, So Cristvo
SE, Brazil.
liany.luna@hotmail.com (16)
997391934,
marinanaves_5@hotmail.com,
mcominetti@ufscar.br (16) 3306-6666, jamesalmada@hotmail.com
Background: Major components identified in ginger (Zingiber officinale Roscoe)
are terpenes and phenolic compounds such as gingerol and shogaol. SSi6 is a
semisynthetic substance synthesized based on [6]-gingerol by chemical
modifications. Aim: This study investigated effects of SSi6 on morphology,
proliferation rate, migration and invasion of triple negative MDA-MB-231 cells and
a non-tumor cell MCF-10A. Methods: For morphology assay, cells were exposed to
different concentrations (6.25-100M) of the substance and photographed in
different times (0-48h). In the proliferation assay, using MTT method, cells were
exposed to crescent concentrations of SSi6 (3.12-100M) for 48h. To evaluate the
effects on migration, a wound healing assay was performed. Cells were treated with
SSi6 (6.25-20M) and photographed in several times (0-24h). Invasion assay was
performed using BioCoat Matrigel Invasion Chambers kit. Cells were treated with
different concentrations of SSi6 (6.25-18.75 M) for 22h. Results: SSi6 affected the
morphology of MDA-MB-231 cells more pronouncedly than in MCF-10A cells. We
identified evidences of cell death since 50 M in 2h of treatment for tumor cells.
SSi6 was also able to inhibit MDA-MB-231 growth and proliferation, with IC50 of
22.90 M and moreover, was able to inhibit cell migration and invasion, suggesting
a relative specificity for tumor cells. Conclusion: Results suggest that SSi6 could be
used as a new potential candidate to be further investigated as an antitumor drug that
could be used to improve current chemotherapy. More studies, mainly in vivo, must
be performed in order to definitely prove its antitumor properties.
J. C. J. Quilles; D. Y. Tezuka; J. F. R. Ribeiro; A. Leito; C. A. Montanari

Medicinal Chemistry Group NEQUIMED
Institute of Chemistry of So Carlos IQSC / University of So Paulo USP
Avenida Trabalhador So Carlense, 400 Centro So Carlos / SP CEP: 13.566590
E-mail:
quilles@usp.br;
daianetezuka@usp.br;
andleitao@iqsc.us.br; carlos.montanari@usp.br
Tel. (+55) 16-33738784 / 17-991156785
jean_francisco@iqsc.usp.br;
Pancreatic cancer has an overall poor diagnostic and it is often lethal due to the lack
of therapeutic alternatives associated with the late detection. In this respect, there are
numerous studies that correlate different macromolecules with the progression and
spread of this type of cancer. One of them is cathepsin L (catL), a cysteine protease
expressed in high amount in cancer cells (including the pancreatic one) turning out to
be a promising target for new putative anticancer compounds. In this work, thirty
dipeptidyl nitrile based compounds were evaluated using biochemical assays based
on the substrate (Z-FR-MCA) cleavage by catL, coupled with cell viability assay
using the MTT colorimetric method against Mia-Paca2 epithelial pancreatic cancer
cells. The enzyme kinetic study displayed potency for these inhibitors in the low
nanomolar to micromolar range. Interestingly, the cell viability was not affected by
most of these compounds, which could indicate that catL may not trigger the cell
death process. The cytostatic profiling for the best compounds tested in the
biochemical assay is underway using a clonogenic assay and cell cycle study. If these
compound turn out to be cytostatic agents, the research of these cysteine protease
inhibitors as a pancreatic tumor arrest agents will turn out to be the main focus,
providing an incitement in a field with scarce scientific data.
Financial support: CAPES/FAPESP 2013/00798-2.

A51
A52
TITLE- CYTOSKELETON MODULATION THROUGH COFILIN-1 MIGHT

HAVE A ROLE IN EPITHELIAL-MESENCHYMAL TRANSITION OF
COLORECTAL CANCER CELLS
METFORMIN AS A CHEMOSENSITIZING AGENT TO CISPLATIN IN

NON-SMALL CELL LUNG CANCER
Annie Cristhine Moraes Sousa-Squiavinato (1) and Jose Andrs Morgado Daz (1)
Structural Biology Group, Cell Biology Program - National Cancer Institute (INCA),
Rio de Janeiro, Brazil.
Tharcisio Citrangulo Tortelli Junior (1*), Rodrigo Esaki Tamura (1), Sarah Milani de
Morais Leandrini (2), Silvina Odete Bustos (1), Bryan Strauss (1), Said Rabbani (2),
Roger Chammas (1)
(1)
Address of the presenting author:

Centro de Pesquisa (CPQ)- Instituto Nacional de Cncer (INCA)
Rua Andr Cavalcanti, 37 - Centro
Cep: 20231-050 - Rio de Janeiro - RJ- Brasil
email:anniecristh@gmail.com, jmorgado@inca.gov.br
(2)
Background- Colorectal cancer (CRC) is a frequently lethal disease, due to the

formation of metastasis, event theoretically defined as the final step of the epithelialmesenchymal transition (EMT). First, epithelial cells lose their intercellular contacts,
down-regulate epithelial proteins and up-regulate mesenchymal proteins leading to
acquisition of migratory and invasive phenotype and development of metastasis. In
order to lose contacts and migrate, cells must rearrange it's actin cytoskeleton.
Cofilin-1 modulates actin dynamics, however its role during EMT is unknown.
Aim- Establish the role of cofilin-1, during EMT in a CRC cell line. Methods- TGF-treated HT-29 were evaluated by expression of epithelial and mesenchymal
markers using Western Blotting, RT-PCR and immunofluorescence for subcellular
localization. In addition, migration, invasio, metalloproteinases activity and RhoA
activity assays were performed. Results- TGF- induced morphological changes in
HT-29, with loss of cell contacts, increased membrane protrusions, stress fibers
(consistent with migratory cells), reduction in the expression of junctional proteins
(E-cadherin and Claudin-3) and increased expression of mesenchymal protein
(Vimentin). Treated cells migrated, invaded and had high metalloprotease activity.
When assessing actin dynamics, TGF- increased gene expression of cofilin-1 and
LIMK-2, pathway (RhoA-Rock-LIMK2-pCofilin1) that was shown to be active in 5
minutes after treatment. Increase in the F/G actin ratio, explained by higher levels of
p-cofilin-1 corroborates the formation of actin fibers. Conclusion- We were able to
establish the EMT model in HT-29 cells. These preliminary results suggest that
cofilin-1 has a role in the EMT.
Financial Support: FAPERJ, CNPq e MS
Institute of Cancer of State of Sao Paulo ICESP Department of

Radiology and Oncology, Faculty of Medicine, University of Sao
Institute of Physics, University of Sao Paulo, Sao Paulo, Brazil
Background: Instead of non-transformed cells, most of cancer cells use the glycolytic
pathway as their main energetic source, even when oxygen is fully available. Tumor
sub-populations that use the mitochondria as main energy source are more resistant
to chemotherapy, due to histone demethylase JARID1b and PGC1 overexpression.
Therefore, changes in metabolic profile of tumor cells, using metformin to induce
glycolysis, may contribute for tumor chemosensitization. Aims: To change cellular
metabolism profile, using metformin, to chemosensitize lung cancer cells against
cisplatin. Methods: A549 (p53 WT) and H1299 (p53 KO) cell line were used on this
study. RT-PCR was used for p53, PGC1 and JARID1b expression and propidium
iodide, for cell death assay. Mitosox, CM-H2DFDA, TMRE and Mitogreen, were
used to measure mitochondria ROS production, hyperpolarization and mass. Lactato
production was measured using Biovision kit K607-100. Results: Metformin
sensitized A549 cells against cisplatin. Chemosensitization was lost when A549 cells
were pre-treated with low dose of cisplatin. Pre-treated A549 cells had
downregulation of p53 and upregulation of JARID1b and PGC1, even after thirty
days of stopping treatment. Metformin chemosensitization did not work on H1299
cells or in A549 cell with p53 inhibition, and p53 transduction in pre-treated A549
cells restored metformin chemosensitization to cisplatin. Pre-treated cells have
higher ROS levels and are more hyperpolarized. Metformin induces lactato
production only on cells that express p53. Conclusion: Pre-treatment with low dose
of cisplatin leads to chemoresistance by p53 loss and mitochondrial
hyperpolarization. Metformin-induced chemosensitization and lactato production
works when p53 are available.
Supported by CNPq
tharcisio.junior@hc.fm.usp.br
Av. Dr. Arnaldo, 251, 8o andar CTO
CEP 01246-000, Cerqueira Cezar, Sao Paulo, SP, Brazil
Phone: +55(11) 3893-3007
Mob.: +55(11) 99306-9784

A53
A54
EFFECTS OF [10]-GINGEROL ON HMT-3522 S1 AND T4-2 CELLS IN

LRECM 3D CULTURE
THE RUTHENIUM COMPLEX, TRANS-RU(THYSMET)(PPH3)2(BIPY)]PF6,

INDUCES APOPTOSIS IN MDA-MB-231 BREAST TUMOR CELLS
Angelina Maria Fuzer1, Sun-Young Lee2, Mina Jahan Bissell2, Mrcia Regina
Cominetti1.
Amanda Blanque Becceneri1, Ceclia Patrcia Popolin1, Anglica Ellen Graminha1,

Angelina Maria Fuzer1, Ana Maria Plutin2, Alzir Azevedo Batista3, Mrcia Regina
Cominetti1.

angelina_fuzer@yahoo.com.br, mcominetti@ufscar.br;
2
Lawrence Berkeley National Laboratory, Life Sciences Division,
symoonlee@lbl.gov, mjbissell@lbl.gov
Background: Many studies have revealed advantages of three-dimensional culture

techniques (3D) over traditional two-dimensional monolayer cultures (2D). Threedimensional cultures better mimic the tumor microenvironment found in vivo, as well
as the interaction of cells with the extracellular matrix (ECM). Different responses
were found for cells in 3D culture compared to those cultured in 2D, such as
increased resistance of tumor cells to various drugs and increased selective
sensitivity for inhibition of proliferation in tumor cells compared to normal cells.
Aims: The aim of this work was to test the effects of [10]-gingerol on breast tumor
cells using laminin-rich extracellular matrix (lrECM) 3D culture. Methods: To verify
whether [10]-gingerol treatment was able to revert the malignant phenotype of T4-2
cells, an immunostaining was performed using anti-6-integrin and anti--catenin
antibodies. Anti-cleaved-caspase 3 antibody was used to verify apoptosis. An
immunoblotting assay was performed to confirm the reversion. Results: The results
showed selectivity of [10]-gingerol against the malignant T4-2 lineage, compared to
non-tumor cells. The mechanism of action involved in this reversion of the malignant
phenotype was the downregulation of EGFR and 1-integrin and the induction of
apoptosis in T4-2 cell line. Conclusion: To the best of our knowledge, this is the first
report of a natural product being able to revert the malignant phenotype in T4-2 cells
and can help to explain the previously observed antitumor effects of [10]-gingerol.
Financial support: FAPESP 2015/08146-0, 2013/00798-2, 2012/18908-6.

amandabecc@gmail.com; ceciliapopolin@yahoo.com.br;
angelicagraminha@yahoo.com.br; angelina_fuzer@yahoo.com.br,
mcominetti@ufscar.br; 2Universidade de Havana, Departamento de Qumica;
3
Universidade Federal de So Carlos, Departamento de Qumica, anap@fq.uh.cu.;
daab@ufscar.br
Background: Cancer is among the leading causes of morbidity and mortality
worldwide. Triple negative breast cancers (TNBC) lack estrogen receptor (ER),
progesterone receptor (PR) and hormone epidermal growth factor receptor 2 (HER2)
and exhibit aggressive nature with higher rates of recurrence and cause high
mortality among breast cancer patients. The majority of drugs currently used in
cancer treatment show no selectivity for tumor cells, making it difficult to treat and
generating unwanted side effects. In the last two decades ruthenium complexes have
been widely studied and gained prominence for cancer treatment due to its unique
characteristics and important results achieved. Aim: This study aims to understand
the effects of the ruthenium complex trans-[Ru(ThySMet)(PPh3)2(bipy)]PF6 on
apoptosis of TNBC MDA-MB-231 tumor cell and in nonmalignant human breast
MCF-10A cells. Methods: Cells were treated with different concentrations (216M) of [Ru(ThySMet)(PPh3)2(bipy)] for 2h and adequately prepared for each
experiment. The apoptotic activity of the complex on MDA-MB-231 and on MCF10A cells were analyzed by flow cytometry using PE-AnnexinV Apoptosis Detection
Kit (BD) and through nuclear fragmentation assay with DAPI. Results: The complex
induces cell apoptosis in all concentrations tested (2-16M) in both cell lines.
Nuclear staining displayed that treated TNBC cells suffered morphological changes
related to apoptosis, as nuclear fragmentation, but MCF-10A did not suffered any
changes. Conclusion: These results show that this complex acts in the cell death
process inducing apoptosis more selectively in TNBC. More studies should be
performed to understand the biological mechanism used by the complex to exert its
functions.
Financial support: FAPESP 2014/25121-8; 2013/00798-2
A55
A56
INTERACTION
OF
ANTIMICROBIAL
PEPTIDES
WITH
GLYCOSAMINOGLYCANS OF THE EXTRACELLULAR MATRIX
MMP ACTIVITY INFLUENCES ADHESION PROPERTIES OF ORAL

SQUAMOUS CELL CARCINOMA IN 3D MATRIX
Letcia Sperandio (1)*; Wagner Alves de Souza Jdice (1); Antonio Carlos Ribeiro
Filho (1); Antonio de Miranda (2); Marcus Buri (2); Edgar Julian Paredes Gamero (1,2).
Grasieli de Oliveira Ramos (1), Bibiana Franzen Matte (2), Jos Ricardo Busatto (2),
Marcos Vinicius Rauber (2), Alan Rick Horwitz (3), Manoel Santanna Filho (2),
Marcelo Lazzaron Lamers (2,4)
1-Interdisciplinary Center for Biochemical Research, University of Mogi das Cruzes,

Av. Dr Cndido Xavier de Almeida Souza, 200. Mogi das Cruzes, SP, Brazil.
2-Department of Biochemistry, Federal University of So Paulo, R. Trs
de Maio 100, So Paulo, SP, Brazil.
The authors declare that there are no competing financial interests
*Corresponding author:
Email: leticiasperandio7@gmail.com
University of Mogi das Cruzes, Av. Dr Cndido Xavier de Almeida Souza 200,
Mogi das Cruzes, SP, Brazil. Cep: 08780-911
+55(11) 4798-7102/ 4798-7103
(1) Universidade do Oeste de Santa Catarina, Joaaba, SC, Brazil

(2) School of Dentistry, Universidade Federal do Rio Grande do Sul, Rua Ramiro
Barcelos 2492, CEP 90035-003, Porto Alegre, Brazil.
(3) Department of Cell Biology, University of Virginia, Charlottesville, Virginia,
USA
(4)Department of Morphological Sciences, Universidade Federal do Rio Grande do
Sul, Porto Alegre, Brazil
bfmatte@gmail.com; +5551 8402-1090; +5551 3308-5011
Background: Antimicrobial peptides (AMPs) have showed possess a potent antitumor activity. Among them there are Gomesin, Protegrin, Tachyplesin and
Polyphemusin. These peptides exhibit cytotoxicity against tumors by various cellular
mechanisms, but the most interesting aspect of their actions is the ability to enter to
the cell by unknown mechanisms. Objective: Thus, the goal of the present study was
to investigate if the extracellular glycosaminoglycans can participate in the entrance
of AMPs in tumor cells. Materials and Methods: Antimicrobial peptide gomesin was
synthesized using t-Boc strategy. The intrinsic fluorescence of tryptophan was
monitored to observed the formation of the complex GAG/MAP peptides using a
LS-55 Spectrofluorometer Perkin Elmer in the wavelength scan of EX = 280 nm and
EM=300-500 nm. The variation in fluorescence of gomesin-Trp was calculated as
F/F0 in absence and presence of GAG. The dissociation constants Kd of GAG
binding to AMPs were determined by a rectangular hyperbola curve using Grafit
software. Results: The dissociation constant (Kd) values for each GAG were: 0.61
M (Heparin); 3.43 M (Dermatan Sulfate); 0.95 M (Chondroitin Sulfate); 1.01
M (Heparan sulfate); 1.48 M (Hialurnico Acid); Dextran Sulfate was used as
control of negative charge (4.50 M). Conclusion: According the results, all GAGs
are able to binding gomesin with high affinity and may be involved to entrance of
peptides in the tumor cell promoting cell death.
During invasion, tumor cells remodel the extracellular matrix (ECM) mainly by
matrix metalloproteases (MMP) activity. Our aim was to evaluate the role of MMP
during Oral Squamous Cell Carcinoma (OSCC) invasion. We analyzed the
distribution of MMPs 1, 2, 9 and 14 by immunohistochemistry staining of human
OSCC biopsies and it was observed an increase of MMPs 1, 9 and 14 mainly in cells
at the invasion zone, suggesting a role for cell migration. To analyze the role of
MMPs during cell migration, the highly invasive OSCC cell line (SCC25) was
platted in a 3D matrix containing fibronectin (10mg/ml) and collagen (0.6mg/ml;
1.2mg/ml; 1.8mg/ml) in the presence/absence of MMP inhibitor (GM0001, 25mM)
and imaged for 20h. We observed that a denser ECM decreased the migration speed,
directionality and the protrusion area, while the use of the MMP inhibitor
pronounced these effects. A possible explanation is that MMP activity is necessary
for a better adhesion process. Then, SCC25 cells were transfected with the adhesion
marker paxillin-GFP, platted in the same conditions and imaged in confocal
microscope with reflectance. It was observed that the inhibition of MMP impaired
the adhesion process in denser matrix. Taken together, the increase on MMP
expression observed in tumor cells at the invasion zone contributes to an increase on
ECM remodeling, which results in physical space for cells to invade and in a better
adhesion process to the substrate, with a consequent improvement of migration
properties of invasive cells.
Finantial support: FAPESP, CNPq and CAPES
The authors declare no conflict of interest.

Funding Support: CAPES, CNPQ, Science without Borders Program, FAPERGS,
UFRGS

A57
A58
ELECTRON MICROSCOPY OF NEOPLASTIC CELLS FOUND IN

MALIGNANT PLEURAL EFFUSIONS (MPE) OBTAINED FROM
PATIENTS WITH BREAST CANCER HISTORY
RHO GTPASES ARE INVOLVED IN PROSTATE CANCER CELL

RESPONSE TO GLUTAMINE DEPRIVATION
Naiane Carlesso Bassani (1), Giovana Tavares dos Santos (1), Ariane Campos (4),
Mariana Simes (1), Lauren Cars (3), Marilda da Cruz Fernandes (1), Joo Carlos
Prolla (2) Claudia Giuliano Bica (1) and Gisele Orlandi Introini (1)
giseleorlandi@gmail.com
(1) Federal University of Health Sciences of Porto Alegre (UFCSPA)
(2) Holly Hospital of Porto Alegre (ISCMPA)
(3) Federal University of Rio Grande do Sul (UFRGS)
(4) State University of Campinas
Breast cancer is the leading cause of cancer deaths among women in the world and
the MPE occurs in about 50% of patients with this cancer. In MPE, we can find (1)
single cells, (2) massive spheres, and (3) non-spheroidal agglomerates (berry-like
clusters). The presence of spheres or agglomerated corresponds to a class of patients
with better prognosis compared to those with isolated neoplastic cells. This work
aims to describe the morphology of neoplastic arrangements using Light and
Electron Microscopy found in MPE, from patients with breast cancer history.
Samples were aspirated using a fine-needle at the Santa Casa de Misericrdia de
Porto Alegre Hospital. After the preparation, the material was observed under an
electron microscope. Considering the findings observed in both massive spheres and
berry-like clusters it was possible to see: (1) an evident nucleolus, suggesting
remarkable transcriptional activity, (2) desmosomes, ensuring cluster structural
integrity (3) vesicles en route and (4) several mitochondria, reflecting intense
metabolic activity. Contradictorily, most of the single cells were exhibiting apoptotic
bodies, rupture of nuclear envelope, and discontinuity of plasmatic membrane.
Chemical messengers from cells and matrix are essential for surveillance; in their
absence, cells kill themselves by activating an intrinsic suicide program, called
anoikis. The circumvention of anoikis accompanies the acquisition of anchorage
independence. A cooperative relationship between cells integrating
agglomerates/spheroids guarantee a long term survival in MPE. So, we propose the
hypothesis that single cells capable of escaping from death became more invasive
and with higher metastatic potential in comparison with clusters.
Financial support for the research project: Rio Grande do Sul Research Foundation
(FAPERGS)
Ethical approval: Ethics Committee of the Federal University of Health Sciences of
Porto Alegre, No. 1074/2010, having continuity for the doctoral project entitled
"Study of the association between pleural effusion and breast cancer" proposed and
approved by the Research Committee of Ethics of UFCSPA number 252516/2013
and ISCMPA number 193,243/2013.
Luciana Bueno de Paiva (1)*, Vanessa Aline Bernusso (1), Joo Agostinho
Machado-Neto (2), Fabiola Traina (2), Sara Teresinha Olalla Saad (1), Mariana
Lazarini (1) (3).
(1) Hematology and Hemotherapy Center, University of Campinas, Campinas, So
Paulo, Brazil
(2) Department of Internal Medicine, University of So Paulo at Ribeiro Preto
Medical School, Ribeiro Preto, So Paulo, Brazil
(3) Institute of Environmental, Chemical and Pharmaceutical Sciences - Federal
University of So Paulo, Diadema, So Paulo, Brazil
* paiva.b.luciana@gmail.com; Mobile phone: 19 981101266; Institutional phone: 19
35218734;
Supported by FAPESP and CNPq
Keywords: cancer cell metabolism, Rho Family of GTPases, ARHGAP21.
Tumor cells present higher glycolysis and glutamine uptake in comparison to normal
cells and these changes in metabolism are essential for cancer progression. The Rho
GTPases RhoC, Cdc42 and Rac1 were associated to glutaminolysis through
glutaminase activation. Our aim was to investigate the Rho GTPase participation in
the response of prostate cancer cells to glutamine deprivation. LNCaP and PC3 cells
were cultured under different glutamine concentrations for 72 hours and
mitochondrial activity was evaluated using MTT. Proliferation, apoptosis and
autophagy were analyzed by FACS. Autophagic proteins and ARHGAP21
expression was evaluated using western blot. Rho GTPase activity was accessed by
pull down and silenced with specific siRNAs. PC3 and LNCaP cells cultured under
reduction of glutamine presented decreased mitochondrial activity and proliferation,
and increased expression of Beclin, LC3 and p62. Glutamine reduction also induced
apoptosis in LNCaP cells, whereas ARHGAP21 was downregulated in both cell
lines. RhoC activity was not changed, while RhoA activity decreased in PC3 cells
cultured under glutamine deprivation. Cdc42 activity was increased in PC3 and
decreased in LNCaP cells. Interestingly, the silencing of RhoA or RhoC reduced
mitochondrial activity of PC3 cells and a further decrease was observed by RhoC
knockdown combined to glutamine deprivation.
Our results indicate that Rho GTPases and their regulators, such as ARHGAP21,
might participate in the cell response to glutamine removal. The increase of Cdc42
may be related to PC3 cell resistance to glutamine deprivation. In addition, RhoC
silencing maybe an additional approach to decrease glutamine deprivation resistance
in cancer cells.
A59
A60
EVALUATION ANTINEOPLASTIC POTENTIAL IN VITRO OF THE

ETHANOLIC EXTRACT ISOLATED OF THE SPECIE SWITENIA
MACROPHYLLA ON TUMOR CELL LINES
SYNTHETIC CHALCONE INDUCES CYTOTOXICITY AND G2/M CELLCICLE ARREST IN COLON TUMOR CELLS
Ingryd Nayara de Farias Ramos (1); Laine Celestino Pinto(1); Leilane de Holanda
Barreto(1); Bruno Moreira Soares (1); Emerson Lucena da Silva (1); Felipe Pantoja
Mesquita(1); Milton Nascimento da Silva (2); Raquel Carvalho Montenegro(1).
Emerson Lucena da Silva (1), Ingryd Nayara de Farias Ramos (1), Felipe Pantoja
Mesquita (1), Lane Celestino Pinto (1), Thatyana Rocha Alves Vasconcelos (2),
Rommel Mario Rodrguez Burbano (1), Raquel Carvalho Montenegro (1).
(1) Biological Science Institute, Federal University of Par, Belm, Brazil.

(2) Department of Chemistry, Federal University of Par, Belm, Brazil.
(1)
Biological Science Institute, Federal University of Par, Belm,
Brazil.
(2)
Department of Organic Chemistry, Fluminense Federal University,
Contacts:
Email: ingrydramos@yahoo.com.br
Phone number: (91) 982020807.
Contacts:
Cancer is a group of diseases that affects millions of people and is the second cause
of death on the world's population. Currently the biology of cancer has been
extensively studied and one of the main lines of research is the development of new
molecules with anticancer potential. As plants and bioactive compounds derivatives
of these have a wide variety of structures and functions, present study evaluated the
antineoplastic potential in vitro of the ethanolic extract, isolated from the species
Swietenia macrophyla (SM-EE) originated from the Amazon flora in neoplastic cell
lines. We evaluated the extracts cytotoxic activity in tumor and normal cells, the
pattern of cell death, cell proliferation analysis by colony formation and genotoxic
potential of tumor in vitro. The results showed that the extract has cytotoxic activity,
proving active on colorectal carcinoma line (HCT-116), with IC50 of 55.87 mg/mL.
It was also observed that the extract induced an increase in the pattern of cell death
by apoptosis in HCT-116 cell line in highest concentrations tested when compared to
the negative control. The SM-EE extract also showed anti clonognic activity,
reducing formation of colonies and induced a significant increase in DNA damage
index in HCT116 cell line, only in the highest concentrations tested. Thus, this
results suggest that a SM-EE extract has potential antineoplastic activity indicating
big prospects with this compound in future cancer therapies. However, additional
tests need to be realized to delimit the exact mechanism of action of the substance.
Funding support: CNPq and UFPA.
Email: lucenaemerson@hotmail.com;
Phone number: (091) 98228-0353.
Colorectal cancer is the fourth cause of deaths in women and third in men
worldwide, for 2016 are expected 34.280 new cases in Brazil. In this perspective,
research of new molecules with antineoplastic activity is necessary. Chalcones have
demonstrated extensive pharmacological potential, such as antineoplastic,
antimalarial, anti-inflammatory, antimicrobial and antioxidant. Thus, aim of this
study was to evaluate in vitro cytotoxic activity induced by synthetic chalcone
(GCCG29) against two colon cancer cell lines (HCT-116 and HT-29) and fetal lung
fibroblast cell (MRC-5). Cell viability of GCCG29 was determined by MTT assay in
three lines, and cell-cycle analysis was performed in HCT-116 by flow cytometry to
determinate population of cells in each cell-cycle phase. Results were obtained from
three independent experiments and evaluated by analysis variance teste (ANOVA)
followed by Bonferroni test using a significant level of 5%. Our data revealed that
GCCG29 inhibits cellular growth in HCT-116 (IC50: 2,2 M), HT-29 (IC50: 8,7 M)
and MRC-5 (IC50: 7,5 M), revealing higher selectivity to HCT-116 than MRC-5. To
investigate effects on cell-cycle distribution, HCT-116 was treated with different
concentrations of GCCG29 (1, 2 and 4 M) for 72 hours. GCCG29 increased
population of cells in G2/M phase after treatment at concentration of 4 M
(p<0.0001), indicating that there was an inhibition of cell growth due to G2/M cellcycle arrest. Therefore, this study indicated that GCCG29 inhibited cancer cells
growth in vitro and caused G2/M cell-cycle arrest in colon cancer cell line. Authors
are grateful to CNPq and UFPA for financial support.

A61
A62
ARHGAP21 EXPRESSION IS DECREASED IN BONE MARROW SAMPLES

FROM MYELODYSPLASTIC SYNDROMES PATIENTS AND IT IS
RESTORED AFTER IN VITRO TREATMENT WITH DECITABINE
EXPRESSION ANALYSIS OF CANCER STEM CELL-RELATED GENES IN

MDA-MB-231 CELL LINE CULTURED IN TYPE I COLLAGEN
MATRICES
Authors: Mariana Ferreira Pissarra (1), Mariana Lazarini (1)(2), Isabella Barbutti
Gonalves (1), Adriana Silva Santos Duarte (1), Sara Teresinha Olalla Saad (1)
Iuri Cordeiro Valado1, Ana Carolina Lima Ralph1, Murilo Vieira Geraldo2, Vanessa
Morais Freitas1
1
University of So Paulo, Brazil.
2
Department of Structural and Functional Biology, Institute of Biology, State
University of Campinas, Brazil.
(1) Hematology and Hemotherapy Center, University of Campinas, Campinas, So

Paulo, Brazil
(2) Institute of Environmental, Chemical and Pharmaceutical Sciences - Federal
University of So Paulo, Diadema, So Paulo, Brazil
Mariana Ferreira Pissarra: institutional phone (19) 3521-8734; mobile phone (11)
97234-8371; email: marianafpissarra@hotmail.com; Adress: Carlos Chagas,480
Cidade Universitria Prof. Zeferino Vaz, Baro Geraldo, 13083-878, Campinas/SP
- Brasil
Mailing addresses:
Iuri Cordeiro Valado (iuricv@usp.br, phone contact: +55 11 3091-7778 / +55 11
9727-88893).
Ana Carolina Lima Ralph (anaralph@usp.br)
Murilo Vieira Geraldo (murilovg@unicamp.br)
Vanessa Morais Freitas (vfreitas@usp.br)
The myelodysplastic syndrome (MDS) is a medical condition characterized by a

defective production of blood cells. The syndrome can vary in its severity and third
of the patients progress to acute myeloid leukemia (AML). Decitabine is one of the
main treatments for high-risk MDS. ARHGAP21 is a RhoGAP protein involved in
hematopoiesis. Aims: Investigate the expression of ARHGAP21 in bone marrow
samples from MDS and AML patients. We also evaluated ARHGAP21 expression in
cell lines and progenitor cells treated or not with decitabine. Methods: Bone marrow
samples were collected from healthy donors (n=21) and untreated patients with MDS
(n=46), AML with myelodysplastic related changes (AML-MRC) (n=8) and de novo
AML (n=32). Leukemia cell lines (U937, K562) and CD34+ cells from two AML
patients were treated with 1uM and 5uM of decitabine for 48 hours. ARHGAP21
expression was analyzed using quantitative PCR. Results: Expression of
ARHGAP21 was decreased in low-risk MDS bone-marrow samples (n=32; p=0.016)
and a lower expression was observed in high-risk MDS bone-marrow samples (n=14;
p=0.048) when compared with healthy donors, whereas ARHGAP21 expression was
not changed in AML cells. Interestingly, ARHGAP21 expression was restored in
U937 and K562 cells treated with 5uM decitabine and in CD34+ cells treated with
1uM decitabine. Conclusion: Our results suggest that ARHGAP21 downregulation
may be an important event in the physiopathology of MDS and that decitabine
treatment is able to recover its expression. It will be important to further investigate
the relation of ARHGAP21 expression and clinical response to decitabine.
Background: Breast cancer (BC) is the most frequent and lethal malignancy among
women and its high recurrence rates may be associated to the emergence of cancer
stem cells (CSCs), which are both highly chemoresistant and metastatic. Along with
CSCs, the tumor-associated extracellular matrix (TAECM) has also a pivotal role in
the maintenance and progression of BC. TAECM is enriched in type I collagen (Col
I) fibers and has been highlighted as an inductor of CSCs and indicator of poor
prognosis in BC. Aims: We hypothesized that Col I matrices would induce CSCreprogramming and enrich for CSC-related genes. Methods: We analyzed the
expression of 84 genes associated with CSC phenotype through a commercial PCR
array. First, MDA-MB-231 cells were cultured for 7 days in tissue culture plates
(control group) or within low- (1,5mg/ml), intermediate (3,0mg/ml) or high
(4,5mg/ml) density Col I matrices. We then proceeded with total RNA extraction,
cDNA synthesis, running of the PCR array and data analysis, according PCR array
manufacturers instructions. Results: We identified 29 genes differentially expressed
in cells grown within Col I matrices, including well-known CSC molecular markers
(CD44, ITGA6). For further evaluation, we chose genes with low Ct values, higher
fold regulation relative to control group, and which biological relevance was not
addressed by similar studies. The selected genes are linked to cell fate determination
(DACH1, PTCH1), pluripotency (NANOG, LIN28A, POU5F1) and cell migration
and metastasis (PLAT, PLAUR, SNAI1). Conclusion: Further validation is ongoing
and may point to collagen I density as an enhancer of CSC phenotype.
Supported by FAPESP and Cnpq
FAPESP and CNPQ.
A63
A64
ISOLATION
AND
IMMUNOPHENOTYPIC
AND
FUNCTIONAL
CHARACTERIZATION OF CANCER STEM-CELLS FROM BONE
MARROW SAMPLES OF PATIENTS NEWLY-DIAGNOSED WITH
MULTIPLE MYELOMA
COMBINING PHOTODYNAMIC THERAPY AND CHEMOTHERAPY:

NANOTECHNOLOGY IMPROVING BREAST CANCER TREATMENT
Paola M. Dantonio (1), Veruska L. F. Alves (1), Rodrigo C. Fernando (1), Daniela
Teixeira (2), Alexandre S. Basso (2), Gisele W. B. Colleoni (1)
(1) Department of Clinical and Experimental Oncology, UNIFESP, So Paulo
(2) Department of Microbiology, Immunology and Parasitology, UNIFESP, So
Paulo
Contact details
Paola M. Dantonio
E-mail: paola.dantonio@hotmail.com
Address: Rua Dr. Diogo de Faria, 824, 5th floor, room 8
Telephone: +55 11 5576-4848 (extension 2569)
Mobile: +55 16 98250-0099
Background: Multiple myeloma (MM) is characterized by infiltration of tumor
plasma cells in bone marrow (BM), secretion of clonal immunoglobulins and tissue
damages. Although overall survival increased in the last years, MM remains an
incurable disease due to high relapse rates. Cancer stem-cells (CSCs) may be related
to drug resistance and disease relapse, and figure as possible therapeutic target.
Boucher et al. (2012) proposed CD19+/CD34+/CD138- antigen expression and the
activity of the aldehyde dehydrogenase 1 (ALDH1) for immunophenotypic
characterization of multiple myeloma CSCs (MM-CSCs). Aims: To isolate and
characterize immunophenotypically, functionally and by gene expression the MMCSCs derived from BM samples of newly-diagnosed MM patients. Methods: BM
aspirates were collected and CD138+ cells were separated by magnetic sorting. The
remaining cells were submitted to sorting by flow cytometry (FACSAria II). Cells
were labeled with anti-CD19, anti-CD34 and anti-CD138 antibodies, in addition to
Aldefluor/ALDH1 reagent. Results: CD34+/CD19+/CD138-/ALDH1+ population
could be isolated in MM samples (median: 1.556, range: 126-16.633, n = 10).
Experiment controls are CD138+ cells (median: 72.904, range: 1.536-580.700, n =
13). Conclusion and Next Steps: Isolated MM-CSCs will be submitted to RNA
extraction and analysis by RT Profiler PCR Array Human Cancer Stem Cells
(Qiagen) to assess gene expression profile. It contains 84 genes involved in several
cellular processes such as cellular proliferation, asymmetric division, cellular
migration, signal transduction and stemness-related genes (NANOG, POU5F, SOX2,
ALDH1A1, CD34 and NOTCH). We expect to find potential targets to improve
current MM treatment. Financial Support: FAPESP 2010/17668-6. Ethical Approval:
CEP 0127/2014.
Natalia Maria Candido (1), Marlia de Freitas Calmon (1), Maryanne Trafani de
Melo (2), Antnio Cludio Tedesco (2), Paula Rahal (1).
(1) Department of Biology; Laboratory of Genomic Studies; Institute of Bioscience,
Language & Literature and Exact Science IBILCE/UNESP; So Jos do Rio Preto,
SP, Brazil
Rua Cristvo Colombo, n 2265, Jardim Nazareth, CEP 15054-000
Telephone contacts: +551732212379 and +5516997826445
na_candido@hotmail.com
(2) Department of Chemistry; Photobiology and Photomedicine Research Group;
Faculty of Philosophy, Sciences and Letters of Ribeiro Preto FFCLRP/USP;
Ribeiro Preto, SP, Brazil
Nanotechnology applied to cancer treatment is evolving rapidly and new techniques,
such as photodynamic therapy, are being implemented to solve limitations of
conventional therapeutic strategies. For this, nanoemulsions (NEs) have shown
advantages over other chemotherapeutic carriers, and its association with
photodynamic therapy has been quite an application newsworthy. Therefore, this
study proposed to investigate the action of NEs in murine metastatic breast cancer
cell line (4T1). Four different nanostructured systems were synthesized and
characterized: empty NEs; NEs containing chloro-aluminium phtalocyanine
(ClAlPc), which is a photosensitizer agent; NEs with doxorubicin (DOX), which is a
widely used chemotherapeutic agent and, lastly, NEs with both ClAlPc and DOX.
These NEs were incubated with 4T1 cells at different concentrations and, after the
incubation and photoactivation by laser, cell uptake analyses, cell viability assays,
cell death and cell cycle evaluation were performed. Confocal microscopy showed
efficient NEs uptake by cancer cells and MTT assay showed biocompatible
formulations, with cell viability around 80 to 100%, considered not toxic to cells in
vitro. However, the photodynamic therapy associated to the chemotherapeutic agent
NE-mediated increased dramatically the cytotoxic levels, demonstrating an excellent
treatment approach. Flow cytometry corroborated the previous results by showing a
significant increase in cell death and cell cycle arrest. So, the biocompatibility
associated with an adequate biodistribution leads to results that favor the use of these
protocols in vivo. Thus, the potential NE-mediated photodynamic therapy combined
to chemotherapy is fairly encouraging and could improve the available
methodologies for breast cancer treatment.
Financial Support: Coordinating for the Improvement of Higher Education Personnel
(CAPES)

A65
A66
BREAST CANCER-DEPENDENT MESENCHYMAL STROMAL CELL

MIGRATION IS PARTIALLY INHIBIT BY ASPIRIN
POTENTIAL
EVALUATION
OF
CYTOTOXIC
2TETRADECYLCYCLOBUTANONE LINEAGE IN CELLULAR BRL 3A STUDIES IN VITRO
Rafaela de Assiz Louback1, Thas Ribeiro de Oliveira2, Mayra dos Santos Carneiro3,
Karina Lani Silva3, Ana Luiza Azeredo Coutinho de Castro1, Maria Clara Calvacante
Cavallari1, Hlio dos Santos Dutra1, Martin Bonamino3, Rafael Lindoso4, Maria
Isabel Doria Rossi1.
1
Instituto de Cincias Biomdicas e Hospital Universitrio Clementino Fraga Filho,

UFRJ; 2Instituto de Bioqumica Mdica, UFRJ, 3Instituto Nacional do Cncer
(INCA) ,4Instituto de Biofsica Carlos Chagas Filho, UFRJ.
Introduction and aims. Mesenchymal stromal cells (MSC) migration to tumor
microenvironment and its contribution to the reactive stroma are induced by proinflammatory cytokines and extracellular vesicles (EVs) that might be a target for
nonsteroidal anti-inflammatory drugs such as Aspirin. Our aim was to investigate the
effect of Aspirin on bone marrow (BMSC) and adipose (ADSC) MSC migration to
luminal and basal breast cancer cell lines.
Methodology. ADSC and BMSC were obtained from patients undergoing plastic
surgery or bone marrow donors. Procedures were approved by the HUCFF-Ethical
Board. Breast cancer cell lines T-47D and MDA-MB-231, and the human fibroblast
cell line (FPH) were from the Rio de Janeiro Cell Bank. Transwell migration and cell
viability assays, and measurement of EVs secretion were performed. Wnt canonical
signaling was investigated in MDA-MB-231 cells transduced with 7TGC (7xTcFeGFP//SV40-mCherry) plasmid.
Results. ADSC and BMSC migrated more efficiently towards soluble factors
secreted by MDA-MB-231. No difference was observed in relation to FPH whose
migration towards tumor cell lines was lower. Aspirin induced cell death at 5.55mM
while after 24h of incubation with 1.39 and 2.78 mM the chemotactic potential of
MDA-MB-231 was reduced. At 2.78mM Aspirin reduced, after 24h, the amount of
EVs produced by tumor cells. However, preliminary results indicate that Aspirin
does not inhibit Wnt canonical signaling in MDA-MB-231 cells.
Conclusion. MSC chemotaxis towards the more aggressive breast cancer cell line is
partially inhibited by Aspirin independently of canonical Wnt signaling. Aspirin
interferes with EVs biogenesis and induces cell death at high concentrations.
Financial Support: CNPq and FAPERJ
The authors have no conflict of interest
.
Barbezan, A. B., (1), Sales, B. R. (2), Martins, R. (1), Bueno, J.B (1), Santelli, G. M.
M. (2), Villavicncio, A. L. C. H (1)
(1) Radiations of the Technology Center of the Institute of Energy and Nuclear
Research, University of So Paulo, So Paulo, Brazil
(2) Department of Cell Biology and the Institute of Biomedical Sciences
Development, University of So Paulo, So Paulo, Brazil
Corresponding author. Tel.: 55 11 3133-9803
E-mail: angelbarbezan@usp.br
Introduction: 2-Tetradecylcyclobutanone (2-tDCB) is a radiolytic product generated
from foods with fatty acids (triglycerides) which was also subjected to irradiation in
parts of the 2-tDCB ingested and excreted by means of the feces and part were
deposited in adipose tissue. Thus, studies have been worked recently only in colon
cells. In this present work, Liver cells BRL 3A line were chosen since the
accumulation of fat in the body is quite common.
Objective: In order to evaluate the possible cytotoxic damage by cell viability test
MTT. It was observed the influence 2-tDCB in different concentrations of different
incubation times in liver cells BRL3A lineage.
Methods: The compound 2-tDCB was solubilized in 2% ethanol where the selected
line is derived from normal rat liver (BRL3A). In addition, they were grown in
culture medium supplemented with 10% fetal bovine serum. Cells were plated at the
density 4X103 cells/ well in a 96 well plate. The cytotoxic effect of 2-tDCB was
evaluated at concentrations of 100, 300 and 500M for 24 to 48 hours. Furthermore,
all the tests were performed according to kit MTT in triplicates and the results were
analyzed by GraphPad Prism.
Results: As a result, the lines treated with 2-tDCB in 24 hours, cytotoxicity appeared
in concentrations from 300M. In the period from 48 hours showed only change in
the concentration of 300M.
Conclusion: In conclusion, the 2-tDCB compound exhibits significant cytotoxicity in
24-hour period in BRL3A line from the concentration of 300M and a slight effect
48h period was observed for 100M concentration. In fact, samples with 300M
showed a little more pronounced. Therefore, future studies will be necessary to
identify the molecular mechanisms where compound in question operates.
Financial Support: IPEN / CNEN and CNPq.
A67
A68
CHARACTERIZATION OF CANINE BREAST TUMOR ( TUBULAR

CARCINOMA) WITH ENPHASIS IN THE EXTRACELLULAR MATRIX
ESTABLISHMENT OF A CULTURE OF CANINE MAMMARY TUMOR

FOR IN VITRO TREATMENT USING AMNIOTIC STEM CELLS
Marcella Giancoli Kato Cano da Silva, Jssica Borghesi, Mariana Ferreira Lima,
Katia de Oliveira Pimenta Guimares, Maria Angelica Miglino, Phelipe Oliveira
Favaron
Jssica Borghesi, Marcella Giancoli Kato Cano da Silva, Mariana Ferreira Lima,
Maria Angelica Miglino, Ana Claudia Carreira Oliveira, Phelipe Oliveira Favaron
University Paulista-UNIP, Sao Paulo-SP, Brazil

Department of Surgery, School of Veterinary Medicine and Animal Science,
University of Sao Paulo (FMVZ-USP), Sao Paulo-SP, Brazil;
*Email: marcellakato@gmail.com
Background: The breast tumor is the most common neoplasm in dogs. For sharing
various anatomical and physiological similarities, dogs are a valuable model for
understanding this type of tumor in humans.
Aims: The objective of this research was to analyze the histopathologic
characteristics of tubular carcinoma in dogs, using light microscopy,
immunohistochemistry, and identification and quantification of collagen in the
extracellular matrix of the tumors.
Methods: Samples of 5 nodes of carcinoma tubular collected in neutering campaigns
in Sao Paulo city were dehydrated in a series of ethanol concentrations (70-100%),
cleared in xylene and then embedded in paraffin. The 5 m sections were stained
with hematoxylin and eosin and Masson trichrome.
Results: Histologically, the cells from tubular carcinoma had a rounded-shape with
an eosinophilic cytoplasm and distinct borders. It was possible to check the tubular
cells structure, as well as the presence of leukocytes, especially lymphocytes in areas
of necrosis. Interlobular fibroblasts vessels and stromal feature occurring in plasma
cell infiltrate. Characteristic areas of fibrosis composed by collagen fibers were
present in the tumoral parenchyma.
Conclusion: The tubular carcinoma in dogs showed similar structural characteristics
comparing with other species. However, considering that the literature for
classification of the different types of tumor are still confuse, herein we intend to do
an in-deep characterization of tubular carcinoma in dogs using histopathological
analysis, as well as expression of markers (vimentin, cytokeratin, E-Cadherin,
PCNA, p53, VEGF, anti-estrogen, anti-progesterone).

University of Sao Paulo (FMVZ-USP), Sao Paulo-SP, Brazil;
University Paulista-UNIP, Sao Paulo-SP, Brazil
NUCEL (Cell and Molecular Therapy Center) and NETCEM (Center for Studies in
Cell and Molecular Therapy), School of Medicine - Chemistry Institute,
Biochemistry Department, Sao Paulo University, Sao Paulo-SP, Brazil.
*Email: jehborghesi@hotmail.com
Background: Treatment of breast cancer in dogs is based on surgery and adjuvant
chemotherapy. Recently, stem cells have been indicated as an alternative for
treatment of cancer due the ability of cell recruitment, releasing of anti-inflammatory
cytokines and pro-apoptotic factors, as well as promoting apoptosis.
Aims: The aim of this study was to establish a culture of canine mammary tumor
cells for future evaluation of its treatment using canine amniotic stem cells (ASC).
Methods: 10 samples of breast carcinoma were collected for cell culture, which were
placed in polypropylene conical tubes after enzymatic digestion (containing DMEMhigh glucose and 0.1% type I collagenase) for 2h at 37C. Thereafter, the digestion
medium was centrifuged, the supernatant discarded and the pellet resuspended in
DMEM-high glucose, 10% FBS, 1% streptomycin/penicillin, and 1% non-essential
amino acids. The cells were analyzed according to morphology and characteristics of
growth. The Project was aproved by the bioethic comission of Scholl of Veterinary
Medicine and Animal Science-USP (Protocol number 6391091215).
Results: 24 hours after to be plated, the first adhered cells were observed. The cells
had a fibroblast-like morphology with elongated cytoplasm and central nucleus. The
cells had a satisfactory and rapid growth capacity, typically observed for tumor cells.
Normally, the cells were expanded after 3 days of the culture until passage 4, and
subjected to freezing assay using 10% DMSO and 90% of FBS.
Conclusion: Due the immunomodulation characteristics of ASC it is expected a
reduction in proliferation of the tumoral cells. We expected to establish a new
treatment using ASC for treatment of canine mammary tumor, since today the
conventional treatment did not present significant results.

A69
A70
ORGANOTELLURIUM COMPOUNDS INDUCE ACTIN CYTOSKELETON

DISARRAY AND MEMBRANE BLEBBING ON MELANOMA CELL LINES
ANTICANCER AND ANTI-INFLAMMATORY ACTIVITIES OF THE

ETHANOLIC CRUDE EXTRACT FROM THE BRAZILIAN RED
PROPOLIS
Adam A. Martens, Stephanie C.C. Santos, Rodrigo L.O.R. Cunha, Glucia M.M.
Santelli
Background Malignant melanomas display high variability, being the tumours with
highest frequency of somatic mutations, which has led to the development of
personalised medicine, in which each tumour is screened for mutation and so drugs
directed to the defective proteins are used. To circumvent this issue, we would like to
propose a drug that would act on melanomas regardless of their genetic background,
and at the same time spare non-cancer cells. For that, we use enantiomeric
organotellurium compounds (RF-13R and RF-13S) in metastatic melanoma cell lines
with different genetic background (HT-144, SK-Mel-19, SK-Mel-28 and SK-Mel147).
Aims The aim of this work was to evaluate the actin cytoskeleton alterations induced
by RF-13R and RF-13S on melanoma cell lines, as well as also possible efficacy
differences between the enantiomers.
Methods Cells were stained with anti-tubulin-TRITC, phalloidin-FITC and DAPI
and morphology was evaluated in LSM510 laser scanning confocal microscope. Cell
movement and formation of membrane blebbing was evaluated in real time
microscope InCell analyser 2200
Results Fluorescent staining shows actin cytoskeleton disarray and some level of
cortical accumulation of actin on all melanoma cell lines, but not on control cell line
NGM, which retained its morphology and display stress fibres. Real-time analysis
showed compromised motility and directionality, as well as different degrees of bleb
formation, varying from small blebs all around the cell perimeter to large single
blebs which expanded and retracted.
Conclusion These organotellurium compounds act specifically on melanoma cell
lines inducing cytoskeletal alterations which compromise invasion processes.
Thais Petrochelli Banzato (1), Fernanda Francetto Juliano (2), Paula Araujo
Monteiro (1), Fabiana Regina Nonato (1), Dbora Barbosa Vendramini Costa (3),
Severino Matias de Alencar (2), Joo Ernesto de Carvalho (4)
(1) DFT, CPQBA, University of Campinas - UNICAMP, Campinas, SP, Brazil
(2) Department of Agri-Food Industry, Food and Nutrition, Luiz de Queiroz
College of Agriculture, University of Sao Paulo (USP), Piracicaba, SP, Brazil
(3) Cancer Biology - Tumor Immunology, The Wistar Institute, Philadelphia-PA,
US.
(4) Faculty of Science Pharmaceutical, University of Campinas - UNICAMP,
Campinas, SP, Brazil
Email: thaisbanzato@gmail.com.br
Inst.: (19) 21392875
Mobile: (19) 997491123
The tumor microenvironment offers multiple targets for cancer therapy, including
inflammation. Thus, co-treatment with drugs that modify the inflammatory
environment can promote a response with adjuvant chemotherapy acting on specific
targets. Natural products are a major source of effective drugs for the treatment of
cancer and often inspire the development of new potential agents. The Brazilian red
propolis, collected from Alagoas State, northeast of Brazil, presents a unique
chemical composition enriched in isoflavones and its biological properties still need
to be explored. This study aimed to investigate the antiproliferative activity of the
ethanolic crude extract from the Brazilian red propolis (EERP) using the ehrlich solid
tumor model, as well as its anti-inflammatory potential, using the carrageenaninduced paw edema (doses used were 50, 100 and 200 mg/Kg, gavage),and croton
oil-induced ear edema (topical application 200mg/Kg), in Balb-c mice. Treatments
with 200 mg/kg and 100 mg/kg inhibited paw edema formation in the first 24 hours
and between 4.5 and 6 hours of experiment, respectively. The highest dose also
decreased the ear edema formation by 53.95%, which was associated with inhibition
of neutrophil migration to the inflamed site, measured by myeloperoxidase level. All
treatments were able to decrease tumor growth in 40%, without signs of toxicity such
as loss of body weight, changes in blood parameters and biometry of organs. This
preliminary study shows that the Brazilian red propolis possesses antiproliferative
and anti-inflammatory activities. Further studies will be conducted to evaluate the
connection between these activities.
FAPESP: 2013/24416-1, Ethical Approval 3922-1
A71
A72
EVALUATION
OF
A
7-KETOCHOLESTEROL
LOADEDPHOSPHATIDYLSERINE LIPOSOME ON INFLAMMATION AND
MACROPHAGE PROLIFERATION
FUNCTIONAL
AND
MOLECULAR
EVIDENCE
FOR
HETEROMERIC ASSOCIATION OF P2Y 1 RECEPTOR WITH P2Y 2
AND P2Y 4 RECEPTORS IN MOUSE GRANULOCYTES
Giovani Marino Favero (1,2), Daniel Fernandes (3), Ana Paula Prestes (3), Louise
Nicolle Bach Kmetiuk (2)Tharcisio Citrangulo Tortelli Junior (1), Roger Chammas
(1)
(1)
Institute of Cancer of State of Sao Paulo ICESP Department of
Radiology and Oncology, Faculty of Medicine, University of Sao
(2)
Departmentof General Biology, Ponta Grossa State University, Brazil
(3)
Department of Pharmaceutical Sciences, Ponta Grossa State
University, Brazil
Antonio Carlos Ribeiro-Filho ( 1 ) , Marcus Vinicius Buri ( 2 ) , Carlos Castilho

Barros(3), Juliana Luporini Dreyfuss ( 2 ) , Helena Bonciani Nader ( 2 ) , Giselle
Zenker Justo ( 2 ) , Rogrio Bastos Craveiro(4), Joo Bosco Pesquero(4), Antonio
Miranda (4), Alice Teixeira Ferreira ( 4 ) , Edgar Julian Paredes-Gamero ( 1 , 2 * ) .
Background: The exposure of phosphatidylserine (PS) is one of the first steps of

programmed cell death. Phagocytosis on cancer microenvironment is well described
in tumors and is associated with malignancy and poor prognosis. Tumor Associated
Macrophages (TAMs) act suppressing the anticancer immune response. The tumor
parenchymal cells are also capable of phagocytosis cells in apoptosis. In a previous
study we observed that 7-Ketocholesterol are capable to inducing autophagy on
melanoma cell. Aims: Evaluate the activities of a 7-ketocholesterol loadedphosphatidylserine liposome on autophagy and phagocytosis of tumor
microenvironment. Methods: Liposomes were constituted by 100mg commercial
Phosphatidylserine and 10 mg of 7-ketocholesterol extracted with chloroform /
methanol (10: 1), dried, resuspended in 10 mL phosphate buffer, homogenized and
sonicated for 20 minutes. The size and Zeta Pontencial(ZP) of liposomes were
evaluated. Anti-inflammatory activity of liposomes was evaluated by Paw Edema
induced by carrageenan. A dependent-dose effect of liposomes on J774 macrophages
was assessed by MTT. Results: PS liposomes presented 141,9nm + 9,101 size with a
-25,2 ZP; PS-7-Ketocholesterol (PS/7KC) liposomes presented 153,9 nm + 10,35
size with a -29,1 ZP. The paw edema was inhibited by both liposomes after 240 min
of the carrageenan induction. The concentration of 26uM/mL of PS and PS/7KC
liposomes stimulated cell proliferation. PS/7KC at the concentrations above
84uM/mL inhibited the cell proliferation. Conclusion: PS liposomes have effects in
vivo and in vitro and must be related to macrophage activity. PS/7KC in high doses
impairs the J774 macrophage growth.
Supported by CNPq and Fundao Araucria
gmfavero@yahoo.com.br
Av. Dr. Arnaldo, 251, 8o andar CTO
CEP 01246-000, Cerqueira Cezar, Sao Paulo, SP, Brazil
Phone: +55(11) 3893-3007
Mob.: +55(42) 9128-0063
Centro Interdisciplinar de Investigao Bioqumica, Universidade de Mogi das

Cruzes, Av. Dr Cndido Xavier de Almeida Souza, 200. Mogi das Cruzes, SP,
Brasil.
2
Departamento de Bioqumica, Universidade Federal de So Paulo, R.
Trs de Maio 100, So Paulo, SP, Brasil.
3
Departamento de Nutrio, Universidade Federal de Pelotas, R. Gomes Carneiro,
n1, 96010-610, Pelotas, RS, Brasil.
4
Departamento de Biofsica, Universidade Federal de So Paulo, R.
Botucatu 862, So Paulo, SP, Brasil.
*Corresponding author: Edgar J. Paredes-Gamero
Email: edgar.gamero@unifesp.br
Universidade Federal de So Paulo
Rua Pedro de Toledo, 669 - 9o andar - Prdio de Pesquisa II
CEP: 04039-032; Tel: +55(11) 55764868; Fax: +55(11) 5573-6407
Background: All hematopoietic cells express P2 receptors, however pharmacological
characteristics such as expression and affinity in granulocytes are unknown.
Aims: The aim of this study was to observe the molecular association among P2Y
receptors.
Methods: Pharmacological characteristics of P2 receptors were evaluated by Ca2+
measurements using Fura-2 fluorophore. P2 receptors expression were analyzed by
flow cytometry and RT-PCR. P2 interaction were shown by coimmunoprecipitation,
western blotting and FRET.
Results: Granulocytes were responsive to P2Y agonists, whereas P2X agonists were
ineffective. Ca2+ increase, elicited by ADP and UTP was dependent on intracellular
stocks and sensitive to G-coupled receptor inhibition. Moreover, MRS2179, a
specific antagonist of the P2Y1 receptor, abolished ADP response. Interestingly,
ADP and UTP exhibited full heterologous desensitization, suggesting that these
agonists interact with the same receptor. The heteromeric association between P2Y1
receptor and the P2Y2 and P2Y4 receptors was shown by immunoprecipitation and
FRET analysis.
Conclusion: Clear evidence of heteromeric association of P2Y receptors was found
during the evaluation of P2 receptors present in mice granulocytes, which could
impact in the classical pharmacology of P2Y receptors in granulocytes.
The experimental approved by the Bioethics Committee of our Institution - Federal
University of So Paulo (1464/03).
Funding Support: "Coordenao de Aperfeioamento de Pessoal de Nvel
Superior (CAPES)", "Conselho Nacional de Desenvolvimento Cientfico
e Tecnolgico" (CNPq) and Fundao de Amparo Pesquisa do Estado
de So Paulo (FAPESP Grant number: 2013/09068-7 and 2015/16799-3)

A73
A74
ADENOSINE PRODUCES A DUAL EFFECT ON CELL VIABILITY IN

ESOPHAGEAL CANCER CELL LINES
NOVEL BENZOTHIAZOLE AFN01 PRESENTS ANTI-PROLIFERATIVE

EFFECTS ON GASTRIC CANCER CELL LINE INDUCING CELL CYCLE
ARREST
Mathias Andr Kunde1, Anglica Regina Cappellari2, Pedro Vargas2, Bianca Regina
Ribas de Abreu2, Jade dos Santos Ferreira Moreira3, Fernanda Bueno Morrone2,3
1
Faculdade de Medicina, Pontifcia Universidade Catlica do Rio Grande do Sul,

Porto Alegre, Brazil;
2
Programa de Ps-Graduao em Biologia Celular e Molecular, Laboratrio de
Farmacologia Aplicada, Pontifcia Universidade do Rio Grande do Sul, Porto Alegre,
Brazil;
3
Faculdade de Farmcia, Pontifcia Universidade Catlica do Rio Grande do Sul,
Porto Alegre, Brazil.
Esophageal cancer (EC) is highly aggressive and considered the sixth most deathly
cancer in the world. There are two main subtypes of EC: esophageal adenocarcinoma
(EAC) and esophageal squamous cell carcinoma (ESCC). Purinergic signaling has
been shown as an important mechanism involved in cancer biology. In this way,
adenosine was described to favoring cancer cell proliferation, angiogenesis and
immune suppression. However, it was also showed to promote cancer cell death.
Therefore, the present study intended to evaluate the effect of adenosine on EC
proliferation and cell viability. We used two different EC cell lines: OE33 and OE21,
representative of EAC and ESCC, respectively. Adenosine was used as treatment in
the following concentrations 0.1, 1 and 5 mM. The cell number was determined by
Tripan Blue exclusion method after 24 hours of the treatment and the cell viability
was evaluated by MTT assay after 24, 48 and 72h. We observed that in OE33
lineage, adenosine promoted an increase in cell proliferation at 0.1 mM and a
reduction of the cell viability at 5 mM after 48 and 72h. In OE21 cells, adenosine
induced an increase of cell viability at 0.1 mM, in 48h and promoted a reduction of
cell viability at 1 and 5 mM after 24, 48 and 72h of treatment. In conclusion, our
results suggest that adenosine can promote an increase of cell proliferation in low
concentrations and in higher concentrations it can promote cell death in both EC cell
lines evaluated.
Key words: Esophageal cancer, adenosine, purinergic system
Supported by: CAPES, CNPq, FINEP
Registered author:
Name: Mathias Andr Kunde
Email: mathias.kunde@acad.pucrs.br
Address: Rua Professor Cristiano Fischer, nmero 2256, apartamento 208 Porto
Alegre/RS - CEP: 91410-000
Phone: 51-9197-5137
Felipe Pantoja Mesquita (1), Laine Celestino Pinto (1), Emerson Lucena da Silva (1),
Bruno Moreira Soares (1), Ingryd Nayara de Farias Ramos (1), Adrhyann Jullyanne
de Sousa Portilho (2), Tassa Mara Thomaz Arajo (2), Andr Salim Khayat (2),
Thatyana Rocha Alves Vasconcelos (3) Rommel Mario Rodrguez Burbano (1),
Raquel Carvalho Montenegro (1).
(1) Laboratory of Human cytogenetic, Federal University of Par, Belm, Brazil
(2) Oncology Research Center, Federal University of Par, Belm, Brazil
(3) Department of Chemistry, Federal University Fluminense, Rio de Janeiro, Brazil
E-mail: felipe_mesquita05@hotmail.com
Phone:(91) 98810-5758
Despite incidence rates has been decreased in developed countries since 20th
century, gastric cancer is the third leading cause of death. Furthermore,
chemoresistance and high toxicity in gastric cancer treatment brings out importance
to searching for new compounds. Benzothiazoles and its analogues have attracted
considerable attention because for their anticancer properties. Thus, aim of this
present study was investigated anticancer potential of novel Benzothiazole AFN01 in
gastric cancer cell line. Previously, it was performed MTT assay to obtain cytotoxic
behavior in gastric adenocarcinoma cell lines (AGP01, ACP02 and ACP03). Cell
line with great cytotoxic activity was selected to perform clonogenic assay and cell
cycle test. Results were obtained from three independent experiments and
statistically evaluated by analysis of variance test (ANOVA) followed by posttests.
AFN01 showed a high cytotoxic activity against AGP01 (IC50 = 1.90 M), ACP02
(IC50 = 1.08 M) and ACP03 (IC50 = 1.80 M). ACP02 cell line was selected to
performed clonogenic assay, which showed that AFN01 reduces replicative cancer
cell capability at 1 M (p<0.001) and 2 M (p<0.0001) after 72 hours treatment
compared to negative control, as well as doxorubicin at 2 M (p<0.0001). Then, cell
cycle analysis reveal that G0/G1 and S phase of cell cycle arrest is induced by
benzothiazole at 1 M (p<0.001) and 2 M (p<0.0001) compared to negative
control. In conclusion, AFN01 is an excellent anticancer agent candidate reverting
proliferative feature, although further studies are required. Funding support:
FAPESPA.
A75
A76
DIFFERENT EFFECTS OF TELOMERASE INHIBITORS ON LUNG

CARCINOMA CELLS AND NON-TUMOR CELL IMMORTALIZED
ORGANOTELLURIUM COMPOUNDS INDUCE INCREASING

FREQUENCY OF MICRONUCLEI IN MELANOMA CELL LINES
Anali D.M.B.Garnique, Glaucia Maria Machado-Santelli.
Stephanie C. C. Santos (1) , Adam A. Martens

Glucia M. Machado- Santelli(1) .
Departamento de Biologia Celular e do Desenvolvimento, Instituto de Cincias

Biomdicas, Universidade de So Paulo, So Paulo, Brasil.
Background: The telomere is a repeat sequence of double-stranded DNA that
protects chromosome ends, the length is maintained by telomerase, whose expression
is low or absent in somatic cells but is present in most cancer cells. As the cell
continues to divide, telomere shortening and undergoes a process called telomere
dysfunction. This DNA breaks activates p53, a tumor suppressor that induces cell
senescence, or other checkpoint responses depending on the cell type. Telomerase is
therefore regarded as an important target in cancer therapy.
Aims: To study the effects of telomerase inhibitors, their interference on cellular
proliferation and its effect on senescence induction.
Methods and Results: LC-HK2 (NSCLC) and hTERT EPR-1 cell lines were treated
with TMPyP4 inhibitors (5M) and Thymoquinone (10 and 40 M) for 24, 72 and
120 h. Treatment with TMPyP4 increased cell proliferation after 120 h and both lines
were strongly adhered to the plate surface. The LC-HK2 line treated with 40 M
thymoquinone for assay of senescence-associated -galactosidase showed a higher
frequency of senescent cells when compared to control where there is also loss of
cell adhesion in both strains. The cell cycle did not change in any of the treatments
with respect to control.
Conclusion: Both lines appear to respond to treatment with telomerase inhibitors by
changing cell morphology and proliferation. Depending on the concentration of the
inhibitor, affects cell adhesion varied shape and survival of the cell.
(CAPES, CNPq, FAPESP)
(1)
(2)
(1)
, Rodrigo L. O. R. Cunha
IN
(2)
Department of Cell and Developmental Biology, Institute of

Biomedical Sciences University of So Paulo, So Paulo, Brazil.
Center for Natural and Human Sciences, Federal University of ABC,
So Paulo, Brazil.
Background DNA damaging agents are commonly used as chemotherapy drugs, for
DNA damage activates checkpoints, and promotes cell cycle arrest or cell death and,
if the damage is irreversible such as the ones detected in micronucleus assay
induce cellular senescence or signal to non-apoptotic cell death pathways. The
tellurium compounds exhibit varied activities in cells malignant, as growth arrest
induction and apoptosis, suggesting promising application as potential agents anticancer. Therefore, we propose to evaluate the potential genotoxic of organotellurium
compounds.
Aims The aim of this work was to evaluate the potential genotoxic effect of
organotellurium compounds RF-13R and RF-13S on melanoma cell lines, as well as
possible differences in responses between the enantiomers.
Methods Cells were treated with organotellurium enantiomers RF-13R or RF-13S in
presence of the cytochalasin- B, a cytokinesis inhibitor
Results SK-Mel-28 treated with RF-13R and RF-13S increased micronucleus
frequencies when compared to the control. RF-13S was more potent . Cells stained
with propidium iodide were analyzed on fluorescence microscope. One thousand
binucleated cells were counted and the number of nuclear alterations (micronuclei,
nuclear bud and nucleoplasmic bridge) was assessed.n inducing formation of
micronuclei. Cell lines HT-144 and SK-Mel-147 showed no relevant difference
between treated and control cells. For all cell lines, frequencies of nuclear bud and
nucleoplasmic bridge were similar in all treatment conditions.
Conclusion The enantiomers showed different potency in inducing micronucleation,
and their effects depended on the cell line used; results for cell line SK-Mel-28
suggest that both compounds display genotoxic effects.

A78
A77
MONOPOLAR MITOTIC SPINDLE AND FAILURE CYTOKINESIS
MODULATE HIPPO PATHWAY
GENERATION AND CHARACTERIZATION OF A KNOCKIN CELL LINE

FOR THE LI-FRAUMENI BRAZILIAN FOUNDER MUTATION TP53R337H
Ana Carolina Chiacetti Rodrigues* (1), Hermano Martins Bellato (1), Fernanda
Cristina Sula Lupinacci (1), Jssica Tayna da Silva (1), Glaucia Noeli Maroso Hajj
(1), Vilma Regina Martins (1), Martn Roff (1).
Maria Oliveira, 1Paula Rezende-Teixeira,1Flavia Belarmino, 1Glacia M MachadoSantelli.

1.Department of Cell and Developmental Biology, University of So Paulo, So
Paulo, Brazil.
Numerical chromosomal change plays a controversial role in cell biology. The
aneuploidy, feature found in most human solid tumors, in the cancer it has been
associated with the worst prognosis of the disease. Desregulation of Hippo signaling
pathway have also been reported in various types of cancers. Therefore, the aim
ofthis study is increase genetic instability by interfering in cell cycle progression and
evaluating the Hippo pathway in this cell population. The MCF-7 breast cancer cell
was sequentially treated with monastrol and blebbistatin determined increase of
population in G2/M, indicating that there was a delay in the cell cycle progression as
shown by flow cytometry. These cells showed altered morphology with cytoplasmic
projections. The expression hippo pathway components was evaluated by qRT-PCR.
There was a marked increase of Lats mRNA expression after treatment with
monastrol and blebbistatin. YAP/TAZ have also been expressed over the control, but
to a lesser rate compared to mRNA Lats. Similar profile was observed in the
blebbistatin treatment and after 48hrs recovery. Expression of actin mRNA was also
higher compared with the control, suggesting a possible involvement of cytoskeletal
in morphological changes. The cellular localization of Lats, YAP and TAZ proteins
was assessed by immunofluorescence and actin staining with phalloidin. The images
were acquired and evaluated by scanning laser microscopy.
(CNPq, CAPES, FAPESP)
(1) Cellular and Molecular Biology Laboratory, International Research Center,

A.C.Camargo Cancer Center, So Paulo, Brazil.
* anachiacetti@gmail.com; +55 11 2189-5000, r. 2977; +55 11 99185-3856
BACKGROUND: Li-Fraumeni syndrome is a rare hereditary condition caused by
germline mutations on TP53. These mutations increase the risk of developing several
kinds of tumors in specific organs, such as adrenocortical gland, brain and breast. In
Brazil there is a founder mutation at codon 337 that leads to the substitution of an
arginine by a histidine (R337H). However, the few studies characterizing the
molecular mechanisms of TP53R337H failed to definitively address the alterations that
are responsible for tumorigenesis in Li-Fraumeni syndrome. AIM: Construction of a
knockin cell line TP53R337H for the characterization of molecular alterations related
to TP53 function. METHODS: CRISPR/Cas9 technology was used to generate a
knockin cell line bearing TP53R337H and immunofluorescence to evaluate expression
and localization of p53 and its transcriptional target MDM2. RESULTS: The U87MG clone (here named as 5M) was obtained using the CRISPR/Cas9 system.
After transfection, clone selection and RFLP-screening, this clone was sequenced
and it was shown to contain an allele with R337H mutation and a knockout allele.
TP53R337H accumulates in the nucleus even in the absence of a TP53-activating
stimulus. Furthermore, MDM2 not only accumulates in the 5M clone, but also is
more translocated into the nucleus in the presence of the R337H mutation.
CONCLUSIONS: These results suggest that the R337H mutation can normally
activate MDM2 expression, however, MDM2-mediated degradation might be
altered.
Supported by FAPESP and CNPq.
Ethical approval: 1050/15 (COPE).
A79
PGE2 INDUCES EPITHELIUM-MESENCHYMAL
COLORECTAL CANCER CELLS.
A80
TRANSITION
IN
JAK1/2 INHIBITION AS ALTERNATIVE TREATMENT FOR MULTIPLE

MYELOMA
rika Elias Ferreira (1) , Jos Andrs Morgado-Diaz (1)

(1) Structural Biology Group, Cell Biology Program, National Institute of Cancer
(INCA), Rio de Janeiro, Brazil.
Mariana Bleker de Oliveira (1); Veruska Lia Fook Alves (1); Angela Isabel Pereira
Eugnio (1); Rodrigo Carlini Fernando (1); Gisele Wally Braga Colleoni (1)
(1) Department of Clinical and Experimental Oncology, UNIFESP, So Paulo-SP.
Address of the presenting author:

Rua Andr Cavalcanti, 37 - Centro, CEP: 20231-050 - Rio de Janeiro - RJ
Contact Details
Address: Rua Doutor Diogo de Faria, 824, Hemocentro, CEP 04037-003, So Paulo,
SP, Brazil.
Email: marianableker@gmail.com
Institutional number: (11) 5576-4848 (extension 2659)
Mobile number: (11) 99356-7049
Contact details:
Tel - +55 (21) 3207-6537 , Mobile - +55 (21) 99996-9674
email - ef.erika@yahoo.com.br or jmorgado@inca.gov.br
Background: The inflammatory process is one of the most important risk factors in
colorrectal cancer (CRC) and the increased expression of COX-2 enzyme is
associated with this process. It is known that prostaglandin E2 (PGE2), the main
product of COX-2, is a potent inducer of tumor progression, increasing the
metastasis ability of cancer cells. During initial steps of cancer progression the cells
adquire an undifferentiated phenotype providing a more migratory and invasive
potential, an event kowns as epithelial-mesenchymal transition (EMT). In this
context, the role that PGE2 plays during the EMT is not completely understood.
Aims: To analyse the role of PGE2 during the EMT development in CRC. Methods:
CRC cells, HT-29, were treated with different concentrations of PGE2 and the
proliferation rates were analyzed after 24, 48 and 72hs. Western blotting (WB) and
immunofluorescense (IF) assays were employed to analyze the expression and
subcellular localization of epihelial and mesenchymal markers. Additionally,
analysis by polymerase chain reaction was conducted to evaluate the PGE2 receptors
profile. Results: 1000nM PGE2 increased the proliferation of HT-29 cells after 48h
of treatment. We observed that PGE2 in this time and concentration induced a
decrease of E- cadherin and claudin 3 expression as well as protein redistribution
from the cell-cell contacts. In addition, IF for beta-catenin sugests translocation of
the protein to the nucleus in the treated cells. Conclusion: HT-29 cell treated with
PGE2 display events related with EMT development and these results are the basis to
determine the mechanistic by which PGE2 induces EMT.
CNPQ, FAPERJ and MS
Disclosure of Conflicts of Interest

The authors declare no conflicts of interest to disclosure.
Background: Constitutive activation of JAK proteins by IL-6 in multiple myeloma
(MM) promotes survival and proliferation of tumor cells and is still an unexplored
therapeutic target in this incurable disease. Aims: To perform functional studies in
MM cell lines treated with JAK1/2 (ruxolitinib) and proteasome (bortezomib)
inhibitors. Methods: JAK1/2 expressions were analyzed by real time PCR in MM cell
lines. In vitro studies were performed using bortezomib (B) and ruxolitinib (R) in
RPMI-8226 cell line, alone and in co-culture with normal stromal cell line HS5.
Apoptosis and cell cycle were assessed by flow cytometry. Results: RPMI-8226
showed expression of JAK1/2 and, after treatment with B+R, presented higher
number of cells in SubG0 phase (p<0.001), with reduction of cells in S (p<0.01) and
G2/M (p<0.001) phases, comparing with wild type. RPMI-8226 presented 50% of
cells in late apoptosis after treatment with B+R, but co-culture protected these cells
from apoptosis (32% after B+R) (p<0.001). The addition of immunomodulatory
drug lenalidomide (L) reversed the protective effect of co-culture and combination of
three drugs (B+R+L) induced 62% of cell death (p<0.05). Conclusion: B+R
combination induced cell cycle alterations and apoptosis in RPMI-8226 (partially
reverted with stromal cells co-culture). This new drug combination has a rational for
MM treatment, but the protective effect of microenvironment is still a challenge to be
overcome. The addition of an immunomodulatory drug, like lenalidomide, added
evident in vitro benefit. Financial support: FAPESP 2010/17668-6 and CNPq
(140107/2014-2). Approved by UNIFESP Ethics Committee (0011/13).

A81
A82
OXATP, AN ANTAGONIST OF P2X7 RECEPTOR, INHIBITED THE CELL

DEATH INDUCED BY ANTIMICROBIAL PEPTIDE GOMESIN.
ROLE OF [10]-GINGEROL IN BREAST CANCER METASTASIS IN VIVO
Wynne Barbosa de Sousa Silva

Paredes Gamero (1,2).
(1)*
; Antonio Carlos Ribeiro Filho
(1)
; Edgar Julian
1-Interdisciplinary Center for Biochemical Research, University of Mogi das Cruzes,

Av. Dr Cndido Xavier de Almeida Souza, 200. Mogi das Cruzes, SP, Brazil.
2-Department of Biochemistry, Federal University of So Paulo, R. Trs
de Maio 100, So Paulo, SP, Brazil.
*Corresponding author:
Email: wynnebarbosass@gmail.com
University of Mogi das Cruzes, Av. Dr Cndido Xavier de Almeida Souza 200,
Mogi das Cruzes, SP, Brazil. Cep: 08780-911
+55(11) 4798-7102/ 4798-7103
Background: The P2X7 receptor, expressed on CHO cells, is related to cell death cell
by membrane permeabilization. In addition, the antimicrobial peptide Gomesin also
promotes membrane permeabilization by overload of Ca2+. In this study the
relationship between P2X7 receptor activation and Gomesin peptide activity was
evaluated. Objective: Establish the relationship between CHO cell death by Gomesin
and P2X7 receptor activation. Materials and Methods: CHO cells were seeded and
stimulated with Gomesin to determinate the EC50 by MTT assay. Cell death was
confirmed by Annexin-V and Propidium iodide assay. Then, OxATP an irreversible
antagonist of P2X7 receptor was used. OxATP was able to reduce the Gomesindependent cell death. Results: The EC50 of Gomesin was 20 M. Gomesin promoted
double label Annexin+Propidium iodide+. The P2X7 receptor inhibition reduced the
cell death triggered by Gomesin. Conclusion: An interaction between Gomesin cell
death mechanism and P2X7 activation could occur to promote cell death in CHO
cells.
FAPESP, CNPq and CAPES for the Financial support.
Ana Carolina B. M. Martin(1); Angelina Maria Fuzer(1); Rebeka Tomasin(2), Amanda

Blanque Becceneri(1); Paulo Cezar Vieira(3); Heloisa Sobreiro Selistre de Arajo(4);
Normand Pouliot(2); Marcia Regina Cominetti(1)
(1)
(2)
(3)
(4)
Department of Gerontology Federal University of So Carlos

Peter MacCallum Cancer Centre
Department of Chemistry Federal University of So Carlos
Department of Physiology Federal University of So Carlos
Contact details:
Ana Carolina Baptista Moreno Martin(1), martin.acbm@gmail.com
Rodovia Washington Luis, Km 235 Federal University of So Carlos Department
of Gerontology
Background: The major cause of death in cancer patients is due the metastases
formation. Triple negative breast cancer (TNBC) affects 20% of women with breast
cancer. This cancer type is very aggressive and has a high propensity to form lung
and brain metastases. Nowadays, there is no effective treatment for TNBC and
therefore, new therapies are required.
The aim of this study was to verify the inhibitory effects of [10]-gingerol in
spontaneous triple negative breast cancer model in vivo.
Methods: MTT assay was performed to verify the inhibitory effects of [10]-gingerol
in cell proliferation. To investigate [10]-gingerol mechanisms of action, apoptosis
and cell cycle assays were performed. For the in vivo data a spontaneous model of
breast cancer metastasis to brain was used.
Results: [10]-gingerol was able to inhibit cell proliferation in different cell lines. In
the murine cell line (4T1Br4), [10]-gingerol induced apoptosis and cell cycle arrest
at sub-G0 phase. [10]-gingerol inhibited lung, bone and brain metastases in an intramammary fat pad spontaneous breast cancer model.
Conclusion: [10]-gingerol showed to be a non-toxic compound with antiproliferative effects, inducing cell apoptosis in vitro and also inhibiting lung, bone
and brain metastases in a spontaneous breast cancer model in vivo. Patent deposit:
BR 10 2015 024093 7. Approved by ethical committee E507 and 3224-1.
Financial support: FAPESP, CNPq, CAPES.
A83
A84
EFFECTS OF CELL-MATRIX INTERACTIONS IN EPITHELIALMESENCHYMAL TRANSITION OF BREAST CANCER CELLS: ROLE OF

INTEGRINS.
Brando-Costa R,M1, Helal Neto E2, Vieira AM1, Magne TM1, Morandi V1, BarjaFidalgo C1.
1
2
UERJ - UNIVERSIDADE DO ESTADO DO RIO DE JANEIRO

FIOCRUZ- FUNDAO OSWALDO CRUZ
Metastasis is a process that involves primary tumor cells invasion and escape from
physical barriers, like extracellular matrix (ECM), to disseminate in distant organ
sites. Genetic and epigenetic changes triggers epithelial-mesenchymal transition
(EMT) in tumor cells, causing metastasis. During tumor progression, tumor cells are
able to remodeling their own matrix, increasing the expression of ECM proteins that
contribute to its growth and invasion. Integrins are responsible for cell-ECM
interactions and transduction of signals. Integrins can crosstalk with tyrosine kinase
receptors, like TGF-b receptor, through non-canonical signaling pathways. However,
the effect of an ECM obtained from mesenchymal cancer cells on tumor epithelial
cells is still unknown. Here we evaluated the mechanisms involved in the contact of
a mesenchymal breast cancer lineage (MDA-MB-211)-produced ECM (MDAECM), with an epithelial breast cancer lineage, MCF-7. Comparative analyses
proteins expression showed that MDAMB-231 expressed more laminin and tenascinC when compared to MCF-7. Although there is no difference in the expression of
fibronectin by two cell types, we detected higher levels of fibronectin in MDA-ECM.
MCF-7 cells cultured on MDA-ECM showed morphological changes, assuming a
mesenchymal and migrating profile. In addition, the contact with MDA-ECM
induced a decrease in E-cadherin levels and increase in N-cadherin, -SMA and
fibronectin in MCF-7. MCF-7 cells cultured on MDA-ECM showed an increase in
FAK, ERK and AKT phosphorylation. These results suggest that MDA-ECM
modifies the epithelial profile of MCF-7, enabling integrin-dependent signaling
pathways that are related to the expression of mesenchymal markers and to MCF-7
migration, contributing to EMT.
Brando-Costa
R.M.
Renata
Machado
Brando
Costa
(renata_machado88@hotmail.com) (21) 2868-8298 - Av 28 de setembro, 87 fds.Vila Isabel - Rio de Janeiro, RJ - Brasil.
RESISTANCE AGAINST EHRLICH ASCITES TUMOR PROGRESSION

CAUSED BY PYCNOGENOL IN MICE
Isabela Machado Barbosa David (1), Flvia de Souza Fernandes (1), Thiago Bezerra
Gaspar Carvalho da Silva (2), Rodrigo Costa Zeferino (3), Maicon Roberto
Kviecinski (1)
(1) Post-Graduate Program in Health Sciences. Universidade do Sul de Santa
Catarina (UNISUL). Palhoa, SC, Brazil. E-mail: isabela.nutre@gmail.com; Phone:
+55 48 32791167 or +55 48 96974123
(2) Medicine School, Universidade do Sul de Santa Catarina (UNISUL). Palhoa,
SC, Brazil.
(3) Laboratory of Experimental Biochemistry, Universidade Federal de Santa
Catarina, Florianpolis, SC, Brazil
Background: Evidences indicate that pycnogenol, a standardized extract of French
maritime pine bark, has chemopreventive potential against carcinogenesis. Aims: To
evaluate a resistance caused by pycnogenol against Ehrlich ascites tumor progression
in mice. Methods: Mice were divided (n=12) and pretreated for 20 days every 24 h
by gavage: one negative control pretreated with excipient, one positive control
pretreated with resveratrol (25 mg/kg) and one group pretreated with pycnogenol
(100 mg/kg). Ehrlich tumor cells (5 x 106) were inoculated intraperitoneally. Weight
and abdominal circumference were measured at the beginning and 10 days after
tumor inoculation, when half of each group was euthanized for the evaluation of
ascitic fluid (volume, compacted cell volume, cell viability, type of cell death, lipid
peroxidation, GSH content and catalase activity). The remaining animals were kept
for timing lifespan. Results: Compared to the negative control, pycnogenol and
resveratrol inhibited weight gain (30 and 27%), abdominal circumference (up to 35
and 30%) and the volume of compacted cells (>15 and 5%). Only pycnogenol
reduced significantly the ascitic fluid volume and the count of tumor viable cells,
increasing the count of non-viable ones and apoptosis (2-fold). Pycnogenol caused
superior increase of lifespan (up to 60%), whereas resveratrol caused <
25%. Pycnogenol and resveratrol caused similar protection against lipid peroxidation
(up to 30%), reducing the activity of catalase (50%) and increasing GSH levels (2fold). Conclusion: From the overall, pycnogenol caused superior resistance against
Ehrlich ascites tumor progression compared to resveratrol. Ethical approval
(15.025.4.01.IV). Authors declare no conflict of interest.
Neto, E. H. Edward Helal Neto (edwneto@gmail.com) 21) 3977-2484 Av

Comandante Guaranys, 447 - Jacarepagu / Cep: 22775-903 - Rio de Janeiro/RJ
Brasil
Vieira A.M. Andreza Maia Vieira (deza.jpa@gmail.com ) - (21) 2334-0499 - Rua
So Francisco Xavier, 524 Maracan.
Magne TM- Tas Monteiro Magne (tais.magne@hotmail.com) (21) 2868-8298 Av 28 de setembro, 87 fds.- Vila Isabel - Rio de Janeiro, RJ - Brasil.
Morandi, V Vernica Maria Morandi Da Silva (veronica@uerj.br) (21) 23340499 - Rua So Francisco Xavier, 524 Maracan.
Barja-Fidalgo, C. Thereza Christina Barja-Fidalgo (barja-fidalgo@uerj.br) (21)
2868-8298- Av 28 de setembro, 87 fds. - Vila Isabel - Rio de Janeiro, RJ - Brasil.

A85
A86
CYTOTOXIC POTENTIAL OF NOVEL CURCUMIN-RESVERATROL

HYBRID SYNTHETIC COMPOUNDS ON MCF7
ROLE OF OBESITY AND DEVELOPMENT OF BREAST TUMORS IN

INFLAMMATORY RESPONSE DURING BCG INFECTION: ANALYSIS OF
INFLAMMATORY MEDIATORS SYNTHESIS AND LIPID BODY
FORMATION IN MACROPHAGES
Rodrigo Machado Pereira(1)*, Guilherme lvaro Ferreira da Silva(1), Matheus de

Freitas Silva(2), Letcia Ferreira Coelho(2), Isadora Mitestainer Guirelli(2), Cludio
Viegas Jnior(2), Marisa Ionta(1)
(1) Institute of Biomedical Sciences, Federal University of Alfenas, MG, Brazil
(2) Institute of Chemistry, Federal University of Alfenas, Alfenas, MG, Brazil
*rodrigomachadopereira@yahoo.com.br, 55-35-32991360 or 55-35-998242841
Antitumor activity of curcumin, a derived from Curcuma longa, has been reported.
Likewise, resveratrol, a polyphenol present in several dietary sources has been
considered as a potential chemotherapeutic agent. Although of pharmacological
potential of these compounds their clinical application has been limited due to their
low solubility and bioavailability. In this frame, organic medicinal chemists have
worked in obtaining new prototypes designed by molecular hybridization between
active natural products. The present study aimed to analyze the cytotoxic potential of
novel compounds that contain curcumin-resveratrol structural analogy domains on
MCF-7 cells. Cell viability was determined by MTS and trypan blue exclusion assay.
Cell cycle progression and apoptosis induction were investigated using flow
cytometry. From eight different hybrid compounds screened on different tumor cell
lines (MCF-7, A549, HepG2 and HT144), two of them significantly reduced cell
viability of MCF-7 cells. IC50 values were 40.49 1.01 M and 19.09 0.67 M,
respectively, for PQM-162 and PQM-163, which were significantly lower in relation
to those found for curcumin (IC50 = 68.25 2.59 M) or resveratrol (IC50 > 100 M).
The combined use of curcumin and resveratrol also were investigated and we
observed a slightly synergistic effect (IC50 = 61.71 2.42 M). PQM-162 and PQM163 capacity of reducing cell viability in MCF-7 was due to, at least in part, their
ability to promote G2/M arrest and cell death. Taken together, our data support that
these innovative synthetic hybrid compounds represent promise chemical structures
for further investigation for their antitumor activity.
Financial support: Fapemig, CAPES, FINEP and CNPq.
Sabrina P. Maciel (1), Gabriel S. C Rodrigues (1), Daniel A. M. Toledo (1), Nathlia
N. Gonalves (1), Fernanda Vandanezi (1), Sayuri Oti (1), Jacy Gameiro (2), Heloisa
D. S. Bizarro (1)
(1) Laboratory of Cell Biology, Federal University of Juiz de Fora, Juiz de Fora,
Brazil.
(2) Laboratory of Immunology, Federal University of Juiz de Fora, Juiz de Fora,
Brazil.
Email: sabrina.pmaciel@gmail.com
Address: Rua Alzira Feliciana Siqueira, 85, bairro Jardim Novo Horizonte, CEP:
37.410-000 Trs Coraes/MG, Brazil
Telephone contact: (32) 2102-3206/218 (institutional); (32) 991388412 (mobile)
Lipid bodies (LBs) are organelles involved in the metabolism of lipids and
intracellular signaling, which increase in number and size in leucocytes during
inflammatory processes, neoplastic and infectious, including obesity, breast cancer
and intracellular infections by Mycobacterium bovis BCG, respectively. The
lipogenesis is associated with poor prognosis in various malignancies, suggesting the
role of LBs in cancer development. This work studied the correlation of breast tumor
and obesity in inflammatory response in the face of M. bovis BCG infection.
Approval of the ethics board certified by the Protocol 039/2012-CEUA, Balb/c
female mice were subjected to diet for 16 days for in vivo stimulation with tumor
cells 4T1 and after 14 days, macrophages from peritoneum and fat tissue were
obtained and cultured in vitro to stimulation with BCG for 24h. The results
demonstrated that macrophages from obese animals have increased numbers of LBs
as well as increased PGE2 production and increased leptin synthesis and cytokines
TNF- and TGF- compared to macrophages from lean animals with tumors.
Macrophages from lean animals showed increased adiponectin and IL-10, antiinflammatory profiles. Our results suggest that the tumor together obesity enhances
the formation of LBs and the synthesis of inflammatory mediators during BCG
infection, demonstrating the importance of the role of LBs as a target for future
therapeutic interventions to influence the balance of the interactions between
pathogen and the host. Support: FAPEMIG; CNPq, PROPESQ/UFJF

A87
A88
CELL INTERACTIONS IN 3D ENVIRONMENT BETWEEN DENDRITIC

CELL-TUMOR HYBRID CELLS AND HUMAN LYMPHOCYTES: AN
ANTITUMOR VACCINE ENHANCEMENT
ANTI-TUMOR EFFECT OF DNA IMMUNIZATION WITH SCFV6.C4 IN

ASSOCIATION WITH FRC OR IFN-
Karen SteponaviciusCcruz (1,2); Alexandre Urban Borbely (2); Vanessa Morais

Freitas (3); Jos Alexandre Marzago Barbuto (1).
(1) Department of Immunology, Institute of Biomedical Sciences, University of Sao
(2) Cell Biology Laboratory, Institute of Biological and Health Sciences, Federal
University of Alagoas, Maceio, Brazil
(3) Department of Cell and Developmental Biology, Institute of Biomedical
Sciences, University of Sao Paulo, Sao Paulo, Brazil.
Background: The behavior and functions performed by dendritic cell-tumor hybrid
cells are still insufficiently known, although it is widely used. Aims: This study
aims to analyze the interaction of these hybrid cells with different subpopulations
of lymphocytes in a three dimensional (3D) environment with or without collagen
III. Methods: Mature DCs (mDC) were fused with tumor cells from patients with
breast cancer or with SKBR-3 cell line and co-cultured with CD4+ and CD8+
lymphocytes in a 3D environment with collagen III, divided in groups and
analyzed through CBA and confocal time-lapse microscopy. Results: Our results
indicated that cells in Tumor Fusion group interacted with both CD4+ and CD8+
lymphocytes. The SKBR-3 Fusion group interacted only with CD4+ lymphocytes.
The CD4+ lymphocytes interacted twice in the SKBR-3 Fusion group than in other
groups. Besides, their interaction was only with a single lymphocyte. CD8+
lymphocytes interaction with Tumor Fusion group resulted in longer interactions
when in comparison with the other groups and hybrid cells had a reduced
migratory speed in comparison with mDCs. No IL-2 was observed in cultures
where the hybrid cells were present and the proliferation of lymphocytes in Tumor
Fusion group was not observed. Conclusion: The 3D co-culture was able to
demonstrate hybrid cells derived from breast cancer had different behavior to other
cells showing the importance of a custom protocol for each tumor type, taking into
consideration their unique characteristics. Funding Support: FAPESP
(2010/18139-7) and CNPq (2009/54599-5). Ethical Approval: Ethics Committee
on Human Research of ICB-USP (078.2010).
Bianca Ferrarini Zanetti (1), Camila Pontes Ferreira (2), Jos Ronnie Carvalho de
Vasconcelos (2), Sang Won Han(1)
(1) Research Center for Gene Therapy, Department of Biophysics, Universidade
Federal de So Paulo, So Paulo, Brazil.
(2) Department of Microbiology, Immunology and Parasitoloy, Universidade Federal
de So Paulo, So Paulo, Brazil.
bianca_zanetti@msn.com; Rua Mirassol, 207, 04044-010, So Paulo, So Paulo,
Brazil. Phones: (+5511) 98218.2105 (mobile); (+5511) 5084.7582 (institutional)
Background: Previously, we created a DNA vaccine against carcinoembryonic
antigen (CEA)-expressing tumors using a surrogate for CEA, scFv6.C4, and its
efficacy was validated in CEA- transgenic mice (CEA2682). Aims: To assess
adjuvant effect of FrC (Fragment C of Tetanus Toxin), GM-CSF (GranulocyteMacrophage Colony-Stimulating Factor), IFN- (Interferon Gamma) and IDUA
(Alpha-L-Iduronidase) in the DNA vaccine with scFv6.C4. Methods: C57bL/6CEA2682 mice were immunized 4 to 5 times by IM electroporation with the plasmid
vector uP-PS/scFv6.C4 alone or in association with vectors coding for FrC, GMCSF, IFN- or IDUA. These vaccinated mice were challenged by subcutaneous
injection of the murine colorectal cell line MC38-CEA (1x105 cells) and tumor
growth was monitored. The humoral and cellular immune responses were assessed
by ELISA and T cell proliferation assay, respectively. Results: The scFv6.C4
immunization gave rise to antibodies able to recognize CEA, and the titer was 4
times higher in comparison to pre-immune sera (p<0,001). When challenged with
MC38-CEA cells, approximately 50% of the scFv6.C4 immunized animals were free
of tumor during 100 days of follow-up, and others had tumor growth rate diminished.
The adjuvants tested in this study did not increase significantly antibody titers,
however, the mice immunized with scFv6.C4 and FrC or IFN- showed an increased
survival rate, but it was not statistically significant. Cell proliferation assay showed
an increased in CD8+ response after tumor challenge. Conclusion: DNA
immunization with scFv6.C4 in association with FrC or IFN- enhanced antitumor
effect. Ethical Approval: CEUA 2013/703012 CIBio 17/2013. Funding Support:
FAPESP.
To whom correspondence should be addressed: Karen Steponavicius-Cruz, Cell

Biology Laboratory, Institute of Biological and Health Sciences, Federal
University of Alagoas, Campus A.C. Simes. Av. Lourival Melo Mota, s/n,
57072-970. Maceio, Alagoas, Brazil. Laboratory Phone: ++558232141704.
Mobile: +558296473987. E-mail: kstepon@hotmail.com

A89
A90
EVALUATION OF METFORMIN ON PROSTATE EPITHELIAL CELLS

AFTER
FATTY ACID AND/OR INSULIN TREATMENT IN HIGH
CONCENTRATION
INHIBITION OF MDA-MB-435 CELL ADHESION AND MIGRATION BY

[10]-GINGEROL AND ITS PROBABLE INTERACTION WITH INTEGRIN
V3
Breno Costa Landim (1), Renata Graciele Zanon (1), Claudio Vieira Silva (1),
Daniele Lisboa Ribeiro (1,2)
Tsuboy, M.S.F.1,2,*; Uliana, C.V.3; Oliveira, T.R.3; Fraga, H.M.4; Fernandez, J.H.4;
Faria, R.C.3; Lima, M.A.5; Cominetti, M.R.1; Selistre-de-Arajo, H.S.2
brenolandim@hotmail.com
(1) Institute of Biomedical Sciences, Histology Department. Federal University of
Uberlandia, Uberlandia, MG, Brazil, 38400-902
(2) Leader
1-Laboratrio de Biologia do Envelhecimento, Departamento de Gerontologia, 2Laboratrio de Bioqumica e Biologia Molecular, Departamento de Cincias
Fisiolgicas, 3-Laboratrio de Bioanaltica e Eletroanaltica, Departamento de
Qumica, Universidade Federal de So Carlos, UFSCar, So Carlos, SP. 4-Centro de
Biocincias e Biotecnologia, Universidade Estadual do Norte Fluminense, UENF,
Campos dos Goytacazes, RJ, 5-Departamento de Bioqumica, Universidade Federal
de So Paulo, UNIFESP, So Paulo, SP.
A clinical relationship between metabolic disorders and obesity is widely described

recently. Metformin is a drug used for diabetes treatment and due to its AMPK
activation and control of cellular metabolism, a protective role of metformin in
cancer has been postulated. The aim of this investigation was to evaluate the effect of
high concentrations of fatty acid and/or insulin and the role of metformin in these
situations in normal prostate epithelial cells (PNT1A). PNT1A cells were treated
with palmitate (100 M, HF) and/or insulin (50U, HI) with or without metformin
(100 M, MET) for 24hrs and 48hrs and analyzed for different assays. Wound
healing analysis showed that HF and HI stimulated high cell migration, mainly after
48 Hrs and that metformin drastically reduced this effect caused by palmitate and
insulin treatments. MTT assay revealed that PNT1A exhibited high cell proliferation
when treated with HF and HI in 24H and 48H. Surprisingly, MET did not decreased
cell proliferation after HF and HI treatment, and MET per se stimulated a
proliferative status in PNT1 cells. Additionally, the caspase 3/7 assay showed that
there is no difference at apoptotic levels in any of treated groups. Thus, the high
concentration of palmitate and insulin can drastically increase cell proliferation and
migration which could not be completely blocked by metformin, at least, not at this
physiological dosage.
Keywords: fatty acid; insulin; cell proliferation; prostate cancer; metformin
Integrin v3 play a significant role in cancer biology and has been involved in the
pathophysiology and progression of malignant tumors. To date, it was demonstrated
that resveratrol (isolated from grapes) is able to bind to integrin v3 and this
binding is crucial for apoptosis induction in MCF-7 cells. In this study, we have
investigated [10]-gingerol (from ginger) for its specificity for integrin v3, using
inhibition of cellular adhesion and migration assays in MDA-MB-435, staining of Factin, differential scanning fluorometry, cyclic voltammetry and docking analysis.
Cell adhesion to vitronectin was inhibited by [10]-gingerol 10 and 50 M while
adhesion to laminin was inhibited only with 50 M treatment (1h). Cell migration to
vitronectin and laminin was inhibited by [10]-gingerol in both conditions (4h).
Staining of F-actin after treatment with [10]-gingerol (1h) showed concentrationdependent increase in F-actin disturbance. These biological effects observed in
MDA-MB-435 can be, at least in part, a outcome of the interaction of [10]-gingerol
with integrin v3, as demonstrated by the change in melting temperature of integrin
v3 exposed to [10]-gingerol, cyclic voltammograms with increase in the peak
current intensities (non-modified magnetic particle vs. integrin-magnetic particle
incubated with [10]-gingerol) and docking analysis, which showed two possible
models of interaction between [10]-gingerol and integrin v3 through MIDAS site.
Taking together, our data suggest that [10]-gingerol could be a molecule candidate to
a new ligand for integrin v3. Further studies are being conducted to confirm the
interaction of [10]-gingerol with integrin v3 in order to understand its potential
anti-metastatic effects.
Financial Support: FAPESP (2013/00798-2 and 2014/08015-0).
E-mail address:
Marcela S F Tsuboy mastetsu@hotmail.com
Universidade Federal de So Carlos, UFSCar, So Carlos, SP
Telefone: (16) 3306-6672 (16) 99793-7694
A91
A92
THE ROLE OF O-GLCNACYLATION ON HUMAN MELANOMA CELL

LINES MIGRATION
NUCLEOPLASMIC RETICULUM
MESENCHYMAL STEM CELLS
Felipe Bouchuid Cato1, Bruno Loureno Diaz1, Wagner Barbosa Dias1, Michelle
Botelho Caarls1
Camila Cristina Fraga Faraco (1); Jerusa Arajo Quinto Arantes Faria (1); Michele
ngela Rodrigues (1,2); Dawidson Assis Gomes (1).
(1) Departamento de Bioqumica e Imunologia, Universidade Federal de Minas
Gerais.
(2) Departamento de Patologia Geral, Universidade Federal de Minas Gerais.
Universidade Federal do Rio de Janeiro, Instituto de Biofsica Carlos Chagas Filho

(Rio de Janeiro Brasil)
Melanoma is the most aggressive skin cancer, characterized by the malignant
transformation of melanocytes. The disease can progress through different stages:
Radial Growth (RGP), Vertical Growth (VGP) and Metastatic Growth Phases
(MGP). Its a deadly cancer, especially if diagnosed at the metastatic stage.
Therefore, its important to understand the mechanisms involved in migration, and
metastasis. The O-GlcNAcylation is a type of intracytoplasmic dynamic
glycosylation, and, previous studies have shown a deregulation of O-GlcNAcylation
in different cancers leading, in most cases, to malignancy and poor prognosis. The
aim of this work is to compare the migration capacity of three different human
melanoma cell lines, and to investigate the role of O-GlcNAcylation on the
melanoma cell motility. The migration of the cell lines (WM983A - VGP; WM983B
MGP; WM852 - MGP) was analyzed initially by a wound healing assay. WM852
presented the fastest migration. WM983A, had the slowest migration, while,
WM983B, had an intermediate time for closing the scratch space. The cell motility
was also determined by the area of phagokinetic tracks on gold sol particle-coated
plates for the melanoma cell lines incubated with an inhibitor of O-GlcNAcase
(OGA), an enzyme that removes the O-GlcNAc from proteins, therefore, increasing
the total O-GlcNAcylation levels. When compared to the control cells without the
drug, the incubation with the OGA inhibitor decreased the motility of all melanoma
lines. These results indicate that the O-GlcNAcylation can play a role on the cell
motility, and may participate in the final stages of melanoma progression leading to
metastasis.
STRUCTURE
IN
TUMOR
AND
Correspondence: milacffaraco@gmail.com. Instituto de Cincias Biologicas. 6627,

Antnio Carlos Av., Pampulha - Cep:31270-901. Institutional number:
+553134092631
Nuclei morphology can be used to distinguish normal from tumor cells and for
cancer grading. Cancer cells have nuclear pleomorphy and irregularities in the
nuclear envelope (NE). One of those irregularities is in the nucleoplasmic reticulum
(NR), a nuclear organelle composed by invaginations of the NE into the
nucleoplasm. Some studies demonstrated that the NR can be prevalent in cancer
cells; therefore it can be used as a marker enabled to differentiate a tumor cell from
another cell type. Our aim was to compare by immunofluorescence assay the
structure of the nucleus and NR of SK-HEP-1 cells, previously classified as a tumor
mesenchymal stem cell (TMSC), and hASC, human adipose mesenchymal stem cells
(MSC). For that, the NE (anti-Lamin B2), the nucleolus (anti-Fibrilarin) and nucleus
of both cell types were stained. Z-stacks images were acquired using the confocal
microscope LSM 880 with Airyscan. Results reveal that both cell types present
structures stained by Lamin B2 inside the nucleus, considered as NR. SK-HEP-1
cells showed more points of NR invaginations which were longer when compared to
hASC. SK-HEP-1 cells showed osteogenic differentiation capacity, which might be
an evidence of their stem cell profile. Collectively, these data suggest that SK-HEP-1
cells might have a TMSC behavior and that we can differentiate them from normal
MSC by comparing nuclear characteristics. This work was approved by UFMG
ethical committee (CAAE: 22912213.3.0000.5149) and supported by CNPq, CAPES
and FAPEMIG.
Keywords: Melanoma, O-GlcNAcylation, Migration

Supported by: CNPq

A93
A94
GLIOMA-SYNTHESIZED MELATONIN AS A POSITIVE PROGNOSTIC

FACTOR
EVALUATION OF ANTITUMOR ACTIVITY OF AUY922 IN GASTRIC

ADENOCARCINOMA CELL LINES
Gabriela S. Kinker (1), Sueli M. Oba-Shinjo (2), Claudia E. Carvalho-Sousa (1),

Sandra M. Muxel (1), Suely K. N. Marie (2), Regina P. Markus (1), Pedro A.
Fernandes (1).
(1)
(2)
Department of Physiology, Institute of Bioscience, University of So Paulo,

So Paulo, Brazil.
Department of Neurology, School of Medicine, University of So Paulo, So
Paulo, Brazil.
Presenting author: Gabriela S. Kinker, Department of Physiology, Institute of

Bioscience, University of So Paulo, Rua do Mato, 321, tv 14, room 317, So
Paulo, SP 05508-090, Brazil. E-mail: gabriela.kinker@usp.br.
Institutional
telephone: +55 11 30910983. Mobile: +55 11 970664160
Gliomas, the most common primary brain tumors in adults, exhibit poor responses to
standard treatment and are associated with high cancer-related morbidity/mortality.
Recent studies have shown that melatonin, an indolamine synthesized mainly, but
not exclusively, by the pineal gland, induces cytotoxicity in glioma-initiating cells
and reduces the invasion of glioma cell lines, inhibiting the nuclear factor B
(NFB) oncopathway. Given that C6 rat glioma cells produce melatonin, we
investigated the correlation between the capacity of gliomas to synthesize/metabolize
melatonin and their overall malignancy. Using three human glioma cell lines (HOG,
T98G and U87MG), we demonstrated that tumor-synthesized melatonin exerts an
autocrine anti-proliferative effect. Accordingly, the most aggressive cell line
(U87MG), which synthesized/accumulated less melatonin, presented the highest
sensitivity to exogenous melatonin. Based on The Cancer Genome Atlas RNAseq
data of 351 glioma patients, we designed a predictive model of the content of
melatonin in the tumor microenvironment, the ASMT:CYP1B1 index, combining the
gene expression levels of melatonin synthesis and metabolism enzymes. The
ASMT:CYP1B1 index negatively correlated with tumor grade, as well as with the
expression of pro-proliferation and anti-apoptotic NFB target genes. More
importantly, the index was a grade- and histological type-independent prognostic
factor. Even when considering only high-grade gliomas, a low ASMT:CYP1B1
index, suggestive of decreased melatonin and enhanced aggressiveness, was strongly
associated with poor survival, which was further confirmed using an independent
dataset (n = 264). Overall, our data reveal the prognostic value of the melatonergic
system of gliomas and provide insights into the therapeutic role of melatonin.
Adrhyann Jullyanne de Sousa Portilho(1), Tassa Maira Thomaz Arajo(1,2), Felipe

Pantoja Mesquita (1), Aline Damasceno Seabra(1,2), Abigail Nayara dos Santos
Silva (2), Marceli Carolini Alves Almeida (2), Luciana Gonalves Quintana (2),
Antnio Andr Conde Modesto(2), Paulo Pimentel de Assumpo(2), Smia
Demachki(2), Rommel Mario Rodrguez Burbano(1,2), Andr Salim Khayat(1,2).
1
2
Human Cytogenetics Laboratory, Federal University of Par, Belm, PA.

Oncology Research Center, Federal University of Par, Belm, PA.
EmaiL: dry_cnbio@hotmail.com
Phone: (91) 98193-8462
Hsp90 is a major molecular chaperone that plays a pivotal role in assisting correct
folding and functionality of its client proteins in cells. A large number of Hsp90client proteins play crucial roles in establishing cancer cell hallmarks. Hsp90
inhibitors, such as AUY922, can be potential and effective cancer chemotherapeutic
drugs with a unique profile and have been examined in clinical trials, including for
gastric cancer. Thus, the present study aimed at identifying the antitumor effect of
AUY922 (Novartis, USA) in two gastric cancer cell lines, ACP02, stablished from
primary tumor, and AGP01, stablished from peritoneal carcinomatosis, in an attempt
to compare the results between them. We performed MTT assay, cell cycle assay,
scratch assay and ethidium bromide/acridine orange differential staining. Statistical
analyses were done by means of two-way ANOVA, using Prisma program. AUY922
showed cytotoxic activity in AGP01 (IC50 = 2,27 M) and ACP02 (IC50 = 2.75
M) and induced apoptosis in AGP01 at 2.27 M and 4.54 M (p<0.001), and
ACP02 at 2.75 M and 5.5 M (p<0.001). AUY922 also induced G2/M arrest at
both concentrations of the two cell lines (p<0.001). Moreover, this drug inhibited cell
migration of ACP02 six hours after treatment at both concentrations (p<0.05) and
AGP01 six hours after treatment at the highest concentration (p<0.05). Results
showed that antitumor effect of AUY922 was similar in both cell lines, except for
migration, since this drug was more eficient in inhibiting cell migration of cell line
stablished from primary tumor (ACP02) than those obtained from metastasis
(AGP01).
Financing: CAPES.
Financial support: FAPESP (PAF: 2010/52687-1; RPM: 2013/13691-1; GSK:

2014/27287-0), CNPq (RPM: 480097/2013-5; GSK: 162670/2014-1).
Conflict of interest: The authors declare that they have no conflict of interest.
A95
ABIRATERONE ACETATE AND PI3K INHIBITOR (BEZ235) DECREASE

INFLAMMATION AND TUMOR GROWTH IN THE PROSTATE OF RATS
SUBMITTED TO CHEMICAL CARCINOGENESIS
Bianca Facchim Gonalves1, Joyce Zalotti Brandt 1, Cristiane Figueiredo Pinho1,
Wagner Jos Fvaro2, Silvana Gisele Pegorin de Campos3, Wellerson Rodrigo
Scarano1*
1
Sao Paulo State University UNESP, Institute of Biosciences, Department of

Morphology, Botucatu, SP, Brazil
2
University of Campinas UNICAMP, Institute of Biology, Campinas, SP, Brazil
3
Sao Paulo State University UNESP, Institute of Biosciences, Letters and Exact
Sciences, So Jos do Rio Preto, SP, Brazil
The antiandrogen Abiraterone acetate (Abi) has been used to treat prostate cancer
(PCa), but it can lead to tumor resistance and activation of additional pathways such
as PI3K/Akt. Thus, the association of this therapy with BEZ235 appears as an
alternative strategy to control PCa progression. This study aimed to evaluate the
effect of Abi and BEZ on PCa. Male adult Fischer 344 rats were inoculated with
MNU (15mg/kg) in the dorsolateral prostate (DLP) and received testosterone
cypionate twice a week for 220 days, subcutaneously. After tumor-induction animals
were allocated into four groups: I- Tumor-induced control; A (Abi-14mg/kg); B
(BEZ-45mg/kg) and AB (Abi+BEZ). Treatments were performed by gavage for 10
days. DLP was removed, weighed and submitted to histopathological analysis for
lesions incidence and multiplicity, epithelial proliferative index (Ki67) and Western
blot. DLP weight decreased in AB group compared to control. Drugs treatment
decreased proliferative index and the incidence and multiplicity of premalignant and
malignant lesions. Oppositely, atypical hyperplasia increased in A and B compared
to control and AB. Inflammatory-related disorders such as reactive hyperplasia and
intraluminal inflammation decreased in treated groups, while stromal inflammation
was significantly reduced in AB compared to control. TNF- expression decreased in
A and AB, while IL-6 decreased in all treated groups, suggesting reduction of
inflammation. Histopathological data and western blot analysis suggest that Abi and
BEZ act on reduction of cell proliferation and inflammatory process, contributing to
the reduction of incidence and progression of prostatic lesions. Financial Support:
FAPESP (2013/22604-5;2014/14202-7; Protocol CEUA: 559).
The authors declare that they have no conflict of interest.
Keywords: Prostate cancer, Abiraterone acetate, BEZ235, N-methyl-N-nitrosurea,
Fischer 344 rats.
*Correspondence: Wellerson Rodrigo Scarano, PhD, Rua Professor Doutor Antonio
Celso Wagner Zanin, s/n. Institute of Biosciences of Botucatu - UNESP,
Department of Morphology, CEP:18618-689. Botucatu, SP, Brazil. Email:
scarano@ibb.unesp.br; Phone: +55 14 3880047
A96
RESTORATION OF TGF- SIGNALING PATHWAY MODULATES
MicroRNAS EXPRESSION PROFILE IN PAPILLARY THYROID CANCER
Kelly Cristina Saito (1), Cesar Seigi Fuziwara (1), Edna Teruko Kimura (1).
Sciences, University of So Paulo, So Paulo, Brazil.
Disclosures: The authors declare no conflict of interests that could prejudice the
impartiality of this study
Corresponding author: Kelly Cristina Saito. Department of Cell and Developmental
Biology. Institute of Biomedical Sciences. University of So Paulo. Av. Prof. Lineu
Prestes 1524 room 414. CEP 05508000. Butant. So Paulo, SP.Brazil. Telephone:
(55) 11 3091-7304. e-mail: saito@icb.usp.br
Background: TGF- anti-proliferative pathway is activated by TGF- binding to
membrane receptors triggering SMAD2/3 phosphorylation, which in turn, combine
with SMAD4 to nuclear translocation. Papillary thyroid carcinoma (PTC) escape to
TGF- action due to reduced SMAD4 levels, effect partially attributed to microRNA
miR-146b up-regulation. On the other hand, biogenesis and maturation of several
microRNAs are controlled by SMADs.
Aims: The objective of the present study was to evaluate the global miRNA
expression modulated by TGF-/SMADs pathway in PTC cells.
Methods: SMAD4 was transfected in BCPAP cells (pBabe-SMAD4-Flag-puro) and
gene expression were analyzed by Real Time-PCR and proliferation rate by
cytometry. Global microRNA expression was evaluated by Affymetrix GeneChip
miRNA 3.0 Array and data processed in Expression and Transcriptome Analysis
Console (TAC) Software to obtain differentially expressed microRNA. MicroRNAs
sequences and target prediction were retrieved from miRBase and miRWalk
webtools.
Results: SMAD4 mRNA level in BCPAP-SMAD4 cells was increased 3-fold,
resulting in reduced cell number (30%-24h, 50%-48h, and 30%-72h). Genechip array
(3 times-fold change) revealed 58 modulated microRNAs, 19 down and 39 upregulated. Among deregulated microRNA, in silico analysis identified the RNASmad Binding Element (SBE) sequence (5`-CAGAC-3`) in the pre-microRNA
sequence of miR-1299, miR-877, miR-150 and miR-589. Interestingly, target
prediction of miR-150 revealed its potential regulation in proliferative pathways
members, such as, BRAF, CCND1 and CCND2 and this microRNA was downregulated in PTC.
Conclusion: The microRNAs global expression revels a new set of microRNA
potentially regulated by TGF-/SMAD, suggesting a new important role of SMADs
in thyroid tumorigenesis.
Information on ethical approval and funding support: All procedures were conducted
in accordance to the guidelines of Ethical Committee for Animal Research of
Institute of Biomedical Sciences, University of So Paulo, Brazil (134/10).Supported
by FAPESP, CNPq and NapmiR.

A97
A98
NOTCH PATHWAY IS ENHANCED BY MODULATION OF MicroRNAs IN

TSH INDUCED GOITER.
TARGETING AURORA A KINASE ACTIVITY IN LUNG CANCER CELLS

DRIVEN BY ONCOGENIC KRAS IMPAIRS LUNG CANCER INITIATING
CELL FUNCTION
Dbora Guimares Nadale de Souza (1), Thiago Maciel dos Santos de Oliveira (1),
Cesar Seigi Fuziwara(1) e Edna Teruko Kimura (1)
(1) Department of Cellular Biology and Development, Institute of Biomedical
Sciences, University of So Paulo, Brazil
Luiza Coimbra Scalabrini (1), Tatiana Correa Carneiro-Lobo (1), Lutero Augusto
Hasenkamp (1), Daniela Sanchez Bassres (1).
(1) University of So Paulo, So Paulo, SP, Brazil
Corresponding author: Dbora Guimares Nadale de Souza, Department of Cellular

Biology and Development, Institute of Biomedical Sciences, University of So
Paulo, Brazil. Address: Av. Professor Lineu Prestes, 1524; room 414. Telephone:
(11) 3091-7304 (11)97719-9111 email: dgnsouza@usp.br
Background: Goiter is the thyroid growth frequently observed in the population. The
TSH stimulation is a main inductor of thyroid follicular cell proliferation. The
implication of others signaling pathway, such as, induced growth factor and
microRNAs can contributed to the benign goiter physiopathology.
Aims: To investigated the role of microRNAs global expression and its potential
targets in proliferative signaling pathways in TSH induced goiter.
Methods: Female Wistar rats (n=6) were treated with methimazole (0.05%) in
drinking water. After 5 days thyroid were removed and total RNA was extracted
from pooled thyroid tissue using Trizol (n=3). Another set was processed to
immunohistochemistry (n=3). The global miRNA analysis was performed using rat
genome PCR-Array/miRNome (Qiagen). Differentially expressed miRNAs were
processed in Pcr Data Analysis Software and miRNAs targets were searched
using TargetScan program.
Results: Analysis of PCR-Array containing 653 miRNAs showed 84 differentially
expressed miRNAs, which 18 were down-regulated and 66 were over-expressed. In
silico analysis of targets modulated miRNAs showed an enrichment of Notch
pathway components, such as, Notch1 and Numb mRNAs. Notch1 is the receptor
that promotes cell proliferation and targeted by miRNAs miR-34c-3p and miR-30c1-3p that are down-regulated 7.0-fold and 2.4-fold, respectively. In addition, miR146b-5p that is over-expressed 2.1-fold, targets Numb, a repressor of Notch pathway.
Indeed, immunohistochemistry showed increased Notch 1 expression in goiter
samples.
Conclusion: TSH induced goiter reveals an enrichment of microRNAs targeting
Notch signaling showing a new role of Notch involved with goiter.
Activating mutations in KRAS are prevalent in cancer and RAS signaling is

enhanced in cancer initiating cells (CICs), which are self-renewing tumor cells able
to initiate tumor formation, sustain tumor growth and drive tumor dissemination.
However, therapies targeting oncogenic RAS were sofar ineffective, and
identification of KRAS targets with therapeutic potential is warranted. Given that we
have recently shown that Aurora kinase A (AURKA) is a KRAS target in lung
cancer, and that AURKA has been implicated in promoting CIC function, we
hypothesized that targeting AURKA would impair KRAS-positive lung CIC
malignant function. We targeted AURKA in KRAS positive lung cancer H358 and
A549 cells by RNA interference (RNAi) or with an Aurora A inhibitor (AI II) and
analyzed tumorsphere formation, clonogenic growth and self-renewal, expression of
stem cell surface markers by flow cytometry and expression of stem cell
transcription factors by qPCR. A549 and H358-derived tumorspheres were able to
self-renew and, when compared to the parental cell lines, had increased
clonogenicity in vitro and tumorigenicity in vivo. Tumorsphere cells displayed
increased expression of stem cell surface markers CD44, CD24, CXCR4 and
ABCG2, as well as increased expression of stem cell transcription factors Sox2,
Oct4, Nanog and Bmi1. AURKA targeting decreased the expression of these factors,
tumorsphere formation and growth in serial cultures, and reduced clonogenic growth
of tumorsphere forming cells. Our results suggest that AURKA inhibition therapy
reduces KRAS-positive lung CICs, and might, therefore, contribute to a sustained
therapeutic effect in KRAS-induced lung cancer.
Financial support: FAPESP, CAPES, FAMRI
Presenting author email: scalabrini.luiza@gmail.com
Presenting author phone number: (11)3091-2172 (laboratory); (11)95813-1313
Conflict of interest: All authors disclose any financial and personal relationships with
other people or organizations that could be perceived to bias this work.
Information of ethical approval: All procedures were conducted in accordance with
guidelines of Ethical Committee for Animal Research of Institute of Biomedical
Science, University of So Paulo, Brazil (134/10). Support: CNPq, FAPESP and
NAPmiR

A99
A100
PHARMACOLOGICAL AND BIOCHEMICAL STUDIES FOR ORAL

ADMINISTRATION OF A TOXIN ISOLATED FROM THE VENOM OF A
SOUTH AMERICAN RATTLESNAKE CROTALUS DURISSUS TERRIFICUS
WITH ANTITUMOR EFFECT
Joana D. Campeiro(1), Marcelo P. Marinovic(1), Fernando Cintra Carapeto(2), Caroline
Dal Mas(1), Marcela Nering(1), Gilles Landman(2), Eduardo B. Oliveira(3), Mirian A. F.
Hayashi(1)
(1) Department of Pharmacology, Federal University of So Paulo, So Paulo,
Brazil;
(2) Department of Pathology, Federal University of So Paulo, So Paulo, Brazil;
(3) Faculty of Medicine of Ribeiro Preto, University of So Paulo, Ribeiro Preto,
Brazil.
Background: Crotamine is a small basic polypeptide isolated from the venom of a
South American rattlesnake Crotalus durissus terrificus. Lower concentrations of
crotamine are innocuous for non-tumoral cells, although presenting cytotoxicity
against highly proliferating tumoral cells. In vivo treatment with crotamine injected
intraperitoneally reduces the tumor growth and increase the lifespan of mice with
subcutaneous tumors.
Aims: This work evaluated the viability of use of native crotamine as antitumoral
agent by oral delivery in C57Bl/6J mice animal model engrafted with subcutaneous
B16-F10 melanoma.
Methods: Crotamine-treated group received orally 10 g of crotamine/animal/day,
for 21 days, and control untreated sham group received the vehicle as placebo. At the
treatment end, the animals were euthanized for serum and tissues collection for the
analysis of biochemical parameters (including lipid analysis), and histological
evaluations.
Results: After oral treatment for 21 days, a significant reduction in tumoral growth
with no evidence of toxic effect or lesion in tissues such as liver and kidney was
observed. However, soon after the first days of treatment, a reduction in blood
glucose level was observed and significant difference in weight gain was noticed at
the end of treatment. The lipid profile analysis also showed reduction of total
cholesterol, triglycerides and LDL, with an elevation of LDL levels.
Conclusion; The results confirm the viability of using crotamine as antitumoral by
oral delivery, besides suggesting a possible beneficial role of this polypeptide in
metabolic profile.
THE POTENTIAL EFFECTS ANTITUMOR

DIAPOCYNIN IN HUMAN CELLS
OF
APOCYNIN
AND
Adriana Arruda Matos (1), Flvia Amadeu de Oliveira (1), Cintia Kazuko Tokuhara
(1), Ana Paula Campanelli (1), Valdecir Farias Ximenes (2), Rodrigo Cardoso de
Oliveira (1)
(1) Department of Biological Sciences, Bauru School of Dentistry, University of So
Paulo, Bauru, So Paulo, Brazil.
(2) Department of Chemistry, Faculty of Sciences, University of the State of So
Adress contact:
Alameda Octvio Pinheiro Brisolla 9-75
Cidade Universitria
Bauru-So Paulo-Brasil
Email: adriana-matos@usp.br
Telephone: +55 14 3235 8246 or +55 14 981258448
Background: Osteosarcoma is the most common tumor associated with other
malignancies. In recent decades there has been a notable advance in the treatment of
this disease, however more studies are needed to understand its mechanisms of
action. In this way, apocynin and diapocynin have been used as inhibitors of
processes involved in cancer progression. Understanding the mechanisms by which
these compounds interact with cells may contribute in the understanding of the
development of new therapeutic strategies for bone diseases. Aims: To evaluate the
potential antitumor effects of apocynin and diapocynin in osteosarcoma human cells.
Methods: Human osteosarcoma cell (SaOS-2) and normal human osteoblasts (HOS)
were treated with different concentrations of apocynin and diapocynin (10, 25, 50
and 100 g/mL). After treatment, the cytotoxic effect was evaluated by MTT
reduction assay. The proliferation and apoptosis were analysed by flow cytometry
using CFSE and Annexin V-PI, respectively. Reactive oxygen species (ROS)
production was evaluated by the fluorescent probe DCF. Moreover, cell migration
capacity was assessed by scratch assay. Results: Apocynin and diapocynin, at
concentrations of 50 and 100 g/mL, reduced SaOS-2 viability, but did not affect
HOS. Furthermore, the compounds inhibited cell proliferation and increased the
percentage of apoptotic cells in SaOS-2. Moreover, apocynin and diapocynin also
promoted the reduction of ROS production and inhibited SaOS-2 migration.
Conclusion: Apocynin and diapocynin inhibited viability, proliferation, ROS
production and migration of osteosarcoma cells, nevertheless did not promote
alterations of normal osteoblasts; therefore, these compounds appear to be potential
candidates against osteosarcoma. Funding support: CAPES.
Ethical approval number: 6530090914. Funding support FAPESP, CNPq e CAPES.

A101
A102
CELL CONTRACTILITY MODULATES MIGRATORY BEHAVIOR IN

ORAL SQUAMOUS CELL CARCINOMA
DisBa-01, A RECOMBINANT RGD - DISINTEGRIN, INHIBITS ACTIN

POLYMERIZATION IN BREAST TUMOR CELLS
Grasieli de Oliveira Ramos (1); Maurcio Tavares Tamborindeguy (2,3); Manoel

SantAna Filho (2); Karen Newell-Litwa (4); Alan Rick Horwitz (4); Marcelo
Lazzaron Lamers (2,5)
Rafael Luis Bressani Lino (1); Wanessa Fernanda Altei (1); Ana Carolina Baptista
Moreno Martin (2); Heloisa S. Selistre-de-Arajo (1).
(1) Universidade do Oeste de Santa Catarina, Joaaba, SC, Brazil

(2) Basic Research Center, Dentistry School, Federal University of Rio Grande of
Sul, Rua Ramiro Barcelos 2492, CEP 90035-003, Porto Alegre, Rio Grande do Sul,
Brazil
(3) Department of Biotechnology, Federal University of Rio Grande do Sul, Porto
Alegre, Rio Grande do Sul, Brazil
(4) Department of Cell Biology, University of Virginia, Charlottesville, Virginia,
USA
(5) Department of Morphological Sciences, Institute of Basic Health Sciences,
Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
mauriciotamborindeguy@gmail.com; +5551 9787-4178; +5551 3308-5011
Oral squamous cell carcinoma (OSCC) is the most prevalent cancer in oral cavity
and presents a high mortality index due to tumor invasion and metastasis. The nonmuscle myosin II isoforms (NMII) are involved in the regulation of cell migration,
which is the main feature of cell invasion. We hypothesized that the modulation of
NMII activity is impaired in OSCC which might contribute to the invasive behavior
of this tumor. The aim of the study is analyze the role of NMII and cell contractility
in tumor invasion. We analyzed the expression and distribution of NMIIA, NMIIB
and NMIIC isoforms in OSCC biopsies and cell lines and it was observed that all
NMII isoforms are overexpressed at the invasion zone and in a highly invasive
OSCC cell line. We also analyzed the effects of NMII-related contractility in tumor
cell dispersion by using genetically encoded phosphomimetics isoforms or drugs that
increase/decrease cell contractility. It was observed that an increase on contractility
resulted in cell detachment from spheroids, while a decrease in contractility resulted
in better cell-cell adhesion. Taken together, our results indicate that NMII is involved
in tumor cell dissemination and that modulation of cell contractility might be an
interesting tool for cancer treatment. Experimental design and informed consent
procedures were approved by the Ethical Committee of UFRGS and of Hospital de
Clnicas de Porto Alegre. All patients in this study provided written informed
consent. Authors declare no conflict of interest. Funding Support: CAPES, CNPQ,
FAPERGS, UFRGS.
(1) Departamento de Cincias Fisiolgicas, Universidade Federal de So Carlos, So

Carlos, SP, Brasil; E-mail: rafael.bressani.lino@gmail.com; Telephone: +55 (16)
3351-8333; Mobile: +55 (16) 99754-5278.
(2) Departamento de Gerontologia, Universidade Federal de So Carlos, So Carlos,
SP, Brasil;
The migration process and the adhesion receptors have been considered key targets
for cancer treatment due to the possibility of limiting invasion and metastasis.
Adhesion receptor blockers, such as the snake venom disintegrins, have therapeutical
potential due to their effects as inhibitors of cell migration. However, the molecular
mechanisms of disintegrin action in the migration process are not completely
elucidated. The aim of this study was to evaluate the effect of DisBa-01, a
recombinant RGD-disintegrin, in the morphology and viability of murine breast
tumor cells (4T1BM2) and fibroblasts (L929). After incubation with DisBa-01 (1,
10, 100 and 1000nM) for 6h, cells were stained with phalloidin and analyzed on
automated fluorescence microscope. DisBa-01 did not affected L929 cell adhesion
but it completely inhibited actin polymerization in 4T1BM2 cells without cell
detachment. In a colony formation assay, cells treated with DisBa-01 decreased both
the number and size of colonies. DisBa-01 did not affect 4T1BM2 cell proliferation
tested by MTT assay. In addition, the ability of DisBa-01 to induce apoptosis was
measured by flow cytometry analysis using PE Annexin-V and 7AAD methods. No
apoptotic cells were observed after DisBa-01 treatment, corroborating the hypothesis
that DisBa-01 is not cytotoxic. In conclusion, DisBa-01 acts on breast tumor cells
morphology as well as colony formation without interfering in their viability. Since
DisBa-01 binds to v3 integrin with high affinity, its effects could be due to the
higher level of expression of this integrin in 4T1BM2 cells (98%) compared to L929
cells (62%).
Support: FAPESP, CAPES, CNPq.
A103
A104
DisBa-01, A RGD-RECOMBINANT DISINTEGRIN, INHIBITS THE

PROLIFERATION AND CELL ADHESION INDUCED BY ESTRADIOL IN
HUMAN TUMOR BREAST CELLS MCF-7
ACTION OF LINSEED SEED OIL IN MCF-7 BREAST CANCER CELLS
Tas Marolato Danilucci (1); Ana Carolina Baptista Moreno Martin (2); Wanessa
Fernanda Altei (1); Heloisa Sobreiro Selistre de Arajo (1)
(1)
Departamento de Cincias Fisiolgicas, (2) Departamento de Gerontologia;

Universidade Federal de So Carlos, UFSCar, So Carlos, SP, Brasil;
E-mail: tais_danilucci@hotmail.com
DisBa-01 is a high-afnity v3-integrin binding protein with anti-metastatic and

anti-angiogenic effects. Estrogens are associated with increased proliferation and
decreased apoptosis of tumor cells. Several studies have demonstrated a crosstalk
between the signaling cascades induced by both estradiol and integrins. Therefore,
the aim of our study was to investigate whether DisBa-01 can inhibit the
proliferation and cell adhesion induced by estradiol. MCF-7 cells were stimulated
with -Estradiol (10-7M) and treated with DisBa-01 (500nM and 1000 nM), and
allowed to grow for 24, 48 and 72 h. Cell proliferation was determined by MTT
assay. DisBa-01 inhibited proliferation induced by estradiol in 48 and 72 hours at
concentrations 500nM and 1000nM. In the absence of estradiol, DisBa-01 did not
affect cell proliferation. For adhesion of cells, fibronectin was immobilized on
culture plates. Cells previously stimulated with -Estradiol for 72 h were treated with
DisBa-01 (500nM and 1000 nM) for 30 min and labeled with calcein. Labeled cells
were transferred to the plate (1x106 cells/well) and incubated for 1 h. The analysis
showed that DisBa-01 inhibited adhesion to fibronectin only in the cells prestimulated with estradiol. DisBa-01 also inhibits the proliferation and cell adhesion
induced by estradiol stimulation. Considering that the target of DisBa-01 is v3
integrin, we suggest that the binding of this disintegrin could decrease the estradiol
effects in estrogen dependent tumor cells. More studies are under development in our
lab to elucidate the correlation of these cell pathways. Financial support: CAPES and
FAPESP.
J. G. Liria1, V. D. Oliveira1, C. Pacheco-Soares2, N.S. da Silva1

Laboratory of Tissue and Cell Biology, Development and Research Institute UNIVAP, Sao Jose dos Campos - SP, Brasil
2
Laboratory of Dynamics of Cellular Compartments, Development and Research
Institute UNIVAP, Sao Jose dos Campos- SP, Basil
e-mail: nsoares@univap.br
Among functional foods, linseed seeds is the best known source as essential fatty
acids -3 and -6, also having fiber and phenolic compounds known to exert
antioxidant activity [1]. The term plasma applies to a gas containing neutral species
and electrically charged as electrons, positive or negative ions, atoms and molecules
[2]. The atmospheric plasma has been shown to be an effective antimicrobial and
flexible process with application to a wide variety of food [3]. The identification of
possible cell behavior changes after exposure of MCF-7 cells to golden linseed seed
oil treated with atmospheric plasma was the aim of this study. The seeds were
separated into groups according to plasma irradiation time of 5, 10, and 15min.
Parameters used: distance from the torch=1.5cm, argon gas=5L/min air=5L/min,
power=20W, discharge current=20mA. The temperature attained on the linseed
during the irradiation process did not exceed 60C. After irradiation, the oil of
linseeds seed was extracted in hydraulic press. 10g of linseeds seed were required for
obtain 1 ml of oil. The extracted oil was added in amounts of 1, 10 and 50L in 96
well plates with 5x106 cells per well of MCF-7 strain and incubated for 24h at 37 C
and 5% CO2. The cell viability was carried out by analyzing formazan crystals
formed (MTT) where this reduction occurs mainly in metabolically active cells.
After incubation, the linseed seed oil was removed, 0.5mg/mL of MTT was added to
the cultures, which were incubated for 1h at 37C followed by 30min gentle agitation
in 200mL DMSO. The plates were read with a 570-nm filter in an ELISA
Spectracount Reader. The results show that linseed seed oil stimulated cell growth,
being most significant with a volume of 10L and plasma irradiation for 10min. In
conclusion, the linseed seed oil after plasma irradiation has a stimulating effect on
cancer cells.
Supported by FAPESP 2013/20054-8
12-
3-
References
GALVO, E.L., SILVA, D.C.F., SILVA, J.O., MOREIRA, A.V.B., SOUZA,
E.M.B.; Avaliao do potencial antioxidante e extrao subcrtica do leo da linhaa.
Cinc. Tecnol. Aliment., Campinas, 28(3): 551-557, jul-set, 2008.
NASCIMENTO NETO, A.B. Desenho e construo de um prottipo gerador de jato
de plasma frio presso atmosfrica para aplicaes biomdicas. 2013. 77 f.
Dissertao (Mestrado em Engenharia Mecnica) Universidade Federal do Rio
Grande do Norte. Natal. 2013.
NIEMIRA, B.A.; Cold Plasma decontamination of foods. Annual review of food
science and technology. v.3. p. 125-42. 2012.
4-

A105
A106
CHARACTERIZATION OF HUMAN CELL CULTURES OBTAINED FROM

ADRENAL NODULES TISSUE OF PRIMARY MACRONODULAR
ADRENOCORTICAL HYPERPLASIA WITH ARMC5 MUTATION
EFFECT OF PGE2 AND EP RECEPTOR ANTAGONISTS ON THE

MIGRATION OF HUMAN GLIOMA CELLS
Cavalcante, IP1, Nishi M2, Zerbini MCN3, Chambo JL4, Almeida MQ2,5, Lotfi CFP1*,
Fragoso MCBV2,5 *
1
Institute of Biomedical Sciences, Department of Anatomy, 2Laboratory of Medical

Investigation LIM/42, 3Department of Pathology, 4Department of Urology, 5Adrenal
Unit, Discipline of Endocrinology & Metabolism, University of Sao Paulo, SP,
Brazil
*The authors contributed equally to this work.
Disclosure: The authors have nothing to disclose.
Contact: isadoracavalcante@gmail.com
Background: Primary macronodular adrenocortical hyperplasia (PMAH) is a rare
cause of Cushings syndrome. The mechanisms causing hypercortisolism and adrenal
hyperplasia are not fully clarified. The cortisol production from adrenal cortex
mediated by aberrant receptors and autocrine/paracrine regulation of intra-adrenal
ACTH have been considered. Additionally, germline/somatic ARMC5 mutations
have been described as main cause of PMAH. Aim: Characterization of PMAH cell
cultures through morphological, functional and molecular analyses. Methods: Cell
cultures from adrenal nodules of 6 unrelated patients with PMAH were analyzed for:
1) ARMC5 sequencing; 2) Oil Red O staining; 3) ARMC5 immunofluorescence; 4)
aberrant receptors, steroidogenic enzymes, POMC and ARMC5 genes expression by
qPCR. Results: 1) ARMC5 germline mutations (n=6) associated or not with somatic
or LOH; 2) accumulation of lipid droplets in the cytoplasm of cells; 3) ARMC5
expression seems to be prevalent in the cytoplasm; 4) expression of HSD3B2,
CYP17A1, MC2R and ectopic receptors were significantly different in PMAH cells
when compared to normal adrenal cells; POMC expression was similar in all
cultures. Conclusions: The detection of lipid droplets and MC2R, HSD3B2 and
CYP17A1 genes expression imply the presence of steroidogenic cells in the cultures
obtained. Expression of steroidogenic enzymes in two cultures was lower than in
H295R cells, which can be related to ARMC5 mutations. PMAH cells expressed
various ectopic receptors, with AVP1aR and GIPR being mostly expressed. In
summary, the cell cultures were characterized for further studies to elucidate the role
of ARMC5 in PMAH.
*Gomes RN, Feitoza F, Colquhoun A.

Dept. of Cell and Developmental Biology, Biomedical Sciences Institute, University
of So Paulo, Brazil
Glioblastoma (GBM) is the most common brain cancer in adults. The highly
aggressive nature of GBMs is characterized by their diffuse infiltration into the
normal brain parenchyma and interaction with the ECM components in the brain.
The concentration of PGE2 found in gliomas often correlates with the tumour grade.
While PGE2 is known to stimulate migration in various tumors, less is known about
its effects in glioma. The aim of this study was to analyze the role of PGE2 and their
receptors, EP 1-4, in the control of cell migration in U-251MG and U-87MG human
glioma cell lines. The expression of ECM molecules involved in migration was also
studied.
Initially, U-251MG and U-87MG human glioma cells were treated with PGE2
(10M) for 48 hours and proteins involved in ECM were analyzed by RT-qPCR.
Next, we examined the effect of PGE2 on the migration of both cell lines using a
transwell assay. Finally, antagonists of EP2 (AH6809: 1M) and EP4 (L161982:
1M) were used. Changes were found in the expression of laminin, fibronectin, type
IV collagen and v, 3 and 5 integrins in both cell lines. Addition of exogenous
PGE2 increased cell migration while the antagonists AH6809 and L161982 reduced
migration in U-251MG and U-87MG.
The present study, demonstrated that PGE2 affects gene expression of ECM
molecules and plays an important role in glioma cell migration. This effect is
partially mediated by activation of the EP2 and EP4 receptors.
Financial support: CAPES and FAPESP
Funding: Fapesp 2015/50192-9. This study has been approved by the ethics
commitee 1288/CEPSH CAAE:49449815.7.0000.5467 Plataforma Brasil: 1.151.314
A107
A108
GLIOBLASTOMA CELLS INTERCONNECT IN THE ABSENCE OF HDAC

ACTIVITY AND TGF SIGNALING
HUMAN CERVICAL CANCER CELL LINES EXPRESS PAFR AND ITS

CONTROLS TUMOR CELL PROLIFERATION
Aline Cristina de Menezes (1), Maria Ceclia Nunes (1), Mariana Cabanel (1), Ktia
Carneiro (1)
Barbara Dalmaso(1) Ildefonso Alves da Silva Junior(1) and Sonia Jancar(1)

(1). Department of Immunology, Institute of Biomedical Science, University of So
(1)
Laboratrio de Proliferao e Diferenciao Celular, Universidade Federal do

Rio de Janeiro (UFRJ)
Aline Cristina de Menezes, Laboratrio de Proliferao e Diferenciao Celular,

Universidade Federal de Uberlndia. Instituto de Cincias Biomdicas, Avenida
Carlos Chagas Filho, 373 Bloco F, 2andar- sala F2-01, 21941-902 Ilha do
Fundo- Rio de Janeiro-RJ. Tel.: (55) 21 3938-6483/ 21 99914-9566. Email:
aline_crismenezes@yahoo.com.br
Background: Glioblastoma (GBM) is the most aggressive brain tumor presenting
poor prognosis. GBM is characterized by a highly heterogeneous cell population
maintained by cancer stem cells (CSC) which are resistant to conventional treatment.
Alternative strategies for GBM treatment include the inhibition of Histone
Deacetylase (HDAC) activity by decreasing cell viability/proliferation and
clonogenic capacity of CSC. Recent studies from our group have shown that the
inhibition of HDAC activity impacts on cell shape and function in a TGF dependent
fashion. Aims: To investigate whether HDAC activity is necessary for TGF
signaling transduction in tumor cell shape and function in vitro. To approach this
question we have used GBM U87MG under HDAC and TGF inhibition and
investigated biological parameters related to tumor aggressiveness such as cell
motility, gene expression and cell morphology. Methods: GBM U87MG cells were
incubated with 10 nM of HDAC (TSA) or/and 50 mM of TGF inhibitors
(SB431542) for 72 hours and processed for qPCR, cell cycle progression, migration
assay and morphological analysis. Results: Our results showed that TSA treated cells
were able to form tunneling tubes and presented a decreased migration rate and cell
viability. In contrast TGF inhibition did not form tunneling tubes but indeed
presented decreased cell motility coupled to changes in gene expression pattern.
Conclusion: Our results suggest that tunneling tubes formation would be a strategy to
bypass a decrease in cell motility and keep the long range signaling status among
tumor cells in the absence of HDAC activity and TGF signaling.
Presenting author contact: Barbara Dalmaso

Phone number +55(11)30917744 or +55(11)973739598
e-mail: barbaradalmaso@hotmail.com
BACKGROUND: The receptor for Platelet-activating factor (PAFR) is expressed in
a wide range of cells, including some tumor cells. It was shown that cysplatin
increases PAFR expression in B16F10 melanoma cells, and exerts a cytoprotective
effect on treatment-resistant tumor cells.
AIM: In this work we examined the effect of PAFR antagonist in tumor cell
proliferation and the expression of PAF receptor in cisplatin-treated human cervical
cancer cell lines.
METHODS: The human carcinoma cell lines C33, SiHa and HeLa were cultured
(RPMI 10% FBS, 5x104 cells/well). Cell proliferation was evaluated by flow
cytometry and expressed as MFI CFSE. A group was treated with the PAFR
antagonists (CV 3938 10M) added to the cultures every second day. Another group
was treated with Cysplatin (50M) for 4h. The expression of PAFR gene expression
in tumor cells was analyzed by real-time PCR, compared with non- treated group and
expressed as CT in relation to a non-tumoral human cheratinocyte HaCat.
RESULTS: The tumor cell lines C33, Siha and HeLa showed higher expression of
PAFR mRNA compared to the non-tumoral HaCat cell line. When blocking the
PAFR with CV proliferation of the carcinoma cells, but not of HaCat, was
significantly reduced, suggesting that during tumor cells culture, PAFR-ligands are
generated and stimulate proliferation. Cysplatin treatment further increased PAFR
expression and proliferation of the carcinoma cells.
CONCLUSION: Cisplatin enhances expression of PAFR. The proliferation is
dependent on PAFR activation, possibly by ligands generated by cysplatin induced
cell stress.
Financial support: FAPESP and CNPq
Funding supporting: CNPq/CAPES

A109
A110
EXPLORING THE ROLE OF CXCR4 EXPRESSION ON OSTEOSARCOMA

CELLS CHEMOTHERAPY RESISTANCE
LOSS OF TUMOR SUPPRESSOR miR-654-3p DURING PROGRESSION TO

AGGRESSIVE PAPILLARY THYROID CANCER
Gabriele O. Ribeiro1,2; Juliana Souza2, Maria Eugnia L. Duarte2; Anneliese FortunaCosta2; Amanda S. Cavalcanti2
Murilo Vieira Geraldo(1,3), Helder Takashi Imoto Nakaya(2), Edna Teruko

Kimura(1)
(1)Department of Cell and Developmental Biology, Institute of Biomedical Sciences,

University of Sao Paulo; Sao Paulo, Brazil.
(2)Department of Clinical Analyses and Toxicology, School of Pharmaceutical
Sciences, University of Sao Paulo, Sao Paulo, Brazil.
(3)Department of Structural and Functional Biology, Institute of Biology, State
University of Campinas, Campinas, Brazil.
Master Program in Musculoskeletal Sciences, National Institute of Orthopedics and

Traumatology, Rio de Janeiro, RJ, Brazil
2
Research Division, National Institute of Orthopedics and Traumatology, Rio de
Janeiro, RJ, Brazil
Osteosarcoma (OS) is an aggressive pediatric tumor of growing bones that, despite
surgery and chemotherapy intervention, is prone to relapse and give rise to
metastasis. Some evidences point out to the participation of the chemokine receptor
CXCR4 and its ligand CXCL12 in OS metastasis development. Nevertheless, it is
still not know whether the cells that scape from the primary tumor after
chemotherapy either express high levels of CXCR4 or present chemotactic response
towards CXCL12 stimulus. Although OS cell lines are a widely used in vitro model,
they do not comprise the OS heterogeneity observed at the clinical level. Here we
established primary OS cells to investigate the in vitro effect of the chemotherapeutic
agents Doxorubicin, Cisplatin and Methotrexate on CXCR4 expression and function.
OS primary cells were isolated by enzymatic digestion of tumor biopsies samples
and their tumorigenic identity was confirmed through the evaluation of typical OS
chromosomal aberrations. FACS analysis showed that the percentage of OS cells
expressing CXCR4 ranged from 5% to 45%. Interestingly, OS cells cultured under
hypoxia condition (mimicking the tumor microenvironment) showed more CXCR4
positive cells than cells cultured under normoxia. Moreover, the proportion of OS
CXCR4 positive cells increased after in vitro chemotherapeutic treatment, indicating
that these cells are indeed more chemotherapy resistant. We concluded that OS
primary cells are heterogeneous regarding CXCR4 expression. Furthermore, CXCR4
expression seems to be related to chemotherapy resistance and it can be modulated
by O2 levels.
Presenting author:
Murilo Vieira Geraldo,
Av. Bertrand Russel, s/no Cx. Postal 6109, CEP 13083-865, Campinas, SP
email: murilovg@unicamp.br, phones: (+55)19 3521-6114; (+55)11 98589-8180
Background: Papillary thyroid carcinoma (PTC) is the most prevalent form of
thyroid cancer. Although highly curable, 5% of PTCs present resistance to
radioiodine therapy and poor prognosis. MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression implicated in several types of cancer,
including PTC. However, the role of miRNAs in aggressive PTC remains unclear.
Aims: To identify miRNAs involved in progression of PTC. Methods: Normal and
tumor thyroid cell lines and thyroid tissue samples from animal model of PTC
progression were used for large-scale analysis of miRNA expression and functional
analyses. Large data set of miRNA and mRNA expression from human PTC was
downloaded from The Cancer Genome Atlas (TCGA) project. miRWalk and DAVID
webtools were used for in silico construction of regulatory network. Results: We
identified the downregulation of 28 miRNAs situated at the imprinted locus 14q32 in
PTC cell lines and animal samples, which was confirmed in human PTC samples
from the TCGA project. The regulatory network potentially modulated by these
miRNAs suggests the influence on key cancer-related biological processes, such as
cell adhesion, migration and angiogenesis. Functional analyses show that
overexpression of miR-654-3p, downregulated during PTC progression, markedly
inhibited cell proliferation and migration in vitro and restored the expression of
tumor- and metastasis-suppressor genes. Conclusion: We identified the global
downregulation of miRNAs from the 14q32 region during aggressiveness
progression of PTC. MiR-654-3p presented tumor suppressor properties in vitro and
could therefore be explored as new therapeutic strategy for PTC. Ethical approval:
Ethical Committee, ICB-USP (n#134/p93/b2). Funding support: NAPmiR, FAPESP
and CNPq.
Keywords: microRNA; papillary thyroid carcinoma; 14q32
The authors declare no conflict of interest
A111
SHH
SIGNALING
PATHWAY
IN
GLIOBLASTOMA PROLIFERATION
A112
THE
MODULATION
OF
Tania Cristina Leite de Sampaio e Spohr1,2; Gabriela Basile Carballo1,2,; Jessica

Honorato1,; Jos Marques Brito Neto2; Vivaldo Moura-Neto1
(1) Instituto Estadual do Crebro Paulo Niemeyer, Secretaria de Sade do Estado do
Rio de Janeiro/RJ - Brasil
(2) Instituto de Cincias Biomdicas, UFRJ, Rio de Janeiro - Brasil
(email: spohr@icb.ufrj.br - telephone: 21-2277-9393 / 21-99699-2548)
Background: Glioblastoma (GBM) is a very aggressive cancer which maintenance is
related to cancer stem cell (CSC). The process of tumor malignancy involves the
reexpression of some genes involved in embryonic development such as Shh, Notch
and Wnt, in which tumor cells recover embryonic properties. It is believed that CSC
are responsible for GBM radio and chemoresistant properties, resulting in high
mortality, and Shh signaling pathway could regulate those properties.
Aims: To study the role of developmental genes in tumor progression, especially the
Shh signaling pathway.
Methods: GBM11 cultures were maintained for 8 days in the presence of rh-Shh
[1ng/ml] or rh-Shh+GANT61[20M] (Shh pathway inhibitor) or GANT61 alone,
and we evaluated the proliferation using MTT assay. Immunocytochemistry and
Western-blotting were preformed after 8 days of treatment.
Results: GBM 11 cells express Shh signaling pathway components mRNA and
proteins (Smo and Gli1), but their proliferation is not affected after cyclopamine
treatment. rh-Shh treatment increased GBM11 proliferation, decreased patched
expression and increased Shh and Oct-4 expression. Interestingly, rh-Shh+Gant 61
treatment or Gant 61 alone, we observed that the effects above were reverted.
Conclusion: Shh signaling pathway is important to modulate GBM proliferation and
maintenance of CSCs properties of GBM.
Funding Support: CAPES, FAF/ONCO, FAPERJ, CNPq, INNT-INCT-MCT, PRSADE - ASSOCIAO BENEFICENTE DE ASSISTNCIA SOCIAL E
HOSPITALAR
Ethical Approval: DAHEICB 015
CYCLOPAMINE, A POSSIBLE TUMOR INHIBITOR?

Gabriela Basile Carballo1,2; Jos Marques Brito Neto2; Vivaldo Moura Neto1; Tania
Cristina Leite de Sampaio e Spohr1,2.
(email: gabscarballo@gmail.com - telephone: 21-2277-9393 / 21-98849-4013)
(1) Instituto Estadual do Crebro Paulo Niemeyer, Secretaria de Sade do Estado do
Rio de Janeiro/RJ - Brasil
(2) Instituto de Cincias Biomdicas, UFRJ, Rio de Janeiro - Brasil
Background: Glioblastoma (GBM) is a very aggressive tumor and its maintenance
and resistance to therapies are related to the presence of tumor stem cells (CTTs).
GBM process of malignancy involves the reexpression of some genes involved in
embryonic development as Shh, Notch, Wnt, wherein the tumor cell recovers
embryonic properties. Studies demonstrated that Shh signaling pathway is involved
in chemo and radiotherapy resistance of CTTs, as well as its ability to self-renew.
Aims: Analyze the role cyclopamine on Shh pathway and its influence on GBM
proliferation
Methods: GBM cultures were maintained for 8 days in the presence of different
concentrations of cyclopamine and the proliferation was evaluated by the MTT
assay, in three different days. Immunocytochemistry, western-blotting and RT-PCR
to CTT markers were preformed after 8 days of treatment.
Results: In all GBM lines tested, cyclopamine decreased proliferation in vitro but
this effect was not observed in control cultures such as human fibroblast. We also
observed an increased expression of Oct-3/4 and Sox-2 in the glioblastoma cell lines,
observed by Immunocytochemistry, Western-blotting and RT-PCR.
Conclusion: Based on the results obtained, the Shh pathway may be involved in
maintenance of glioblastoma CTT and cyclopamine may be a possible tumor
proliferation inhibitor.
Information on ethical approval: DAHEICB 015
Funding support: CAPES, FAF/ONCO, FAPERJ, CNPq, INNT-INCT-MCT, PRSADE - ASSOCIAO BENEFICENTE DE ASSISTNCIA SOCIAL E
HOSPITALAR

A113
A114
THE ROLE OF GALECTIN -3 IN THE MODULATION OF TUMOR

MICROENVIRONMENT OF BREAST CANCER
INCREASED CELL PROLIFERATION IN CASTRATED RAT PROSTATE

AFTER SMOOTH MUSCLE CELLS CONTRACTION BLOCKADE BY
ALPHA BLOCKADE
Authors: Juliana Pena Gonalves (1), Anneliese Fortuna (2), Renato Sampaio(3),
Tatiana Lobo Coelho de Sampaio (4), Maria Isabel Doria Rossi (4)
(1) Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro,
Rio de Janeiro, Brazil
(2) Medical Biochemistry Institute, Federal University of Rio de Janeiro, Rio de
Janeiro, Brazil
(3) Faculty of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
(4) Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de
Janeiro, Brazil
Contact: jupgoncalves@biof.ufrj.br
Phone: 55 21 3938-6535
Cell phone: 55 21 97107-1457
Galectin-3 is a lectin expressed by different types of cancers and is found both
extracellularly and intracellularly, regulating various processes involved in tumor
progression. A possible role for gal-3 produced by tumors in the recruitment of other
cells to form the tumor-associated stroma has not been established. Adipose derived
mesenchymal stromal cells (ADSCs) can migrate to the tumor stroma, where they
can contribute to tumor progression and metastasis.
We seek to clarify the role of gal-3 expressed by tumor cells in the homing of
adipose derived mesenchymal stromal cells (ADSCs) to tumor sites.
ADSC were obtained from HUCFF patients after approval of the Ethical Board.
Migration were evaluated in transwell assay. MDA-MB-231 breast cancer cell line
were transduced with miRNA for gal-3.
After the silencing of gal-3 expression in MDA-MB 231, the migratory stimuli was
reduced, resulting in 14% less migration of ADSCs, compared to the control cells
(expressing gal-3). Addition of recombinant gal-3 to the cultures of silenced tumor
cells increased 25% ADSC migration. Evaluation of the pro-inflammatory molecules
produced by tumor cells revealed that gal-3 silencing led to a decrease in the
expression of cytokines and chemokines. This reduction can be involved in the
decrease migration of ADSC. However, treatment with recombinant protein further
reduced those levels, in an apparent conflict with the increased migration observed.
This may suggests different actions, depending on its cellular location.
We conclude that gal-3 expressed by tumor cells can contribute to ADSC attraction,
by indirect mechanisms. Financial support: CNPq and FAPERJ
Isabela Gaseta Ferraz Paiva (1); Caroline Nascimento Barquilha (1); Srgio
Alexandre Alcntara dos Santos (1); Ketlin Thassiani Colombelli (1); Ana Carolina
Lima Camargo (1); Flvia Bessi Constantino (1); Luis Antonio Justulin Junior (1);
Sergio Luis Felisbino (1).
(1) Department of Morphology, Institute of Biosciences, Sao Paulo State University
(UNESP), Botucatu, SP, Brazil. isabela.gaseta@gmail.com; +55 14 3880 0481; +55
19 991420909.
Background: Androgen ablation promotes a marked reduction in the epithelial
compartment of the prostate gland followed by major reorganization of the stroma,
which became thick and plentiful of collagen fibers. It has assumed that smooth
muscle cells (SMCs) contraction plays a major role in glandular atrophy and stromal
remodeling after castration. Aims: We evaluated the effect of SMCs contraction
blockade by doxazosin on prostate involution induced by castration. Methods:
Ventral prostate (VP) from castrated and castrated plus doxazosin treatment
(25mg/kg/day) rats were evaluated by morphological and morphometric analyses
after 3, 7 and 10 days after castration. VP sections were stained by H&E and
immunostained for cell proliferation marker Ki-67. Results: Doxazosin treatment
reduces the rate of glandular atrophy induced by castration as showed by statistically
significant higher glandular weight observed after 10 days of treatment (71 mg in
vehicle-castrated vs 127mg in castrated plus doxazosin). Ki-67 immunostaining
showed rare proliferative cells in vehicle-castrated prostate (less than 0,1%), while in
the dox-treated castrated prostates we observed a mean of 3% of proliferative cells.
Conclusion: SMCs contraction contributes greatly for the process of prostate weight
loss induced by castration. Surprisingly, SMC relaxing also induced epithelial cell
proliferation in an androgen-suppressed condition. Since doxazosin treatment alone
is well known to induce apoptosis on prostate epithelial cells, we believe that some
growth(s) factor(s) from SMC and/or fibroblasts may are involved in this epithelial
cell proliferation. Elucidation of the nature of these factors can contribute for a better
understanding of castration-resistant prostate cancer onset.
Financial support: So Paulo Research Foundation (FAPESP) (2013/08830-2)
Ethics Committee: 139/09
The authors declare that they have no conflict of interest.
A115
A116
4-NEROLIDYLCATHECOL (4-NC) INDUCES ANTIPROLIFERATIVE

EFFECTS IN NAVE AND VEMURAFENIB-RESISTANT MELANOMA
CELLS IN VITRO AND IN XENOGRAFT MODEL
ROLE OF ADAMTS-1 (A DISINTEGRIN AND METALLOPROTEINASE

WITH THROMBOSPONDIN MOTIFS 1) IN NUCLEUS OF NORMAL-LIKE
AND TUMORAL HUMAN BREAST CELLS
Dbora Kristina Alves-Fernandes (1); Fernanda Faio-Flores (1); Silvana Sandri (1),
rica Aparecida de Oliveira (1), Walter Turato (1), Silvia Berlanga de Moraes
Barros (1); Silvya Stuchi Maria-Engler (1).
Suly Vieira da Silva ; Mara de Assis Lima1; Vanessa Morais Freitas1

1
Department of Cell and Developmental Biology, Biomedical Sciences Institute,
University of So Paulo
(1)
Skin Biology Group, Clinical Chemistry and Toxicology Department,

School of Pharmaceutical Sciences, University of So Paulo,
FCF/USP, Brazil
debora.kalves@usp.br
Phone: 55 11 3091 3631
Mobile: 55 11 9 7251 3463
Melanoma accounts for only 4% of skin malignancies, but it has a high mortality
rate. Though the specific inhibitors have revolutionized melanoma treatment, the
most patients are subject to resistance, justifying the constant search for new
therapeutic compounds. Recent data from our laboratory indicated that 4nerolidylcathecol (4-NC), extracted from Pothomorphe umbellata, induces apoptosis
in melanoma cells by ROS production, DNA damage and p53 upregulation and
showed an antiproliferative effect in reconstructed skin. We aim to evaluate the 4NC efficacy in overcoming resistance to BRAF inhibitors in melanoma cell lines as
well as in cutaneous melanoma in xenograft models. 4-NC showed cytotoxicity (IC50
~30M), colony formation and migration decrease in vitro assays in nave and
vemurafenib-resistant (R) melanoma cells. SK-MEL-103 melanoma cells (5x105
cells) were injected s.c. in female BALB/c nude mice. Once the tumors appeared,
treatments were initiated by i.p. injection for 3 times a week for 3 weeks with 4-NC
(10mg/kg), DMSO or no treatment. 4-NC was able to significantly reduce growth of
xenograft tumors at least 4 times compared to controls, with complete tumor
remission in one animal. P53, p16 and cleaved PARP expression were increased in
the tumors of treated animals indicating apoptosis and cell cycle arrest. MMP2 and
MMP14 gene expression were decrease in the same samples, demonstrating a
possible role of 4-NC also in vivo in the inhibition of melanoma invasion.
Approval CEUA/FCF/70-2012USP/Brazil.
Financial support: FAPESP (2014/06959-0); CNPq (385282/2011-7).
Keyword Melanoma; 4-nerolidylcathecol; vemurafenib-resistant cells; xenograft
model.
Presenting author: Dbora Kristina Alves Fernandes (Poster presentation)
Background: Mammary tumors are the second most frequent type of neoplasm in
worldwide among women. Quantitative and qualitative changes occur in
extracellular matrix resulting from remodeling, originated from the activity of
proteases secreted by mammary cells in microenvironment. Metalloproteinases
belonging to the ADAMTS (a disintegrin and metalloprotease with thrombospondin
motifs) family are known proteases secreted by cells with proteolytic activity on
proteoglycans from extracellular matrix. However, our group observed that also
found in the nuclei of the three mammary cell lines studied here. Aims: The
objective of the study is to find out ADAMTS-1 function in the nucleus, whom are
its partners and if this protease located within nucleus could exercise regulatory
effect on tumor cells behavior, particularly in human breast cells lines. Methods:
Western blots and immunofluorescence were carried out to compare ADAMTS-1
levels in cytoplasmic and nuclear fractions in MCF-10A, MCF-7 and MDA-MB-231
cells, when were treated with heparin and importazole. Cells were sequentially
treated with a detergent and high-salt solution to remove the cytoplasmic and
nucleoplasmic components, and immunofluorescence were performed to assess
subcellular localization of ADAMTS-1. Results: Our data showed that ADAMTS-1
is not located in cell nucleus when were treated with heparin and importazole. Cells
attached on a coverslip show that ADAMTS-1 is predominantly in nucleus even after
DNA elimination. Conclusion: Our findings indicate that: ADAMTS-1 loses nuclear
localization when secretion is stimulated or when importin--mediated nuclear
import is blocked and ADAMTS1 can be a chromatin binding protein.
Approved (CEP-ICB N 708/15).
Support: FAPESP 2015/09845-9

A117
A118
ANALYSIS OF PGD2 SYNTHESIS AND ITS IMPACT ON GLIOMA CELL

ACTIVITY
CYTOTOXIC ACTIVITY OF RUTHENIUM(II)/CINAMIC ACID COMPLEX

ON A549 CELLS
*Ferreira MT, Gomes RN, Colquhoun A
Guilherme lvaro Ferreira da Silva(1), Graciana Yokota Garavelli(1)*, Marina

Montingelli Ortega(2), Jlia Scaff(2), Marilia Imaculada Frazo Barbosa(2), Antonio
Carlos Doriguetto(2), Marisa Ionta(1)*
*Department of Cell and Developmental Biology, University of So Paulo, So

Paulo, Brazil.
(1)
Email: matthewferreira@usp.br; Phone: (11) 3091-7261 (Institution) (11) 984739209 (Cellular)
BACKGROUND: The World Health Organization classifies glioblastoma (GBM) as
a type IV astrocytoma, making it one of the most fatal tumors that exists. Despite
the advances in conventional treatments that improve a patients length of survival,
the overall trajectory of the disease remains unchanged. GBM cells produce
significant levels of prostaglandins, including prostaglandin D2 (PGD2). PGD2
possesses pro- and anti-tumorigenic properties. AIM: A more complete
understanding of PGD2 activity in GBM could yield more effective treatments
against GBM. METHODS: Through RT-PCR, immunohistochemistry, and HPLC
tandem mass spectrometry, we were able to confirm the synthesis of PGD2 in GBM
cell lines. We treated GBM cell lines with physiological and supra-physiological
concentrations of exogenous PGD2 over 72 hours and observed their effects on cell
count, apoptosis, mitosis, migration and viability.
RESULTS: Our results
demonstrate that PGD2 possesses contradictory functions in GBM in a concentrationdependent manner (M PGD2 vs. nM PGD2) on cell count, migration and apoptosis.
CONCLUSION: The literature has demonstrated that PGD2 acting through its
particular receptors, and at various concentrations, can produce opposing responses
within a single system. This established conflict accompanied by our results suggest
that although PGD2 likely aids GBM growth and survival because of lower
endogenous concentrations and strong binding affinity to DP2, there still exists the
possibility that through higher concentrations and via the DP1 and PPAR receptors,
PGD2 could be a useful target for glioma treatment. Future studies are needed to
determine if PGD2 is a viable target for GBM treatment.
(1) Institute of Biomedical Sciences, Federal University of Alfenas, Alfenas, MG,

Brazil
(2) Institute of Chemistry, Federal University of Alfenas, Alfenas, MG, Brazil
*gygaravelli@gmail.com, 55-35-32991360 or 55-35-998952650
Metal-based complexes have been widely investigated for their antitumor activity.
Cisplatin and its derivatives contributed for improving therapeutic proposes for
cancer therapy; however the use of these compounds is limited due to their severe
side effects. In this frame, ruthenium based compounds represent an alternative to
platinum-based compounds therefore they are versatile chemical structures that
present low toxicity. The present study aimed to investigate the antitumor potential
of ruthenium(II)/cinamic acid complex [Ru(cinamic)(dppb)(bipy)]PF6, here called
CINAM, on A549 cells. Lung cancer is the most common of all malignant tumors
being the average five-year survival ranges from 7 to 10% in developing countries.
Proliferative behavior was evaluated through cell viability (MTS and trypan blue
exclusion) and clonogenic assays; and cell cycle analysis was performed by flow
cytometry. We demonstrated that CINAM effectively reduced cell viability of A549
cells after 24h of treatment at 4 M. Cytotoxic activity of CINAM was higher (IC50
= 3.31 0.16 M) than cisplatin (IC50 = 61.87 7.75 M), a chemotherapeutic agent
commonly used in lung cancer therapy. Cells were arrested in G0/G1 phase when
CINAM was used for 24h at 2 M. We also observed increase of sub-G1 population
in cultures treated for 24h at 2 M and 4 M. Our results indicate that
ruthenium(II)/cinamic acid complex has a great cytotoxic activity on A549 cells and
therefore it represents a promising chemical structure for further studies to
investigate its antitumor potential against lung cancer.
Financial support: Fapemig, CAPES and CNPq
Funding: Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP);

Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES).
The institutional ethics committee approved this project.
A119
A120
THE ROLE OF RECK SUPER EXPRESSION ON HPV ASSOCIATED

TUMORIGENESIS
LIPOXIN A4 SELECTIVELY SHIFTS THE TUMOR-ASSOCIATED

MACROPHAGE PROFILE LEADING TO CONTROL OF TUMOR
PROGRESSION
Suellen Herbster (1)*, Marina Trombetta-Lima (2), Andressa Paladino (1), Paulo
Thiago de Souza-Santos (3), Mari Cleide Sogayar (2), Ana Paula Lepique (4),
Enrique Boccardo (1).
(1) Oncovirology Lab, Department of Microbiology, Institute of Biomedical
Sciences, University of So Paulo, So Paulo, Brazil.
(2) Cell and Molecular Therapy Center (NUCEL) NETCEM, School of Medicine,
(3) Molecular Carcinogenesis Program, Brazilian National Cancer Institute, Rio de
Janeiro, Brazil.
(4) Immunomodulation Laboratory, Department of Immunology, Institute of
Biomedical Sciences, University of So Paulo, So Paulo, Brazil.
*Institutional address: Professor Lineu Prestes Avenue, number 1374, room 239.
Zip code: 05508-900.
City: So Paulo SP.
Country: Brazil.
Institutional Telephone: + 55 11 3091 7292.
Mobile: + 55 21 96981 3232.
Email: sueherbster@gmail.com
All authors have disclosed any financial or personal relationships with individuals or
organizations that could be perceived to bias this project.
BACKGROUND: The mechanisms of HPV-induced carcinogenesis include
alterations in extracellular matrix (ECM) components and its remodeling modulators,
such as matrix metalloproteinases (MMP) and its regulators Tissue Inhibitors of
Metalloproteinases (TIMPs) and REversion-inducing Cysteine-rich protein with
Kazal motifs (RECK). RECK inhibits MMP-2, MMP-9 and MMP-14 (MT1-MMP)
activation. RECK expression is frequently lost in human cancers which correlates
with increased angiogenesis and metastasis. We have shown that HPV16 E6 and E7
oncoproteins expression is associated with MMP-9 up-regulation and RECK downregulation. Moreover, we observed an inverse correlation between RECK expression
and lesion grade in clinical samples.
AIM: To study the effect of RECK in HPV associated tumorigenesis.
METHODS: RECK super expression (RECK+) in cervical cancer derived cell lines
SiHa (HPV16) and C33A (HPV negative) was established by lentiviral transduction.
These cells were inoculated s.c. in nude mice to study tumor kinetics and tumor cell
populations. In parallel, these cells were used to analyze proliferation, migration and
clonogenic potential in vitro.
RESULTS: Animals inoculated with either SiHa RECK+ or C33A RECK+ showed
an extended tumor-free survival period when compared with animals receiving
control cells. SiHa RECK+ tumors presented higher relative number of endothelial
cells (CD31+), monocytes (CD45+) as well as decreased relative number of viable
tumor cells (+EGFP).
CONCLUSIONS: Our observations indicate that RECK super expression affects the
tumorigenic potential of both HPV positive and negative cervical cancer derived
cells in vivo.
ETHICAL APPROVAL AND FINANTIAL SUPPORT: CEUA (protocol 138, sheet
13, book 03), FAPESP (2013/27006-9) and INCT-HPV (FAPESP 2008/57889-1 /
CNPq 573799/2008-3).
Natlia Mesquita De Brito; 1Rafael Loureiro Simes,1 Vernica Morandi; 2Iolanda

Margherita Fierro; 3Ivan Maurice Roitt; 1Christina Barja-Fidalgo.
(1) Departamento de Biologia Celular, Universidade do Estado do Rio de Janeiro
(UERJ), Rio de Janeiro, RJ, Brazil.
(2) Departamento de Farmacologia Psicobiologia, Universidade do estado do Rio de
Janeiro (UERJ), Rio de Janeiro, RJ, Brazil.
(3) School of Health & Social Sciences, Middlesex University, London.
Natlia Mesquita de Brito (natymesq@hotmail.com) Institucional phone: +55 21
2868-8298; Mobile: +55 21 993915863 - Av 28 de setembro, 87 fds.- Vila Isabel Rio de Janeiro, RJ - Brasil.
In tumor microenvironment, pro-inflammatory macrophages (M1) acquire antiinflammatory and pro-tumor characteristics exhibiting low cytotoxic properties and a
deficient modulation of NO and ROS production. These tumor-associated
macrophages (TAMs) are derived from LY6Chigh inflammatory monocytes that
accumulate in tumors renewing the population of TAMs. Lipoxins are lipid
mediators with anti-inflammatory and pro-resolution activities in mononuclear cells,
but their effects on TAMs remain to be elucidated. This study tested the hypothesis
that ATL-1, a synthetic analog of 15-epi-lipoxin A4, could modulate the TAM
activity being performed according to guidelines of Ethical committee
(CEUA/077/2012/, UERJ). Firstly, monocyte derived macrophages were
differentiated into TAM after incubation with MV3 conditioned medium.
Contrasting to observed in M1 profile, ATL-1 selectively decreased M2 surface
markers in TAM, an effect dependent of VEGF signalling. In parallel, ATL-1
triggered ROS production and stimulated TAM to generate NO by increasing
iNOS/arginase ratio . These alterations in TAM profile induced by ATL-1 increased
their cytotoxic properties and led to loss of antiapoptotic effects of TAM on
melanoma cells. In addition, ATL-1 inhibited endothelial cell tubulogenesis activated
by TAM, a crucial step in the angiogenic process. Finally, ATL-1 inhibits tumor
progression in vivo, which is accompanied by alterations in TAM and the profile of
LY6Chigh inflammatory monocytes on the spleen, blood and bone marrow. These
results suggest that ATL-1 down-modulates the tumor progression stimulated by
TAM, inducing the shift from an M2- to an M1-like profile in vitro and in vivo,
triggering tumor cell apoptosis and the impairment of the tumor progression.
Supported by CAPES, CNPq, FAPERJ and SR-2/UERJ.


A121
A122
ECTI, A POLYSPECIFIC PROTEASE INHIBITOR, DECREASES

CELLULAR PROLIFERATION OF KASUMI-1 LEUKEMIC CELLS:
CORRELATION WITH METALLOPROTEINASE-9 ACTIVITY AND
NITRIC OXIDE RELEASE.
DIFFERENTIAL EFFECTS OF LEMON GRASS (Cymbopogon citratus)

ETHANOLIC EXTRACTS ON THE NUCLEOLAR ACTIVITY IN EHRLICH
ASCITES CELLS
Luciana de A. L. Costa, Ricardo A. Silva, Heron F.V. Torquato, Fabrcio P. Batista,

Edgar J. Paredes-Gamero, Misako U. Sampaio, Maria Luiza V. Oliva.
Departament of Biochemistry, Federal University of So Paulo, So Paulo, Brazil.
e-mail: luciana_luz16@yahoo.com.br, phones: +55 11 5576-4445 (1099)
and +55 11 98404-4731
Protease inhibitors act on blood coagulation, digestive and inflammatory processes,
tissue remodeling, proliferation, apoptosis and cell cycle. Isolated from
Enterolobium contortisiliquum seeds, EcTI is a serine protease and metalloproteinase
inhibitor of the Kunitz family of plant inhibitors. Polyspecific, EcTI also inactivates
chymotrypsin, human plasma kallikrein (HuPK), plasmin, human neutrophil elastase
(HNE) and Factor XIIa. The aim of this work was to investigate the correlation
between antiproliferative effects of EcTI in Kasumi-1 leukemic cell line and
metalloproteinase-9 activity and nitric oxide (NO) release. EcTI purification
involved DEAE-Sepharose, trypsin-Sepharose, and molecular exclusion (FPLC
system) chromatographies. Cell viability and antiproliferative effects were measured
by MTT assay and BrdU incorporation, respectively, after cells were treated with
different concentrations of EcTI (50-100 M) for 24 or 48 h. The mettaloproteinase
activity was determined by zymography and the protein expression by western blot.
EcTI (100 M, 48 h) decreased 54.4% Kasumi-1 cells viability and proliferation
(58.6%). Metalloproteinase activity decreased 37.7% and a western blot confirmed a
reduced protein expression of MMP-9 - 24 and 42% - using EcTI 50 and 100 M,
respectively. Furthermore, EcTI (100 M) increased NO release after 24 h from 700
nM to 2,000 nM. In conclusion, EcTI is an antiproliferative agent on Kasumi-1
leukemic cells and this activity might be due to metalloproteinase expression and
high NO release. Thus, this study may contribute to the investigation of the
functional activity of proteases and their involvement in the pathophysiological
process of leukemia.
Keywords: Antiproliferation, cell viability, metalloproteinase and protease inhibitor.
Supported by: FAPESP (Proc. 2009/53766-5 and 2013/13239-1), CNPq and CAPES.
Letcia de Souza Giordano1,2, Marilanda Ferreira Bellini1,2, Wilson Aparecido

Orcini2, Rita Luiza Peruquetti1,2,3.
1
Centro de Cincias da Sade, University Sagrado Corao, Bauru, Sao Paulo,

Brasil.
2
Laboratrio de Biologia Molecular e Citogentica, University Sagrado Corao,
Bauru, Sao Paulo, Brasil.
3
Programa de Ps-graduao em Odontologia Sade Coletiva, University Sagrado
Corao, Bauru, Sao Paulo, Brasil.
Correspondent author
+5516981401987
information:
rita.peruquetti@usc.br;
Phone
number:
Introduction: Chronotherapy consists of restricting the administration of therapies to

certain periods of the day based on the different levels of cell activity within this 24
hours period. It is promising cancer therapy due to the circadian effects on the
progression of the cell cycle. It has been also shown that nucleolar activity is
increased in cancer cells due to the high synthetic levels on those cells. Aims: The
aim of this study was to identify if the timing of the administration of C. citratus
ethanolic extracts have influence on the (1) tumor progression; (2) nucleolar activity.
Methods: 20 male mice were treated for 15 days: TC-L (water; 1 hour after lights go
on); TC-D (water; 1 hour after light go off); EECc-L (100 mg/kg of C. citratus
extracts; 1 hour after lights go on); EECc-D (100 mg/kg of C. citratus extracts; 1
hour after lights go off). All the animals were inoculated with 103 Ehrlich ascites
cells in the 7th experimental day. Animal body weight was measured 3 times during
the experimental period. Fluid from the peritoneal cavity was collected to the
following analysis: (1) Quantification of inflammatory cells; (2) Quantification and
identification of cell cycle stages of Ehrlich ascites cells; (3) Quantification of
nucleolar fragments of Ehrlich ascites cells. Results and Discussion: When compared
to TC-D the group EECc-D showed (1) high number of inflammatory cells; (2) lower
number of ascites cells and those cells showed reduction of mitotic activity; (3)
lower number of nucleolar fragments. Conclusion: EECc-D showed better results in
the tumor progression. Financial Support: CNPq. Ethics committee approved:
8706290315. Keywords: Circadian rythms; Cymbopogon citratus; Nucleolus;
Ehrlich ascites cells.
A123
A124
NEW IMIDAZACRIDINE DERIVATE DELAYS TUMOR GROWTH IN

MELANOMA: IN VITRO AND IN VIVO APPROACHES
ALKYL PHOSPHOLIPID AND ALKYL TRIAZOLE DERIVED

COMPOUNDS INDUCE ANTI-PROLIFERATIVE ACTIVITY AND
APOPTOSIS IN ACUTE LYMPHOCYTIC LEUKEMIA (ALL) CELL
MODELS IN VITRO
Renata Virgnia Cavalcanti Santos (1), Silvina Odete Bustos (2), Mardonnny Bruno
de Oliveira Chagas (1), Maria do Carmo Alves de Lima (3), Mara Galdino da Rocha
Pitta (1), Ivan da Rocha Pitta (4), Michelly Cristiny Pereira (1), Roger Chammas (2),
Moacyr Jesus Barreto de Melo Rgo (1).
(1) Laboratrio de Imunomodulao e Novas Abordagens Teraputicas, Ncleo de
Pesquisa em Inovao Teraputica Suely Galdino, Universidade Federal de
Pernambuco, Recife, PE, Brasil.
(2) Laboratrio de Oncologia Experimental, Instituto do Cncer do Estado de So
Paulo, Universidade de So Paulo, So Paulo, SP, Brasil.
(3) Grupo de Pesquisa em Inovao Teraputica, Ncleo de Pesquisa em Inovao
Teraputica Suely Galdino, Universidade Federal de Pernambuco, Recife, PE, Brasil.
(4) Centro Avanado de Inovao em Sade, Ncleo de Pesquisa em Inovao
Teraputica Suely Galdino, Universidade Federal de Pernambuco, Recife, PE, Brasil.
E-mail: renata.vcsantos@gmail.com
Telephone contact: (+5581) 32718485 / (+5581) 997512747
Melanoma is the skin cancer with the lowest incidence, but the highest
aggressiveness. The current resistance to chemotherapy in clinical reduces the
efficiency of treatments, becoming urgent to search for new molecules. The acridine
nucleus present in the imidazacridines has an essential anti-proliferative role, to
topoisomerases I and II. This work aimed to evaluate the antitumor activity of AC05,
an imidazacridine derivate, against melanoma. The cytotoxicity of AC05 was
analyzed either in peripheral blood mononuclear cells, from healthy volunteers, as in
the melanoma lineage UACC62, for concentrations 10, 50 and 100M, through the
MTT in vitro assay. For an in vivo approach, it were used female balb/c nude mice (7
weeks old / n=24). 1x106 cells of UACC62 were injected subcutaneously into the
animals, which were randomly assigned in two groups. The mice were treated by
gavage with AC05 (30mg/kg) or vehicle (0,2% carboxymethylcellulose) for 5 days
followed by 2 days without intervention, during 2 cycles. Statistical significances
were analyzed by Mann-Whitney non-parametric test. The AC05 compound did not
modify the viability of healthy cells (IC50>100M), however the derivate presented
a substantial cytotoxicity against UACC62 cells (IC50=28,29 0,6M). In the mice
model, the compound significantly delayed tumor growth in treated group (v=0,015
0,0041cm3) compared to control group (v=0,16 0,043cm3), p0,05. Together, the
results suggest that AC05 presents a considerable therapeutic potential against
melanoma cancer.
Funding support: INCT_if, CAPES, CNPq and FACEPE.
Ethical Approval: Project FSPF 168/15 - Ethics Committee on Animal Use of So
Paulo University.
Larissa de Oliveira Passos Jesus1*, Heron Fernandes Vieira Torquato2, Vanessa Silva
Contijo3, Edgar Julian Paredes Gamero1, 2, Wagner Alves de Souza Jdice1.
1

Cruzes (UMC), So Paulo, Brazil.
2
Departamento de Bioqumica, Universidade Federal de So Paulo (UNIFESP), So
Paulo, Brazil.
3
Laboratrio de Fitoqumica e Qumica Medicinal, Universidade Federal de Alfenas
(UNIFAL), Minas Gerais, Brazil.
*lari.opj@hotmail.com; +55 (11) 98017-4357 / +55 (11) 4798-7102
Background: Alkyl phospholipid compounds such as Miltefosine have recently
emerged and demonstrate interesting activity against different species of parasites,
some pathogenic bacteria and fungi, as well as cancer cells. However, due to the
drug's gastrointestinal toxicity and hemolytic activity, the search for novel analogous
molecules is needed.
Aims: This study aimed to evaluate cytotoxicity, anti-proliferative ability and
apoptosis induction potential of a class comprised of 20 alkyl phospholipid and alkyl
triazole derived compounds in human acute lymphocytic leukemia (ALL) Jurkat and
CCRF-CEM cell lines.
Methods: All compounds were tested for their cytotoxic effect on Jurkat and CCRFCEM cell lines at concentrations through 25-150 M at 24h and 48h. The better
compounds were used at IC50 concentrations to perform apoptosis assay on cell lines
at 12hs. Cells were labeled with an anti-active caspase-3 antibody and the percentage
of cells with active caspase-3 was determined by flow cytometry.
Results: The compounds C9 and C21 presented the better cytotoxic activity. The IC50
were 70 M and 40 M for C9, and 80 M and 60 M for C21, at Jurkat and CCRFCEM, respectively. The presence of active caspase-3 following exposure to the
compounds suggests activation of apoptosis signaling.
Conclusion: The results indicate that the C9 and C21 compounds are cytotoxic
against ALL models and their apoptosis-inducing characteristics make them possible
an anti-leukemic agent.
Keywords: human acute lymphocytic leukemia, alkyl phospholipid, alkyl triazole,
Jurkat Cells, CCRF-CEM.
Supported by: CAPES, CNPq, FAPESP.

A125
A126
BONE-METASTATIC
POTENTIAL
OF
MCF10A/CD90+
AND
Hs578T/shCD90 BREAST CANCER CELL LINES AND THEIR ROLE IN
OSTEOPOROSIS
ASSOCIATION OF INTERLEUKIN 2 POLYMORPHISMS WITH GASTRIC

CANCER IN PATIENTS WITH H. PYLORI INFECTION
Camila Musumecci Guimares Azzi (1,2); Gabriella Christina G. M. de Paula (1);

Carlos DeOcesano-Pereira (1); Miriam Lemos (1); Mari Cleide Sogayar (1,3); Ana
Claudia Oliveira Carreira (1,2)
(1)
(2)
NUCEL/NETCEM (Cell and Molecular Therapy Center), Internal Medicine

Department, Medical School, University of So Paulo, So Paulo, Brazil
University of So Paulo, So Paulo, Brazil
(3) Biochemistry Department, Chemistry Institute, University of So Paulo, So
Paulo, Brazil
To whom correspondence should be addressed: Camila Azzi
(cah.musumecci@gmail.com), NUCEL Universidade de So Paulo, Rua Pangar,
100, Cidade Universitria, So Paulo, SP, Brazil. 05360-130, Phone: +55 11 26480230 - Mobile Phone: +55 11 94197-4020
Background: Breast cancer (BC) is the most common cancer among women, more
than half developping bone metastases. We previously described two stem cell
markers which are differentially expressed in breast cells: CD90 in highly malignant
Hs578T and CD14 in normal MCF10A cells. Recently, we generated CD90+
overexpressing MCF10A and shCD90 knocked-down Hs578T cells, which display,
respectively, a malignant and a more normal phenotype (submitted to publication).
Aims: To develop an animal model of osteoporosis in rats in order to test the bone
metastatic potential of MCF10A/CD90+ and Hs578T/shCD90 breast cell lines in
vivo. Methods: Osteoporosis was induced by oophorectomy in 12-week-old female
Rowett nude rats. Osteoporosis was analyzed by bone densitometry (at 40 days) and
histological analysis of femurs. In vivo metastasis was induced through injection of
106
cells
(MCF10A-wildtype,
Hs578T-wildtype,
MCF10A/CD90+
or
Hs578T/shCD90). After 35 days, rats were euthanized, and the lungs and other
organs (liver, bones, brain, skin, adrenal glands) were collected for histological
analysis of metastatic sites. Results: The Rowett rats developed osteoporosis at 40
days and their weight increased in comparason with control and sham groups.
Metastasis analysis is underway. Conclusion: An osteoporotic model was established
in rats. We intend to improve the knowledgement of the BC biology and their
correlation with the osteoporosis using an animal model, which may lead to the
development of new clinical and therapeutic protocols in the future.
Ethical approval: Ethics Committee for Animal Use of the Medical Veterinary
School, University of So Paulo
Support: CAPES, CNPq, FINEP, BNDES, MCT, MS-DECIT.
Luanna M Zabaglia1 , Mayara L Sallas1, Jessica L Melchiades1, Wilson A Orcini1,

Elizabeth Chen2, Marilia A C Smith2, Spencer L M Payo1 , Lucas T Rasmussen1
1
Laboratrio de Biologia Molecular e Citogentica, Universidade do Sagrado
Corao, Bauru, So Paulo, Brasil.
2
Departamento de Morfologia, Universidade Federal de So Paulo, Escola Paulista de
Medicina (UNIFESP/EPM), So Paulo, Brasil.
Correspondent author information: luannamunhoz@gmail.com Fone number: +55
(14) 21077069 e +55 (14) 996476812
Background: It is known that the immune response of the host is considered a key
event in the pathogenic process that leads to gastric disease. Helicobacter pylori (H.
pylori) infection induces the production of interleukin 2 (IL-2) in the gastric mucosa,
which increases the inflammation magnitude and may influence in the development of
gastric pathologies. Aims: To investigate a possible association among the IL-2
polymorphisms +114 T>G (rs2069763) and -330 T>G (rs2069762) with development
of gastric cancer and correlating with the presence of H. pylori. Methods: Gastric
biopsies were obtained from 113 patients with gastric symptoms; 62 of them with
normal gastric tissue and 51 with gastric cancer. For characterization of the
polymorphisms +114 T>G and -330 T>G was used PCR-RFLP. Results: Using the co
dominant model, were compared patients with normal gastric tissue and patients with
gastric cancer, according to the presence of H. pylori, and a high risk of gastric cancer
was found in subjects with IL-2 -330 GG genotype (OR = 6,43, 95% CI: 1,47-28.10,
p:0,026 adjusted by presence of H. pylori). When was analyzed polymorphism IL-2
+114, similar results were found. Subjects carrying -330 TT genotype showed a high
risk of gastric cancer (OR= 5.97, 95% CI: 1.60-22.27, p: 0,013 adjusted by presence of
H. pylori). Conclusion: Therefore, our results showed that -330 GG and +114 TT
genotypes are significantly associated with high risk in the developing of gastric
cancer in patients with H. pylori infection.
Financial Support: FAPESP 2015/11613-9 e 2015/11371-5. Ethical Approval:
1.215.180.
A127
A128
THE METALLOPEPTIDASE PHEX MODULATES OSTEOPONTIN (OPN)

IN SQUAMOUS CELL CARCINOMA (SCC)
THREE DIMENSIONAL BREAST CANCER CELL CULTURE: PHENOTYPE

INDUCTION CD44+/CD24-/low/Aldh1+, OXIDATIVE STRESS, EXPRESSION
OF STAT3 AND REPAIR GENES BRCA1, BRCA2 AND RAD51
Raquel L. Neves (1,2), Gabrielly M. D. Chiarantin (1,2), Fbio D. Nascimento (3),

Joo B. Pesquero (1), Helena B. Nader (4), Ivarne S. Tersariol (4,5), Marc McKee
(6,7), Adriana K. Carmona (1) and Nilana M.T. Barros (1,2)
(1) Department of Biophysics, Federal University of So Paulo, So Paulo, So
Paulo, Brazil. (2) Department of Biological Sciences, Federal University of So
Paulo, Diadema, So Paulo, Brazil. (3) Program for Biotechnology and Innovation in
Health, Anhanguera University, So Paulo, So Paulo, Brazil.
(4) Department of Biochemistry, Federal University of So Paulo, So Paulo, So
Paulo, Brazil. (5) Interdisciplinary Center for Biochemical Research, Mogi das
Cruzes University, So Paulo, So Paulo, Brazil. (6) Faculty of Dentistry, McGill
University, Montreal, Quebec, Canada. (7) Department of Anatomy and Cell
Biology, Faculty of Medicine, McGill University, Montreal, Quebec, Canada.
* Raquel L. Neves - Department of Biophysics, Federal University of So Paulo
Address: Street Pedro de Toledo 669. So Paulo, SP - 04039-032, Brazil
Institucional telephone: 55-11-55764450 (1977); Mobile telephone: 55-11998865943 - E-mail: raquel.neves@unifesp.br
Osteopontin (OPN) is a multifunctional phosphoglycoprotein strongly associated to
bone mineralization and tumor biology. Recently, our group identified OPN as the
first physiological substrate of the metallopeptidase PHEX (phosphate-regulating
gene with homologies to endopeptidases on the X chromosome). Mutations in PHEX
gene are responsible for the most prevalent form of rickets (1:20.000), the X-Linked
Hypophosphatemic (XLH), characterized by defective renal phosphate handling,
aberrant vitamin D metabolism and osteomalacia. PHEX studies have been focus in
bone and teeth, however its expression are also detected in others tissues including
lung and ovary. Considering the absence of studies relating PHEX and tumors, the
present work investigates PHEX involvement in squamous cell carcinoma (SCC)
OPN modulation. Real-Time PCR analyses showed a co-expression of PHEX and
OPN mRNA in SCC cells and immunoblotting assays demonstrated that the
recombinant PHEX can degrade OPN fragment present in cell extract (SCC-OPN).
Studies of cellular localization showed an absence of membrane-PHEX protein in
SCC at 12h of cell culture. The enzymatic activity assays demonstrated that
exogenous PHEX was inactivated by endogenous SCC cysteine proteases.
Additionally, the treatment with E64 (cysteine proteases inhibitor) promoted the
rescue of PHEX in SCC membrane, concomitant with the increased of OPN
degradation fragments. Finally, the super expression of PHEX in SCC (SCC
secPHEX) increased the degradation of OPN fragments and reduced the
proliferation/migration of these cells (SCC secPHEX).These results highlight the
involvement of PHEX in OPN modulation not only in bone, but also in tumor.
Ethical approval: 103323
Support by FAPESP and CNPq
Layane Duarte e Souza (1), Elisa Avelino (1), Isis Salviano (1), Juliana Rodrigues (1),
Israel Felzenszwalb (1), Andre Luiz Mencalha (1)
(1) Department of Biophysics and Biometry, Rio de Janeiro State University, Rio de
Janeiro, Brazil.
Corresponding author: layaneds@hotmail.com
Institutional: +55 021 2334-2058
Mobile: +55 022 99955-2860
Background: Breast cancer resistance is associated with STAT3 overexpression, DNA
repair, Reactive Oxygen Species (ROS) and cancer stem-cells (CSC). Although
STAT3 is important to tumorigenesis little is known about it role in regulation of DNA
repair gene expression. Besides, CSC, characterized by CD44+, CD24-/low and
ALDH1+, is related to aggressiveness and tumor recurrence associated with ROS.
Elucidate the interplay among STAT3, DNA repair and CSC could help better
comprehension of tumor biology.
AIMS: This study aims to evaluate regulation of double breaks DNA repair genes by
STAT3 in CSC using MDA-MB-231 breast cancer cell line in three dimensional
culture.
Methods: STAT3 target genes BRCA1, BRCA2 and RAD51 was predicted by
identifying promoter binding sites by bioinformatics tools. Gene expression was
evaluated by RT-qPCR in MDA-MB-231 treated with Stattic, a STAT3 inhibitor. CSC
establishment, cells were cultivated using 1,2% alginate matrix spheres. ROS levels
was accessed in 3D versus 2D culture by FACS analyses.
Results: RAD51 mRNA reduced 5-fold and BRCA1/BRCA2 mRNA did not alter in
STAT3-inhibition. STAT3 and CD44 mRNA increased 6-fold and 5-fold,
respectively, in 3D culture compared to 2D. CD24 did not amplified in 3D culture and
ALDH1 amplified only in 3D culture. The ROS levels was 16% lower in 3D compared
to 2D culture.
Conclusion: Our data suggest RAD51 as STAT3-target gene. Alginate 3D culture
amplified CSC markers as CD44+/CD24-/ALDH1+ suggesting a stem-cell phenotype
and ROS differences, compared to 2D culture, suggest distinct culture model can
modulate intracellular metabolism.
Funding: FAPERJ, CNPq and CAPES.

A129
A130
EVALUATION OF PAFR-INDUCED CHEMORESISTANCE UNDER

HYPOXIA IN MELANOMA
NATURAL BACTERIOPHAGE T4 AND M13 IN THE MEDIA INTERACT

WITH PC-3 HUMAN PROSTATE CANCER CELL AND ALTER GENE
EXPRESSION
Mayara DAuria Jacomassi(1*), Renata F. Saito(1), Matheus Ferracini(1), Roger

Chammas(1)
(1)
(1) Institute of Cancer of State of Sao Paulo - ICESP, Faculty of Medicine,

University of Sao Paulo, Sao Paulo, Brazil
(2)
Melanoma is the most aggressive form of skin cancer and its incidence is
increasing worldwide. Due to its notorious chemoresistance, metastatic melanoma
has been considered a clinical challenge. The comprehension of molecular and
cellular events that contributes to melanomas chemoresistance is important to
improve current therapies and prevent tumor recurrence. Previous in vivo studies
from our group showed that PAFR activation favors melanoma growth by
protecting to cisplatin-induced cell death. Furthermore, the expression of PAFR
was increased in hypoxic tumor areas. It is already known that tumor cells
undergoing hypoxia are more resistant to treatment and that it might contribute to
tumor recurrence. Therefore, we sought to evaluate whether PAFR activation is
involved in chemoresistance upon hypoxia. Human melanoma cell line SKmel 37
was exposed to hypoxia followed by reoxygenation in the presence of PAFR
antagonist, WEB2086 under cisplatin treatment. Cell death was assessed by
propidium iodide staining and flow cytometry analyses .We observed that cells
exposed to hypoxia were more resistant to cisplatin-induced cell death than cells in
normoxia. Moreover, PAFR blockage with WEB2086 sensitized cells in hypoxia
and the combined treatment was synergic. We also evaluated PAFR protein levels
by Western Blot. Interestingly, neither hypoxia nor cisplatin alter PAFR levels.
Thus, we are evaluating if PAFR ligands are being generated upon these
conditions. These results show that PAFR activation in hypoxic conditions is
important to cell survival and triggers chemoresistance.
Supported by FAPESP and CAPES
*Correspondence Author
mayjacomassi@gmail.com
+55 11 38933549
+55 11 997291992
Av. Dr. Arnaldo 251
01246-000 So Paulo SP Brazil
(2)
(3)
Laboratory of Extracellular Matrix, Departament of Morphology; Institute of

Biosciences; Sao Paulo State University (UNESP) Botucatu, SP, Brazil. Zip Code
18618- 689
* Corresponding author: swapnil_sanmukh@ibb.unesp.br + 55 14 3880 0498 + 55 14
998171954
Background: The use of bacteriophages for targeting cancer cells in vitro and in vivo
has emerged as a promising tool. Drugs and small interfering RNAs delivery are
among its possible applications. Aims: Here, we study the interaction of two different
types of Bacteriophages with human PC-3 cells and its possible effects on the
expression of selected genes. Methods: Seventy percent confluent PC-3 tumor cells
were exposed to 1X108 lytic bacteriophage T4 and filamentous bacteriophage M13.
Vehicle exposed cells were used as control. After 24 horus of exposure, the cells were
harvested and the RNAs were extracted and used for qPCR. The expression profile of
integrin alpha V, integrin alpha 5, integrin beta 1, integrin beta 3, integrin beta 5, Hsp
27, Hsp 90 and androgen receptor (AR) were analysed. Results: The expression of
integrin alpha V, integrin alpha 5, integrin beta 3 and integrin beta 5 were increased
markedly; whereas, Hsp90 and AR was decreased significantly from control. Hsp 27
gene was only decreased significantly from control after M13 exposure and integrin
beta 1 were unchanged. Phospho-AKT and PI3K was also increased in these cells after
bacteriophages exposure. Conclusion: Our results suggest the natural bacteriophages
can interacts with cell surface receptors integrins and mediate gene expression
changes. Future studies could show if these interactions may occur in vivo and if
natural bacteriophages could work as natural nanoparticles for drug delivery and gene
therapy for cancer treatment.
Financial support: CAPES (963-14-2); FAPESP (2013/08830-2)
A131
A132
EVALUATION OF TOXICITY ASSOCIATED WITH PACLITAXEL

NANOPARTICLES EXPERIMENTAL ANALYZING THE WEIGHT OF
NONHUMAN PRIMATE SPECIES CEBUS APELLA AS A TOOL FOR
CANCER THERAPY
EFFECTS OF FATTY ACID AND GLUCOSE IN THE OXIDATIVE STRESS,

CELL PROLIFERATION, MIGRATION AND APOPTOSE OF NORMAL AND
TUMORAL PROSTATE CELLS
Dejair da Silva Duarte (1), Nayara Cristina Lima de Oliveira (1), Danielle
Cristinne Azevedo Feio (1), Jos Augusto Pereira Carneiro Muniz (3), Patrcia
Danielle Lima de Lima (2), Rommel Mario Rodrguez Burbano (1)
(1)
Swapnil Ganesh Sanmukh1*, Nicolly Cezar Cruz, Caroline Nascimento Barquilha,

Srgio Alcantara dos Santos1, Helga Nunes1, Ana Carolina Lima Camargo, Bruno
Martinucci1, Bruno Oliveira da Silva Duran1, Flvia Karina Delella, Luis Antonio
Justulin Junior, Srgio Luis Felisbino1
(1) Human Cytogenetics Laboratory, Institute of Biological Sciences, Federal

University of Par, Belm, Par.
(2) Laboratory of Molecular Biology, Center Biological and Health Sciences, State
University of Par, Belm, Par.
(3) National Primate Center, Evandro Chagas Institute, Ministry of Health, Belm,
Par.
(4)
Presenting author: Dejair da Silva Duarte
Address: Lane So Pedro, N: 97, edifice Victor V, apartment 602
E-mail: dejair_duarte@outlook.com ; Telephone contact: (mobile): (91) 983988099 (institutional): 3201-8188
Nayara Cristina Lima de Oliveira, Email: nayara.oliveira@icb.ufpa.br
Telephone: mobile: (91) 8018-5409 Institutional: 3201-8188
Danielle Cristinne Azevedo Feio, Email: daniellefeio@yahoo.com.br
Telephone: Institutional: 3201-8188
Patrcia Danielle Lima de Lima, Telephone: Institutional: (91) 3276-3387
Jos Augusto Pereira Carneiro Muniz, Telephone: Institutional: (91) 3213-0411
Rommel Mario Rodrguez Burbano, Email: rommel@ufpa.br ; Telephone:
Mobile: (91) 8836-4667 Institutional: 3201-8188
The chemotherapy placement system, called LED, has the ability to concentrate in
tumor tissue after injection into the bloodstream, thus being able to direct tumor
drugs incorporated into the nanoemulsion. The purpose of this study is to evaluate
the chronic toxicity of nanoparticles associated with Paclitaxel chemotherapy
(LDE-PTX) analyzing the weight of non-human primate species Cebus apella.
This study used 15 specimens were divided into three groups: negative control
(NC); Experimental (EXP 1 and EXP2) where animals received the LDE-PTX for
intravenously at two different doses of 175 mg/m2 and 250 mg/m2, respectively;
and the positive control (CP1 and CP2) animals received the drug intravenously in
commercial form at the same doses used in the experimental group, respectively.
And during periods of treatment their weights were measured. Data were
submitted to analysis of variance ANOVA with Bonferroni post-test with
significance set at p <0.05, by BioEstat5.3. This study is to show that the
specimens treated with paclitaxel in commercial form (CP1) had mean values of
their weights (kg) lower than the specimens of the negative control and drugtreated in an experimental way at both concentrations (EX1 and EX2). The results
of this analysis of the experimental drug toxicity can be used for contribute to
knowledge and reduced side effects of future patients for treating cancer and for
improving the use of LDE-PTX. The project was approved by the ethics committee
in research with experimental animals UFPA (BIO008-11) and funded by the
National Council for Scientific and Technological Development.
Breno Costa Landim (1), Lvia Prometti de Rezende (1), Maria Raquel Untherkircher
Galheigo (1), Renata Graciele Zanon (1), Daniele Lisboa Ribeiro (1)
(1) Institute of Biomedical Sciences. Federal University of Uberlandia, Uberlandia,
MG, Brazil, 38400-902
daniele.ribeiro@ufu.br
Recent clinical investigations have shown a strong relation between obesity and
prostate cancer. To achieve this issue, this investigation evaluate the effect of high
concentrations of fatty acid and/or glucose in normal and tumor prostate cells. PNT1A
and PC3 cells were treated with high concentration of palmitate (100 M, HF) and/or
glucose (12,4 mmol/l, HG) for 24hrs and 48hrs and analyzed for different assays. The
results showed that there is a good cell migration in the healing area in both cell types
after 48H of all treatments. However, only in PC3 the migration was more
influenciated by HF and HG when compared to control treatment. MTS revealed that
PNT1A cells exhibited high proliferation when treated with HF and HG in 24H, while
PC3 increased proliferation only after HG- 48H treatment. Comparing cell lines, PC3
had greater cell proliferation than PNT1 only after HG-48H. Additionally, there is no
difference on apoptosis in any of treated groups. The analysis of oxidative stress
showed that treatments did not affect lipid peroxidation, except in PNT1 after HF-48H
which exhibited high levels of TBARs. Regarding nitrite production and total
antioxidant activity, HF had more impact than other treatments, and normal cells are
more affected by oxidative stress only after 48 hrs, while the tumor cells are affected
within the first hours of treatment. Thus, the high concentration of palmitte and glucose
negatively influences normal and tumoral prostate cells and stimulates celullar
activities related to carcinogenesis such as cell proliferation, migration and oxidative
stress.
Keywords: fatty acid; glucose; cell proliferation; prostate cancer.

A133
A134
ERK 1/2 AND SURVIVIN COULD CONTRIBUTE TO IMATINIB

RESISTANCE IN CML CELL LINE INDEPENDENT OF BCR-ABL
TLR4 DELETION ATENUATES BROWNING AND INFLAMMATION

PROCESSES IN WHITE ADIPOSE TISSUE DURING CANCER CACHEXIA
Danielle Cardoso da Silva1,2, Miguel Angelo Moreira3, Raquel Ciuvalschi Maia2,

Flavia da Cunha Vasconcelos2
Felipe O. Franco (1); Felipe S. Henriques (1); Juliana C. Vieira (1); Magno A. Lopes
(1); Kaltinaitis B. Santos (1); Pamela V. Knobl (1); Luana G. Leal (1); Claudio S.
Shida (2); Adilson Guilherme (3); Miguel L. Batista Jr. (1)
1- Programa de Ps-Graduao em Oncologia, Instituto Nacional de Cncer

(INCA), Rio de Janeiro, Brasil
2- Laboratrio de Hemato-Oncologia Celular e Molecular, Programa de HematoOncologia
Molecular,
INCA,
Rio
de
Janeiro,
Brasil.
E-mail:
cardoso.ds7@gmail.com. Phone: +5521 981094509
3- Coordenao de Pesquisa - Diviso de Gentica - INCA, Rio de Janeiro, Brasil
About 25% of Chronic Myeloid Leukemia (CML) patients develop resistance to
tyrosine kinase inhibitor (TKI) treatment. Amidst several molecular mechanisms,
Bcr-Abl-independent activation of MAPK/Erk signaling and survivin
overexpression were shown to be important mechanisms of imatinib (IM)
resistance. Still, the need to better comprehend the mechanisms that underlie
resistance and develop new therapeutic strategies lingers. Toward studying aspects
of resistance, the CML cell line resistant to IM, K-IM, was developed in our lab
and this study aims at unraveling the resistance mechanisms present in K-IM cell
line. K-IM cells showed neither mutations in BCR-ABL gene (direct sequencing)
nor efflux protein expression or activity (flow cytometry). There was an increase in
BCR-ABL and BIRC5 (survivin) mRNA levels (RT-qPCR). K-IM was more
resistant to the TKIs IM and dasatinib compared to K562 and K562-Lucena cell
lines (MTT assay). Cells were arrested in G0/G1 phase after treatment with IM in
all cell lines, however, in K-IM cells, apoptosis may not have been triggered since
there was no increase in DNA fragmentation or annexin V/propidium iodide
staining (flow cytometry). Bcr-Abl IM-induced inhibition drastically reduced Erk
1/2 phosphorylation and survivin protein levels (western blotting) in K562 and
K562-Lucena but not in K-IM suggesting that they may contribute to resistance
independent of Bcr-Abl. The new cell line K-IM could be used as a model to study
Bcr-Abl-independent molecular resistance mechanisms and also as a tool for drug
development targeting resistant patients.
Funding Support: CNPq/FAPERJ/Programa de Oncobiologia/UFRJ/FAF
1- Laboratory of Adipose Tissue Biology, Integrated Group of Biotechnology,

University of Mogi das Cruzes, Mogi das Cruzes, So Paulo, Brazil; 2- Department of
Biomedical Engineering, Federal University of Sao Paulo, Sao Jose dos Campos, So
Paulo, Brazil; 3- Division of Endocrinology, Metabolism, and Diabetes, University of
Massachusetts Medical School, Worcester, Massachusetts, United States of America;
Email address: felipe.franco.sp@gmail.com
Phone number: +55 (11) 48987087
Background and aims: Cancer cachexia (CC) is a wasting syndrome characterized by
systemic inflammation, body weight loss, atrophy of both white adipose tissue (WAT)
and skeletal muscle. Recently, CC was associated with early white-to-brown adipose
tissue remodeling (browning) in wild-type mice. In this work we determined whether
Toll Like Receptor 4 (TLR4) deletion modifies adipose tissue remodeling (browning
and inflammation) during CC.
Methods: Male C57BL/6 mice (6-8 week-old), TLR4 wild-type (WT) and knockout
(TLR4-/-) were subcutaneously inoculated with 3.5105 Lewis Lung Carcinoma cells or
vehicle-saline (control) (according to be The Brazilian College of Animal
Experimentation). Adipose tissues from subcutaneous (SCAT) and mesenteric
(MEAT) depots were collected on day 28th after tumor-cells inoculation. Light
microscopy (histology and immunohistochemistry), qPCR (gene expression), western
blot (protein expression) and ELISA (serum inflammatory cytokines) were used.
Results: Body weight loss was evident in both tumor groups, which showed a
reduction of 12.27% in Tumor-WT and 6.82% in Tumor-TLR4-/-. UCP-1 gene and
protein expressions decreased (p<0.01) in SCAT Tumor-TLR4-/- group when compared
with Tumor-WT group. MEAT demonstrated a decrease in TNF, CD68 and CD3
positive cells in Tumor-TLR4-/- when compared with Tumor-WT. IL-1 (p=0.0348)
and INF (p=0.028) decreased in TLR-/- groups when compared with WT-groups.
Conclusion: Taken together, these results suggest that TLR4 deletion reduces browning
and inflammation (WAT and systemic). Thus, TLR4 may play an important role in
browning which makes TLR4 a potential therapeutic target in CC.
Grants: FAPESP (2015-19259-0) and CAPES.
A135
A136
MELK EXPRESSION BY PROSTATIC EPITHELIAL CELLS LNCaP AND

PC3 IS DOWN REGULATED BY FIBRONECTIN AND TESTOSTERONE
TREATMENT
IL-6 ALTERS THE MIGRATORY BEHAVIOR OF HEAD AND NECK

TUMOR CELLS
Nicolly Cezar Cruz, Caroline Nascimento Barquilha, Bruno Martinucci, Maira

Smaniotto Cucielo, Flavia Karina Delella, Sergio Luis Felisbino (1)
(1) Department of Morphology, Institute of Biosciences, So Paulo State
University (UNESP), Botucatu, SP, Brazil. niihccruz@hotmail.com; +55 14 38800474 +55 11 973440072
Background: The prostate cancer (PCA) is the second mostly frequent and the
second higher morbidity and mortality rate among men. Integrating RNAseq data
from tumors arising in two established mouse model of prostate cancer with
published human prostate cancer expression we identified the serine/ threonine
kinase enzyme MELK (Maternal Embryonic Leucine zipper Kinase) as an
important therapeutic target for prostate cancer., once it plays an role in the cell
cycle regulation, proliferation and apoptosis. Aims: Here we characterize the
pattern of gene expression of MELK in two prostatic epithelial cell lines LNCaP
and PC3. Methods: Seventy percent confluent LNCaP and PC3 cells were treated
or not with fibronectin, flutamide and testostorone, separately, supplemented in the
media. After 24 and 96 hours the cells RNAs were extrated and MELK expression
were analyzed by qPCR. MELK subcellular location were also analyzed by
immunoflourescence. Results: MELK location migrates from the cytoplasm to
nucleus in these cells after testosterone exposure. Quantitative PCR showed similar
expression of MELK by both cells in regular culture conditions and did not change
after flutamide exposure. MELK expression was significant decreased in these
cells after exposure to fibronectin and testosterone. Conclusion: Our results
suggest that cellular events related to MELK are down regulated by androgen and
extracellular matrix attachment, such as apoptosis. Future studies should address if
MELK inhibition has the potential to be a effective strategy for targeting prostate
cancer cells.
Financial support: So Paulo Research Foundation (FAPESP) (2015/09947-6 2013/08830-2)
Leonardo Francisco Diel(1), Alessandro Menna Alves(1,3), Grasieli de Oliveira

Ramos(1,2), Lisiane Bernardi(1), Alan Frederick Horwitz(5), Marcelo Lazzaron
Lamers(1,4)
(1) School of Dentistry, Federal University of Rio Grande do Sul, Rua Ramiro
(2) School of Dentistry, Universidade do Oeste de Santa Catarina, Joaaba, SC, Brazil
(3) School of Dentistry, Centro Universitrio UNIVATES, Lajeado, RS, Brazil
(4) Department of Morphological Sciences, Federal University of Rio Grande do Sul,
Porto Alegre, Brazil
(5) Department of Cell Biology, School of Medicine, University of Virginia,
Charlottesville, Virginia, United States of America.
leocvr@bol.com.br , marcelo.lamers@ufrgs.br
During cancer progression, tumor cells develop different skills, as the ability to migrate
in the host tissue, an important step for invasion and metastasis. This capability can be
modulated for several factors present in the tumor microenvironment, such as
interleukins (ILs). In the current literature there are few evidences about the role of ILs
in the migratory behavior of head and neck squamous cell carcinoma (HNSCC) tumor
cells. The aim of this study was to evaluate the effect of different ILs in the migratory
behavior of a HNSCC tumor cell line (SCC25). It was observed that SCC25 showed
low expression of e-cadherin and high expression of n-cadherin, suggesting that this
cell line is in epithelial-mesenchymal transition (EMT).. After,,it was observed that
SCC25 are responsive to different ILs (IL6, IL1B, TNFa). To analyse the role of IL on
cell migration, it was performed time lapse videos (24 hours) with SCC25 in
fibronectin (2g/ml) and the analysis of Rac1 activation, the main RhoGTPase related
to control the migration process. It was observed that only IL-6 was capable to
modulate migratory behavior of SCC25 increasing migration speed and enhancing
directionality, which correlated with an increase on Rac1 activation. Furthermore,
when STAT3 inhibitor was used, even in the presence of IL-6, SCC25 lines returned to
migration patterns similar to control. Therefore, it can be concluded that IL-6 enhances
migratory ability of HNSCC tumor cell line probably due to the increases in Rac1
activation.
The experimental design of this study was approved by COMPESQ UFRGS
Financial support: CAPES, CNPq.

A137
A138
GBM-MICROGLIA INTERACTION MODULATED BY WNT PATHWAY
SHIKONIN
AND
TEMOZOLOMIDE
COMBINATION
REDUCES
EPITHELIAL-TO-MESENCHYMAL TRANSITION IN GLIOBLASTOMA
CELLS
Diana Matias (1,2), Luciane Rosrio (1), Luiz Gustavo Dubois (1), Bruno Pontes
(2), Valria Ferrer (1), Jos Garcia Abreu (1), Flvia Lima (1), Vivaldo Moura
Neto (1,2)
(1) - Instituto Estadual do Crebro Paulo Niemeyer (IEC), Rio de Janeiro, Brazil
(2) - Instituto de Cincias Biomdicas da Universidade Federal do Rio de Janeiro,
Brazil
Diana Matias: dimtias@gmail.com Institutional phone: +552122779393 Mobile:
+5521967249527
Background: Glioblastoma (GBM), an extremely aggressive and deadly brain
tumor, is known for its striking heterogeneity and capability to communicate with
microenvironment components, such as microglia. The microglia-tumor interaction
can contribute to increased invasiveness. The Wnt pathway is one of the most
crucial signaling cascades in tumor progression, playing a part in regulating cancer
cells migration and invasiveness.
Aims: Elucidate the role of Wnt/catenin signaling in microglia and GBM
crosstalk.
Methods: We analyzed and observed GBM-microglia interaction using co-cultures
to observe the modulation of tumor cells invasiveness by the presence of
microglia. For this purpose, we used time-lapse imaging, immunofluorescence and
western blot.
Results: We observed that the conditioned medium obtained from glioblastoma
cells induces translocation of -catenin in microglial cells nuclei. This observation
is even more prominent in microglial cells treated with the conditioned medium of
GBM cells, which had been treated with Wnt3a. Moreover, microglia enhanced the
expression of Wnt target proteins, such as connexin 43 and ARG-I.
Conclusion: Our evidences revealed that Wnt/-catenin pathway plays important
role in GBM-Microglia crosstalk and Wnt3a belong to the arsenal of factors that
recruits microglia cells to tumor cells.
Financial Support: Faperj, CNPq, INNT-INCT-MCT, CAPES, Pro-Saude Assoc.
Benef. Assist. Soc. e Hospitalar
Diana Matias (1,2), Luciane Rosrio (1), Brenno Carneiro (1), Luiz Gustavo Dubois
(2), Vivaldo Moura Neto (1,2).
(1) - Instituto Estadual do Crebro Paulo Niemeyer (IEC), Rio de Janeiro, Brazil
(2) - Instituto de Cincias Biomdicas da Universidade Federal do Rio de Janeiro,
Brazil
Luciane Rosrio: lucianevorosario@gmail.com Institutional phone: +552122779393
Mobile: +5521996717463
Background: Glioblastoma is characterized by their aggressiveness and high resistance
to conventional therapies. Therefore, new therapeutic strategies is a key step to
increase the patients survival. The combination of usual chemotherapeutic drugs with
new molecules exhibiting anti-tumoral properties, such as Shikonin, has been studied.
The purpose of those new therapies would be reduce the migratory abilities of
glioblastoma cells, and this invasiveness regulated by epithelial-to-mesenchymal
transition (EMT).
Aim: The main objective of this work was evaluate the effect of Shikonin and
Temozolomide combination in glioblastoma motility and verify if there is difference in
expression of migratory and invasion-regulatory proteins .
Methods: To attain these objectives, we treated glioblastoma cell lines with Shikonin
alone and in combination with TMZ during 24h. Viability was evaluated by MTT
assay. Expression of cytoskeleton and EMT proteins were assessed by Western blot
and immunocytochemistry.
Results: Shikonin treatment reduced glioblastoma cell lines viability. A reduction of
GFAP and vimentin expression levels was observed in cells treated with Shikonin plus
temozolomide. Moreover, we observed that -catenin, N-cadherin and Slug, proteins
that regulate EMT, were reduced after the combined treatment. Reduced AKT levels
were also detected.
Conclusion: These data suggest that combination of Shikonin and temozolomide
reduces the migration and invasion of glioblastoma cells, which seems related with the
reduction of the EMT in glioblastoma. Shikonin and temozolomide reveal potential for
glioblastoma treatment.
Financial Support: Faperj, CNPq, INNT-INCT-MCT,CAPES, Pro-Saude Assoc.
Benef. Assist. Soc. e Hospitalar
12-
3-
A139
A140
ANDROGEN STIMULATION EFFECTS OF BMYB OCCUPATION OF

THE GENOME IN LNCaP HUMAN PROSTATE EPITHELIAL CELLS
OCT4A EFFECTS ON MEDULLOBLASTOMA CELL AGGRESSIVENESS

INVOLVE ABNORMAL EXPRESSION OF NON-CODING RNAS
Rafaela Rosa-Ribeiro1*, Gary Karpen2, Guilherme Oliveira Barbosa1, Hernandes F.

Carvalho1
Patrcia Benites Gonalves da Silva (1), Carolina Oliveira Rodini (1), Carolini Kaid
(1), Mrcia Cristina Leite Pereira (1), Gabriela Furukawa (1), Daniel Sanzio Gimenes
da Cruz (2), Clarissa Ribeiro Reily Rocha (3), Carla Rosenberg (1), Oswaldo Keith
Okamoto (1)
(1) Department of Structural and Functional Biology. State University of

Campinas, Campinas SP, Brazil
(2) Departament of Molecular and Cell Biology. Lawrence Berkeley National
Laboratory, Berkeley CA, USA.
* Contact: rafa.darosa@gmail.com
Enriched consensus BMYB (transcription factor) binding sites were found by in
silico analysis of the proximal promoter of the genes highly responsible to
androgen deprivation in the rat ventral prostate (VP). Bmyb expression increases
five-fold in the VP three days after castration (qRT-PCR). Immunofluorescence
has localized Bmyb expression to epithelial cells within the gland. Aiming at
understanding how androgen regulation affects BMYB, LNCaP prostate cancer
epithelial cells were grown under 1nM R1881 (synthetic androgen) or vehicle
(DMSO) for 6, 12 and 24 hours. Highest difference in BMYB expression
(evaluated by qRT-PCR) and activation (evaluated by Western blotting and
immunofluorescence) was found between vehicle and R1881 groups at 12h. The
same groups were submitted to BMYB ChIP-seq experiment. Presence of R1881
promoted LNCaP cells proliferation and BMYB was strongly associated to genes
involved with cell cycle. However, in vehicle LNCaP cells, BMYB is highly
expressed and activated, binds to canonical and non-canonical motifs and
associates with genes, which do not characterize enrichment ontologies. Intergene
association of BMYB with the DNA reveals a probable function related to
chromatin structure or large-scale genome organization of regulated genes in
vehicle group. Comparing ChIP-seq of R1881 results with existing transcriptome
data, was found five percent of genes in common, suggesting that BMYB is coopted to the promoters of AR-regulated genes. In conclusion, the results indicate
that MBYB is engaged in classical functions related to cell cycle (R1881), and
might assume new functions in high-order chromatin structure, compatible with
gene expression regulation and chromatin organization.
FAPESP:2009/16150-6;2012/07512-4;2013/23123-0 CEUA/UNICAMP:2917-1
(1) Centro de Pesquisa sobre o Genoma Humano e Clulas-Tronco. Departamento de

Gentica e Biologia Evolutiva, Instituto de Biocincias, Universidade de So Paulo.
So Paulo, SP, Brazil p.benites@hotmail.com (11)96850-7240 / (11)3091-0972
(2) Departamento de Patologia, Faculdade de Medicina Veterinria e Zootecnia,
Universidade de So Paulo. So Paulo, SP, Brazil
(3) Departmento de Microbiologia, Instituto de Cincias Biomdicas, Universidade de
So Paulo. So Paulo, SP, Brazil
Medulloblastoma is the most common malignant pediatric brain tumor. The expression
of typical pluripotency genes is correlated with poor prognosis in medulloblastoma and
OCT4 expression was shown capable of discriminating patients with poor survival
outcome. Despite this prognostic value, direct evidences of OCT4 contribution to more
aggressive traits in medulloblastoma are missing. In this context, we investigated the
role of Oct4A isoform on pro-tumorigenic features of medulloblastoma in vitro and in
vivo and evaluated molecular alterations that could be responsible for acquisition of a
more aggressive phenotype in medulloblastoma cells. Medulloblastoma cells
overexpressing OCT4A displayed enhanced cell proliferation and cell cycle alterations.
Increased clonogenic activity, tumorsphere generation capability and subcutaneous
tumor development were also observed, and these effects were OCT4A expression
level-dependent. Evaluation of cell mobility in vitro showed loss of cell adhesion and
greater 3D-spheroid invasion. In an orthotopic model of medulloblastoma, OCT4A
overexpressing cells generated more developed, aggressive, infiltrative and metastatic
tumors. OCT4A overexpression was associated with chromosomal instability but copy
number aberrations varied in frequency and type according to the cell line, with little
association with differently expressed genes. Interestingly, marked differential
expression of non-coding RNAs, including newly discovered, still poorly
characterized, long non-coding RNAs and multiple small nucleolar RNAs were
observed in medulloblastoma cells with OCT4A overexpression. Altogether, our
findings support the relevance of pluripotency-related factors in the aggravation of
medulloblastoma traits classically associated with poor clinical outcome, and
underscore the prognostic and therapeutic value of OCT4A in this challenging type of
pediatric brain cancer.

A141
SULFIREDOXIN: A POTENTIAL
ADVANCED PROSTATE CANCER
A142
THERAPEUTIC
TARGET
FOR
Caroline Nascimento Barquilha*, Nicolly Cezar Cruz, Isabela Gaseta Ferraz Paiva,
Nilton Jos dos Santos, Swapnil Ganesh Sanmukh, Bruno Martinucci, Flvia Karina
Delella, Srgio Luis Felisbino
(1)Department of Morphology, Institute of Biosciences, So Paulo State University
(UNESP), Botucatu, So Paulo, Brazil.
*caroline.barquilha@gmail.com; +55 14 38800481; 14 982206043.
Background: Despite the advance of anti-hormonal therapies against prostate cancer
(PCa), new therapeutic approaches have been investigated for treatment of advanced
stage tumors that are resistant to androgen deprivation. Oxidative stress is involved
with cancer development, playing a role in the progression and metastasis of PCa.
Aims: Here, we investigated new therapeutic targets for PCa, focusing on enzymes
related with oxidative stress defense. Methods: Samples of different stages of
prostate cancer progression from two types of knockout mice were used to generate
their transcriptomes, using next generation RNA sequencing. The list of
differentially expressed genes from mice transcriptomes were crossed with published
human prostate cancer expression data to pinpoint cancer-associated genes
expression changes that are conserved between the two species, with special
attention to oxidative stress-related genes. Results: Cross-species analysis
demonstrated an increased expression of sulfiredoxin (Srxn1), mainly in the mouse
advanced stage tumor and human higher Gleason Score. In addition, human data
bases also demonstrated that patients with Srxn1 upregulation have lower survival
rates than the others patients (P<0,000122). Immunohistochemistry for Sulfiredoxin
in mice tumor samples confirmed increased expression of this enzyme in the
undifferentiated adenocarcinoma. Quantitative PCR showed PC3 androgenindependent cancer cells express higher levels of Srxn1 than LNCaP and RWPE-1
cells, and this expression increases with H2O2 treatment. Conclusion: Taken
together, our results suggest that inhibition of Sufiredoxin has the potential to be an
effective strategy for targeting advanced prostate cancer.
Ethical Protocol: CEEA 613/2014
Financial Support: So Paulo Research Foundation (FAPESP) 2013/08830-2
ERK SIGNALING PATHWAY IS ACTIVATED BY PrP-HOP COMPLEX IN

GLIOBLASTOMA STEM CELLS PROLIFERATION
Mariana Brando Prado1, Rebeca Piatniczka Iglesia1 and Marilene Hohmuth Lopes1
1
Department of Cell and Developmental Biology; Institute of Biomedical Sciences;

University of Sao Paulo; Sao Paulo, Brazil. Zip code 05508-900.
Presenter contact details:
Address: Av. Prof. Lineu Prestes, 1524 - Cidade Universitria "Armando Salles
Oliveira", Butant - So Paulo - SP - CEP 05508-900.
E-mail: maribrandaop@gmail.com
Phone Numbers: (11) 98228-6287(mobile)/ (11) 26488256(Lab)
Glioblastoma multiforme (GBM) is an aggressive tumor derived from glial cells,
presenting a high rate of recurrence and lethality. Studies show that the GBM is
maintained by a subpopulation of stem-like cells, called glioblastoma stem cells
(GSCs). These cells are chemo and radio resistant and are able to remain in a
quiescent state, therefore representing an important target for GBM treatment. Our
group has demonstrated a central role of cellular prion protein (PrP) and its main
ligand, the co-chaperone stress inducible protein one or heat-shock organizing
protein (STI1/HOP) in GBM growth in vivo and in the maintenance of GSCs
proliferation in vitro. Also, the inhibition of PrP-STI1 complex by a specific peptide
is able to delay tumor growth and increase animal survival in immunodeficient mice.
In this study we investigated the role of extracellular signal-regulated kinase (ERK)
pathway in the control of GSCs proliferation mediated by HOP-PrP interaction. To
address this issue GSCs population knocked down to PrP expression and their
respective counterparts were used as study model. Our western blotting experiments
showed that the recombinant HOP treatment activates ERK signaling pathway in
GSCs in a PrP-dependent manner. Furthermore, preliminary BrdU incorporation
assays showed an increase in GSCs proliferation after treatment with recombinant
HOP, which was impaired with ERK pathway inhibition. Therefore, our study
demonstrates that PrP-HOP complex is capable to promote GSCs proliferation
through ERK pathway activation. Hence, the combinational of disruption of HOPPrP interaction or ERK pathway would be a rational and effective strategy for the
glioblastoma treatment.
Financial support: FAPESP
A143
A144
MODULATION
OF
THE
EXPRESSION
OF
MATRIX
METALLOPROTEINASES AND THEIR INHIBITORS UPON CD90
MALIGNANCY MARKER DEPLETION IN MAMMARY CARCINOMA
IGF-1
SIGNALING
AND
WNT/BETA-CATENIN
PATHWAY
INTERACTION DURING
THE PROGRESSION OF COLORECTAL
CANCER
Adauto Spindola Jr (1), Caio Vinicius Carriel Dalmasso(1), Aline Ramos Maia
Lobba (2), Ana Claudia Oliveira Carreira (1), Marina Trombetta-Lima (1), Mari
Cleide Sogayar (1,2).
Cssio Dejair Fleming de Moraes (1,2), Jos Andrs Morgado-Daz (2), Wallace
Martins de Arajo (2)
(1) NUCEL/NETCEM-Faculty of Medicine, University of So Paulo, So Paulo,

Brazil.
(2) Chemistry Institute, University of So Paulo, So Paulo, Brazil.
e-mail: adauto.spindola@gmail.com, Tel: +55 (11) 26480227 Cel: +55 (11)
999911522
Rua Pangar- 100 Butant - CEP: 05360-130 - So Paulo-SP-Brazil
Background
Breast cancer is the main cause of death related to cancer among women, therefore,
the functional analysis of genes involved in tumor progression, and the search for
prognostic
biomarkers
are
of
extreme
importance.
CD90 is a malignancy marker in several tumor types, being classically involved in
cellular processes such as apoptosis, cell-cell and cell-matrix interactions.
We recently described that CD90 is highly expressed in aggressive mammary
carcinoma cell lines, with its overexpression leading to increased Epidermal Growth
Factor Receptor (EGFR) expression. EGFR signaling is known to increase Matrix
Metalloproteinases (MMPs) activity, which, in turn, leads to ECM degradation and
facilitates cellular invasion.
Aims
To analyze MMPs and their inhibitors upon CD90 depletion in the Hs578-T human
breast carcinoma cells.
Methods
Hs578-T cells were subjected to CD90 knockdown by shRNA. MMPs (MMP-2,
MMP-9, MMP-14) and their inhibitors (TIMP-1, TIMP-2, TIMP-3, TIMP-4 and
RECK) expression was analyzed by qRT-PCR and Western blotting. In addition,
conditioned media collected from the cells were subjected to zymography assays to
analyze MMP-2 and MMP-9 expression.
Results
Our data indicate that MMP-2, MMP-14 and TIMPs are not significantly modulated
in this model. On the other hand, EGFR, RECK and MMP-9 expression decreases
upon CD90 downregulation.
Conclusion
One possible mechanism by which CD90 is involved with tumor aggressiveness is
through modulation of the EGFR pathway and increased MMP expression, a
phenotype which is partially reversed upon CD90 downregulation.
(1) Federal University of the State of Rio de Janeiro (UNIRIO), Center of Biological
and Health Sciences, Biomedical Institute. E-mail: cassiodfleming@gmail.com
(2) Group of Estructural Biology, Cancer Biology Program, INCa. E-mail:
jmorgado@inca.gov.br
Background: Colorectal cancer (CRC) is a public health problem worldwide. It
results from mutations in oncogenes and tumor suppressor genes, which leads to the
deregulation of different pathways responsible for events such as differentiation,
proliferation, migration and survivor. In this context, the Wnt/-catenin pathway is
chronically active in CRC preventing the formation of the -catenin destruction
complex resulting in the accumulation of cytoplasmic -catenin and consequently
translocation to the nucleus, where it activates target genes of Tcf/Lef responsible for
the events above described. On the other hand, it is known that the Insulin-like
growth factor 1 (IGF-1) binds to its receptors activating RAS/MEK/ERK and
PI3K/Akt signaling. Aims: To evaluate the interaction between the signaling
mediated by IGF-1 and the Wnt/-catenin pathway in the progression of CRC.
Methods: HCT-116 and HT-29 cells lines were incubated with IGF-1 and Wnt3a.
Crystal violet and wound healing assays were performed to measure proliferation
and migration. Wnt/-catenin activity was assessed by measuring luciferase activity.
Results: Wnt3a induced the activation of TCF/LEF in both cells and IGF-1 did not
change -catenin activity. The treatment with IGF-1 increased cell proliferation in
HCT-116 cells. Cell migration of both cells was decreased with the treatment of
Wnt-3a and IGF-1. However, when HCT-116 cells were treated with Wnt3a and
IGF-1 together the cell migration increased. Western blot showed a decrease in the
expression of E-cadherin. Conclusions: IGF-1 signaling and the Wnt/-catenin
pathway interaction contributes with the progression of colorectal cancer.
Acknowledgements: CNPq, FAPERJ and Ministrio da Sade - Brasil.
Funding support. BNDES, CAPES, CNPq, FAPESP, FINEP, MCTI, MS-DECIT

A145
A146
MORPHOLOGICAL CHARACTERIZATION OF EXOSOMES DERIVED

FROM GLIOBLASTOMA CELL LINES
EXPRESSION PROFILE OF TELOMERE-ASSOCIATED GENES IN NONSMALL CELL LUNG CANCER
Isabel Porto-Carreiro (1), Sheila Martins (1) and Vivaldo Moura-Neto (1)
Camila Baldi (1), Mrcio de Carvalho (2), Rogrio Antonio de Oliveira (3), Tomas
Tokar (4), Igor Jurisica (4), Patricia P. Reis (5) and Maria Isabel Nogueira Cano (1)
(1) Laboratrio de Biomedicina do Crebro, Instituto Estadual do Crebro Paulo

Niemeyer (IECPN), Rio de Janeiro, Brazil.
(1)
iportocarreiro@gmail.com. +55 21 2462-1117 / +55 21 98793-5224 (cell phone) (2)
Glioblastomas (GBMs) are highly malignant brain tumors with a poor prognosis due
to their high rates of proliferation, invasiveness, vascularization and
radio/chemoresistance. Tumor cells communicate one with each other and with the
surrounding normal tissue by releasing extracellular vesicles, in special exosomes.
Exosomes are 40-100nm vesicles derived from the endocytic pathway upon fusion of
multivesicular bodies (MVBs) with plasma membrane. We have isolated exosomes
from human glioblastoma cell lines that were immortalized in our laboratory from
tumors obtained by surgical resection. Culture supernatants of different GBM lines
were harvested and exosome purification was performed by differential
ultracentrifugation. Analysis of the 100,000xg pellets by electron microscopy
revealed the typical cup-shaped membrane vesicles of 40-100nm in diameter, which
(3)
are the main morphological features of exosomes. Electron microscopy was also
done with GBM cultures. Cells prepared for transmission electron microscopy
(TEM) presented hallmarks of tumor cells: a large number of mitochondria and
endoplasmic reticulum profiles as well as disassembled Golgi apparatus. TEM
analysis also showed some intracellular organelles that resemble MVBs, with their
intraluminal vesicles inside, whose diameter is compatible with purified exosomes.
GBM observed at scanning electron microscopy (SEM) presented a great quantity of
extracellular vesicles associated with cell surfaces, suggesting either secretion events
or incorporation of previously secreted vesicles process. These results show that our
GBM lines secrete typical exosomes, which can contribute in tumor communication.
This theme will be explored further and increase the knowledge by which
glioblastoma progress.
Funding Support: CAPES, FAF/ONCO, FAPERJ, CNPq, INNT-INCT-MCT
Ethical Approval: DAHEICB015
(1) Dept. of Genetics, Institute of Biosciences, So Paulo State University (UNESP),

Botucatu, SP, Brazil.
(2) Faculty of Medicine Veterinary and Animal Science, FMVZ, So Paulo State
University (UNESP), Botucatu, So Paulo, Brazil.
(3) Dept. of Biostatics, Institute of Biosciences, So Paulo State University
(UNESP), Botucatu, SP, Brazil.
(4) Princess Margaret Cancer Centre, University Health Network, Toronto,
Ontario, Canada.
(5) Department of Surgery and Orthopedics, Faculty of Medicine, So Paulo State
University, UNESP, Botucatu, So Paulo, Brazil. Experimental Research Unity
(UNIPEX), Faculty of Medicine, So Paulo State University, UNESP, Botucatu, So
Paulo, Brazil
E- mail: camilabaldin.s@gmail.com
Lung cancer is a leading cause of cancer mortality worldwide, with ~1.6 million
patient deaths every year. Efforts have been made to identify molecular markers that
may be useful as therapeutic targets in Non-Small Cell Lung Cancer (NSCLC), the
most common type (~85%) of lung cancer. Telomeres have been considered a
potential target due to their role in genome stability and cell proliferation. Human
telomeres are nucleoprotein complexes composed by repetitive DNA associated with
proteins of the shelterin complex, maintained by the action of telomerase. Recent
data showed that the protein component of telomerase (TERT, telomerase reverse
transcriptase) and some of the shelterin components are altered in cancer. However,
the mechanisms of telomere deregulation associated with NSCLC development and
progression are not understood. Therefore, our aim is to verify deregulated
expression of telomere-associated genes and ncRNAs associated with telomere
regulation and maintenance in NSCLC, because we believe that they could be novel
biomarkers associated with NSCLC. Assessing our previously generated RNA-Seq
data in 30 NSCLC tumors and 10 matched histologically normal lung tissues we
showed that a subset of these genes is up or down-regulated in tumors compared to
the normal lung samples. We are currently conducting Q-PCR gene expression
analysis to validate the expression of telomere-associated genes in independent
samples set. Further, we plan to investigate alterations in telomere length of patients
with NSCLC and compare to age-matched, healthy individuals. Studies such as this
will shed light into the role of telomeres in NSCLC development and progression.
Research Ethics Board # 4319/2012, Supported by: CAPES
A147
A148
RESPIRATORY MODULATION RATE AFFECTS SENSIBILITY OF

METABOLIC
INHIBITORS
IN
NEURONAL
AND
TUMORAL
PHENOTYPES OF SHSY-5Y NEUROBLASTOMA LINEAGE
VIMENTIN ALTERATIONS AND AUTOPHAGY INDUCED BY RBAc-PDT

IN MELAN-A AND B16-F10 CELLS
Ivi Juliana Bristot, Marco Antnio De Bastiani, Daiani Vargas, Patrcia Schnhofen,
Bianca Pfaffenseller, Bianca Wollenhaupt de Aguiar, Fbio Klamt
Laboratory of Cellular Biochemistry, Department of Biochemistry, Federal
University of Rio Grande do Sul (UFRGS), 90035-003, Porto Alegre (RS) Brazil.
Lucas Augusto Lopes Genez, Sheila Maria Brochado Winnischofer, Glaucia Regina
Martinez*
Department of Biochemistry and Molecular Biology, Federal University of Paran,
Curitiba, PR, Brazil
*Correspondence to: grmartinez@pq.cnpq.br
Background Many different types of tumor cells present aberrant energetic

metabolism, characterized by high glucose uptake and lactate release even on aerobic
conditions (Warburg Effect). Understanding the metabolic differences between
normal and tumoral cells is essential for the development of effective anti-cancer
treatments.
Aims We evaluated the effect of mitochondrial consumption rate modulations over
the resistance/sensitivity of several anti-metabolic drugs.
Methods We first compared differences between proliferative (tumoral phenotype)
and retinoic acid (RA)-differentiated (neuronal phenotype) SH-SY5Y human
neuroblastoma cells in proliferative rate, neuronal morphology and gene expression
levels of neuronal and metabolic markers. Basal mitochondrial oxygen consumption
rate (OCR) was determined using OROBOROS Oxygraph-2K. Concentrations of
cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and potassium cyanide
(KCN) associated with the increase (FCCP) and inhibition (KCN) in 50% of OCR
were used in co-treatments with 2-Deoxyglucose (2-DG) / 3-Bromopyruvate (3-BP)
(glycolysis inhibitors) and Rotenone (ROT) / Antimycin A (AA) (mitochondrial
inhibitors).
Results RA-differentiated SH-SY5Y (neuronal phenotype) cells demonstrated low
proliferative rate, a pronounced neuronal morphology, high expression of genes
related to synapse vesicle cycle and of dopaminergic markers. In contrast,
proliferative SH-SY5Y cells (tumoral phenotype) showed an increase in metabolic.
Surprisingly, OCR modulations were enough to induce cell death in the tumoral
phenotype, and were able to significantly sensitize SH-SY5Y cells to 3-BP and 2-DG
toxicities, both glycolysis inhibitors tested.
Conclusion Modulation of mitochondrial respiration rates in combination with antimetabolic drug could be a new therapeutic venue for the management of human
brain tumors.
Photodynamic therapy (PDT) is a non-invasive treatment that requires the exposure

of cells or tissue to a photosensitizing drug (PS) inducing cell death. Rose Bengal is
an important photosensitizer with high efficiency of singlet oxygen generation,
which may cause alteration of organelles like mitochondria and the cytoskeleton by
photodamage. Vimentin is a cytoskeletal protein that is overexpressed in cancers and
generally appears to be related to the growth and metastasis of tumors. Thus, the
main objective of this study was the investigation about the influence of vimentin in
the process of cell death in melanocytes (Melan-a) and melanoma cells (B16-F10)
after treatment with PDT, using Rose Bengal Acetate (RBAc) as PS. The viability
was assessed by MTT metabolism, by crystal violet (CVS) labeling and neutral red
uptake (NRU). The results showed that both cell lines showed reduced viability by
MTT and CVS, while the NRU analysis showed an inverse result; this pattern is a
strong indicator of cell death by autophagy. To confirm, we analyzed the expression
of proteins related to autophagic process, beclin-1 and LC3 by western blotting.
Also, the effect of PDT in B16-F10 resulted in an increase in the relation vimentin
phosphorylated at serine 56/vimentin, 18 hours after PDT. For Melan-a cells, no
variation in the relation vimentin phosphorylated serine 56/vimentin was observed,
indicating that these two cell lines have different alterations in the cytoskeleton after
PDT. This study provides a better understanding of cell death related to cytoskeletal
changes after PDT in melanocytes and melanoma cells.
Keywords: Melanoma, Phosphorylation, Photodynamic Therapy, Rose Bengal,
Vimentin.
Funding: CNPq, CAPES, INCT de Processos Redox em Biomedicina, FINEP.
Funding MCT/CNPq (#306439/2014-0) fellowship and CNPq/MS/SCTIE/DECIT

Pesquisas Sobre Doenas Neurodegenerativas (#466989/2014-8) grant.

A149
A150
THE EFFECTS OF IMIDAZOLIC SALTS IN ORAL CARCINOMA CELLS
LPSF/AC05 AN ACRIDINE DERIVATIVE THAT INDUCES CELL CYCLE

ARREST AND INIBITION OF TOPOISOMERASE II IN HUMAN
LEUKEMIA AND LYMPHOMA CELLS
Paloma Santos de Campos (1), Natalia Koerich Laureano (1), Natalia ngela Bortoli
(1), Lisiane Bernardi (1), Manoel Santanna Filho (1), Pantelis Varvaki Rados (1),
Henri Stephan Schrekker (2), Marcelo Lazzaron Lamers (3)
(2) Chemistry Institute, Universidade Federal do Rio Grande do Sul, Porto Alegre,
Brazil
(3) Department of Morphological Sciences, Universidade Federal do Rio Grande do
nataliakoerich@hotmail.com
(51) 3308-5011 (51) 8302-8988
Imidazolic salts can be found in natural products and are isolated from the roots of
Lepidium meyenii. Several studies have shown that the development of diverse
imidazolic salts, together with the oligomers, have antioxidant, antifibrotic,
antitumor, antibacterial and antifungal properties. The objective of this study was to
evaluate the effect of different imidazolic salts on the behavior of oral squamous cell
carcinoma cell line (CAL27). It was tested 5 different compounds (C4MlmCl,
C10MlmCl, C16MlmCl, C16MlmMeS, C18MlmCl) at several concentrations
(control, 2.5g/ml, 5g/ml, 10g/ml and 20g/ml) and we analyzed cell proliferation
and cell-cell cohesion. It was observed that all compounds decreased cell
proliferation at 2.5g/ml. During analysis of cell-cell cohesion (spheroids formation),
Cal27 (1x10 cells) were plated on 96 well-low adherent plates (1.5% agarose) in the
immediate or later (24h) presence of the compounds and pictures were taken after 24,
48, 72 and 96h after drug incubation. It was observed that compounds containing
16C in the structure showed the best effect in the inhibition of spheroid formation
and maintenance of spheroid integrity, suggesting changes in the regulation of cellcell adhesion process. These preliminary results indicate a potential role of
imidazolic salts in the complementary treatment of Oral Squamous Cell Carcinoma.
Funding Support: CAPES, CNPQ, FAPERGS, UFRGS
Costa, V. C. M.1; Chagas, M. B. O.1; Lima, M. C. A.; Pitta, M. G. R.1; Pitta, I.R.1;
Rego, M. J. B. M.1
1
Laboratory for Immunomodulation and New Therapeutic Approaches, Federal

University of Pernambuco (UFPE).
Laboratory for Planning and Drug Synthesis, Federal University of Pernambuco
(UFPE).
Background: Leukemias and lymphomas are neoplasms characterized by abnormal
lymphocytes accumulation in bone marrow and lymph nodes. This uncontrolled
proliferation is related mainly with alterations in cell cycle control and activity of
enzymes, such as topoisomerases. Acridines and its derivatives has the ability to bind
with DNA bases pairs there by inhibiting topoisomerases. Thus, the use of bioactive
molecules such as new acridine derivative (LPSF/AC05) is a promising strategy for
cancer treatment.
Aims: To evaluate the effects of LPSF/AC05 on cell cycle and the topoisomerase II
activity.
Methods: The leukemia (CCRF-CEM and Jurkat) and lymphoma (Raji and Toledo)
cell lines were treated with LPSF/AC05 (30 to 60 M). Then, the cells were stained
with propidium iodide and analyzed by flow cytometry. In order to evaluate
topoisomerase II inhibition it was used Human Topoisomerase II assay kit in the
presence of LPSF/AC05 and DMSO control. Statistical analysis was performed
using Students t-test (p<0.05).
Results: LPSF/AC05 did not alter significantly the cell cycle in leukemic cells. On
the other hand in Raji cells there was a significantly arrest on G0/G1 and S
(p=0.0076 and p=0.043). In Toledo the cycle arrest happened on G0/G1 (p=0.0169).
Moreover, LPSF/AC05 significantly inhibits the activity of topoisomerase II at 50
and 100M (p=0.0168 and p=0.0127) when compared to positive control etoposide.
Conclusion: LPSF/AC05 induced cell cycle arrest in G0/G1 in lymphoma cell lines
and inhibit the activity of the enzyme topoisomerase II, which prevents DNA
replication leading to tumor cell death.
Funding support: INCT_if, CNPq, FACEPE

A151
A152
ROLE OF DERMCIDIN IN VEMURAFENIB-INDUCED RESISTANCE TO

CUTANEOUS MALIGNANT MELANOMA
SYNDECAN AND SYNTENIN SIGNALING AS A POTENTIAL

THERAPEUTIC TARGET FOR ADVANCED PROSTATE CANCER
Jennifer M. Neyra1 and Jose E. Belizario1
Nilton Jose dos Santos (1)*, Caroline Nascimento Barquilha (1), Isabela Gaseta
Ferraz Paiva (1), Luis Antonio Justulin Jr (1), Srgio Luis Felisbino (1)
Department of Pharmacology of Institute of Biomedical Sciences, University of Sao

Paulo,Av. Lineu Prestes, 1524 CEP 05508-900 - Sao Paulo, SP, Brazil
Cutaneous melanoma is the skin neoplasia with higher mortality and unpredictability
in its evolution. In disseminated disease, conventional chemotherapy has low
effectiveness, except, vemurafenib (Zelboraf; Plexxikon/Roche) which was approved
for the treatment of BRAF-mutated metastatic melanoma. The DCD protein was
identified among the nine gene signature for prediction of the melanoma patient
clinical diagnostic. In previous studies we have demonstrated in a cohort of 42/155
patients (36%) that the expression of DCD protein correlated to lower overall
survival (median survival 36 months). The aim of this study is to investigate the
cooperation of DCD and BRAF/RAS pathways. G-361 melanoma cell line display
BRAF V600E mutation and DCD gene overexpression and is an excellent model for
therapeutic studies. In vitro assays we show that vemurafenib (1-5 mM) induced cell
death to G361 cells in a dose-time-dependent manner. In vivo studies, we showed
that the growth of G361 melanoma xenografts in NUDE mice is inhibited after
knockdown of DCD. The association of vemurafenib and autophagy inhibitor
chloroquine has been tested at different doses and experimental schedules in G361
tumor xenografts. Chloroquine (CQ) is a classical antimalarial and anti-inflammatory
drug that inhibits lysosomal acidification and promotes apoptosis in melanoma cells.
Next, we will establish G361 cell line clones resistant to Vemurafenib after longterm treatment in vitro and in vivo and perform PCR and western blot assay to
measure the expression of DCD in resistance cell lines. Our ultimate goal is to prove
that DCD expression is a better predictor of resistance to pharmacological targeted
therapy. Support CAPES and FAPESP.
The development of assay in vivo started with the kinetic growth melanoma tumor in
the xenografted nude mouse to evaluate principality the rate of growth of tumor. We
injected 10 ^ 6 cells G361 via s.c. in the back of the mice and the tumor progression
was accompanied from its appearance to a volume of about 1500 mm3, then proceed
to surgery these and immunohistochemical characterization, phenotypic study. The
test of treatments were in tumors with 50mm cubic.
(1) Department of Morphology, Institute of Biosciences So Paulo State University

(UNESP), Botucatu, So Paulo, Brazil
*Correspondent Author: Nilton J. Santos Department of Morphology, Institute of
Biosciences UNESP, Botucatu, Sao Paulo, Brazil. Street Professor Dr. Antonio
Celso Wagner Zanin, s/n Botucatu, SP Zip Code: 18.618-689.
njsantos@outlook.com.br Phone: (18) 98146 0460 / (14) 3880-0502
Background: Prostate cancer (PCa) is the most common malignancy in men and the
second leading cause of male cancer-related deaths in the Western world. Studies
emphasize the role of cell-extracellular matrix interaction on initiation and
progression of PCa. AIMS: To identity by global expression analysis new target
genes for PCa therapies, with special attention to genes related to extracellular matrix
signaling. METHODS: Two established mouse models for prostate cancer: Pten
knockout and Trp53 and Rb1 double knockout in the prostatic epithelial cells,
exclusively, were produced and exhibited prostate cancer at different stages of
progression. Normal and tumoral samples from ventral, lateral, dorsal and anterior
prostatic lobes were submitted to histopathological analysis and next generation
sequencing to obtain their transcriptomes. RESULTS Analysis of these
transcriptomes showed altered expression of several extracellular matrix genes, such
as Syndecan family. Gene expression and immunhistochemistry showed that
Syndecan 1-4, and their cytoplasm binding protein Syntenin exhibited a dual pattern,
showing a reduced expression at early stage of prostatic intraephitelial neoplasia and
an increased expression in advanced poorly-differentiated adenocarcinomas.
CONCLUSION: These results show that syndecans proteoglycan family and their
binding proteins plays a role on prostate cancer progression towards advanced stages
and can be potentials new therapeutic targets for PCa treatment.
Financial support: FAPESP 2015/26175-7; FAPESP 2013/08830-2; CNPq
305391/2014-3.
Approved by the CRUK Institute Ethics Committee of the University of Cambridge
and also by the Ethics Committee on Animal Experimentation of the Institute of
Biosciences of Botucatu (Protocol 613/2014).

A153
A154
PEPTIDES FROM TRANSCRIPTION FACTOR Brn-2 INHIBIT CELL

MIGRATION IN B16F10-Nex2 CELLS
A ROLE OF LIPID RAFTS IN THE INTERNALIZATION AND

INTRACELLULAR TRAFFICKING OF AN ANTITUMOR MOLECULE,
AMBLYOMIN-X
Alves, VRG; Travassos, LR; Arruda, DC

University of Mogi das Cruzes
Katia Luciano Pereira Morais1,2; Giampietro Schiavo3, Maurcio Barbugiani

Goldfeder1, Juliana Mozer Sciani 1; Ana Marisa Chudzinski-Tavassi1
Background: The transcription factor Brn-2 when overexpressed can regulate the
levels of MITF and PDE5A, promoting tumor metastasis. We propose that peptides
from Brn-2 could have inhibitory effects on the development of melanoma, including
effects related to migration and invasion inhibition and can be helpful to the
development of promising anticancer drugs.
Aims: Determination of the effects of transcription factor Brn-2-derived peptides on
cell migration.
Methods: B16F10-Nex2 were treated with the peptides at different concentrations, to
determine the range of toxic and non-toxic peptide levels. Cell viability was
evaluated by MTT assay. To determine the effect on cell migration of peptide
concentrations that did not affect proliferation, B16F10-Nex2 cells were plated onto
12-well plates. After 24 h growth, vertical wounds were made in all wells. Cells were
treated with peptides 1, 3, 4, 6 and 7. Cell migration was determined in both treated
and untreated cells by subtracting the distance between cell-fronts after 2, 4 and 24 h
from that measured at the beginning of incubation (0 h).
Results: We showed that the peptides did not affect cell proliferation of B16F10Nex2 cells even at the highest concentration used. Internal peptides of transcription
factor Brn-2, no. 1, 3, 4 and 7, showed inhibitory effects on tumor cell migration
unlike peptide no. 6, that did not.
Conclusion: Based on the results of tumor cells migration inhibition, the Brn-2
peptides can be further investigated as anti-invasive or anti-metastatic reagents as
well as agents disrupting the epithelial-to-mesenchimal transition.
Funding support: CNpq, FAPESP and FAEP.
Biochemistry and Biophysics Laboratory, Butantan Institute, SP, Brazil;

Department of Biochemistry, Federal University of So Paulo, SP, Brazil; 3Institute
of Neurology, University College London, LD, United Kingdom.
2
Background: Amblyomin-X is a Kunitz-type protease inhibitor that was identified

through the transcriptome analysis of the salivary gland from Amblyomma
cajennense tick. The recombinant form of this protein triggers apoptosis in different
tumor cell lines, and promotes regression of tumor growth and reduction of
metastasis. Recents studies have suggested Amblyomin-X internalization by lipid
rafts regions and recycling endosomes as intracellular destination. However, further
works are needed to clarify the exact intracellular trafficking. Aims: verify
Amblyomin-X intracellular trafficking in human tumor cells.
Methods:
Amblyomin-X was directly labeled with the amine-reactive dye through a
commercial kit and was called 488-Amblyomin-X. After SK-Mel-28 and Mia-PaCa2 tumor cell lines treated with 488-Amblyomin-X, cells were incubated with Alexa
Fluor 555 Transferrin or Alexa Fluor 555 Cholera Toxin Subunit B. Then, cells were
fixed and the slides were mounted and visualized by confocal microscopy. The same
procedure was performed to check caveolin-1 co-localization, but with
permeabilization step and incubation with the appropriate antibodies. Results:
Amblyomin-X labeled protein was able to co-localize Cholera Toxin Subunit and
caveolin-1 in both tumor cells. Also, some points of co-localization with transferring
were observed. Conclusion: Amblyomin-X internalization and intracellular
trafficking occurs via the non-exclusive lipid raft pathway, considering some points
of co-localization with transferring. Collaborating with previous work, these
evidences suggest a slow intracellular trafficking pathway and possible involvement
of caveossomos. Also, these data along with future studies will help in understanding
the structural state of this protein and how to reach its intracellular targets.
Supported by CNPq, Fapes, BNDES and Unio Qumica Farmacutica
A155
A156
ANTI-MIGRATORY EFFECTS OF SYNTHETIC NITRONES IN MDA-MB231 BREAST CANCER CELLS
ASYMMETRIC DIVISION IN MEDULLOBLASTOMA CANCER STEM

CELLS
Wanessa Fernanda Altei (1), Lucimara J. Martins (2), Ralph G. Costa (2), Fernando
Coelho (2), Adriano Defini Andricopulo (3), Heloisa S.Selistre-de-Araujo (1)
1)
(1) Laboratory of Biochemistry and Molecular Biology, Department of Physiological
Sciences, Federal University of So Carlos, Brazil.
(2) University of Campinas, Chemistry Institute, Department of Organic Chemistry,
Campinas, Brazil.
(3) Laboratory of Medicinal and Computational Chemistry, Physics Institute, Sao
Paulo University, Sao Carlos, Brazil.
Beatriz Araujo Cortez (1), Lucas Carvalho Price (1), Oswaldo Keith Okamoto (1)
wanaltei@gmail.com (16) 981361383

Despite the advances in therapies and drug development, most cancer deaths are due
to metastasis resistant to conventional treatments. Therefore, the need for novel
antimetastatic agents has become critical for patients survival. Aiming to find new
molecules acting on migration of metastatic cells, we evaluated eight synthetic oxinitrones for its ability to inhibit cancer cell migration. Nitrones have the general
chemical formula X-CH=NO-Y and are widely employed to prepare several
biologically active compounds. Tested compounds also have a spyrohexadienone
motif, known by its relevant biological profile. Briefly, tumor breast cancer cells
MDA-MB-231 were seeded in 24-well culture plate and grown to 80-90%
confluence. The center of the cell monolayers were scraped with a sterile
micropipette tip to create a denuded zone. Compounds were incubated with cells at 1
M and their effect on migration inhibition were evaluated through the measure of
wound closure after 22 h. The two nitrones containing a benzyl substituent with
deactivating groups in p-position showed pronounced cell migration inhibition (> 60
%). However, benzyl substituent with electron donating groups decreased the
migration inhibition activity. Considering that nitrones have traditionally been used
as spin trap compounds capturing free radicals, the dynamic of electrons in
structures is crucial. Therefore, we suggest that the pattern of substitution in benzyl
ring could be responsible for their action on cell migration inhibition. Cytotoxicity,
apoptosis and cell cycle have been investigated by flow cytometry to elucidate the
effect of these compounds.
(1) Human Genome and Stem Cell Research Center, Department of Genetics and
Evolutionary Biology, Institute of Biosciences, University of Sao Paulo.
Medulloblastoma is the most common malignant brain tumor in children up to age
four. Cancer stem cells (CSC) in these tumors were reported in 2003 and they
correlate with tumor aggressiveness, might being important to tumor initiation and
progression. Efforts have been done to understand CSC behavior and its contribution
to cancer development, and recent data shows that they can undergo asymmetric cell
division (ACD). However, CSC display mechanisms to increase the rates of
symmetric divisions, generating cells with high proliferative capability that
contribute to tumor growth. The present work aims to investigate ACD in
medulloblastoma cells and define how these divisions occur and are regulated.
DAOY, D283 and P13-USP-Med (medulloblastoma cell lines) were cultured as
tumorspheres for 7 days, when the CSC population was enriched (identified by
CD133, Nestin and Sox2). Spheres and dissociated cells were cultured for 48h and
mitosis were analyzed by confocal laser scanning microscopy. The segregation
pattern of proteins related to ACD, proliferation and differentiation was investigated.
For the first time ACD was described in medulloblastoma. Cortical NuMA
localization was a marker for ACD in all conditions, and the segregation pattern of
Numb and CD133 (stem cells markers) were observed. The frequencies of ACD
varied depending on the culture medium: more ACDs were observed in 1% FBS than
in 10% FBS or in the presence of growth factors (N2, B27, EFG and FGF). These
results show that ACD rates can be modulated and allow us to regulate ACD to study
its relation to tumor aggressiveness.
Grant: FAPESP 2014/10519-6

A157
A158
MicroRNA-146b-5p UPREGULATES MIGRATION AND INVASIVENESS

OF PAPILLARY THYROID CARCINOMA CELLS, TARGETING RHOKINASE-1
MicroRNAs 221/222 INHIBIT MIGRATION OF PAPILLARY THYROID

CARCINOMA CELLS
Cilene R. de Lima1, Murilo V. Geraldo2, Cesar S. Fuziwara1, Edna T. Kimura1,

Marinilce F. Santos1.
1

University of Sao Paulo-USP, Sao Paulo/SP - Brazil.
2
Department of Functional and Structural Biology Biology Institute, State
University of Campinas, Unicamp, Campinas/SP - Brazil.
Cilene R. de Lima1, Caroline A. O. Xavier1; Murilo V. Geraldo2, Edna T. Kimura1,

Marinilce F. Santos1.
1

University of Sao Paulo-USP, Sao Paulo/SP - Brazil.
2
Department of Functional and Structural Biology Biology Institute, State
University of Campinas, Unicamp, Campinas/SP - Brazil.
cilima77@usp.br (55) (11) 3091-7227
cilima77@usp.br
Background: In tumors, more pronounced migratory phenotypes usually increase
metastatic potential of cancer cells. MicroRNAs (miRs), post-transcriptional
regulators of gene expression, have been studied in different cancers and their
differential expression is frequently associated with tumor progression and
metastasis. In papillary thyroid carcinoma (PTC), miR-146b-5p is highly expressed
and positively correlated to the degree of malignancy. Aim: to investigate the role of
miR-146b-5p on the migratory and invasive behavior of thyroid cells. Methods:
experimental procedures were performed with non transformed thyroid (PCCL3) and
PTC cell lines (TPC-1 and BCPAP) overexpressing and suppressing miR-146b-5p.
Migration was studied using transwell, wound healing and time-lapse assays. Gelatin
degradation assays and F-actin staining were also employed. Potential targets were
evaluated by western blotting. Results: Overexpression of miR-146b-5p in PCCL3
cells led to increased migration and invasion with the presence of large lamellipodia.
The opposite effect (decreased migration and invasion) was observed in PTC cell
lines after inhibition of miR-145b-5p, with cells showing simultaneous processes in
different directions. Gelatin degradation was inhibited only in TPC-1 after miR146b-5p suppression. The inhibition of miR-146b-5p in PCCL3 also inhibited
migration and invasion, and overexpression of this miR in PTC cells increased both
processes, without effects on cell morphology or gelatin degradation. By western
blotting, the predicted target ROCK1 was downregulated by miR-146b, but not
ROCK2 or RhoA GTPase. Conclusion: miR-146b-5p upregulates migration and
invasion of thyroid cells, targeting potentially targeting ROCK1.
Background: MicroRNAs (miRs) are important biological regulators involved in a

wide range of biological roles, including tumor cell migration and invasion.
Increased migration augments the tumor metastatic potential and aggressiveness. In
papillary thyroid carcinoma (PTC), miR-221/222 are highly expressed, being
positively correlated with malignancy and aggressiveness. Their role, however, is not
yet fully understood. Aim: to investigate the role of miR-221/222 on migration and
invasion of thyroid cells. Methods: experimental procedures were performed with
non transformed thyroid cell line (PCCL3) and PTC cell lines (TPC-I and BCPAP,
with different oncogenic backgrounds) overexpressing and suppressing both miR221/222. Migration was studied using transwell (with and without Matrigel) and
time-lapse assays. F-actin arrangement was also analyzed. Results: In transwell
assays (directional migration), PCCL3 overexpressing (~3X) both miR-221/222
showed decreased migration and invasion (~50-60%), as well as reduced cell
spreading. The exact opposite effect was observed in TPC-1 and BCPAP, which
express high levels of miR-221/222, after inhibition of both miRs. Transfected cells
also showed very large lamellipodia and lack of polarity. The miR-221/222
suppression in PCCL3 increased migration and invasion, and their overexpression in
PTC cells decreased both processes. Curiously, in time-lapse assays (random
migration), PTC cells showed reduced speed (~40%) after miRs suppression, with no
differences in cell directionality. Conclusion: in non transformed and tumor thyroid
cells miR-221/222 exert an anti-migratory effect, although lamellipodia formation
was stimulated. This effect is much more evident in directional migration, under
chemotactic stimuli.
Support by FAPESP, CAPEs and PRP-USP (NAPmiR).

Supported by FAPESP, CAPEs, CNPq, PRP-USP (NAPmiR).
A159
A160
EFFECTS OF ATP IN HEMATOPOIETIC AND LEUKEMIC STEM CELLS
EVALUATION OF ANTITUMORAL POTENTIAL OF ANNONA

CRASSIFLORA FRACTIONS IN HUMAN CERVICAL CANCER CELL
LINES: PRELIMINARY RESULTS
Roberto Theodoro de Araujo Jnior (1), Edgar Julian Paredes-Gamero (1,3)

(1) Departamento de Bioqumica, Universidade Federal de So Paulo, So Paulo,
Brazil;
(3) Centro Interdisciplinar de Investigao Bioqumica, Universidade de Mogi das
Cruzes, So Paulo, Brazil.
Endereo: Rua Pedro de Toledo, 669 9 andar Vila Clementino.
(11) 5576-4868, ramal 1312.
e-mail: araujo.rtj@gmail.com Tel.: (19) 9 82783643.
Hematopoiesis is the process by which all lineages of blood cells are generated
hierarchically from immatures cells present in bone marrow. Hematopoietic Stem
Cells (HSC) are the apex of this hierarchy, which are the only the self-renewing cells
capable of production of all lineages of blood cells. Leukemia usually results from
aberrant hematopoietic process initiated by Leukemic Stem Cells (LSC), a
population of cells possess extensive proliferative capacity and the potential for selfrenewal been able to maintain the leukemia. Recurrence is the major problem in
Leukemia treatment, this is attributed of Leukemic Stem Cells (LSC). Conventional
chemotherapy does not affect LSC, therefore, new therapeutic strategies are
necessary to target this population. Thus, in this study the ability of extracellular
ATP to promote the differentiation of HSC and LSC through activation of P2
receptors were evaluated. Murine leukemic cells C1498 were treated with different
concentrations of ATP (0,1 - 1 mM) and LSC were quantified by flow cytometer.
ATP was able to induce a reduction (30%) of cell viability at the highest
concentration (1 mM) after 72 h. The LSC population decreased after 72 h and the
expression of undifferentiated marker BMI decreased. Normal HSC population
decreased after treated with ATP (1 mM) after 12 h, and expression of Ki67 and
PU.1 increased. Thus, our data suggest that ATP induce differentiation in murine
LSC and HSC by activating P2 receptors.
Acknowledgments
FAPESP, CAPES and CNPq.
NOTES
The authors declare no competing financial interest.
Marcela Nunes Rosa (1), Carla Carolina Munari (1), Viviane Aline Oliveira Silva
(1), Rosy I. M. de Azambuja Ribeiro, (2) Rui Manuel Reis (1,3)
(1) Molecular Oncology Research Center, Institute for Research and Education,
Barretos Cancer Hospital, Brazil.
(2) Experimental Pathology Laboratory, Federal University of So Joo del-Rei,
Brazil
(3) Life and Health Sciences Research Institute, University of Minho, Portugal.
nr.marcela@gmail.com.
Telephone: (17) 3321-6600. Extension: 7093/7094. Mobile: (16) 99299-9600
Rua Antenor Duarte Vilela, 1331, Dr. Paulo Prata, CEP: 14.784-400. Barretos-SP
Background: Natural products represent important sources of new anticancer drugs.
The Brazilian ora is considered to be one of the most diverse in the world. In our
previous studies, the partitions hexane, chloroform (C) and ethyl acetate (D) from
Annona crassiflora (Brazilian Cerrado native) showed cytotoxic potential on cervix
cancer cell lines (CCCL). However, the fractions responsible for this activity need to
be investigated.
Aims: To evaluate the antitumoral activity of fractions from C and D partitions of A.
crassiflora on human CCCL.
Methods: Cervix cancer cell lines (Siha, HeLa and CaSki) were plated into 96-well
microplates and treated for 72 hours with different concentrations of 21 fractions
prepared from chloroform or ethyl acetate partition of A. crassiflora leaves. Cell
viability was evaluated by colorimetric assay (MTS). The cytotoxic activity was
assessed using the parameter of 50% inhibition of cell line growth (IC50). Moreover,
DNA damage and cell cycle-related proteins expression was evaluated 24 hours after
treatment and Western blotting assay was carried out.
Results: The most pronounced cytotoxicity was observed in 7C24 fraction, with IC50
values of 16 (HeLa), 34 (SiHa) and 35 g/mL (CaSki). Western blotting analysis
showed that 7C24 induces poly (ADP-ribose) polymerase (PARP) cleavage, H2AX
phosphorylation and alteration on p21 expression in HeLa and SiHa cells.
Conclusion: The A. crassiflora has a potential antitumoral effect on CCCL.
However, further studies are necessary to investigate the compounds responsible for
this activity.
Number of the ethical approval: CEP-HCB 1.252.699
Funding support: FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013-FPXIIBIOPLAT), FAPEMIG and Barretos Cancer Hospital.

A161
A162
LOCALIZATION AND FUNCTION OF DYNLL2 AND MYOSIN-VA IN THE

PRIMARY CILIUM
EXPRESSION PATTERN OF CDKN2A AND CDKN2B GENES AND THEIR

REGULATORS IN CANINE MAMMARY TUMORS
Talita Sanches Perez, Rui M. Patrcio da Silva Jr., Carmen L. Pontes, Roy E. Larson,
Enilza M. Espreafico
Thamirys Aline Silva Faro (1), Danilo do Rosrio Pinheiro (1), Francisco Canind
Ferreira de Luna (1), Wallax Augusto Silva Ferreira (1), Lucien Roberta Valente
Miranda de Aguirra (2), Washington Luiz Assuno Pereira (2), Rommel Mario
Rodriguez Burbano (3), Maria Lcia Harada (1), Brbara do Nascimento Borges (1)
Department of Cell and Molecular Biology, Faculty of Medicine of Ribeiro Preto,

University of So Paulo, Ribeiro Preto, So Paulo, Brazil.
Email: talitaperezsanches@hotmail.com; Institutional Phone: (16) 3315-3044.
The primary cilium is a cell surface projection present in nearly all types of
vertebrate cells. It plays essential sensory roles, so that ciliary defects trigger a wide
range of human diseases. Primary cilia are anchored to the cell by the basal body,
made from the mother centriole in a coordinated fashion with the cell cycle.
Ciliogenesis and the primary cilium function are dependent on the intraflagelar
transport machinery (IFT) involving a variety of molecular motors. DYNLL paralogs
(DYNLL1 and 2) act as hub proteins that bind to many different targets. DYNLL1
acts as dynein light chain and is essential for cilia function. DYNLL2 serves as
myosin-Va light chain. Despite refined information on DYNLL2 structure, little is
known of its subcellular localization and dynamics in living cells. Here, we
investigated the localization and function of DYNLL2 and myosin-Va in melanoma
cells with focus on the primary cilium. Our results show that EGFP-DYNLL2
strongly localizes to the basal bodies and to a lesser extent along the ciliary
projection, colocalized with the ciliary marker ARL13B. Interestingly, using a
monoclonal antibody directed to a phosphorylated site in the globular tail of myosinVa, we also found staining for phosphorylated myosin-Va in the basal bodies, as well
as along the extension of the primary cilium. Knockdown of myosin-Va resulted in a
significant decrease in the number of ciliated cells in culture. These results support
the hypothesis that myosin-Va and DYNLL2 play a role in the primary cilium.
Financial support: FAPESP, CAPES, CNPq, FAEPA-HCFMRP
(1) Molecular Biology Laboratory, Biological Sciences Institute, Federal University

of Par, Belm, Par, Brazil
(2) Animal Pathology Laboratory, Animal Health and Production Institute, Federal
Rural University of Amazon, Belm, Par, Brazil
(3) Human Cytogenetics Laboratory, Institute of Biological Sciences, Federal
University of Par, Belm, Par, Brazil
E-mail: bnborges@ufpa.br; Telephone: +5591 3201-7585/99994-1592
Background: Mammary cancer is the most common malignant tumor in female dogs,
accounting for 50% of all type of cancer that affect these animals. Among the
commonly altered genes, are those codifying regulatory proteins of the cell cycle,
such as CDKN2A and CDKN2B. The expression levels of these genes are negatively
regulated by proteins codified by BMI-1, MYC and TXB2 genes, and deregulation
on their expression may lead to cancer. Aims: To evaluate the expression pattern of
these five genes and check if this pattern is modulated by methylation changes in
canine mammary tumors in dogs from the North region of Brazil (Belem, Par).
Methods: Eighty-five samples of tumoral and non-tumoral tissues from 40 animals
were collected at the Federal Rural University of Amazons Veterinary Hospital. All
procedures were approved by UFRAs Ethics Committee. For the methylation
analysis, DNA samples were subjected to bisulfite sequence PCR. The mRNA
expression was evaluated using Taqman assays. Results: When comparing tumoral
with the non-tumoral samples, we observed that BMI-1, MYC and CDKN2A
showed no change on the gene expression and in the methylation pattern. However,
TXB2 presented a slight decrease in mRNA expression while CDKN2B showed an
increase. In both cases, the observed changes were not correlated with
clinicopathological data nor methylation status. Conclusion: Deregulation of MYC,
BMI-1 and CDKN2A do not seem essential for triggering mammary cancer in
canines, while CDKN2B and TXB2 mRNA expression are probably regulated by
other genetic (TGF-b1 pathway activation) and epigenetic (as miRNAs) mechanisms.
Financial Support: CNPq, CAPES, UFRA and UFPA
A163
A164
EMC1 IS INVOLVED IN PRO-TUMORIGENIC PATHWAYS IN BREAST

CANCER
SERUM LEVELS OF GALECTIN-7 ARE PREDICTIVE OF LYMPH NODE

METASTASIS IN PATIENTS WITH GASTRIC CANCER
Marcela Motta de Castro (1), Lucas Goedert (1), Jessica Rodrigues Plaa (2); Enilza
Maria Espreafico (1)
Antnio Felix da Silva Filho (1); Mario Rino Martins (2); Kamila de Melo Vilar (1);
Maira Galdino da Rocha Pitta (1); Leuridan Cavalcante Torres (3); Moacyr Jesus
Barreto de Melo Rgo (1)
(1) Department of Cell and Molecular Biology and Pathogenic Bioagents, University
of So Paulo, Ribeiro Preto, Brazil.
1.
(2) Oncology, Stem Cell and Cell Therapy Program, University of So Paulo,
Ribeiro Preto, Brasil.
2.
marcelamottadecastro@gmail.com, 55 16 33153044, 55 16 988563468.
3.
Previous work of our laboratory has demonstrated initial evidence of EMC1

involvement with breast cancer progression by inducing proliferation, migration and
cell survival. In this way, is essential to identificate specific metabolic pathways in
which EMC1 participates. Using TCGA (The Cancer Genome Atlas) freely available
breast cancer data, we performed several differential expression analyses using
TCGAbiolinks package in R programming environment. As preliminary results, we
first confirmed EMC1 enriched expression in breast tumors compared to adjacent
normal paired samples. Indeed, EMC1 high expressing patients have decreased
survival compared to patients with low EMC1 levels. Interestingly, EMC1
expression is higher in those patients triple negative for HER2, progesterone and
estrogen receptors. To get insight into the involvement of EMC1 with specific gene
networks, we divided TCGA patients into two groups of EMC1 expression (EMC1
High and Low groups). Differential expression analysis was performed and the
identified genes were submitted to KEGG pathway enrichment analysis. As results, it
was observed the enrichment of pro-tumorigenic pathways, such as focal adhesion
and MAPK signaling pathway, which may explain EMC1 induced tumor
progression. Additionally, EMC1 promoter is bound by MYC and its overexpression
correlates with MYC amplification, which gives strong evidence that EMC1 is
activated by this aggressive oncogene. Collectively, our in silico analyses using
TCGA data identified EMC1 correlation with aggressive breast cancer pathways,
providing support that EMC1 may be involved in tumor malignancy.
(1) Laboratrio de Imunomodulao e Novas Abordagens Teraputicas (LINAT),

Departamento de Bioqumica, Universidade Federal de Pernambuco (UFPE)
(2) Real Instituto de Cirurgia Oncolgica e Hospital de Cncer de Pernambuco
(HCP)
(3) Instituto Medicina Integral Prof. Fernando Figueira (IMIP)
Author presenting: Antnio Felix da Silva Filho
Address: Laboratrio de Imunomodulao e Novas Abordagens Teraputicas,
Universidade Federal de Pernambuco. Av. Prof. Mores Rgo, 1235 - Cidade
Universitria, Recife - PE, 50670-901.
Email: tonifelix4@gmail.com Telephone: (81) 997097087
In contrast to other galectins such as galectin-1 and -3, the function of galectin-7
(Gal-7) is still largely unknown. Gal-7 is a prototype galectin, a protein with affinity
for -galactoside residues with a single carbohydrate recognition domain in its
biological structure, whose expression was found increased in many tumor types
including breast, head and neack, cervical and bladder. However, there is no research
that verifies the Gal-7 expression in serum from gastric cancer patients. Gastric
cancer is the second leading cause of cancer death and remains a major clinical
challenge due to poor prognosis and limited treatment options. In this context, this
research aimed to evaluate serum levels of Gal-7 in gastric cancer patients and its
association with clinical manifestations. So, 48 gastric cancer patients (27 males,
mean age 61.6 years, and 21 females mean age 61.5 years) were assessed. Gal-7
levels were measured by ELISA and results wer e analyzed by Mann-Whitney and
Pearson Correlation test in Action Stat software. There was a positive correlation
between Gal-7 serum levels and lymph node involvement (R= 0,7827 and p =
0,0044). There was no statistical significance in the comparison between Gal-7 levels
and the factors sex, age, smoking, alcohol consumption, race and tumor
differentiation. The results demonstrate the potential of this glycoprotein as a
biomarker of stomach cancer.
Key-words: Gal-7, stomach cancer, glycoprotein, biomarker.
Financial support: FACEPE and INCT-if

A165
A166
RMEL3, A BRAFV600E-ASSOCIATED LONG NONCODING RNA,

INDUCES MELANOMA MALIGNANCY THROUGH THE MAPK AND
PI3K PATHWAYS
MODULATION OF ANTI-PROLIFERATIVE AND PRO-APOPTOTIC

EFFECT ON HEPATOCARCINOMA CELLS BY DODAC/PHO-S
LIPOSOMES
Lucas Goedert (1,2,*), Cristiano G. Pereira (1,*), Jason Roszik (3,*), Jessica R. Plaa
(2,4), Cibele Cardoso (1), Guo Chen (3), Wanleng Deng (3), Vashisht Gopal YennuNanda (3), Wilson A. Silva Jr. (2,5), Michael A. Davies (4), Enilza M. Espreafico (1)
Arthur Cssio de Lima Luna (1,2); Jos Roberto de Assis Santos Filho (1); Durvanei
Augusto Maria (1,2)
(1) Department of Cell and Molecular Biology, Faculty of Medicine of Ribeiro

Preto, University of So Paulo, Ribeiro Preto, So Paulo, Brazil.
(2) National Institute of Science and Technology in Stem Cell and Cell Therapy and
Center for Cell-Based Therapy, Ribeiro Preto, So Paulo, Brazil.
(3) Department of Melanoma Medical Oncology, The University of Texas MD
Anderson Cancer Center, Houston, Texas, United States of America.
(4) Clinical Oncology, Stem Cell and Cell Therapy Program, Ribeiro Preto Medical
School, Ribeiro Preto, So Paulo, Brazil.
(5) Department of Genetics, Ribeiro Preto Medical School, and Center for
Integrative System Biology CISBi-NAP/USP, University of So Paulo, Ribeiro
Preto, So Paulo, Brazil.
*These authors have contributed equally to this work. Contact Information:
goedertlucas@gmail.com Phone: (55)16-33153344; (55)48-91498675.
Previous work identified RMEL3 as a lncRNA with enriched expression in
melanoma, however its characterization and relevance to this disease was unknown.
In this way, by using TCGAbiolinks Bioconducter R package, we have performed
bioinformatics analyses according to RMEL3 expression in a set of melanoma
patients from The Cancer Genome Atlas (TCGA), followed by experimental
validation. Firstly, we confirmed RMEL3 enriched expression in melanoma and
demonstrated its association with the presence of BRAFV600E. RMEL3 siRNAmediated silencing markedly reduced (95%) colony formation in different
BRAFV600E melanoma cell lines. Multiple genes of the MAPK and PI3K pathways
found to be correlated with RMEL3 in TCGA samples were experimentally
confirmed. RMEL3 knockdown led to downregulation of activators or effectors of
these pathways, including FGF2, FGF3, DUSP6, ITGB3 and GNG2. It was also
observed that high RMEL3 expression correlates with global hypomethylation and
with a set of differentially expressed microRNAs that may contribute to regulation of
the MAPK and PI3K components. RMEL3 knockdown induces gain of protein
levels of tumor suppressor PTEN and the G1/S cyclin-Cdk inhibitors p21 and p27, as
well as a decrease of pAKT (T308), BRAF, pRB (S807, S811) and cyclin B1.
Consistently, knockdown resulted in an accumulation of cells in G1 phase and
subG0/G1 in an asynchronously growing population. Thus, TCGA data and
functional experiments demonstrate that RMEL3 is required for MAPK and PI3K
signaling, and its knockdown decrease BRAFV600E melanoma cell survival and
proliferation.
(1) Biochemistry and Biophysical Laboratory, Butantan Institute, University of

Sao Paulo, Sao Paulo, Brazil
(2) Department of Medical Sciences, Medical School, University of Sao Paulo, Sao
Paulo, Brazil
Presenting author: Biochemistry and Biophysical Laboratory, Butantan Institute,
1500, Vital Brasil Avenue, Sao Paulo 05503-900, phone: +55112627-9750, Brazil,
Email: arthurluna@usp.br
Background: In a recent report, PHO-S (Synthetic Phosphoethanolamine) was
encapsulated in DODAC (Dioctadecyldimethylammonium Chloride) liposomes by
our group. In vitro studies demonstrated the efficacy of DODAC/PHO-S liposomes
on inhibiting cell proliferation and inducing cytotoxicity in Hepa1c1c7 murine
hepatocellular carcinoma.
Aims: Therefore, we aim to describe programmed cell death pathways whereby the
DODAC /PHO-S liposomal formulation induces cytotoxicity in Hepa1c1c17 cells.
Methods: Liposomes 0.3 2.0 mM (DODAC/PHO-S) were prepared by
ultrasonication. The cell cycle phases, protein expression and types of cell death were
analyzed by flow cytometry. The internalization of liposomes, mitochondrial
electrical potential and evaluation of lysosomal stability was evaluated by confocal
laser scanning microscopy.
Results: There was observed a significant increase in the population of Hepa1c1c7
cells with cell cycle arrest at the S and G2/M phases. The internalization of the
liposomes was observed with only 3 and 6 hours of treatment. The cells treated with
DODAC/PHO-S significantly reduced the mitochondrial electrical potential and
promoted changes in the lysosomes. The liposomes promoted increased in the
expression of proteins DR4 receptor, caspases 3 and 8, cytochrome c, p53, p21, p27
and Bax. There was also a decrease in the expression of proteins Bcl-2, cyclin D1,
CD90, CD44.
Conclusion: Thus, the overall results showed that the liposomes were effective,
activating the intrinsic and extrinsic pathways of programmed cell death.
Information on ethical approval and funding support: Sao Paulo Research
Foundation, process number: 2015/02950-1. Ethics Committee on Animal Use
(CEUA) FMUSP, protocol number 112/14.
Funding Support:
This work was supported by grants to EME from FAPESP (2014/18189-5) and
CNPq (311347/2011-8 and 506780/2013-9). FAPESP provided fellowships to LG
(2014/07726-0) and CGP (2010/16097-5 and 2012/24056-2). CAPES provided
fellowship to LG and JRP. CNPq provided fellowship to CC. LG, JRP, WASJ are
members of Center For Cell-Therapy, CEPID/FAPESP (grant #2013/08135-2).
MAD is supported by NIH/NCI 1R01CA154710-01. The MD Anderson RPPA Core
is supported by NCI # CA16672.
A167
A168
INDUCTION OF PROGRAMMED CELL DEATH BY DODAC/PHO-S

LIPOSOMES ON B16F10 CELLS
EVALUATION OF WNT/BETA-CATENIN AND SONIC HEDGEHOG

SIGNALING
PATHWAYS
DURING
TONGUE
MALIGNANT
TRANSFORMATION IN WILD-TYPE AND GALECTIN-3 DEFICIENT
MICE CHALLENGED BY THE CARCINOGEN 4NQO
Jos Roberto de Assis Santos Filho (1); Arthur Cssio de Lima Luna (1,2); Durvanei
Augusto Maria (1,2)
(1)
Biochemistry and Biophysical Laboratory, Butantan Institute,
(2)
Department of Medical Sciences, Medical School, University of Sao
Paulo, Sao Paulo, Brazil
Presenting author: Biochemistry and Biophysical Laboratory, Butantan Institute,
1500, Vital Brasil Avenue, Sao Paulo 05503-900, phone: +55112627-9750, Brazil,
Email: joserobertoasfilho@gmail.com
Background: Liposomes of Dioctadecyldimethylammonium Chloride/ Synthetic
phosphoethanolamine (DODAC/PHO-S) was synthesized by our group. In vitro
studies demonstrated that DODAC/PHOS inhibited cell proliferation and induced
cytotoxicity B16F10 murine melanoma cells.
Aims: Therefore, we aim to to analyze the proteins involved in cell death after
treatment with DODAC/PHO-S liposomes.
Methods: Liposomes (DODAC/PHO-S), at concentrations of 0.3 - 2.0 mM, were
prepared by ultrasonication. The expression of receptor DR4, caspases-8 and -3 and
cytochrome c was analyzed by flow cytometry.
Results: The results demonstrated that DODAC/PHO-S liposomes, at 0.3 -2.0 Mm,
was able to induce S and G2/M phase arrest in B1610 cells. DODAC/PHO-S
lipossomal formulation promoted increased of caspase-8 and -3, receptor DR4 and
cytochrome c free, demonstrating the effectiveness this liposomes to promote cell
death. Corroborating the previous findings, the liposomal formulation of
DODAC/PHO-S 1:1 (0.3 and 2.0 mM) was significantly more effective when
compared to PHO-S, in promoting cell death by apoptosis in B16F10 cells.
Conclusion: The DODAC/PHO-S liposomes was effective in promoting cytotoxicity
in B16F10 cells, inducing the increased of the pro-apoptotic proteins. The data
demonstrated that DODAC/PHO-S liposomes is effective in promote the activation
of programmed cell death.
Information on ethical approval and funding support: This study was funded by So
Paulo Research Foundation, process number: 2015/02950-1. This project were
approved by the Ethics Committee on Animal Use (CEUA) - FMUSP. protocol
number 112/14- CEUA
Debora de Oliveira Santos (1), Adriano Mota Loyola (2) , Srgio Vitorino Cardoso
(2) , Roger Chammas (3) , Fu-Tong Liu (4) , Paulo Rogrio de Faria (1)
(1) Uberlandia Federal University, Biomedical Science Institute, Department of
Morphology, Uberlandia, Brazil
(2) Uberlandia Federal University, School of Dentistry, Uberlandia, Brazil
(3) Sao Paulo University, School of Medicine, So Paulo, Brazil
(4)Department of Dermatology, University of California, Davis, School of Medicine,
Sacramento, CA, USA
debora_olivsantos@hotmail.com.br/ (34)98827-5942
Introduction: Oral squamous cell carcinoma (OSCC) is one of the most deadly
cancer worldwide. Currently, there are limited clinical tools aiding clinicians to
establish its early diagnosis, and genetic and epigenetic events leading to the
pathogenesis of OSCC remain unsolved. The use of carcinogen-induced knocked out
mouse models would help to improve its early detection and also determine the role
of proteins in this process. Aims: To evaluate Wnt/beta-catenin and Sonic Hedgehog
pathways in dysplasias and carcinomas developed in tongue of wild-type (WT) and
galectin-3 deficient (KO) mice. Methods: 37 WT and 38 KO mice were challenged
with 4NQO and killed at different times. Tongues were removed, processed, and
submitted to an immunohistochemical approach to study Wnt-1, Wnt-3A, Shh and
Gli-3 proteins. Kruskal-Wallis test was employed. Results: Dysplasias and
carcinomas from WT and KO mice were negative for Wnt-1. Wnt-3A expression
was higher in WT than KO mice (p>0.05). Shh expression was significantly higher
in both lesions from WT mice (p<0.0001), and in tongue malignant transformation
only in the WT group. Gli-3 immunoreactivity decreased from dysplasia to
carcinoma in WT mice, but increased in KO mice (p>0.05). Conclusion(s): Activated
Wnt/beta-catenin pathway was seen in both groups of mice, whereas the Sonic
Hedgehog pathway was associated with tongue malignant transformation only in WT
mice. Ethical approval: The animal study was managed following an animal protocol
approved by the Committee on Animal Experimentation of the UFU (protocol:
n067/10).
Funding support: Research Supporting Foundation of Minas Gerais (FAPEMIGCDS-APQ-00397-09).

A169
A170
RUTHENIUM(II)/PIPERONILIC ACID COMPLEX INDUCES CELL

CYCLE ARREST AND APOPTOSIS IN CELLS DERIVED FROM NONSMALL CELL LUNG CANCER
TCTP IN MELANOMA MALIGNANCY: USE OF SERTRALINE AS A NEW

THERAPEUTIC APPROACH
Guilherme lvaro Ferreira da Silva(1), Marco Aurlio Banionis(1)*, Marina

Montingelli Ortega(2), Jlia Scaff(2), Marilia Imaculada Frazo Barbosa(2), Antonio
Carlos Doriguetto(2), Marisa Ionta(1)*
1(1) InstituteofBiomedicalSciences, Federal Universityof Alfenas, Rua Gabriel
2Monteiro da Silva, 700, zip code 37130-000, Alfenas, MG, Brazil
3(2)InstituteofChemistry, Federal Universityof Alfenas, Rua Gabriel Monteiro da
Silva, 700, zip code 37130-000, Alfenas, MG, Brazil
*marcobanionis94@gmail.com, 55-35-991337223 or 55-32-3299-1360
Lung cancer is the leading cause of cancer deaths worldwide. Advanced disease
stage is the most relevant factor influencing mortality. Platinum complexes (cisplatin
and its analogues) have been used for lung cancer treatment; howeverthe serious side
effects have been limited their clinical application. In this frame, the ruthenium based
compounds represent an alternative to platinum-based compounds due to their
chemical versatile and low toxicity. The present study aimed to investigate the
effects of ruthenium(II)/piperonilic acid complex [Ru(pipe)(dppb)(bipy)]PF6, from
hereon called PIPE, on A549 cells. Different methodological approaches were used
in order to evaluate antiproliferative and cytotoxic activity including cell viability
assays (MTS, trypan blue), clonogenic assay and mitotic index determination. Cell
cycle analysis and protein expression profile were assessed by flow cytometryand
immunoblot, respectively. We demonstrated that PIPE effectively reduced cell
viability of A549 cells in a concentration- and time-dependent manner. Cells were
arrested in G0/G1 when PIPE was used at 9 M for 24h and colony forming capacity
was also drastically reduced. These effects were related to downregulation of cyclin
D1. Furthermore, we showed that PIPE (18 M)increased sub-G1 population and it
also induced caspase 3 activation and p53 phosphorylation. Taken together, our
results demonstrate that PIPE inhibits cell proliferation of A549 cells and has proapoptotic activity supporting further in vivostudies for its antitumor activity.
Boia- Ferreira, M.1; Hamasaki, A. E. 1; Basilio, A. B. C. 1; Matsubara, F. H. 1;

Baldissera, A. B1; Vieira, P. F.2; Appel, A. H. 1; Gremski, L. H. 1; Chaim, O. M. 1;
Veiga, S. S. 1; Senff- Ribeiro, A. 1
(1) Federal University of Paran
(2) State University of Ponta Grossa
The aim of this study was to evaluate the role of TCTP on melanoma malignancy in
order to propose new therapies. TCTP is a multifunctional protein overexpressed in
several tumor types. Silencing TCTP was shown to be able to induce tumor
reversion, the loss of the malignant phenotype. TCTP and p53 present a negative
feedback loop with reciprocal repression. Sertraline is an antidepressant (SSRI class),
which interacts with TCTP and decreases its cellular levels. We studied the silencing
of TCTP in a murine melanoma model (B16F10) by RNAi, transfected cells
presented lower proliferation and migration than control. Decreased levels of TCTP
were confirmed by RT-PCR analysis and immunoblotting. When B16F10 was
compared to B16F1 (less invasive and tumorigenic) the expression of TCTP mRNA
in was 1.83-fold higher than that from B16-F1 cells. TCTP expression is related to
phenotypic differences of murine melanoma cell lines. Sertraline was evaluated in
vitro (viability and clonogenic assay) and in vivo on the B16F10 model and the
results highlight its effect on inhibiting tumor growth (~75%) and on the response to
the chemotherapeutic agent dacarbazine, which was improved. When human
melanoma cells (A2058 and MeWo) were treated with sertraline a decrease in
malignant features (viability, migration and clonogenicity) was also observed as well
as a decrease in TCTP protein levels and increase in p53 protein levels. Thus,
sertraline and the TCTP-p53 axis should be considered a new approach for the
improvement of melanoma treatment.
This work was supported by: CNPq, CAPES, UFPR, Fundao Araucria.
Financial support: Fapemig, CAPES and CNPq

B CELL BIOLOGY OF INFLAMMATION
B1
B2
IMMUNE CELLS OF THE MUCOSA IN THE ILEUM ARE ALTERED

DURING ACUTE TOXOPLASMA GONDII INFECTION
LNI2013 OIL OF THE AMAZON FLORA HAS AN ANTI-INFLAMMATORY

AND HEALING ACTIVITY IN SKIN
Larissa Carla Lauer Schneider (1), Joquebede Caroline Pessoa Nascimento (1),
Matheus da Silva de Novaes (1), Giovana Alves Santos (1), Evandro Jos Beraldi
(1), Stephanie Carvalho Borges (1), Dbora de Mello Gonales SantAna (1), Nilza
Cristina Buttow (1)
(1)
Bastos, A.C.*(1); Gomes, M.F. (1), Santos G C Q (1), da Silva J K do R (2), Maia
JGS (2), Bastos GNT (1)
(1) Department of Morphological Sciences, State University of Maring (UEM),

(2)
Maring, Brazil.
(3)
E-mail: lari_uem@yahoo.com.br
Phone: 55 44 3011 4928
Mobile: 55 44 9101 4332
The oral Toxoplasma gondii infection activates both the innate and the adaptive
immune responses in the gastrointestinal tract. Immune cells of the lamina propria,
which include dendritic cells, lymphocytes and neutrophils, produce chemokines and
cytokines that are necessary to attack the parasite, besides secreting substances that
reduce the inflammation and protect the tissue. We evaluated the presence of
immune cells stained with peroxidase (ICs), intraepithelial lymphocytes (IELs) and
Paneth cells (PCs) in the acute phase of toxoplasmosis. Male Wistar rats (60 days
old) were randomly distributed in 9 groups (n = 5): not infected (control group), 6,
12, 24, 48 and 72 hours, 7, 10 and 15 days after the infection with inoculation of
5000 sporulated T. gondii oocysts (ME-49 strain, genotype II). For the quantification
of PCs and IELs, ileum segments were processed by routine histological techniques
and stained with HE. For the ICs evaluation, ileum segments were embedded in
OCT, cut by cryostat and the tissue sections subjected to immunohistochemistry with
peroxidase. All animal procedures were approved by the Ethics Committee for
Animal Experimentation of the UEM (Protocol n. 079/2013). The number of PCs
increased significantly only in the group GI48, while the IELs density gradually
increased starting after 24 hours of infection. The ICs, however, increased only after
10 days of infection. These results show that T. gondii generates both an innate and
an adaptive response during the acute infection by promoting increase in the density
of defense cells in the mucosa of the ileum.
Funding support: CAPES.
(1) Neuroinflammation Laboratory, Federal University of Par - UFPA, Belm,

Brazil
(2) Natural Products Engineering Laboratory, Federal University of Par - UFPA,
Belm, Brazil
*Address: Neuroinflammation Laboratory, Federal University of Par - UFPA,
Institute of Biological Sciences, Rua Augusto Corra, 01, CEP: 66075-110, Belm,
Brazil.
*E-mail: alinecostabastos@gmail.com
Introduction: The inhabitants of the Amazon region use the LNI2013 oil as a healer.
Therefore, the study of this oil is important for the treatment of pressure ulcers. Aim:
Evaluate the healing and anti-inflammatory activities of LNI2013 oil in ischemia and
reperfusion model. Methods: The extract is involved in a process to obtaining a
patent. The pressure ulcers were induced by the method described by Peirce in 2000.
Sixteen male Wistar rats were randomly allocated in four groups: control (saline),
diethylamine diclofenac, dersani and LNI2013. The oil was used topically. The
lesion area was measured by ImageJ software, exudates volume, number of
migratory cells measured and histological staining were investigated. Results: The
Ischemia and reperfusion process promoted an area of 8715.32%, while the groups
treated with diethylamine diclofenac, dersani, LNI2013 promoted 791.58%,
66.503.68%, 43.5521.23% respectively. The exudate volume in the control group
was 4.251.25ml, already the groups treated with diethylamine diclofenac,
dersani and LNI2013 were 1.370.47mL; 1.680.23mL; 1.51.00mL respectively.
The number of migratory cells (nx10^6/mL) to the lesion area in the control group
was 143,7539,79, while in the groups diethylamine diclofenac , dersani and
LNI2013 were 13.59.14; 71.2512.33; 12.59.96 respectively. The histological
analysis to HE of the group treated with LNI2013 showed the best restructuring of
skin when compared with the controls groups. Conclusion: The results suggest that
the LNI2013 oil has healing and anti-inflammatory activities and it could be a
promising drug for treatment of pressure ulcers.
CONCEA:123-13. Sources of research support: CNPq, UFPA.

B3
B4
INCREASE OF CYTOKINE EXPRESSION IN PERIARTICULAR

INFLAMMATORY PROCESS OF ARTHRITIS ZYMOSAN-INDUCED BY
LOW-INTENSITY INFRARED LASER
IMMUNOMODULATORY EFFECTS OF GALECTIN-1 ON ATOPIC

DERMATITIS EXPERIMENTAL MODEL
Mab Pereira Corra (1)*; Frans Eberth Costa Andrade (2); Cristiane Damas Gil (1,2)
Lcia Mara Janurio dos Anjos (1)*, Adenilson de Sousa da Fonseca (2), Jacy
Gameiro (3), Flvia de
Paoli (1).
1. Department of Morphology, Federal University of Juiz de Fora, Juiz de Fora,
Minas Gerais, Brazil.
2. Department of Biophisics and Biometry, Estate University of Rio de Janeiro,
Brazil.
3. Department of Parasitology, Microbiology and Immunology, Federal University
of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil.
*Correspondence to: Lcia Mara Janurio dos Anjos, Departamento de Morfologia,
Instituto de Cincias Biolgicas, Universidade Federal de Juiz de Fora, Rua Jos
Loureno Kelmer, s/n Campus Universitrio, So Pedro, Juiz de Fora, Minas
Gerais,
36036900,
Brazil.
Tel:
+55
(32)
2102-3212.
email:
luciamara.anjos@ufjf.edu.br.
Background: Low-level light therapy (LLLT) is a phototherapy treatment used in
various diseases such as arthritis due to their ability to biostimulation, accelerating
tissue repair and decreasing inflammatory processes. Aim: Investigate whether
LLLT, recommended in device guidelines, could modify gene expression of proinflammatory (IL-1 and TNF-) and anti-inflammatory (IL-10 and TGF-)
cytokines, in infiltrated cells of arthritic ankle connective tissue. Methods:
Inflammatory process was induced in mice ankle by zymosan administration. The
animals were exposed to two different fluences of LLLT: 3J/cm2 and 30J/cm2.
Morphological analysis by H&E staining and gene expression of cytokines by RT qPCR were performed after animal euthanasia. Results: The inflammatory process
was decreased after laser treatment with the 30J/cm2 fluence, and the expression of
cytokines was altered in all groups irradiated. The LLLT 30J/cm2 group showed
increased of IL-1 gene expression (P<0,01) while the LLLT 3J/cm2 group showed
increased of IL-10 gene expression (P<0,001). Conclusion: Our results suggest that
low-intensity laser 30J/cm2 fluence was able to modify the inflammatory process of
arthritis-induced zymosan by decreasing the inflammatory cells, probably by
biostimulation since increased pro-inflammatory cytokines expression. Even thought
the low-intensity laser in 3J/cm2 fluence had not decreased the inflammatory process,
showed increase expression of IL-10, anti-inflammatory cytokines, which could
change the progression of joint damage by induction or modulation of regulatory
immune response. Ethical approval: CEUA- UFJF-039/2014. Funding support:
FAPEMIG / CNPQ.
(1) Postgraduate Program in Genetics, Instituto de Biocincias, Letras e Cincias

Exatas (IBILCE), So Paulo State University (UNESP), So Jos do Rio Preto, SP,
Brazil;
(2) Postgraduate Program in Structural and Functional Biology, Department of
Morphology and Genetics, Federal University of So Paulo (UNIFESP), So Paulo,
SP, Brazil;
*Contact: mabiiic@gmail.com/ (17) 98205-5622
BACKGROUND
Galectin-1 (Gal-1), a 14.5 kDa protein that contributes to homeostasis of the
inflammation, has been poorly investigated in the skin inflammatory diseases,
especially atopic dermatitis (AD).
AIMS
This study evaluated the role and expression of Gal-1 in AD murine experimental
model.
METHODS
Ovalbumin (OVA)/Alum-immunized male Balb/c mice (days 0 and 7) were
challenged with 250g OVA diluted in 50l Johnsons Baby oil on days 11, 14-18
and 21-24. Pharmacological treatments with rGal-1 (0.3 or 3g/mice) or
dexamethasone (Dex; 1 mg/kg) were administered i.p. on days 21-24. After 24 hours
of the last OVA challenge, clinical symptoms, plasma IgE anti-OVA levels,
histopathology and immunohistochemistry to detect Gal-1 expression in skin were
performed. Ethical approval protocol: CEUA/UNIFESP n. 1906060115.
RESULTS
AD mice exhibited high plasma levels of IgE anti-OVA compared to control groups
(naive and sham) showing the efficacy of experimental model. Treatment with rGal1 (3g) and Dex abrogated the clinical symptoms of AD at mice that showed similar
skin thickness to control animals. AD induced upregulation of endogenous Gal-1
expression in the dermis associated with increased mast cell number and eosinophil
migration. rGal-1 (0.3 and 3g) and Dex administration reduced significantly
eosinophil migration into dermis compared to untreated AD mice as well as
downregulated Gal-1 levels in the skin as detect for immunohistochemistry.
CONCLUSION
These results point to anti-allergic properties of Gal-1 through regulation of clinical
symptoms and recruitment of inflammatory cells, especially lymphocytes and
eosinophils, and might impact on the development of new strategies for skin
inflammatory diseases.
Keywords: Low-level laser therapy, cytokines, zymosan, arthritis.

FINANCIAL SUPPORT
FAPESP (2015/9858-3) and CNPq.

B5
B6
MURINE STRAIN DIFFERENCES IN INFLAMMATORY ANGIOGENESIS

OF INTERNAL WOUND IN DIABETES
INVOLVEMENT OF THE ANNEXIN A1-FPR ANTI-INFLAMMATORY

SYSTEM IN THE OCULAR ALLERGY
Mariana Prado Marmorato*; Alexandre Dantas Gimenes; Tamires Barbosa Lucena

da Rocha; Sonia Maria Oliani; Cristiane Damas Gil
Simone Aparecida de Almeida, 1 Laura Alejandra Ariza Orellano, 1Luciana Xavier

Pereira, 1Celso Tarso Rodrigues Viana, 1Paula Peixoto Campos, 2Silvia Passos
Andrade, 1Monica Alves Neves Diniz Ferreira
1
Departments of General Pathology and 2Physiology and Biophysics Institute of

Biological Sciences, Federal University of Minas Gerais. Av. Antnio Carlos 6627Campus Pampulha, Cx Post 468, CEP 31270-901, Belo Horizonte/MG, Brazil
Tel: 55 31 3409 3038; Fax: 55 31 34092879
Department of Morphology and Genetics, Federal University of So Paulo

(UNIFESP), So Paulo, SP, Brazil;
2
Department of Biology, Instituto de Biocincias, Letras e Cincias Exatas (IBILCE),
So Paulo State University (UNESP), So Jos do Rio Preto, SP, Brazil.
*Contact: marmorato.mariana@gmail.com / (11) 97361-0708
Background: Genetic susceptibility is associated with inflammation,

neovascularization, and diabetes phenotypes. However, to what extent this
susceptibility influences inflammatory angiogenesis in internal injuries in diabetes
has not been fully investigated. Aims: Evaluate the influence of genetic background
in internal injury repair process in diabetic mice. Methods: Using the subcutaneous
implantation of a synthetic matrix as an internal wound model in Swiss, C57/BL6
and Balb/c mice, we have studied inflammation, angiogenesis, and cytokine
production in the fibrovascular tissue induced by implants in diabetic animals.
Results: The hyperglycemic levels (mg/dl) after the diabetogenic treatment were
455.0 15 in Swiss, 393.0 22 in C57/BL6, and 190.0 10 in Balb/c mice.
Angiogenesis was highest in Swiss implants from nondiabetic animals and lowest in
Balb/c implants. However, the angiogenic inducers VEGF and nitric oxide (NO)
were highest in the non-diabetic Balb/c mice. Strainrelated differences were also
observed in the angiogenic parameters in implants from diabetic mice. Hb content
and number of vessels decreased more than 40% in Swiss implants. In contrast, Hb
content did not alter in implants from Balb/c diabetic mice and the number of vessels
decreased. VEGF levels increased in implants from Swiss and C57/BL6 diabetic
mice, but decreased in Balb/c implants. The levels of pro-inflammatory markers
intra-implant also varied among the strains in both conditions. In the hyperglycemic
environment, the inflammatory parameters were more pronounced in implants from
Balb/c mice compared with C57/BL6 implants. Overall, the inflammatory markers
increased in implants from diabetic Swiss. Conclusion: These findings demonstrate
the major contribution of the genetic background in the pattern of inflammatory
angiogenesis components of internal injury, in both normoglycemic and
hyperglycemic animals.
Key words: angiogenesis; diabetes; genetic background; internal wound
BACKGROUND
Annexin A1 (ANXA1) is a potent anti-inflammatory protein effective in terminating
inflammatory response, and its role in allergic settings has been poorly studied.
AIMS
The purpose of this study was to reveal the protective role of mimetic peptide of
ANXA1 (Ac2-26) in experimental model of allergic conjunctivitis (AC) and to
highlight the potential involvement of members of the formyl peptide receptor (Fpr)
family in this process.
METHODS
Ovalbumin (OVA)/Alum-immunized Balb/c mice (days 0 and 7) were challenged by
eye drops containing OVA (250g) on days 14-16, and two groups received
pharmacological treatments with Ac2-26 (1mg/kg) and Fpr antagonist Boc2
(100g/animal) intraperitoneally during challenged days. Plasma IgE anti-OVA
levels, histopathology, immunohistochemistry and immunoblot were performed 24
hours postchallenge. Ethical approval protocols: CEUA/UNIFESP 1975251115.
RESULTS
Plasma IgE anti-OVA levels increased significantly in the OVA-immunized wild-type
and ANXA1-null mice, supporting the efficacy of AC model. Ac2-26 treatment
reduced the proportion of mast cell (MC) degranulation in the conjunctiva (35%)
compared with untreated AC group (55%) and supported by the increased expression
of mouse MC protease 6 in eye homogenates. The lack of endogenous ANXA1
increased the degranulation (57%) of peritoneal MCs in AC group compared to
control (32%). Additionally, AC elevated levels of Fpr1 and 2 in the epithelium of
bulbar conjunctiva which was exacerbated in ANXA1-null mice. Boc2 treatment
abrogated Ac2-26 effect through increasing MC degranulation and eosinophil influx
in the conjunctiva.
CONCLUSION
Collectively, data provide in vivo support to anti-allergic effects of ANXA1-Fpr
system and may serve as a therapeutic target in this ocular disorder.
FINANCIAL SUPPORT
FAPESP (2012/50641-0; 2015/22944-6).

B8
B7
EXOGENOUS GALECTIN-1 ALLEVIATES ALLERGIC INFLAMMATION
IN RODENT MODELS OF CONJUNCTIVITIS
Tamires Barbosa Lucena da Rocha (1)*; Claudia Mello-Bosnic (2); Mariana Prado
Marmorato(1); Sonia Maria Oliani (2); Cristiane Damas Gil (1,2)
(1) Department of Morphology and Genetics, Federal University of So Paulo
(UNIFESP), So Paulo, SP, Brazil;
Brazil.
*Contact: tamires.blr@gmail.com/ (11) 99679-4904
BACKGROUND
Galectin-1 is a -galactoside-binding protein with diverse biological activities in the
pathogenesis of inflammation but its role in allergic inflammation is uncertain.
AIMS
This study evaluated the effect of treatment with recombinant galectin-1 (rGal-1) in
two rodent models of allergic conjunctivitis (AC).
METHODS
eye drops containing OVA (250 g) on days 14-16, and two groups received
pharmacological treatments with rGal-1 (0.3 g) intraperitoneally or ocular
instillation during challenged days. In another experiments, rGal-1 (0.3 or 3 g) and
disodium cromoglycate were instilled on control and compound 48/80-challenged
eyes of Wistar rats. Clinical score, plasma IgE anti-OVA levels, histopathology,
immunohistochemistry and immunoblot were performed 4-6 hours postchallenge.
Ethical approval protocols: CEUA/UNIFESP 7611050814 and CEUA/UNESP
092/2014.
RESULTS
Both treatments with rGal-1 (i.p. and instillation) reduced significantly IgE antiOVA plasma levels, upregulated its endogenous expression as well as diminished
leukocyte migration in palpebral conjunctiva compared with untreated AC group.
Additionally, ocular instillation of rGal-1 elevated IL-10 anti-inflammatory cytokine
levels in plasma and intact mast cells in eyes evidenced by upregulation of mouse
mast cell protease 6 and IFN-gama levels. This effect of rGal-1 on mast cell
degranulation was confirmed in 48/80-challenged rat eyes. Both doses of rGal-1 (0.3
and 3 g), as well as disodium cromoglycate, exhibited inhibitory effects on
compound 48/80, a mast cell activator, through decreased clinical signs of
conjunctivitis associated with reduction of 50% of mast cell degranulation in
palpebral conjunctiva.
CONCLUSION
In conclusion, rGal-1 exogenous administration represents an important new strategy
for ocular allergic inflammation.
SMOOTH MUSCLE FOAM CELL FORMATION INDUCED BY MT-III A

GROUP IIA PHOSPHOLIPASE A2 ISOLATED FROM BOTHROPS ASPER
SNAKE VENOM
Karina C Giannotti1, Elbio Leiguez1, Mariana N Viana1, Neide Galvo do
Nascimento1, Priscila Signor Motta1, Thas L Arajo2, Renata H A Hernandez1,
Bruno Lomonte3, Catarina Teixeira1
1Laboratrio de Farmacologia, Instituto Butantan, So Paulo/SP.
2Laboratrio de Biologia Vascular, Instituto do Corao (Incor), So Paulo/SP.
3Instituto Clodomiro Picado, Universidad de Costa Rica, Costa Rica.
Background:Snake venom secreted phospholipases A2 (sPLA2) have structural and
functional homology with mammalian sPLA2s, whose levels are elevated in
inflammatory diseases like atherosclerosis. In this disease, vascular smooth muscle
cells (VSMCs) accumulate lipid in lipid droplets (LDs). Aims: To investigate the
effect of the snake venom GIIA-sPLA2s MT-III on VSMCs evaluating LD formation
and activation of diverse factors involved in lipid accumulation. Methods: VSMCs
obtained from male Wistar rats (CEUAIB 1024/13) were incubated with DMEM
(control) or MT-III (1-12 h). LD formation was quantified after cell staining with
OsO4. ABCG1, ABCA1, PLIN2, PLIN3, COX-1, COX-2 and PGE2 distribution and
protein expression were analysed by immunofluorescence and western blotting.
Results: Incubation of VSMCs with MT-III (0.4 to 0.8 M) significantly increased
LDs numbers from 1 up to 12 h. MT-III did not affect COX-1 expression, but
increased COX-2 protein content at 3 and 6h,and ABCG1 expression at 3h of
incubation. Moreover, MT-III induced PLIN2 and PLIN3 recruitment without
changing their protein expression. In addition, COX-1, COX-2 and PGE2 pools were
colocalized to LDs in MT-III-stimulated cells. Conclusions: These data show the
ability of a venom GIIA sPLA2 to induce LDs biogenesis in VSMCs and to increase
the expression of the inflammatory COX-2 in these cells. Increased expression of the
lipid efflux protein ABCG1 in the early phase of VSMCs response reinforce the
excess of lipid accumulation caused by MT-III. Furthermore, these results evidence
that LDs may constitute intracellular sites for the synthesis of prostanoids in VSMCs
stimulated by GIIA sPLA2s.
Finantial support: FAPESP

FINANCIAL SUPPORT
FAPESP 2014/17753-4; 2015/9858-3; CNPq 308144/2014-7.

B9
B10
COMPARISON OF IN VITRO AND IN VIVO METHODS FOR THE

EVALUATION OF LEUCOCYTE ADHESION
GALECTIN-3: ITS ROLE IN OCULAR ALLERGY AND POTENTIAL AS A

PREDICTIVE BIOMARKER
Angelica Aparecida Antoniellis Silveira1; Rafaela Mendona1, Ceclia Souto

Seguin1, Daiana Morelli Vital1, Venina Marcela Dominical1, Flvia Costa Leonardo1,
Fernando Ferreira Costa1; Nicola Conran1
Frans Eberth Costa Andrade (1)*; Mab Pereira Corra (2); Cristiane Damas Gil (1,2)
Vascular Inflammation Laboratory, Center of Hematology and Hemotherapy,

School of Medical Sciences, University of Campinas (UNICAMP). 480, Carlos
Chagas St., Campinas- SP, Brazil.
(1) Postgraduate Program in Functional and Structural Biology, Department of

Morphology and Genetics, Federal University of So Paulo (UNIFESP), So Paulo,
SP, Brazil;
Brazil.
Telephone: +55 (19) 3521-8611 - e-mail: angelicasilveira_mg@hotmail.com

*Contact: frans_eberth@hotmail.com (17) 99721-3494
Background: Leukocyte recruitment to sites of inflammation, in vivo, involves cell
activation, rolling and firm endothelial adhesion, before migration. Aim: Evaluate
the capacity of different systems to measure neutrophil adhesion in the presence of
inflammatory (TNF-) and anti-inflammatory stimuli (simvastatin, SIM). Methods:
Neutrophils (separated from healthy individual blood) were incubated with/without
SIM (10M) and stimulated with TNF- (200ng/mL). Adhesion of cells to
fibronectin (FN; 20g/mL) ligand was evaluated under static and microfluidic in
vitro conditions. Static conditions were reproduced in 96-well plates and absence of
cell flow (30 min, 37oC), while the VenaFluxTM Platform was used to provide
microfluidic fluid conditions for the observation of cell rolling/adhesion under shear
stress (0.5d/cm2, 3 min, 37oC) in 400m-diameter microcapillaries, to mimic
vascular conditions. Intravital microscopy of the microcirculation of the cremaster
muscle of TNF- (0.5g)-stimulated C56BL/7 mice was used to determine leukocyte
recruitment in vivo. Results: Under both static and microfluidic in vitro conditions,
TNF- significantly augmented neutrophil adhesion compared to basal, while SIM
abrogated this increased adhesion (Static assay: 14.22.1%, 46.33.2%, 30.73.3%;
basal, TNF-, SIM, respectively, p<0.001, n=7) (Microfluidic assay: 14.62.2,
38.46.2, 26.84 cells adhered/0.186 m2; basal, TNF-, SIM respectively p<0.001,
n=14). In vivo, SIM (30mg/kg) was also able to diminish TNF--induced leukocyte
adhesion to the endothelium (2.20.6, 9.90.4, 4.30.3 cells adhered; vehicle, TNF, SIM respectively, p<0.001, n4). Conclusion: Data indicate that all systems
evaluated, despite employing different experimental conditions, present relative
reproducibility for evaluating agents that stimulate and inhibit leukocyte adhesion,
supporting the use of in vitro assays for observations of leukocyte interactions when
in vivo experimentation is not possible.
Financial Support: FAPESP.

BACKGROUND
Galectin-3 (Gal-3) has emerged as an important regulator of inflammation, however
its molecular mechanisms in allergic responses remain unclear.
AIMS
The potential of Gal-3 as a biomarker of allergic conjunctivitis (AC) and the effect of
immunosuppressive therapy on its expression was evaluated.
METHODS
eye drops containing OVA (250g) on days 14-16, and one group received 0.03%
tacrolimus eye drop during challenged days. Plasma IgE anti-OVA levels,
histopathology and immunohistochemistry were performed 24 hours postchallenge.
Impression cytology of ocular surface was performed to obtain epithelial cells from
bulbar conjunctiva of patients with severe AC, treated or not with 0.03% tacrolimus,
and control, and Gal-3 expression detected by immunocytochemistry. UNIFESP
ethical approval protocols: CEUA-5448030215, CEP-537/2015.
RESULTS
Plasma IgE anti-OVA levels increased significantly in the OVA-immunized wildtype and Gal-3-null mice, supporting the efficacy of AC model. Increased expression
of endogenous Gal-3 was associated with high influx of eosinophils/mast cells in the
conjunctiva of wild-type mice, as well as elevated numbers of blood
eosinophils/neutrophils. Tacrolimus diminished Gal-3 expression, conjunctival mast
cell influx and eosinophil recruitment. Further, the lack of Gal-3 abrogated the
pattern of inflammatory cell recruitment in the AC model compared to sham Gal-3null mice. Human conjunctival epithelial cells from AC patients also exhibited
elevated levels of Gal-3 that was downregulated by tacrolimus associated with
corticosteroid and antihistamine.
CONCLUSION
Data provides further evidence for an upregulation of Gal-3 expression in both AC
model and human disease, suggesting its role in the development of this ocular
pathogenesis.
FINANCIAL SUPPORT
CAPES, FAPESP (2015/9858-3).

B11
B12
CARDIAC COMPLEMENT IN TOLL-LIKE RECEPTOR 4 KO MICE

SUBJECT TO RENAL ISCHEMIA/REPERFUSION
NITRIC OXIDE-DEPENDENT EFFECT OF HYDROXYUREA ON TNF-STIMULATED NEUTROPHIL ADHESION IN VITRO
Junho, C.V.C., Trentin-Sonoda, M., Carneiro-Ramos, M.S.
Cecilia Souto Seguin1, Daiana Morelli Vital1, Flavia Costa Leonardo1, Angelica
Antoniellis Silveira1, Rafaela Mendona1, Flavia Garcia1, Camila Bononi Almeida1,
Fernando Ferreira Costa1, Nicola Conran1
Universidade Federal do ABC, Santo Andr, SP- Brazil

Background: The immune system consists of an array of cells and molecules
distributed throughout the organism; its function is to recognize antigens or
molecular structures, generating an effective response in the pathogens destruction or
inactivation. It can also act via the complement system (CS). The cardiac tissue is a
main target of the immune system and several cardiovascular diseases (CVD) are
preceded by general or local inflammation, contributing directly to cardiac
remodeling and changes in heart tropism. It is known that renal ischemia/reperfusion
(I/R) may result in the development of systemic inflammation and it can reach the
cardiac tissue. Tissue response may be initiated by Toll-like receptors [TLRpathogen-associated molecular patterns (PAMPs) and damage associated molecular
patterns (DAMPs) recognizers] or also by complement system. Within the
inflammatory process, the complement component C3a is released in abundance and
binds to C3aR1 receptor, which might be involved in TLRs activation.
Aim: Since it has been demonstrated that renal I/R is able to induce CH and ablation
of TLR4 prevents such CH development, the aim of this study was to evaluate if
TLR4 -/- had differential expression of complement components when compared to
wild type mice.
Methods: C57BL/6 male mice and C57BL/6 TLR 4 -/- were subjected to surgical
occlusion of left renal pedicle for 60min followed for reperfusion (I/R) for 15 days.
Morphometric analyses were performed using as parameters heart weight, kidneys
weight and body weight. Real time PCR were utilized to analyze gene expression,
and Western Blot to analyze protein expression.
Results: The C3aR component in KO group after 15 days of reperfusion, showed no
change. These data is in accordance with previous data where we have not observed
any modulation in the WT groups after 15 days of reperfusion. However, analysis of
the complement component C3 mRNA levels, showed a significant increase in the
WT I/R group compared to WT sham group (p <0.05), and TLR4 presence was
capable of promoting a bigger increase (almost 100x). The component C5R presents
significant increase after 15 days of reperfusion (p <0.05) as well as Cfb (p<0.05) in
TLR4 KO I/R animals, compared to Sham.
Conclusion: Cardiac gene expression of complement components differs in WT and
TLR4KO mice subjected to renal ischemia/reperfusion, indicating a possible
interaction between these two main responses of innate immunity in our model.
Hematology Center, School of Medical Sciences, University of Campinas

(UNICAMP), Brazil.
1

School of Medical Sciences, University of Campinas (UNICAMP), Brazil. 480,
Carlos Chagas St., Campinas- SP. Telephone: +55 (19) 3521-8611 - e-mail:
ceciliaseguin@gmail.com
Background: Hydroxyurea (HU) is a chemotherapeutic drug used to treat a number
of diseases including chronic myelogenous leukemia, polycythemia vera and sickle
cell disease. Recent data indicate that HU may have acute anti-inflammatory effects
and act as a nitric oxide (NO) donor. Aim: To investigate the effect of HU on the
adhesive properties of TNF--stimulated neutrophils and analyze the participation of
a NO-dependent pathway in this effect. Methods: Neutrophils, isolated from healthy
human donors, were pre-incubated with HU (100 and 1000M; 15 min), ODQ, an
inhibitor of soluble guanylate cyclase (a second messenger for NO) (0.1 and 10M)
and/or PTIO, a scavenger of NO (5-1000 M), followed by a TNF- stimuli
(200ng/ml), and adhesion to fibronectin was evaluated using microfluidic and static
assays. Results: Microfluidic assays showed that TNF- significantly increases
neutrophil adhesion (TNF- increased neutrophil adhesion from basal by
117.525.0%, P<0.01). Pre-incubation of cells with HU significantly decreased
TNF-induced adhesion at 100M and 1000M by 29.914.9% and 33.312.6%,
respectively (compared to TNF--induced adhesion, n=14, p<0.05). ODQ partially
reversed the effect of HU on TNF--stimulated neutrophil adhesion (p<0.01), while
static adhesion assays showed that PTIO also partially reverses the effect of HU on
TNF--stimulated neutrophil adhesion (p<0.05). Conclusion: HU may protect
neutrophils from inflammatory activation, in turn reducing their adhesive properties.
The effect of HU may be mediated by its ability to modulate NO-dependent
pathways, in vitro. The local ethics committee approved this study.
Financial Support: FAPESP/CAPES.
Financial Support: FAPESP


B13
B14
EVIDENCE OF DAMP RELEASE AND INFLAMMASOME FORMATION

IN PATIENTS WITH SICKLE CELL ANEMIA
STUDY OF THE EFFECT OF HYPERBARIC OXYGENATION ON THE

PROLIFERATION OF ENDOTHELIAL CELLS IN VITRO
Rafaela Mendona, Angelica A.A. Silveira, Cecilia S. Seguin, Fernando V. Pericole,

Flavia C. Leonardo, Sara T.O. Saad, Fernando F. Costa, Nicola Conran.
Jlia Carnaz Benincasa, Luiz Henrique de Freitas Filho, Claudio Chrysostomo

Werneck, Selma Giorgio e Cristina Pontes Vicente.
(1) Departament of Structural and Functional Biology, State University of Campinas,

Campinas, Brazil.
(2) Departament of Biochemistry e Tecidual Biology, State University of Campinas,
Campinas, Brazil.
(3) Departament of Animal Biology, State University of Campinas, Campinas,
Brazil.

1School of Medical Sciences, University of Campinas (UNICAMP), Brazil. 480,
Carlos Chagas St. Campinas- SP. Telephone: +55 (19) 3521-8611 - e-mail:
2rafa.mendonca11@gmail.com
3Background: Sickle cell anemia (SCA) is a genetic disease characterized by the
production of an abnormal hemoglobin (HbS), which polymerizes at low oxygen
concentrations, causing the erythrocyte to become sickle-shaped. Ensuing hemolysis
and cellular alterations lead to a chronic inflammatory state and the vaso-occlusive
processes that cause the diseases numerous clinical complications. Aims: Determine
whether inflammasome formation is augmented in the inflammatory cells of patients
with SCA. Methods: Peripheral blood was obtained from healthy control individuals
(CON) and SCA patients in steady state, off (SCA) and on hydroxyurea (HU)
therapy (SCAHU; 15-30mg/kg/day). Plasma IL-1, IL-18 (indicative of
inflammasome formation), TNF- and free heme were measured by ELISA and
colorimetric test. Results: Plasma IL1 and IL-18 were increased in the SCA group,
compared to CON (IL-1: 0.200.13, 0.610.28 pg/ml; IL-18: 30532, 883 222
pg/ml for CON and SCA, respectively, P<0.05, for SCA compared to CON; N12).
Interestingly, plasma IL-1, but not IL-18, was decreased by HU therapy (0.230.48,
780112 pg/ml, for IL-1 and IL-18, respectively, P<0.05 for SCAHU, compared to
SCA [IL-1]). Plasma Heme and TNF- were increased in both SCA groups,
compared to CON (Heme: 18.92 2.71, 60.14 7.71, 40.95 6.15 M for CON,
SCA, SCAHU, respectively; P<0.01, compared to CON; N12), (TNF-; 0.95
0.09, 2.30 0.36, 2.08 0.33 pg/ml for CON, SCA, SCAHU, respectively; P<0.05,
compared to CON; N12). Conclusion: Increased concentrations of circulating IL1, IL-18, Heme and TNF- in individuals with SCA are consistent with the
hypothesis that augmented inflammasome formation may occur in this disease,
providing insight into the mechanisms by which the inflammatory state is
exacerbated in SCA. HU therapy may partially inhibit IL-1 processing in these
individuals.
E-mail: julia_benincasa@hotmail.com. Address: Rua Charles Darwin s/n, bloco N,

3 andar. Phone: 55 (19) 3521-6106. Mobile: 55 (16) 991933888.
The hypoxia caused by the rupture of the vasculature and vasculopaties can limit
wound healing in several tissues including vessel re-vascularization. The hyperbaric
oxygenation (HBO) treatment involves the exposition of the cells to 100% oxygen in
a 2,5ATA pressurized chamber. This process may cause a trophic effect on
vasculogenic stem cells, thus it may participate in neovasculogenesis after
endothelial lesion. Based on that, we studied the effect of HBO on endothelial cells
(EC) HUVEC (human vascular endothelial cells) proliferation and expression of
eNOS (oxide nitric sintase) and I-CAM. Weve treated EC with HBO for 30, 60, 120
and 180 minutes to assess the cell proliferation and determine the appropriate time of
HBO treatment. Treatment times of 30, 60 and 120 minutes did not affect the
proliferation, after 180 minutes though, cell proliferation decreased 50% and cell
morphology was modified when compared to the control. HBO treatment for 60
minutes increased the eNOS expression and did not alter ICAM-1 expression,
whereas the treatment for 120 minutes had the opposite effect, increasing ICAM-1
and maintaining the eNOS expression when observed by immunofluorescence
microscopy. We conclude that HBO treatment for 60 minutes can alter EC activity
without altering its proliferation, stimulating NO production and inhibiting its
inflammatory state by not increasing I-CAM expression. This project was approved
for the ethics committee from UNICAMP (CEUA) (protocol number 4171-1).
Funding Support: Capes, FAPESP.
Financial Support: FAPESP/CAPES.
B15
B16
SYNTHETIC MATRIX AS SCAFFOLD TO PANCREATIC PARENCHYMA

PROLIFERATION IN EXPERIMENTAL DIABETES AND ASSOCIATION
WITH HUMAN ADIPOSE DERIVED STEM CELLS (HASCS)
EFFECT OF HELICOBACTER PYLORI UREASE IN ENDOTHELIAL

CELLS
Pereira, LX ; Almeida, SA ; Orellano, LAA ; Viana, CTR ; Andrade, SP;

Campos, PP
Instituto of Biological Science, Federal University of Minas Gerais, Belo Horizonte,
Minas Gerais, Brazil
Background: Alternative approaches using biomaterials have been studied to
diabetes. Our research group have been observed that intraperitoneal implants of
polyether-polyurethane synthetic matrix (smPP) integrated with the surrounding
environment. Thus, we reasoned that by placing this smPP in interface with pancreas
in mice, we would provide a scaffold for inducing pancreatic parenchyma
proliferation. Besides, the association with cell therapy has been an alternative to
improve the viability of using biomaterials to tissue regeneration. Aims: Evaluating
the growth of pancreatic parenchyma into implants, in association with human
adipose derived stem cells (hASCs), in experimental diabetes. Assess the profile
glycemic of animals, angiogenic and inflammatory parameters of implants, cell
proliferation into implant. Methods: The smPP has been structurally characterized by
computed microtomography (mCT). The smPP was implanted closer the pancreas of
control and diabetic mice. Four days after, the hASCs were injected directly into
implant. At fifteen days after, the implants were collected and processed for analysis.
Glucose tolerance test was carried out in animals. Results: The pores medium
diameter of smPP was 474m. After injection of hASC, the diabetic group
ameliorated their glucose tolerance curve. Pancreatic parenchyma grew into
implants, including cells immunostaining to insulin. Both groups that received
hASCs showed improvement in angiogenic and inflammatory parameters.
Conclusion: smPP was capable of acting as a biological platform for new tissues
growth as pancreatic parenchyma. In addition, it can also function as a model to
identify key features of the newly formed pancreatic tissue, since it is easily
accessed.
Ethical approval: The Ethics Committee of Animal Use (CEUA) of the Federal
University of Minas Gerais (protocol number 43/2015) approved all animal
procedures.
Financial support: CNPq, CAPES, FAPEMIG.
Souza MJ1; Moraes JA1; Nascimento-Silva V1; Helal-Neto E2; Uberti, AF3; ScopelGuerra, A3; Severo, D3; Morandi, V2; Carlini C3; Barja-Fidalgo, C1.
UERJ 1Lab. Cellular & Molecular Pharmacology & 2Lab. Endothelial Cell
Biology; Structural Biology; 3UFRGS-Lab. Toxic Proteins
Souza, MJ - Mariele de Jesus Souza (mari_ele_js@hotmail.com) -(21)2868-8298 Av. 28 de Setembro 87 fds, Vila Isabel, Rio de Janeiro RJ
MORAES, JA - Joo Alfredo de Moraes Gomes Silva (jmoraesbr@yahoo.com) (21)2868-8298 Av. 28 de Setembro 87 fds, Vila Isabel, Rio de Janeiro RJ
Nascimento-Silva, V - Vany Nascimento da Silva (vanysilva@hotmail.com) (21)2868-8298 Av. 28 de Setembro 87 fds, Vila Isabel, Rio de Janeiro RJ
Helal-Neto, E - Edward Helal Neto (edwneto@gmail.com) (21)23340499 - Rua
So Francisco Xavier 524 PHLC Lab.203/204, Maracan, Rio de Janeiro RJ.
Uberti, AF - Augusto Frantz Uberti (afuberti@cbiot.ufrgs.br) - (51) 38087003 - Av.
Bento Gonalves 9500. Prdio 43.431 sala 114, Porto Alegre, RS.
Scopel-Guerra, A - Adriele Scopel-Guerra (adriscopel@gmail.com) - (51) 38087003
- Av. Bento Gonalves 9500. Prdio 43.431 sala 114, Porto Alegre, RS.
Severo, D Deiber Oliveira Severo (deiberos@pq.cnpq.br) - (51) 38087003 - Av.
Bento Gonalves 9500. Prdio 43.431 sala 114, Porto Alegre, RS.
Morandi, V - Veronica Morandi (moarandi.v@gmail.com) (21)23340499 - Rua
So Francisco Xavier 524 PHLC Lab.203/204, Maracan, Rio de Janeiro RJ.
Carlini, C - Clia Carlini (ccarlini@ufrgs.br) (51) 38087003 - Av. Bento
Gonalves 9500. Prdio 43.431 sala 114, Porto Alegre, RS.
Barja-Fidalgo, C - Christina Barja-Fidalgo (cbarja.uerj@gmail.com) - (21)2868-8298
Av. 28 de Setembro 87 fds, Vila Isabel, Rio de Janeiro RJ
Helicobacter pylori urease (HPu) is reported as a key bacterial product that enables
the bacteria to colonize and survive in the stomach, favoring the occurrence of
gastric ulcer and adenocarcinoma. We have demonstrated that HPu has proinflammatory properties, inducing neutrophils activation. The bacteria, H. pylori,
was shown to interact with endothelial cells (EC), affecting many cell functions. We
are now investigating the direct effects of HPu on EC and the signaling pathways
involved in those processes.
EC (HMEC cell line) was treated for different times with HPu (up to 10nM). The
treatment of EC with HPu did not affect cell viability. On the other hand, the
treatment of EC with HPu enhanced cell permeability, causing dissociation of cell
cell junctional cadherins junctions and VE-cadherin phosphorylation; promoted
alterations on actin cytoskeleton dynamics, and also increased FAK phosphorylation.
Additionally, HPu induced ROS and nitric oxide production by EC. HPu also
enhanced ICAM-1 and E-selectin expression, increasing neutrophils adhesion to EC.
The effects of HPu on EC seem to be modulated by integrin signaling and
lipoxygenase metabolites, as they were attenuated in the presence of RGD peptides
and esculetin. Finally, the treatment with HPu induced tubulogenesis and VEGFR-2
expression, evidencing its potential to induce EC differentiation.
The data indicate that HPu actives EC, probably through an integrin and
lipoxygenase pathways, supporting a role as a pro-inflammatory and pro-angiogenic
key molecule in H pylori-related diseases.
Supported by: FAPERJ; CAPES; CNPQ

B17
B18
PROTEASE-ACTIVATED RECEPTORS PAR-1 AND PAR-2 ARE

INVOLVED IN THE INFLAMMATORY RESPONSE IN ODONTOBLASTLIKE MDPC-23 CELLS
HEMOLYSIS AND VASCULAR INFLAMMATION IN THE HEMOLYTIC

DISEASES, SICKLE CELL ANEMIA, HEREDITARY SPHEROCYTOSIS
AND PAROXYSMAL NOCTURNAL HEMOGLOBINURIA
Alvarez, M.M.P.1, Henriques, F.S.2, Batista, M.L. Jr2, Moura, G.E.D.D.1, Nader,
H.B.1, Tersariol, I.L.S.1 and Nascimento, F.D.3
Fernanda Camila Zauli Fabris1, Hanan Chweih1, Wilson Alves F. Junior1, Anglica
Aparecida A. Silveira1, Flvia Costa Leonardo1, Kleber Y. Fertrin1, Rodolfo D.
Canado2, Sara T.O. Saad1, Fernando F. Costa1, Nicola Conran1, Camila B.
Almeida1.
Departamento de Bioqumica, UNIFESP, So Paulo, Brazil; 2Laboratory of Adipose

Tissue Biology, Integrated Group of Biotechnology, University of Mogi das Cruzes,
So Paulo, Brazil; 3Biotechnology in Health Program, Universidade Anhanguera de
So Paulo, So Paulo, Brazil.
Author email: mpalvarez.marcela@gmail.com
Address: Rua 3 maio, 100; Vila Clementino, 4 andar; Disciplina de Biologia
Molecular, departamento de Bioqumica, So Paulo, Brazil.
Telephone contact: Institutional (+55) (11) 55764442 ramal: 1194/ Mobile (+55)
(11) 997661252
Center of Hematology and Hemotherapy, School of Medical Sciences, University of

Campinas (UNICAMP)
2
Department of Hematology and Oncology, School of Medical Sciences, Santa Casa
de So Paulo.
Contacts
Email: Fernanda-fabris@hotmail.com
Phone: (19) 988331239
BACKGROUND. Protease-activated receptors (PARs) are G protein-coupled

receptors, which are activated by proteolytical cleavage of the amino-terminus and
thereby act as sensors for extracellular proteases. PARs activation plays important
roles in the regulation of development, inflammation, immunity, and angiogenesis.
AIM. The aim of the current study was to investigate the expression and role of
PARs in mouse odontoblast-like cells (MDPC-23).METHODS. Regulation of PARs
expression in MDPC-23 cells was investigated by reverse transcriptase-polymerase
chain reaction (RT-PCR) in the absence or in the presence of LPS (0.1 g/ml).
Antibodies against the PAR-1 and PAR-2 were used to investigate the cellular
expression of these receptors using flow cytometry and confocal microscopy.
Ca2+ signalling concentrationresponse curves were obtained using different PARsspecific agonists, thrombin for PAR-1 or trypsin for PAR-2. Cytoplasmic Ca2+ influx
were monitored through changes in the Fluo-4 fluorescence intensity in real time
using Flex Station. After a baseline reading for 30s, cells were exposed to graded
concentrations of PARs agonists. RESULTS AND DISCUSSION. RT-PCR and
immunocytochemical studies, demonstrated expression of PAR-1 and PAR-2 by
MDPC-23. In confocal microscopy immunohistochemical studies of human dentalpulp complex showed positive staining for PAR-1 and PAR-2. Treatment of MDPC23 cells with trypsin, or with rat PAR-2-activating peptide (SLIGRL), caused a dosedependent increase cytoplasmic Ca2+ influx. LPS-induced the mRNA expression of
IL-6, TNF-, PAR-1 and PAR-2 in MDPC-23 cells. CONCLUSION. The results
demonstrate that odontoblasts like cells express PAR-1 and PAR-2 receptors,
suggesting that both receptors are involved in the inflammatory response.
Background: Hemolytic diseases share some hemolysis-induced complications. The

release of heme and hemoglobin into the circulation results in the depletion of nitric
oxide, cell damage, and chronic inflammation. Aims: To compare the hemolytic and
inflammatory profile in patients with sickle cell anemia (SCA), hereditary
spherocytosis (HS) and paroxysmal nocturnal hemoglobinuria (PNH). Methods:
Blood samples were collected from patients and from healthy volunteers (CON).
Plasma levels of heme and tumor necrosis factor alpha (TNF-) were quantified by
colorimetric assay or ELISA. Numbers of reticulocytes (RETC), cell counts and
serum levels of lactate dehydrogenase (LDH), indirect bilirubin (IB), were quantified
using an automation system. Ethical approval:28247514.2.0000.5404; April 2014.
Results: Neutrophil counts were decreased in PNH, compared to CON (1.8 2.1;
4.8 5.7x103uL-1, respectively, P<0.05 N12). RETC and IB were increased in SCA
and HS, compared to CON (RETIC:16.418.0; 7.68.0; 2.02.0%;IB:2.13.8;
1.93.0; 0.30.4mg/dl, respectively, p<0.01 N10). Heme and TNF-, were
increased in SCA, compared to CON (heme:57.868.0; 22.426.0M; TNF:1.72.5; 0.91.2pg/ml; respectively, p<0.05 N11). LDH were increased in SCA
and PNH, compared to CON (409800; 11952600; 130195U/L; SCA, PNH and
CON, respectively, p<0.01 N11). Conclusion: In our study, high levels of heme,
LDH and TNF- observed in SCA are indicative of intravascular hemolysis and
extensive vascular inflammation. Data indicate that intravascular hemolytic events
may be less frequent in HS and less severe than in SCA. Regarding to PNH our
results indicates mild inflammatory processes with high serum LDH only; it is not
possible to state at this time that this inflammation is due to hemolysis.
Keywords: Protease-activated receptors; Odontoblast; MDPC-23

inflammation.
Supported by CAPES, CNPq and FAPESP. CEP N: 2534010915.
Funding support: FAPESP
cells;
B19
B20
ROLE OF THE OBESITY ON THE IMMUNOMODULATION OF THE

EXPERIMENTAL INFECTION BY MYCOBACTERIUM BOVIS BCG
COUPLING AIR POLLUTION AND INFLAMMATION TO DNA REPAIR:

AN IN-VIVO STUDY
Nathlia do Nascimento Gonalves (1), Gabriel S. C. Rodrigues (1), Heloisa D`Avila

da Silva Bizarro (1)
Nilmara de Oliveira Alves (1), Adriana M. de Oliveira Fonoff (1), Wesley Fotoran
(2), Gustavo Satoru (2), Giovanna Mamesso (1), Marlise di Domenico (2), Sarah G.
de Menezes (3), Janana Torres (1), Natlia S. X. Costa (1), Gabriel R. Jnior (1),
Mariana Veras (1), Carlos F.M. Menck (2) and Paulo Saldiva (1)
(1) Laboratory of Cell Biology, Federal University of Juiz de Fora, Juiz de Fora,
Brazil
E-mail: nathaliang.ufjf@gmail.com
Presenter contact details:
Address: Rua Bahia, 212, Bairro Jardim Amrica, CEP: 36.400-000 - Conselheiro
Lafaiete -Minas Gerais, Brazil.
Telephone contact: (31)3721-3401, (31)99989-4012 (mobile)
Obesity is a major health problem that is associated with chronic inflammatory
response of adipose tissue. Tuberculosis is an infectious disease caused by
intracellular bacterias from M. tuberculosis complex. Studies suggest obesity can
modulate immune responses to pathogens and apparently helps patients with
tuberculosis and HIV infection, because HIV positive obese patients have a lower
death rate than lean patients. The aim of our study was to investigate the relationship
between obesity and infection by BCG in mice. We used C57Bl6 mice treated with
high sugar chow. Normal chow was used as control. After three months, the animals
were infected with BCG intrapleurally. The control received saline (#109/2012
CEUA/UFJF). At 24 hours of infection, we observed an increase in fat mass in the
high sugar diet and a significant reduction in leukocyte influx to the pleural cavity in
obese animals, as well as migration of neutrophils and eosinophils in obese mice.
Also, we observed a reduced number of lipid bodies in obese animals compared to
lean. These results suggest that obese animals apparently has a slower development
of infection that would be beneficial to the host, since the pathological symptoms of
tuberculosis are resulting in an exaggerated immune response to the bacterium and
slow evolution can induces less tissue damage.
Support: FAPEMIG; CNPq, PROPESQ/UFJF.
1 University of So Paulo, School of Medicine, So Paulo, Brazil.

2 University of So Paulo, Institute of Biomedical Sciences, So Paulo, Brazil.
3 University of So Paulo, School of Veterinary Medicine and Animal Science, So
Paulo, Brazil.
email:nilmaraoalves@gmail.com
Fone # 55.11.3061.8531 / # 55.11.98790.9690
Particulate matter (PM) from air pollution in So Paulo is, on average, twice higher
than the limit established by the World Health Organization (WHO). The PM
ensemble was designated as a carcinogenic to humans by the WHO. However, which
pathways it may contribute to cancer risk should be studied. It is long known that
exposure to air pollutants can cause various health effects such as increased
inflammatory response and DNA damage. One of the most versatile defense
mechanisms against the accumulation of DNA damage is the nucleotide excision
repair, which includes the XPC protein. The effects of DNA damage caused by PM
regarding the lung inflammatory response are largely unknown. In this study, we
injected intravenously Intercellular Adhesion Molecule-1 (ICAM-1, involved in
neutrophil infiltration) targeted nanoparticles and evaluated the inflammatory
response by in vivo imaging of these particles. Mice were exposed to an accumulated
dose (concentration vs time of exposure) of 600 g.m-3 during 1 hour, which
corresponds to an average concentration of 25 g.m-3 in 24 hours. Analysis on the
day after the exposure has shown a significantly stronger fluorescent signal for antiICAM in the polluted group than the filtered air group. Furthermore, among the
animals exposed to ambient pollution, XPC mice have shown a stronger
inflammatory response relative to wild mice. The expression of pro-inflammatory
cytokines in the lungs is currently being evaluated. These data demonstrate that
inhalation directly to air pollution in So Paulo promotes the acute inflammatory
responses in mice, especially in XPC knockout mice.
Ethical approval: N 173/14
(1)
(2)
B21
B22
IN VIVO AND IN VITRO 5-FLOUROURACIL EFFECTS ON OVARIAN

PREANTRAL FOLLICLES
HUMAN LYSOZYME-EXPRESSING TRANSGENIC GOAT MILK

REDUCES MALNUTRITION-INDUCED INTESTINAL INFLAMMATION
IN WISTAR RATS
Juliana Zani de Almeida (1) , Laritza Ferreira Lima (2), Anna Clara Accioly Ferreira
(3), Hudson Henrique Vieira Correia (2), Naza Arcngela Ribeiro de S (2) ,
Carolina Maside Mielgo (2), Luis Alberto Vieira (2), Valdevane Rocha Arajo (2),
Cludio Cabral Campello (2), Jos Ricardo de Figueiredo (2), Reinaldo Barreto Ori
(1)*
(1)
(1) Laboratory of the Biology of Tissue Healing, Ontogeny and Nutrition,
Department of Morphology and Institute of Biomedicine, School of Medicine,
Federal University of Ceara
(2)
(2) Faculty of Veterinary Medicine, Laboratory of Manipulation of Oocytes and
Preantral Follicles (LAMOFOPA), State University of Ceara (UECE)
(3)
* e-mail: oria@ufc.br, telephone: + 55 85 33668239
(4)
5-fluorouracil (5-FU) chemotherapy may lead to premature ovarian failure and

collapse of a pool of resting preantral follicles. The objective of this study was to
investigate the in vivo and in vitro 5-FU effects on ovarian preantral follicles. In the
in vivo study, either 5-FU (450 mg/Kg) or saline solution was intraperitoneally
administered to C57BL6J young mice (27 days of age) and the experimental mice
were sacrificed after 3 days. The ovaries were collected and the number of follicles
examined to assess their viability and ultrastructure. Protocols were approved by the
UECEs Ethical Committee. In the in vitro experiment, preantral follicles were
isolated and cultured for 12 days, according to the following treatments: control
medium and control medium with 5-FU at 0.1; 0.2; 0.3; 1.0; 3.0; 10.0 or 30.0 mM.
Survival, diameter, follicular growth and chromatin configuration were assessed after
in vitro maturation (IVM). After in vivo 5FU challenge, the primordial, transitional,
and primary follicle number were significantly lower compared to the control.
Following 5FU exposure, granulosa cells showed the presence of large vacuoles and
loss of mitochondrial cristae. 5-FU at a concentrations of 0.3, 3.0, 10.0, and 30 mM
caused significant reductions in the survival follicular rates (76.14%; 76.60%;
73.33%; 76.09%, respectively, p<0.05), in the diameter (<119.6 m) and in follicular
growth (-0.03<m/d) at concentrations greater or equal than 0.2 mM. After IVM a
high percentage (90.91%) change in chromatin configuration at concentrations > 1.0
mM was observed. Altogether, our findings suggest that 5FU deleteriously affects
ovarian preantral follicles.
(1) Laboratory of Molecular Biology and Development, University of Fortaleza,

Avenida Washington Soares, 1321, Edson Queiroz, 60.811-905, Fortaleza, Cear,
Brazil.
(2) Experimental Biology Core, Health Center, University of Fortaleza, Av.
Washington Soares 1321, Edson Queiroz, 60.811-905, Fortaleza, CE, Brazil.
(3) Department of Animal Science and Department of Population Health and
Reproduction, University of California, Davis, CA 95616.
Federal University of Ceara, Rua Cel. Nunes de Melo 1315, 60430-270, Fortaleza,
CE, Brazil.
* email: katiane1002@yahoo.com.br, telephone: (85) 34773030
Malnutrition is still a health issue affecting children living in unsanitary conditions,
which may be related to semi-arid or arid climates in the developing world. In such
places, the goat is an important source of protein to local communities. In this study,
we evaluated the protective role of human lysozyme-transgenic goat milk (TGM) in
undernourished Wistar rats. TGM samples were collected on the 3rd week from a
human lysozyme-transgenic goat during normal lactation and the wild-type milk
(non-TGM) from a non-transgenic goat born at the same month and year. The
lysozyme from the TGM was quantified by western blotting and tested for
antimicrobial activity. We examined the effect of TGM (1mL by oral gavage) to
weanling rats, challenged by malnutrition using a northeast regional basic diet (RBD)
for 42 days compared to non-TGM and the recombinant human lysozyme, rhLZ,
(270 g/mL, Sigma) treatment and nourished controls, receiving the standard chow
diet. We also assessed weight gain weekly and villus morphometry. In order to
evaluate the role of TGM on intestinal inflammation, we have measured jejunal
TNF-, IL-6, TGF- and IL-10 cytokines. Protocols were approved by the
UNIFORs Ethical Committee. The TGM showed similar lysozyme expression than
the human milk and conspicuous antimicrobial activity. In addition, the TGM
ameliorated weight gain in undernourished rats. TGM, non-TGM, and rhLZ reduced
TNF- and IL-6 RNAm following RBD-induced malnutrition, however TGM
showed the most anti-inflammatory effect, also improving IL-10 transcript levels
(p<0.05). Altogether our findings suggest that TGM reduced bacterial-driven
intestinal inflammation following RBD.

B24
B23
EFFECT
OF
RESVERATROL
ON
ROS
PRODUCTION
GRANULOCYTES IS NOT AFFECTED BY HYPERGLYCEMIA
Julyana Almeida Maia (1), Igor de S Carneiro (1), Ana Valesca Pinto de Lima (1),
Jos Nilson Menezes (1), Ramon da Silva Raposo (2), Katiane Queiroz da Silva* (1),
Elizabeth A. Maga (3), Luciana Relly Berlolini (1), Reinaldo Barreto Ori (4)
IN
INVOLVEMENT OF FINE AND ULTRAFINE PARTICLES IN THE

INFLAMMATORY RESPONSE DURING EXPOSURE TO AIR POLLUTION
IN SO PAULO
Volpe CMO*, Villar-Delfino PH, Anjos PMF, Nogueira-Machado JA

Ncleo de Ps Graduao e Pesquisa do Instituto de Ensino e Pesquisa Santa Casa
Belo Horizonte
* Corresponding author at: Ncleo de Pesquisa e Ps-Graduao, Hospital Santa
Casa de Belo Horizonte, Rua Domingos Vieira 590, Santa Efignia, 30150-240, Belo
Horizonte, MG, Brazil.
Tel.: +55 31 3238 8838; fax: +55 31 3238 8838.
E-mail address: carolinevolpe@outlook.com
Background: Resveratrol (RSV) has been proposed as a therapeutic resource for
human diseases such as type 2 diabetes mellitus (T2DM).
Aim: To evaluate the effects of RSV on reactive oxygen species (ROS) production
by granulocytes from T2DM patients primed by chronic hyperglycaemia in vivo.
Methods: Study approved by ethics committee of Hospital Santa Casa Belo
Horizonte (022/2010). T2DM patients (n=20) and non-diabetic control (ND,n=20)
were aged between 40 and 70 years. Granulocytes (G) from T2DM and ND were
purified using Ficoll-Hypaque gradient method. Effects of RSV on ROS production
in granulocytes were quantified using a luminol-dependent chemiluminescence
assay. An aliquot of luminol (10ug dissolved in 0.4 M dimethyl sulfoxide) was mixed
with G suspension (1x106cells/mL). ROS production was measured for 40min
without stimulation or in the presence of RSV (10 M), ZY particles recovered with
complement fragments C3b (ZC3b;13mg/mL), phorbol 12,13-dibutyrate (PDB;104
M). Cellular viability was determined by trypan blue exclusion test. Statistical
analysis was performed by 2 test (p<0.05 considered significant).
Results: RSV suppressed ROS generation in granulocytes from T2DM patients and
ND controls in similar manner (p>0.05). Percentage inhibition values for T2DM
patients and ND controls were, respectively, G+RSV: 80 and 63; G+ZC3b+RSV: 75
and 82; G+PDB+RSV: 69 and 77.
Conclusion: The effect of RSV is not affected by hyperglycemia since ROS
production in granulocytes from T2DM patients and ND controls was inhibited by
the polyphenol in similar manner (p>0.05) under different assay protocols.
Giovanna Mamesso Di Costanzo*(1), Adriana M. de Oliveira Fonoff (1), Mariana

Veras (1), Carlos Menck (2), Paulo Saldiva (1) and Nilmara de Oliveira Alves (1).
(1) University of So Paulo, School of Medicine, So Paulo, Brazil.
(2) University of So Paulo, Institute of Biomedical Sciences, So Paulo, Brazil.
*Institutional:
(11)
3061-8531,
gimcostanzo@gmail.com.
mobile:
(11)
96579-1845,
e-mail
Exposure to particulate matter (PM) is related to the onset and exacerbation of

respiratory diseases. Fine and ultrafine particles can be active carriers of toxic
compounds into the alveoli of the lungs. There are various effects caused by
exposure to urban pollution to human health, such as the increase of acute
inflammatory diseases. The present study evaluated the inflammatory response
caused by atmospheric pollution of So Paulo using wild and knockouts for the
repair gene XPC mice. For this work, animals were exposed an accumulated dose
(concentration vs time of exposure in hours) of 600 g.m-3 during 1 hour, which
corresponds to an average concentration of 25 g.m-3 in 24 hours. The animals were
divided into 3 groups: filtered air, polluted air and lipopolysaccharide inhalation
(positive control). For the analysis of blood cells on a count of leukocytes was
performed by differentiating cell to cell. The results show a decrease of neutrophils
both wide and XPC mice after the exposure of PM in blood cells. Interestingly, in
XPC animals, we noted an increase of monocytes after inhalation of pollutants. In
addition, it was detected the presence of fine and ultrafine particles (0.01 PM 2.5
m) in the atmosphere of So Paulo. Therefore, this study shows that air pollution of
So Paulo cause acute inflammatory effects in the studied animals, particularly in
XPC mice. These effects can be associated with the presence of fine and ultrafine
particles in the air.
Ethical approval: N 173/14
Resources of research support: FAPEMIG, CAPES, CNPq and Rede Mineira de

ToxinaTeraputica 26/12.

B25
B26
IMPACT OF RENAL INSUFFICIENCY IN THE IN VIVO CARDIAC

TISSUE: THE CONTRIBUTION OF CELL APOPTOSIS IN CARDIAC
REMODELING
BETWEEN MICE AND MEN: IS THE WHITE ADIPOSE TISSUE ALL THE
SAME?
Frayli Maltoni Fratoni (1), Mayra Trentin-Sonoda (1), Marcela Sorelli Carneiro
Ramos (1)
1. Centro de Cincias Naturais e Humanas, Universidade Federal do ABC
Main Author: Frayli Maltoni Fratoni
Email: fmaltoni@gmail.com
Phone: (11) 4996-7478 // (11) 99157-6055
Background: The immune system can act in the cardiac tissue through the liberation
of pro and anti-inflammatory cytokines, which has a direct impact on cell survival.
Renal Ischemia (RI) can induce the proliferation of these cytokines, whose action
can modulate the cardiac hypertrophy (growing), and remodeling (growing and
apoptosis balance).
Aim: The aim of this study is the analysis of apoptotic pathways activated in cardiac
hypertrophy induced by RI.
Methods: C57BL/6 male mice and C57BL/6 Cas-1 -/- approved by the ethics
committee were subjected to surgical occlusion of the left renal pedicle for 60min,
followed by reperfusion (I/R) for 15 days. Morphometric analyses were performed
using heart weight (HW), body weight (BW) and tibia length (TL) as parameters.
Real time PCR was used for analysis of hypertrophic and apoptotic genes expression.
Results: Both WT and Cas-1 -/- I/R groups presented cardiac hypertrophy when
compared to Sham groups, especially due to an increase in the HW/BW and the
HW/TL ratios. About the apoptotic components, analysis of the mRNA expression
showed a significant increase on Bcl-2, Caspase-3, Caspase-9 and AIF levels in the
WT I/R mice at 12 days after reperfusion (p<0.05). Further analysis are being made
regarding the Caspase activity and the mRNA levels in the Cas-1 -/- group.
Conclusion: Cardiac apoptotic components are upregulated in WT I/R mice when
compared to Sham mice, leading to a speculation on their level of expression in Cas1 -/- mice, especially considering the promotion of cell death by Caspase-1.
Financial Support: FAPESP
Sally Liechocki (1); Glaucia Souza Almeida (1); Karina Ribeiro Silva (3); Joo
Regis Carneiro (3); Leandra S Baptista (4); Daniel Antnio Lopes Ferreira (2);
Leonardo V Coelho (2); Ana Carolina Nader Vasconcelos Messias (2); Hugo Castro
Faria Neto (1); Jens Rietdorf (1,5); Patricia Torres Bozza (1), Clarissa M Maya
Monteiro (1).
(1) Laboratrio de Imunofarmacologia Instituto Oswaldo Cruz/FIOCRUZ- Rio de
Janeiro, Brazil; (2) Hospital dos Servidores do Estado do Rio de Janeiro-HSE Rio
de Janeiro, Brazil; (3) Departamento de Clnica Mdica da Faculdade de Medicina UFRJ Rio de Janeiro, Brazil, (4) Instituto de Biofsica UFRJ - Rio de Janeiro,
Brazil and (5) CDTS/FIOCRUZ- Rio de Janeiro, Brazil.
Correspondence author: sallyliechocki@yahoo.com.br
Adipose tissue is a dynamic organ with impact in immune system, besides the
regulation of energy homeostasis. Obesity is characterized by elevated expansion of
adipose tissue, accompanied by a moderate chronic inflammatory condition directly
related with the development of others co-morbidities. Here we investigated the
expression of genes and proteins related to lipid droplets and inflammation on
different adipose tissue depots of morbidly obese patients and experimental model of
obesity. Samples of human adipose tissue (hWAT) were obtained during bariatric
surgery and murine white adipose tissue (mWAT) was obtained from mouse
CC57Bl/6 submitted to normal diet (ND) and high-fat diet (HFD). All hWAT depots
analyzed by qPCR assay showed the same expression levels of perilipin, ADRP,
FABP4, ADIPOQ and LEPR. Besides of TNF- content variety among WAT depots
verified by Western Blot, we found interesting results on cytokine analysis of hWAT
lysate, which was observed lower amount of MCP-1 and leptin in omentum. Despite
of adipocyte size in mWAT/ND and distinct hypertrophy as result of HFD, we
observed similarity on perilipin and SREBP-1 protein expression between four
mWAT depots evaluated. Intriguingly, PPAR1 was almost abolished in all
mWAT/HFD mice. Our results show that distinct WAT depots could contribute in a
different manner to molecular alterations on obesity. Moreover, although murine
experimental model of obesity has been very useful on the study of this disease, we
should consider some particularity before making a direct comparison between
murine and human WAT.
Financial support: CAPES, CNPQ, FAPERJ, CDTS and FIOCRUZ. Ethical
approval: HFSE:000.528(human) and L011/2015(animal).

B28
B27
HISTOPATHOLOGICAL AND INFLAMMATORY EFFECTS ON THE
LIVER OF C57BL6J MICE SUBMITTED TO THE REGIONAL BASIC DIET
Maria Josiane S. Santos (1), Kildere Marques Canuto (1), Cristhyane Costa de
Aquino (1), Conceio da Silva Martins (2), Dulce Maria Nascimento Coelho (2),
Ana Carolina Bencio Alves (1), Jardlon Albino Costa (1), Bruno Bezerra da Silva
(3), Flvia Almeida Santos (4), Gerly Anne de Castro Brito (2), Maria Izabel
Florindo Guedes (3), Reinaldo Barreto Ori (1)*
(1)
(2)
(3)
(4)
Laboratory of the Biology of Tissue Healing, Ontogeny and Nutrition,

Department of Morphology and Institute of Biomedicine, School of
Medicine, Federal University of Ceara, Fortaleza, CE, Brazil
Microscopy and Imaging Core, Department of Morphology and Institute of
Biomedicine, School of Medicine, Federal University of Ceara, Fortaleza,
CE, Brazil
Molecular biology and biotechnology laboratory, State University of Ceara
(UECE), Fortaleza, CE, Brazil
Laboratory of Natural Products, Department of Physiology and
Pharmacology, Institute of Biomedicine, School of Medicine, Federal
University of Ceara, Fortaleza, CE, Brazil
* email: kilderekanuto@yahoo.com.br, telephone: (85) 33668239

Malnutrition is still considered endemic in many developing countries. In Brazil, the
prevalence of malnutrition has declined; however, malnutrition may cause growth
and cognitive deficits. The regional basic diet (RBD) has been produced to mimic the
Brazilian Northeastern dietary characteristics. This study aimed to investigate the
effect of RBD on liver histopathology and inflammation in C57BL6/J mice,
compared to the nourished group, receiving a standard chow diet. Animals were
weighed daily. After 40 days, blood was drawn for ALT serum levels to assess liver
function. The liver was obtained for histopathology and to assess TNF- and IL-10
transcription by qRT-PCR and IL-1 and the ionized adapter calcium protein-1
(IBA-1) immunohistochemistry for Kupffer cells. We also assessed reverse
cholesterol transport markers (apoE, apoA-1 and LCAT). Protocols were approved
by the UECEs Ethical Committee. The RBD decreased body weight gain compared
with the nourished group (p<0.05). No statistical difference was found in serum ALT
levels between experimental groups. In histological sections, we identified greater
IBA-1 immunolabeling and a significant increase in the number of positive-IBA-1
cells in the undernourished group. Greater TNF- (p<0.0001) and IL-10 (p=0.001)
mRNA expression was found in RBD-challenged mice. We also found significantly
increase in apoA mRNA levels in the undernourished group (p=0.04), compared with
the control. Altogether our findings suggest that RBD-induced malnutrition leads to
liver inflammation, indicated by a stronger IL-1 immunolabeling and increased
number of IBA-1 labeled liver cells, as well as, increased expression of TNF- and
IL-10 transcripts and altered apoA-I RNAm levels.
ATORVASTATIN DOES NOT AFFECT ROS PRODUCTION IN

GRANULOCYTES IN DAG-PKC-NADPH OXIDASE SIGNALING
PATHWAY
Anjos, P.M.F., Volpe, C.M.O., Villar-Delfino, P.H., Nogueira-Machado, J.A.
Ncleo de Ps-Graduao e Pesquisa do Instituto de Ensino e Pesquisa da Santa
Casa de Belo Horizonte
Background: Atorvastatin (ATV) is a kind of statin and it is known to inhibit the
cholesterol biosynthesis. Furthermore, it has been reported that ATV decrease the
NADPH-oxidase system activity.
Aims: To evaluate the effect of ATV on reactive oxygen species (ROS) production
in granulocytes from healthy individuals.
Methods: Study approved by ethics committee of Hospital Santa Casa Belo
Horizonte (022/2010). Granulocytes (G), collected from 10 healthy individuals were
aged between 40 and 70 years, were obtained from venous blood samples and were
purified using Ficoll-Hypaque gradient method. ROS production was determined by
a luminol-dependent chemiluminescence assay. An aliquot of luminol (10g
dissolved in 0.4M dimethyl sulfoxide) was mixed with G suspension
(1x106cells/mL). ROS production was measured for 20min without stimulation or in
the presence of phorbol 12,13-dibutyrate (PDB;10-4M) and ATV (20g/100L).
Cellular viability was determined by trypan blue exclusion test. Statistical analysis
was performed by Mann-Whitney test (p<0.05 considered significant).
Results: Our preliminary results expressed in RLU (median) were G+PBS= 3018;
G+ATV= 1684; G+PDB= 19967; G+PDB+ATV= 7022; G+ATV+PDB= 21597. The
results demonstrate that G+PDB and G+ATV+PDB significantly increased (p<0.05)
ROS production compared to their controls, G+PBS and G+ATV, respectively.
Conclusion: ATV does not inhibit ROS in DAG-PKC-NADPH oxidase signaling
pathway.
Resources of research support: FAPEMIG, CAPES, CNPq and Rede Mineira de
Toxina Teraputica 26/12.

B29
B30
THE APOE COG 1410 MIMETIC PEPTIDE MODULATES THE WNT/

CATENIN PATHWAY DURING THE INTESTINAL IEC-18 CELL
MONOLAYER HEALING AFTER 5-FLUOROURACIL CHALLENGE
EFFECT OF ACUTE EXPOSURE TO MARIJUANA SMOKE IN THE

MYOCARDIUM
Ticiana Maria Rangel de Paula Pessoa (1)*, Igor de S Carneiro (1), Luciana Relly
Bertolini (1), Reinaldo Barreto Ori (2), Jos Garcia Ribeiro Abreu Jnior (3)
(1)
(2)
(3)
Laboratory of Molecular Biology and Development, University of Fortaleza,

Fortaleza, Cear, Brazil.
Laboratory of the Biology of Tissue Healing, Ontogeny and Nutrition,
Department of Morphology and Institute of Biomedicine, School of
Medicine, Federal University of Ceara, Fortaleza, CE, Brazil
Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio
de Janeiro, RJ, Brazil
*email: ticianapessoa@unifor.br; telephone: (85) 33668239

Intestinal mucositis is one of the side effects of the 5-fluorouracil (5-FU)
chemotherapy. Apolipoprotein E (ApoE) has anti-inflammatory effects on brain
injury models. This study examined the effect of the ApoE COG1410 mimetic
peptide during IEC-18 cell monolayer healing, following 5-FU challenge and the
Wnt/-catenin pathway involvement. IEC-18 monolayers were exposed to 5-FU
(1mM) in glutamine-free medium with or without the COG1410 peptide (0.3, 1.0,
and 3.0 uM). Cell viability was assessed 12, 24 and 48h by using the MTT assay and
migration by using the monolayer wound model after 24h. Gene expression analyses
were performed after 24h by RT-qPCR for -catenin, axin, APC, TCF4, LEF, c-myc
and cyclin D1. In glutamine containing standard media, the COG1410 (1.0 and 3.0
uM) increased IEC-18 cell viability (p<0.0001) after 12 and 24h compared to the
untreated 5FU-challenged group (p<0.0001). In 12, 24 and 48h assays, the COG1410
increased significantly cell viability after 5-FU, compared to the untreated
challenged control in glutamine-free medium (p<0.0001). After 24h post-5-FU, cell
migration was reduced in the challenged groups when compared to controls. The
COG1410 (1M), increased the number of migrating cells, following 5-FU injury. In
the healing response to 5-FU, an increased gene expression for axin, APC and TCF4
was found when compared to controls, regardless of peptide treatment. However,
COG1410 increased LEF expression. 5-FU challenge reduced -catenin, c-myc and
cyclin D1 transcripts, effects which were significantly improved by COG1410.
Altogether our findings suggest a COG1410 protective role after 5-FU challenge, by
modulating the Wnt/-catenin pathway.
Janaina Iannicelli Torres (1), Giovanna Mamesso Di Costanzo (1), Iran Augusto Neves
da Silva (2), Daiana Aparecida Souza Viana (2), Caio Cesar Silva Viana (2), Mariana
Matera Veras (1 and 2) and Adriana Morgan de Oliveira Fonoff. (1)
(1) Laboratory of Experimental Air Pollution (LIM05), Department of Pathology,
School of Medicine, University of So Paulo.
(2) Departamento of Anatomy of Domestic and Wild Animals, School of Veterinary
Medicine and Animal Sciences, University of So Paulo, So Paulo, Brazil.
Contact: Adriana Morgan de Oliveira Fonoff
drimorgan@usp.br
Introduction: Cardiovascular diseases (CD) are the non-communicable diseases that
are responsible to huge number global death worldwide. Obesity, weight changes,
sedentary lifestyle, age, metabolic factors, high cholesterol levels and tobacco
smoking are risk factor for CD. Increasing marijuana smoking habit appears as a new
possible risk factor and has recently been added to the list of potential trigger for
myocardial infarction.
Aims: Assess the effects of acute marijuana exposure on myocardium inflammatory
response in young mice
Methods: Young adult C57mice (n=6, 51 days/age) were daily exposed (nose-only)
to either marijuana smoke [0.4g of Marijuana] (Group MA) or filtered air (Group
FA) during 10 minutes for 6 consecutive days. After completion of the exposure,
hearts were collected, fixed and routinely processed for immunohistochemistry.
Immunoreactivities for IL-6, IL-1 and TNF- were measured as total volume of
positive stained tissue within the myocardium. Fixed hearts were cut into 5-m
sections and stained with hematoxylin and eosin by standard procedures. Sections
underwent immunohistochemical staining with the antibodies against IL-6 (1:500),
IL-1 (1:200) and TNF- (1:3000) - (Abcam, Cambridge, MA).
Results: Our data indicate that the immunoreactivity of acute inflammatory markers
in the MA myocardium is higher, however data fail to achieve statistical
significance. Possibly because the number of animals have been reduced.
Conclusion: Our data suggest that cardiovascular health of young adults may be
compromised by marijuana smoking habit.
Key words: Myocardial, cannabis, young, inflammation, cytokines.
Granted by FAPESP
Ethics Committee - CEUA n. 036/16

B32
B31
EXPOSURE TO CANNABIS SATIVA SMOKE CAUSES INFLAMMATORY
RESPONSE AND APOPTOSIS IN MICE: AN IN VIVO IMAGING STUDY
Janaina Ianicelli Torres (1), Giovana M. Di Constanzo (1), Adriana M.Oliveira
Fonoff (1),Nilmara de Oliveira Alves(1), Gustavo Satoru (3), Wesley Fotoran (3),
Carlos F.M. Menck (3), Mariana Matera Veras (1,2);
(1) Department of Pathology, School of Medicine, University of Sao Paulo,
(1)
Sao
Paulo, Brazil;
(2)
(2) Department of Surgery, School of Veterinary Medicine and Animal Sciences,
University of Sao Paulo, Sao Paulo, Brazil.
(3) Institute of Biomedical Sciences (University of So Paulo, So Paulo, Brazil)
Corresponding author: Department of Pathology, School of Medicine University of
Sao Paulo. Dr. Arnaldo Avenue, 455, 1st floor - room1220, Cerqueira Csar, 01246903. Sao Paulo, SP, Brazil. phone: +5511 30618531 mobile: +5511 986299385. Email address: janainaiannicelli@hotmail.com.
Background: Marijuana (Cannabis sativa) is one of the most widely abused
substances throughout the world. In this context, it is an urgent need to investigate
the effects of the use of these substances on health.
Aim: To evaluate inflammatory response and cell death after exposure to Marijuana
smoke using in vivo imagining.
Methods: Balb/c mice (n=6) were exposed daily (nose-only) to either Marijuana
smoke [0.2g of Marijuana] (Group MA) or filtered air (Group FA) during 5 minutes,
from 21 to 90 post natal day. After, we injected intravenously Intercellular Adhesion
Molecule-1 (ICAM-1, involved in neutrophil infiltration) and Annexin-V (involved
in cell death by apoptosis) targeted nanoparticles and evaluated these responses by in
vivo imaging using IVIS spectrum.
Results: The data showed a significantly stronger fluorescent marker for anti-ICAM
in the Group MA than the Group FA. We also observed that the inhalation of
Marijuana smoke induced apoptotic effects. Furthermore, among the animals of
Group MA, female mice were more sensitive regarding both the inflammatory
response and cell death. In particular, imaging analysis indicated that these effects
are co-localized in the liver.
Conclusions: Our results showed that inhalation of smoke of Marijuana in real world
conditions of use and dose cause an increase both inflammatory response and
apoptosis in mice. These results are pioneer and relevant to discuss the implications
of its use with emphasis on their effects on health.
PRODUCTION OF PGE2 BY VASCULAR SMOOTH MUSCLE CELLS

UPON STIMULATION BY A SNAKE VENOM METALLOPROTEINASE IS
DEPENDENT ON COX-1 AND COX-2 PATHWAYS
Mariana Viana (1), Karina Cristina Giannotti (1), Jos Maria Gutirrez (2), Catarina
Teixeira (1)
(1) Laboratory of Pharmacology, Butantan Institute, So Paulo Brazil;
(2) Clodomiro Picado Institute, San Jose - Costa Rica.
mariana.viana@butantan.gov.br
Background: During atherosclerotic processes, matrix metalloproteinases (MMPs)
induce migration and proliferation of VSMCs by unknown mechanisms. Snake
venom metalloproteinases (SVMPs) are relevant tools for studies on the actions of
MMPs in VSMCS. Aims: To analyze the effects of the snake venom
metalloproteinase BaP1 in VSMCs, evaluating: a) cell migration, b) release of PGE2,
PGI2 and TXA2, c) protein expression of COX-1 and -2, mPGES-1, EP2 and EP4
receptors and their participation in PGE2 release and d) participation of PGE2 in
VSMC migration. Methods: VSMCs isolated from male Wistar rat aortic artery
(CEUAIB 1128/13) were incubated with BaP1 (5, 25, 50nM) or DMEM (control) for
1- 48 h. VSMCs migration was evaluated by transwell assay, released mediators by
EIA and protein expression of COX-2 and PGE2 receptors measured by western
blotting. Results: BaP1, at 50nM induced VSMCs migration (24h up to 48h
incubation). This metalloproteinase (50nM) induced release of PGE2 and protein
expression of both COX-2 and mPGES-1 in VSMCs, from 1 up to 48 h incubation.
However, PGI2 and TXA2 levels and EP4 and EP2 receptor expression were
unchanged by BaP1. Treatment of VSMCs with COX-1 and COX-2 inhibitors, but
not with EP2R nor EP4R antagonists, abrogated PGE2 release. All of these
treatments did not affect BaP1-induced VSMCs migration. Conclusion: BaP1 is able
to directly stimulate VSMCs for migration, PGE2 release and COX-2 protein
expression. COX-1 and -2 are involved in PGE2 production induced by BaP1 in
VSMCs.
Support: CAPES, CNPq and FAPESP.
Ethics Committee approval CEUA n 039/16.

Funding support: CNPq and CAPES.

B33
B34
ANTI-ANGIOGENIC, ANTI-PROLIFERATIVE AND ANTI-MIGRATORY

EFFECTS OF CHEMICALLY MODIFIED HEPARINS
EFFECT OF JARARHAGIN C, A DESINTEGRIN-LIKE PROTEIN

PURIFIED FROM BOTHROPS JARARACA VENOM IN MICE IMPLANT
SPONGE MODEL
Sousa, M.E.P.1; Russo, T.A. 1; Nader, H.B.1, Dreyfuss, J.L.1,2

1. Department of Biochemistry, Discipline of Molecular Biology, Laboratory Carl
Peter von Dietrich; 2. Department ofHealth Informatics, Interdisciplinary Group for
Science in Health Technology, Paulista School of Medicine, Federal University of
Sao Paulo
Background: Angiogenesis is the formation of new blood vessels from pre-existing
vasculature. Angiogenesis is a complex process constituting multiple steps.
Extracellular matrix degrading enzymes secreted by endothelial cells (ECs) degrade
the basement membrane, allowing the migration and proliferation, resulting in the
formation of endothelial cell capillary tubes. Heparin and its related polysaccharides
have important functions as regulators of vessel growth and regression. This
regulatory activity may be explained by the interaction of heparin with growth
factors and other bioactive molecules.
Aims: Evaluate the effects of chemically modified heparins observing their possible
potentials as anti-proliferative, anti-migratory and anti-angiogenic agent in cultured
ECs.
Methods: The effect of O-N-desulfated and 2-O-desulfated heparins in proliferation,
formation of capillary-like structures, cytotoxicity, adhesion and migration were
assessed in ECs cultures.
Results: The treatment of ECs with O-N-desulfated heparin showed an antiproliferative and anti-adhesive potential, and an increased migratory effects upon
ECs cultures. The 2-O-desulfated heparin demonstrated an anti-proliferative, antiadhesive and anti-migratory effect in ECs cultures. No effects were found regarding
the tube formation on capillary-like structures assay.
Conclusion: The treatment of ECs with chemically modified heparins aims to
prospect a new drug to treat angiogenesis. These two heparins, the O,N-desulfated
and 2-O desulfated heparins have shown anti-proliferative, anti-adhesive and antimigratory effects on ECs but no effects on endothelial morphogenesis and capillary
assembly. We need further studies to determine the efficacy of these heparins on
inhibiting EC proliferation and angiogenesis. In vivo studies using choroidal
neovascular models will be used in our study.
Simone Ramos Deconte (1), Bruno Antonio Ferreira (1), Patricia Bianca Clissa (2) ,
Fernanda Arajo de Assis(1).
(1) Institute of Biomedical Sciences ICBIM-UFU, Federal University of Uberlndia UFU, Uberlndia, Brazil.
(2) Department of Health, Laboratory of Immunopathology, Butantan Institute So
Paulo, Brazil.
Contact - Simone Ramos Deconte, Federal University of Uberlndia - UFU,
Uberlndia, Brazil. Par Street, 2A Superior Block, Umuarama Campus,38405-000,
institutional (5534)3225-8472, mobile (5534)99335-0524, srdufu@gmail.com
Background: Jararhagin C is a 28KDa protein with a desintegrin-like structure
formed from a proteolytic cleavage of Jararhagin. Their functions are still being
studied, but there are evidences that this desintegrin-like protein can act altering the
inflammatory response. Aims: We investigated the effects of 9 days treatment of
Jararhagin C on key components of inammatory angiogenesis in polyesterpolyurethane sponges, used as a framework for a brovascular tissue growth.
Methods: Jararhagin C treatment was realized by intra-implant administration in
male Balb-c mice aged 78 weeks at 20, 200 and 2000ng doses. The implants
collected at day 9 post-implantation were processed for the assessment of
hemoglobin, myeloperoxidase (MPO), N-acetylglucosaminidase (NAG) and collagen
content. Results: Jararhagin C treatment did not change the vascularization in
implants, as showed by the results in the hemoglobin content. After Jararghin C
treatment the neutrophil content (MPO) was increased at 200ng dose (p<0.1) and
2000ng dose (p<0.01). Macrophage activity (NAG) was increased at 200ng (p<0,5)
and 2000ng (p<0,01) doses treatments. Collagen deposition was increased at 20 and
200ng doses (p<0.05) compared to control group. Conclusion: The results showed
the effectiveness of Jararhagin C in modify some parameters of the main components
of inflammatory response. The pro-inflammatory effects of Jararhagin C revealed in
this study can be associate with the capacity of desintegrin-like/cysteine-rich
domains directly or indirectly activate inflammatory response by association with
integrins receptors in the cell membrane.
Financial support: CNPq, FAPESP.

This project was approved by ethics committee UFU (158/13) and had the funding
support of CAPES, CNPq and FAPEMIG.

B36
B35
EFFECTS OF PURIFIED EXTRACT ALFA-ZINGIBERENE IN MURINE
MODEL OF CHRONIC INFLAMMATION
Simone Ramos Deconte (1), Ricardo Ferreira Silva (1), Bruno Antonio Ferreira (1),
Joo Henrique Ghilardi Lago (2), Fernanda de Assis Araujo (2).
(1) Institute of Biomedical Sciences ICBIM-UFU, Federal University of Uberlndia UFU, Uberlndia, Brazil.
(2) Institute of Environmental Sciences, Chemical and Pharmaceutical, Federal
University of So Paulo - UNIFESP, Diadema, Brazil.
Contact - Simone Ramos Deconte, Federal University of Uberlndia - UFU,
Uberlndia, Brazil. Par Street, 2A Superior Block, Umuarama Campus,38405-000,
institutional (5534)3225-8472, mobile (5534)99335-0524, srdufu@gmail.com
Background: Inflammation is a complex physiological process, which has specific
symptoms to an offending agent. The Casearia sylvestris (C.sylvestris) is popularly
used as an alternative therapy for anti-inflammatory, antitumor property in fighting
bacterial infections and in case of snakebites, which are due to the chemical
composition of the oils present in the plant extract. Considering the importance of
chemical and biological C. sylvestris, it becomes feasible the use of herbal medicines
as an alternative in the treatment of diseases. Aim: To evaluate the effects of purified
extract (alpha-zingiberene) extracted from C. sylvestris, in a murine model of chronic
inflammation. Methods: polyester-polyurethane implants were implanted in the
subcutaneous tissue of swiss male mice. Alpha-zingiberene extract (doses of
10,100,1000ng) was administered by daily intraimplant injection. Implants were
collected 9 days after surgery and processed for hemoglobin (HB), myeloperoxidase
(MPO), N-acetyl--D-glucosaminidase (NAG) and collagen. P values <0.05 are
considered significant. Results: HB content changed in the group treated with 10 ng
dose against the control group (p <0.001). The enzymatic activity of MPO identified
a pro-inflammatory potential in all concentrations (p <0.001). The enzyme NAG
showed that the extract decreased the activity of macrophages, extending the acute
phase of inflammation (p <0.05). In the repair, the extract decreased deposition of
extracellular matrix in doses of 100 and 1000 ng, slowing the process (P <0.05).
Conclusion: Alpha-zingiberene extract has ability to regulate inflammatory activity
prolonging the acute phase of inflammation and lowering collagen deposition in
higher doses, thereby retarding the repair.
Ethical approval: 153/13 CEUA-UFU
Funding support: CAPES, CNPq, FAPEMIG
TREATMENT WITH NOVEL OXAZOLIDINONE DERIVATIVES

MODULATES THE EXPRESSION OF TRANSCRIPTIONAL FACTORS
INVOLVED IN IMMUNE RESPONSE IN PERIPHERAL BLOOD
MONONUCLEAR CELLS
Wanessa Layssa Batista de Sena (1); Maria do Carmo Alves de Lima (2); Marina
Galdino da Rocha Pitta (2); Maira Galdino da Rocha Pitta (1); Moacyr Jesus Barreto
de Melo Rgo (1); Michelly Cristiny Pereira (1)
Federal University of Pernambuco, Recife, Brazil
(2) Laboratrio de Planejamento e Sntese de Frmacos (LPSF), Federal University
of Pernambuco, Recife, Brazil
Presenting author:
e-mail: wanessasena@live.com
Phone: +55 81 99564-7183
Background. Inflammatory response consists in a process mediated by many cells
and molecules. Since treatments used in order to control inflammatory process can
lead to serious side effects, it is important to search new therapeutic strategies with
high immunomodulatory activity and low side effects. Oxazolidinone derivatives are
a class of molecules with different biological properties described, including antiinflammatory activity.
Aims. Evaluate the modulation of transcriptional factors involved in immune
response promoted by three oxazolidinone derivatives LPSF/NBs.
Methods. Peripheral Blood Mononuclear Cells (PBMC) were obtained from healthy
volunteers and placed in 24-well plates (2x106 per well). Cells were stimulated with
PMA and IONO (100ng/mL and 1g/mL, respectively) and treated with LPSF/NBs
derivatives (LPSF/NB-2, LPSF/NB-3 and LPSF/NB-5) at 50 and 100M. PPAR,
RORC and FOXP3 mRNA levels were measured by Real-Time PCR using 18S
ribosomal as endogenous control. Statistical analysis were performed using Students
t-test and differences were considered significant when p<0.05.
Results. PBMC treated with LPSF/NB-2, LPSF/NB-3 and LPSF/NB-5 derivatives at
50M showed a significant reduction of FOXP3 transcripts when compared to
untreated cells (p=0.0061, 0.0150 and 0.0325, respectively). Treatment with
LPSF/NB-5 also significantly reduced RORC (50M, p=0.0402) and PPAR
(100M, p=0.0264) mRNA levels when compared to control.
Conclusion. Our findings showed that oxazolidinone derivatives LPSF/NBs are
capable to reduce gene expression of transcriptional factors involved in immune
response, suggesting its immunomodulatory potential.
Ethical approval. CEP/CCS/UFPE N 11006/12.
Funding support. FACEPE, CNPq and INCT_if.

B37
B38
IL-1 PRODUCTION BY THE C-TYPE LECTIN BLL IN PERITONEAL

EXUDATE CELLS: ROLE OF CARBOHYDRATE RECOGNITION ON THE
INFLAMASSOME ACTIVATION
IN VIVO BIOCOMPATIBILITY OF DIAMOND-LIKE CARBON FILMS

CONTAINING TITANIUM DIOXIDE NANOPARTICLES
C.C. Wachesk1, J.A. Portes2, S.H. Seabra2. P.G. Theofilo2, P. Singh3, V.J. TravaAiroldi4, A.O. Lobo1, F.R. Marciano1
Aranda-Souza, M.A.1, Silva, A.C. 1, Higino, T.M.M. 1, Correia, M.T.S., Figueiredo2,

R.C.B.Q. 1
1.
2.
Laboratory of Biomedical Nanotechnology - IP&D / UNIVAP So Jos dos

Campos, SP, Brazil.
2
Laboratory Technology in Cell Culture UEZO - Rio de Janeiro, RJ, Brazil,
3
Laboratory of Biomedical Vibrational Spectroscopy - IP&D / UNIVAP - So Jos
dos Campos, SP, Brazil.
4
Laboratory Associated of Sensors and Materials - INPE - So Jos dos Campos, SP,
Brazil.
Centro de Pesquisas Aggeu Magalhes / FIOCRUZ

Universidade Federal de Pernambuco - UFPE
Lectins are proteins that are able to recognize and bind to specific carbohydrate
domains on the cell membrane. The C-type lectins are a widely distributed protein
class with different functions and usually bind to Galactose/Mannose residues.
Moreover, are involved in several immune-related response and other physiological
functions. Macrophages express a wide variety of pathogen-associated molecular
pattern receptors and hold surface rich in sugar-rich glycoconjugates that cannot be
related to the activation pathway of the immune response. Thus, the aim of this work
was investigate the mechanism of the C-type-induced immunomodulation on
macrophage. Peritoneal exudate cells (PECs) were plated at 106 cells/mL
(37C/5%CO2) and treated with BLL or BLL bonded to Galactose for 24h. Then,
incubated with MTT for 3h, solubilized in DMSO and the formazan precipitate was
determined at 540 nm. The supernatants were incubated with Griess reagent for the
quantification of the nitrite concentration and also stained utilizing BD CBA Mouse
Th1/Th2/Th17 Cytokine Kit. Additionally, the cells were submitted to transmission
electronic microscope processing for evaluation of ultrastructural alterations. BLL
treatment of murine PECs did not cause significant changes in the cell viability. In
addition, cells incubated only with BLL showed significant production of NO. Its
samples presented significant increase on proinflammatory cytokine production as
IL-1, INF-, TNF, IL-6 and IL-17) as well anti-inflammatory cytokine IL-10.
Cytokine release were decreased with the Galactose-inhibited BLL treatment. And
IL-1 release was abolished more than other samples when the PECs were
stimulated with sugar-bound BLL. These findings suggest that the lectin-induced
INF- production promotes the stimulus of Th1 response and enhances macrophage
activation and the ultrastructure analysis demonstrated that the cells treated with
lectin presented abnormal structures, possibly a fibrilar aggregates. Overall, our
results suggest that the specific interaction lectin-oligosaccharide on the macrophage
membrane may lead to inflamassome activation in macrophages via increase of IL1 production.
*Cristiane da Costa Wachesk NANOBIO, IP&D, UNIVAP, Av. Shishima Hifumi

2911 Urbanova CEP 12240-000, SJCampos, SP, Brazil. cris_cw@hotmail.com
(12) 981247203
The coating of implants has generated much interest in order to improve boneintegration and to avoid adverse tissue reactions such as infection and inflammation
[1]. Diamond-like carbon (DLC) is a metastable form of amorphous carbon
containing significant fraction of sp3 bonds [2-3]. The incorporation of titanium
dioxide (TiO2) nanoparticles directly into the bulk forms a hybrid coating [4]. The
purpose of the current manuscript is to investigate the in vivo biocompatibility of
DLC and TiO2-DLC films. The films were deposited using plasma enhanced
chemical vapor deposition (PECVD) technique. The biological characterization was
performed using two different techniques: (i) cytometry (performed after intraperitoneal lavage of CF1 mice, with anti- F4/80 as biomarker of the obtained
macrophages); and (ii) indirect assessment of the produced NO by analyzing nitrite
in the supernatant of macrophage culture (after 24 and 48 h by the Griess reagent).
The samples were surgically inserted into the peritoneal cavity of CF-1 mice and
maintained for 7, 15 and 30 days. The in vivo results demonstrated that the
interaction of macrophages with the DLC coating does not induce inflammatory cell
reactions. No significant increase in nitric oxide production and cytotoxicity was
observed in case of the DLC and TiO2-DLC as compared to the stainless steel. The
TiO2-DLC and DLC coatings were found to be biocompatible when analyzed cell
metabolism.
Keywords: diamond-like carbon, titanium dioxide, biocompatibility, oxide nitric,
macrophages, in vivo.
REFERENCES
[1] Goodman, S.B. et al. The future of biologic coatings for orthopaedic
implants. Biomaterials, v. 34, n. 13, p. 3174-3183, 2013.
[2] Robertson, J. Diamond-like amorphous carbon. Materials Science and
Engineering R: Report, v. 37, p. 129-281, 2002.
[3] Ramrez, R.M.A. et al. An evaluation of the tribological characteristics of DLC
films grown on Inconel Alloy 718 using the Active Screen Plasma technique in a
Pulsed-DC PECVD system. Surface & Coatings Technology, v. 284, p. 235-239,
2015.
[4] Wachesk, C.C. et al. Cell viability and adhesion on diamond-like carbon films
containing titanium dioxide nanoparticles. Applied Surface Science, v. 266, p. 176181, 2013.

C CELL CYCLE AND PROLIFERATION
C1
C2
IMPAIRMENT
IN
NEURAL
CELL
PROLIFERATION
AND
DIFFERENTIATION IN RESPONSE TO HYPERHOMOCYSTEINEMIA
BRASSICA OLERACEA VAR. CAPITATA INCREASE THE CELLULARITY

AND BLOOD VESSELS IN THE SKIN WOUNDS OF WISTAR RATS
Manuela Sozo Cecchini*, Gilian Fernando Bourckhardt, Maria Lusa da Silveira

Hahmeyer, Yara Maria Rauh Mller, Evelise Maria Nazari.
Mariurea Matias Sarandy (1), Monica Maria Lopes do Carmo* (2), Rmulo Dias
Novaes (3), Joo Paulo Viana Leite (4), Reggiani Vilela Gonalves (5)
Departamento de Biologia Celular, Embriologia e Gentica, Universidade Federal de

Santa Catarina, Santa Catarina, Brazil.
(1) Department of General Biology, Federal University of Viosa, MG, Brazil. (2)
Department of Medicine and Nursing, Federal University of Viosa, MG, Brazil. (3)
Department Structural Biology, Federal University of Alfenas, MG, Brazil. (4)
Department of Biochemistry and Molecular Biology, Federal University of Viosa
MG, Brazil. (5) Department of Animal Biology, Federal University of Viosa MG,
Brazil
*manucecchini@hotmail.com
High levels of homocysteine (Hcy) are characterized as a metabolic disorder named
hyperhomocysteinemia, which is related to the occurrence of congenital anomalies.
Specifically, in the nervous system (NS), this condition is associated with DNA
damage and neuronal cytotoxicity. This study evaluated the effect of Hcy on cell
cycle, differentiation and survival of neural cells in midbrain. Thus, fertilized eggs of
Gallus domesticus were treated with 20 mol D-L Hcy/50 L saline solution at E2
and analyzed at E6 (Ethics Committee 175/CEUA/PROPESQ/2014). Control
embryos received 50 L saline solution. Immunohistochemistry showed a significant
reduction in cell proliferation (p < 0.05) in the ependymal layer of the midbrain at
E6. Regarding the proteins involved in cell cycle, Hcy induced an increase in p53
expression (p < 0.01), and a decrease in both proteins, p21 (p < 0.01) and cyclin-E (p
< 0.01), accompanied by a increase in -H2AX (p < 0.001). The analysis of neuronal
differentiation showed a significant decrease in -tubulin III expression (p < 0.01)
after Hcy treatment. However, an increase in GFAP expression (p < 0.01), like as
reactive gliosis, was observed in treated-embryos. Moreover, a significant reduction
of proteins involved in neuronal survival BDNF (p < 0.0001) and glial survival
GDNF (p < 0.0001) was found in embryos exposed to Hcy. These results
demonstrated that 20 mol D-L Hcy did not induce congenital anomalies occurrence
in the midbrain, but this treatment was able to affect the proliferation and
differentiation processes, essentials for the normal embryonic development.
Phone: (+55) 31-3899-4375. monica.maria@ufv.br
Support: CAPES
The cutaneous repair is a process that involves cellular and extracellular mechanisms
(1). The treatment with herbal medicines has been effectively used as healing agents
of skin wounds and can be used in the development of new drugs (2). The aim of this
study was to analyze the effects of the of Brassica oleracea var. capitata in cell
proliferation of skin wounds of Wistar rats.Five circular wounds of 12 mm in
diameter were made in the skin by surgical incision and the treatments were applied
at the wound site daily for 20 days. The animals were divided in four groups: Balsam
(B. oleracea); ointment (B. oleracea); sunflower oil (Helianthus annuus) and control
(0.9% saline).The samples were processed and embedded in paraffin. Sections of 4
m were made and subsequently stained with hematoxylin and eosin (H&E) for the
analysis of cells and blood vessels. The groups treated with B. oleracea showed a
greater number of cells on days 16 and 20 compared to the other groups. There was
an increase density of blood vessels in B. oleracea ointment group compared to the
other groups. Thus, it is clear that the B. oleracea var. capitata is effective for the
treatment of skin wounds, mainly because this plant stimulated the cell proliferation
and formation of new vessels distributing oxygen and nutrients to the cells and
promoting an effective healing. We would like to thank the FAPEMIG for the
financial support (edital PPM00868-15).
References:
Tang, J., Liu, H., Gao, C., Mu, L., Yang, S., Rong, M., Lai, R. (2014). A small
peptide withpotential ability to promote wound healing. PLoS ONE 9(3): e92082.
Xing, W., Guo, W., Zou, C.-H., Fu, T.-T., Li, X.-Y., Zhu, M., Xu, X. (2015).
Acemannan accelerates cell proliferation and skin wound healing through
AKT/mTOR signaling pathway. Journal of Dermatological Science, 79(2), 101109.
C3

C4
A CYCLIN D2-DERIVED PEPTIDE ACTS ON SPECIFIC CELL CYCLE

PHASES THROUGH ERK1/2 ACTIVATION TO CAUSE CELL DEATH OF
BREAST CANCER CELLS
Lilian C. Russo1, Christiane B. Araujo2, Leo K. Iwai2, Emer S. Ferro3, Fabio L.
Forti1
1. Department of Biochemistry, Institute of Chemistry, University of So Paulo, So
Paulo- SP, 05508-000, Brazil
2. Special Laboratory of Applied Toxinology (LETA), Center of Toxins, Immune
Response and Cell Signaling (CETICS), Butantan Institute, So Paulo-SP, 05503000, Brazil
3. Department of Pharmacology, Institute of Biomedical Sciences, University of So
Paulo, So Paulo-SP, 05508-000, Brazil
Intracellular peptides formed by the ubiquitin-proteasome system participate and
regulate many biological functions inside the cells. A peptide named peptide 5
(pep5), derived from Cyclin D2 protein, was fused to cell-penetrating peptide (cpp)
and showed to provoke cell death in different tumor cell lines and reduces the
volume of rat C6 glioblastoma tumors in vivo. Here we use the human MDA-MB231 breast cancer cells, for evaluating whether pep5 has preferential effects along the
cell cycle. Fluorescent labeled pep5 monitored by real time confocal microscopy
enters MDA-MB-231 cells three min after the treatment and localized into the
nucleus and cytoplasm. Flow cytometry analysis of pep5-induced cell death was
higher when MDA-MB-231 cell population was in G1/S transition or in S phase
compared to asynchronous cells. At the same conditions, as confirmed by
immunoblotting, pep5 triggered a specific and permanent phosphorylation in
ERK1/2, but not in JNK, p38, AKT, p53 or AMPK. Affinity chromatography
followed by mass spectrometry, employed for searching and identificating pep5
protein targets, identified CLIC1 and Plectin as the only two proteins interacting with
pep5 in both asynchronous and synchronized MDA-MB-231 cells. These two
interactions could help to explain the long-lasting ERK1/2 phosphorylation, as well
as the cytoskeleton perturbation of MDA-MB-231 cells, which stress fibers integrity
is strongly affected by pep5 treatments. These data suggest that pep5 has potential
therapeutic properties for evoking anti-proliferative actions of specific types of
cancers, such as breast, prostate, neuro- and glioblastomas.
Wnt/-catenin SIGNALING PATHWAY IN HYPERPLASTIC PANCREATIC

BETA CELLS OF PREDIABETIC MICE
Daniela Aparecida Maschio (1), Helena Cristina de Lima Barbosa-Sampaio (2) Carla
Beatriz Collares-Buzato (1).
(1) Department of Biochemistry and Tissue Biology, (2) Department of Structural
and Functional Biology Physiology. (1,2) Institute of Biology, University of
Campinas (UNICAMP), Campinas, SP.
The canonical Wnt pathway, that has the -catenin as co-activator protein and Wnts
as activators, has been considered a signaling pathway with possible involvement on
cell biology. Recent studies suggest a role for this pathway in controlling the
function and mass of cells. In this study, we have investigated the Wnt/-catenin
pathway in hyperplastic islets of prediabetic C57 male mice (fed a high-fat diet for
60 days, HFD 60d group) and in animals displaying normal-sized islets (exposed to
the same diet for only 30 days, HFD 30d group). HFD 60d-fed mice became obese
and prediabetic displaying hyperglycemia, hyperinsulinemia and peripheral
resistance to insulin. This metabolic condition was accompanied by compensatory
hyperplasia of cells which was not verified in HFD 30d animals. We observed
nuclear translocation of active--catenin, by immunofluorescence, and an increase
in protein and gene expression of -catenin in islets of prediabetic mice in
comparison with controls. In contrast, none of these changes were seen in the
animals of HFD 30d group relative to its control. A preliminary screening by RTPCR of the 18 Wnts described in mice demonstrated the expression of the Wnt
subtypes 2b, 3, 3a, 4, 7a, 7b, 8b, 9a and 9b in islet homogenates of both prediabetic
and control mice. In conclusion, the canonical Wnt pathway seems to be activated
during the compensatory hyperplasia of beta cells in prediabetic mice and may
involves Wnts that are expressed by islet cells. Ethics Committee on Animal
Use/UNICAMP/(Protocol. 3443-1).
Financial support: CNPq/FAPESP.
Fapesp, Capes, NAPPS

C5
C6
THE EFFECT OF MEMBRANES DOPED WITH EXTRAT OF ILEX

PARAGUARIENSIS ON CELL PROLIFERATION DURING THE WOUND
SKIN HEALING PROCESS
NOX INHIBITION CAUSES REDUCTION IN THE THICKNESS OF THE

DEVELOPING CEREBELLUM EXTERNAL GRANULE CELL LAYER
Fellipe Antonio Canhetti Correa1, Ana Luiza Glauser Fontes 1,Nauana Hay Paiva 2,
Flvio Luis Beltrame3, Maria Albertina de Miranda Soares4, Jos Rosa
1
Gomes4,Airton Vicente Pereira3
2
(1) Medical student from the State University of Ponta Grossa (UEPG), Ponta
Grossa, Paran, Brazil.
(2) Masters degree in Biomedical Science from the State University of Ponta Grossa
(UEPG), Ponta Grossa, Paran, Brazil.
(3) Department of Pharmaceutical Sciences (DEFAR), Sector of Biological and
Health Sciences (SEBISA), UEPG, Ponta Grossa, Paran, Brazil.
(4) Department of Structural Biology, Molecular and Genetics (DEBIOGEM) Sector
Of Biological And Health Sciences (SEBISA), UEPG, Ponta Grossa, Paran, Brazil.
The skin is the largest organ of the body, which primarily serves as a protection
barrier to maintain the homeostasis. Therefore, the development of wound treatment
is an important area for the health care. We aimed to evaluate the membranes of
sodium alginate and chitosan associated with the extract of Ilex paraguariensis on
the cell proliferation during the wound skin healing process. Twelve rats were
distributed into 4 groups according to the membranes used: sodium alginate (A),
sodium alginate doped with I. paraguariensis extract (AI), a bilayer of sodium
alginate/chitosan (AC), and a bilayer of sodium alginate/chitosan doped with the I.
paraguariensis extract (ACI). A lesion of the 2cmX2cm was produced on the dorsal
skin of Wistar male rats and covered with the membranes for 12 days. Before death,
all rats were injected i.p. with BrdU (1mL/Kg) to evaluate the proliferative index.
After histology and immunohistochemistry, the distance between the marginal
regions of epithelial lesions and the cell proliferation of the hair follicles, epithelium
and the superficial connective tissue of the lesions were evaluate. The groups A, AC
and ACI showed an increase of the proliferative index in the lesioned skin. We
conclude that the alginate membrane and its association with chitosan and the extract
may be efficient to accelerate the cell proliferation during the wound skin healing.
CEUA-05/2012
Ocanha, S. G.*1, Mazzonetto, P. C.1, Porcionatto, M. A.1

(1) Neurobiology Laboratory, Department of Biochemistry, Universidade Federal de
So Paulo (UNIFESP)
*sarah.ocanha@hotmail.com; (11) 99271-3103; Rua Maicatira, 36, So Paulo, SP,
Brasil
patricia_ccamacho@yahoo.com.br;
marimelia.porcionatto@gmail.com
The cerebellum is the region in the central nervous system responsible for motor
coordination and balance. It is an excellent model to study central nervous system
development as it gathers important cellular events such as proliferation, migration
and differentiation of neuronal precursors. The Nox3N64Y mutated mice lineage has
lack of motor coordination and increased proliferation of cerebellar granule neuron
progenitors during early postnatal development (up to 15 days after birth), as well as
disorganized Purkinje cell layer, due to a mutation in the gene that codes for NOX3
protein. The mutation causes the substitution of asparagine (N) by tyrosine (Y) at
position 64 of the protein. NOX3 is a NADPH oxidase that belongs to a family of
transmembrane proteins which main function is to reduce molecular oxygen to form
reactive oxygen species (ROS). ROS produced by NOX can modulate cell signaling
in various physiological processes including proliferation. Our goal is to study the
participation of ROS in the control of proliferation of cerebellar granule cell
precursors by inhibiting NOX. Nox3N64Y and Balb/c mice, when treated with the
NOX inhibitor apocynin, presented a decrease in thickness of the external granule
cell layer (EGL) when compared to control groups suggesting that ROS may lead to
increased proliferation of neural precursors. Furthermore the Nox3N64Y mice had
increased thickness of EGL when compared to BALB/c mice, suggesting increased
proliferation of neural precursors in these animals.
Financial support: CAPES, CNPq and FAPESP
KEYWORDS: skin injury, wound repair and I. paraguariensis

C7
C8
ALLIUM CEPA AS ENVIRONMENTAL POLLUTION BIOINDICATOR IN

TANGAR DA SERRA REGION - MT
Maressa Floriano Camargo (1); Andrielle dos Anjos Barbosa (1); Dayane Castro
Silva (1); Anderson Fernandes de Miranda (2); Alexandro Czar Faleiro* (2)
(1)
(1) Department of Biological Sciences, State University of Mato Grosso, Tangar da

Serra, Brazil;
(2) Faculty of Agricultural Sciences, Biological and Health, State University of Mato
Grosso, Tangar da Serra, Brazil
* Presenting/Corresponding author
Address: State University of Mato Grosso - UNEMAT
Faculty of Agricultural Sciences, Biological and Health
Rodovia MT-358 Km 7 Cx. Postal 287 Jardim Aeroporto
Tangar da Serra MT Brazil
Zip Code: 78300-000
*E-mail: acfaleiro@unemat.br
*Phone: +55 65 3311-4930
*Cellphone: +55 651 9957-0606
Tangar da Serra is located in Mato Grosso Southwest region, being an agricultural
region. The aim of this study was to evaluate the effects caused by the inappropriate
disposal of chemical and organic waste in watercourses. Water samples of Queimap River were collected at the beginning and the end portion of the river during the
month of June/2015 (dry season). Allim cepa bulbs were placed in contact with
distilled water (control) and with water collected from both points of the river in
order to obtain roots. Roots were collected from at least three different bulbs for each
of the sampling point. Samples were placed in vials containing Carnoy fixative (3:1)
and incubated at room temperature for 12h. The post-fixed roots were hydrolysed in
5N HCl at room temperature, washed in distilled water and stained by Feulgen
staining for 1 h. After staining, the meristematic region of the root apex was placed
on a slide, 45% acetic acid was added and then overlaid with coverslips and
subjected to crushing technique. The mitotic index and metaphase were used for the
evaluation of changes for the presence of abnormal mitotic figures and cell division
rate. Statistical analysis showed significant differences between control and treated
samples, indicating the presence of mutagenic substances in the water samples
collected. The variability test conducted previously showed no significant differences
between the bulbs and therefore the differences are related to treatment.
Financial support: FAPEMAT
Keywords: Mutagenic; abnormal mitosis; mutations


C9
C10
ROLE OF S-NITROSOGLUTATHIONE REDUCTASE ON CONTROLING

PROTEIN S-NITROSYLATION DURING MYOGENESIS IN PRIMARY
CULTURES OF MUSCLE PROGENITOR CELLS
CANTHIN-6-ONE INDUCES CELL CYCLE ARREST IN KASUMI-1 CELLS
Aline Miyoko Sakaguchi Yamashita (1), Maryana Tavares de Campos Ancillotti (1),
Martha Meriwether Sorenson (1), Cludia dos Santos Mermelstein (2), Leonardo
Nogueira (1).
1
2
Instituto de Bioquimica Mdica Leopoldo de Meis - IBqM - UFRJ

Instituto de Cincias Biomdicas - ICB - UFRJ
Heron F. Vieira Torquato1*, Edgar J. Paredes-Gamero1,2, Domingos T. de Oliveira

Martins3
1
Department of Biochemistry, Federal University of So Paulo (UNIFESP), So

Paulo, Brazil.
2
Cruzes, Mogi das Cruzes, Brazil.
3
Department of Basic Sciences in Health, Faculty of Medicine, Federal University of
Mato Grosso (UFMT), Cuiab, Brazil.
E-mail: yamashita@bioqmed.ufrj.br Phone: (21) 39386783 Mobile: (24) 992297879

Professional address: Av. Carlos Chagas Filho, 373, Prdio do CCS - Bloco H (Sala
012)- Cidade Universitria, Ilha do fundo, Rio de Janeiro, Rio de Janeiro, Brazil.
During myogenesis, myoblasts proliferate, differentiate, and fuse to form myotubes.

Nitric oxide has an important role during this process, controlling the transition
between myoblast proliferation and fusion. Although this mechanism was thought to
be restricted to the soluble guanylyl cyclase (sGC)-cGMP-protein kinase G (PKG)
signaling pathway, we demonstrated that the S-nitrosylation/denitrosylation
equilibrium (including low-molecular-weight molecules and proteins) also controls
both myoblast proliferation and fusion, independently of the sGC-cGMP-PKG
pathway. Also we showed that S-nitrosoglutathione reductase (GSNOR) has a
critical role in modulating this equilibrium during myogenesis. Although we have
identified this new pathway, it is not clear yet if proteins are in fact S-nitrosylated
during the time course of myogenesis. Furthermore, it is not known whether
myogenesis-related signaling proteins are targets for S-nitrosylation when GSNOR is
inhibited. For this, we used primary cultures of skeletal muscle progenitor cells from
chick embryos. After 48 h, when most myoblasts are being fused into myotubes,
cultures were treated with GSNOR inhibitor (GSNORi), NOS inhibitor (L-NAME)
or S-nitrosocysteine for 0.5-24 h. Protein S-nitrosylation was measured by
Saville/Griess method or by resin-assisted capture of S-nitrosoproteins (SNORAC).
Treatment with GSNORi produced a transient increase in total S-nitrosylated
proteins, with a peak at 2-4 h and return to baseline after 24 h. This response is
consistent with a NOS-dependent S-nitrosothiol production when GSNOR is
inhibited. These data suggest that during the transition between proliferation and
fusion protein S-nitrosylation is impaired by GSNOR.
Chemotherapeutics represent the major approach for the treatment of cancers,

however, due ineffectiveness of treatment in some cases, it becomes necessary to
develop new drugs. Canthin-6-one is a natural product isolated from various plant
genera. The present study was aimed to evaluate antiproliferative effects of Canthin6-one in Kasumi-1 cells. Distribution of cell cycle, BrdU assay and intracellular
proteins labeling (Ki-67, p-RB, cyclin B, phosphorylation of histone 3) was
determined by flow cytometry analysis. Kasumi-1 cells (105 cells/mL) were treated
with canthin-6-one (7 and 45 M) for 24 h. Cell cycle after treatment showed an
increase of G0/G1 phase when stimulated with 7 M, whereas 45 M induced an
increase of G2/M phase after 24 h. Cell cycle arrest was confirmed by BrdU assay,
where 7 M Canthin-6-one induced an increase of G1 phase and 45 M induced
G2/M arrest. Additionally, the protein Ki-67, which is strictly expressed during cell
proliferation, showed lower expression in both concentrations. Furthermore, canthin6-one reduces phosphorylation of RB protein, in both treatments. In order to
differentiate G2 phase of mitosis, phosphorylation of histone 3 (p-H3), a marker of
mitosis, and cyclin B, a cyclin expressed in G2/M phases, were quantified. Canthin6-one (7M) promoted decrease in G2 phase (H3-/Cyclin B1+); whereas the
concentration of 45 M increase G2 phase (H3-/Cyclin B1+). These results show that
Canthin-6-one can affect cell cycle and may be used as a pharmacological prototype
for the development of novel anti-leukemic agents.
Heron F. Vieira Torquato: Department of Biochemistry, Federal University of So

Paulo (UNIFESP), Av. Pedro de Toledo, no. 669, So Paulo, SP, 04039-401, Brazil.
e-mail: heron.fvt@gmail.com / +55 11 5576 4868
Finantial Support: CNPq, CAPES, FAPESP

NOTES: The authors declare no competing financial interest.
Support: CNPq
Ethical approval: CEUA-UFRJ #DAHEICB092-05/16

D CELL DEATH
D1
D2
CITOTOXICITY AND GENOTOXICITY EFFECTS OF PHOTODYNAMIC

THERAPY
A NOVEL THIOPHENE DERIVATIVE INDUCED APOPTOSIS IN BREAST

CANCER CELLS BY A CASPASE-7-INDEPENDENT MECHANISM
Bruno H Godoi 1, Carlos DGO Moraes1, Isabel CS Carvalho1 Newton S da Silva2

and Cristina Pacheco-Soares1.
Michelly Cristiny Pereira (1), Flaviana Alves dos Santos (1), Tiago Bento de
Oliveira (2), Francisco Jaime Bezerra Mendona Junior (2), Maria do Carmo Oliveira
de Lima (3), Marina Galdino da Rocha Pitta (3), Ivan da Rocha Pitta (3, 4), Moacyr
Jesus Barreto de Melo Rgo (1), Maira Galdino da Rocha Pitta (1)
Institute of Research and Development IP&D, University of Paraba Valley

UNIVAP, Laboratory Dynamics of Cellular Compartments, So Jos dos Campos,
So Paulo, Brazil.
(1)
2
Institute of Research and Development IP&D, University of Paraba Valley
UNIVAP, Laboratory of Cell Biology and Tissue, So Jos dos Campos, So Paulo,
Brazil.
(2)
E-mail: bh-godoi@bol.com.br, carlosdailtongom@gmail.com
(3)
Photodynamic therapy (PDT) can induce reactive oxygen species formation and cell
(4)
death. The heat shock proteins (HSP) are expressed in stressed cells. In cancer, HSPs
can confer resistance or sensitivity to certain treatments. The aim of this study is(5)
to
evaluate the effects of PDT in expression of HSPs and genotoxicity in cells of
laryngeal cancer. HEp-2 cells were cultured and were treated with photosensitizer
(AlPcS4) at a concentration of 10M and incubated for 1h, after the cells was
irradiated by semiconductor diode laser, with application continuous mode;
wavelength 670 nm; energy density of 4.5J/cm2; power 35mW; application area
1.0cm2. Finally, the cells were incubated for 24 and 48 hours. Immunofluorescence
assay showed GRP-78 expression only in the PDT group after 24h. Comet assay
showed significant increases in percentage of DNA in the comet tail at 24h
(p<0.0001) in PDT group. In addition when compared the percentage of DNA in the
comet tail only the PDT group showed difference between the periods of 24h and
48h (p=0.0324). The micronucleus assay showed significant decrease in
micronucleus formation in PDT group (p=0.0008) at 24h. However, at 48h, the PDT
group showed significant increase in number of micronucleus (p=0.008).
Furthermore, when compared number of micronucleus both groups showed
difference between the periods of 24h and 48h: the Control group showed a decrease
in the number of micronucleus (p = 0.0240) and the PDT group increased
(p<0.0001). It can be concluded that PDT induced cytotoxicity and genotoxicity in
cancer cells.
(1) Laboratory for Immunomodulation and New Therapeutic Approaches (LINAT),

Department of Physiology and Pharmacology, Federal University of Pernambuco
(UFPE), Recife, PE, Brazil.
(2) Laboratory Synthesis and vectoring molecules, Department of Biological
Sciences, State University of Paraba(UFPB), Joo Pessoa, PB, Brazil.
(3) Laboratory for Planning and Drug Synthesis (LPSF) Federal University of
Pernambuco (UFPE), Recife, PE, Brazil.
(4) Centro Avanado para Inovaes em Sade (CAIS), Instituto Suely Galdino
(ISG)
Background: Breast cancer is the most incident cancer in women worldwide and the
treatment remains a major barrier. Thus, the design and synthesis of more efficient
agents with less side effects are extremely important. Thiophenes are a class of
heterocyclic compounds with Sulphur atom that display different pharmacological
activities. Aims: To investigate the cytotoxicity activity of some thiophene
derivatives in breast cancer cells and evaluate cell death mechanisms. Methods:
Cytotoxicity assays were performed against MCF-7 cells using MTT method and the
analysis of cell death with cytometer by cleavage of caspase 7 and by Acridine
Orange/DAPI staining. Results: All thiophene derivatives SB-44, SB-83 and SB-200
reduced the cell viability in breast cancer cells, showing IC50 lower than 30 M and
SB200 compound showed the best cytotoxicity effect in MCF-7 cells (IC50 14.86M,
descriptive value). The SB-200 derivative treatment induced an increased proportion
of acridine orange/Hoechst double stained cells (35.35% versus 3.14%, p=0.0002)
compared to non- treated cells, with apoptosis morphological alterations independent
of caspase 7 activation (p>0.05). Conclusion: The present study reported anticancer
activity of thiophene derivatives in breast cancer cells and SB-200 derivative had the
best cytotoxic activity and appeared to induce apoptosis by a caspase independent
mechanism. Further studies should be performed to investigate cell-signaling
proteins involved in thiophene cell death mechanisms.
Financial support: FAPESP (2013/20054-8).
Information on ethical approval and funding support: not applicable; Brazilian

National Research Council (CNPq), the Research Foundation of Pernambuco State
(FACEPE), National Institute for Science and Technology in Pharmaceutical
Innovation (INCT_if) and CAPES for student fellowships.
D3
D4
CELL-BASED ASSAYS OF KINASE INHIBITORS IN TRYPANOSOMA

CRUZI, LEISHMANIA AMAZONENSIS AND LEISHMANIA CHAGASI
PARASITES
R18H PEPTIDE FROM Brn-2 TRANSCRIPTION FACTOR INDUCES

CYTOTOXIC EFFECTS AND CELL DEATH IN VITRO IN B16F10-Nex2
MELANOMA CELLS
D. Y. Tezuka; J. C. J. Quilles; A. Leito
Fernanda Fernandes Miranda da Cunha (1), Katia Cristina Ugolini Mugnol (2),
Filipe Menegatti de Melo (3), Renato Arruda Mortara (3), Luiz Rodolpho Raja
Gabaglia Travassos (3), Denise Costa Arruda (1)
Medicinal Chemistry Group NEQUIMED

Institute of Chemistry of So Carlos IQSC / University of So Paulo USP
Avenida Trabalhador So Carlense, 400 Centro So Carlos / SP CEP: 13.566590
E-mail: daianetezuka@usp.br; quilles@usp.br; andleitao@iqsc.us.br
Tel. (+55) 16-33738784 / 17-991156785
Chagas and Leishmaniasis are neglected tropical diseases, which affect millions of
people worldwide, and still do not have an effective treatment. Kinases are enzymes
responsible for transferring phosphate groups which are highly correlated signals that
regulate cell growth and differentiation a likely target for the study and development
of new molecules. In this study we were tested in vitro eighteen compounds selected
using in silico tools based on known mTOR inhibitors. The cellular assay using the
colorimetric method (MTT) was performed analyzing the activity of the set after
incubation for 72 hours (promastigote form of Leishmania spp.) and for 120 hours
(epimastigote form of T. cruzi Y strain) at a concentration of 100 M. The best
inhibitors for T. cruzi were Neq0440 and Neq0474, while Neq0438 showed activity
of interest only to L. amazonensis. Concentration-response curves were obtained for
these compounds, together with cytotoxicity using fibroblast cell line (Balb/C 3T3
clone A31). The results showed that Neq0438 and Neq440 were the most
promissing, with potency in the micromolar range, without cytoxicity in the
mammalian cell. There molecules could be used as chemical prototypes for novel
compounds with improved potency. In addition, the results showed that the
molecules have specificity among parasites, including both species of leishmania.
This work was supported by FAPESP project 13/18009-4.
(1) Ncleo Integrado de Biotecnologia, Universidade de Mogi das Cruzes. So

Paulo, Brasil.
(2) Centro Interdisciplinar de Investigao Bioqumica, Universidade de Mogi das
Cruzes. So Paulo, Brasil.
(3) Departamento de Microbiologia Imunobiologia e Parasitologia, Universidade
Federal de So Paulo. So Paulo, Brasil.
Fernanda Fernandes Miranda da Cunha
Avenida Governador Adhemar de Barros, n2029 casa 91 Vila Rubens
Mogi das Cruzes, SP - CEP: 08735-075
cunha.ffernandes@gmail.com
Telephone institutional: (11) 4798.7106 Telephone mobile: (11) 97106.1703
Background: Natural and synthetic peptides have shown antitumor effects involving
different mechanisms known as necrosis, apoptosis, necroptosis, autophagy and
inhibition of angiogenesis. The Brn-2 protein is expressed in melanocytes and
overexpressed in melanoma cells. We have shown that internal peptides from Brn-2
transcription factor induced cytotoxic effects in melanoma cells. Because these
peptides are from a transcription factor we propose that they could interfere in the
Brn-2 cell signaling pathway as inhibitors. In fact, we believe that new antitumor
drugs are essential to the progress of cancer chemotherapy.
Aims: Determination of the cytotoxic activity in vitro and evaluation of cell death
effects induced by R18H.
Methods: B16F10-Nex2 cells were incubated with R18H peptide and viability was
determined by MTT assay. Moreover, treated and untreated cells were analyzed by
fluorescence microscopy to determine cell death. DNA degradation was observed by
TUNEL assay, chromatin condensation by staining with Hoechst 33342, increase of
superoxide anions by dihydroethidium and membrane permeabilization by propidium
iodide (PI) nuclear staining. In addition, phosphatidylserine translocation in cells
stained with annexin V and PI were analyzed by FACS.
Results: R18H peptide exerted cytotoxic effects in B16F10-Nex2 cells in vitro
(p<0,005). Melanoma cells treated with R18H showed DNA degradation, chromatin
condensation, propidium iodide incorporation, phosphatidylserine translocation and
superoxide anion production.
Conclusion: We have shown that R18H peptide display antitumor effects in vitro and
induce cell death in B16F10-Nex2 cell line. It represents an important evidence of
the anti-tumor activity of Brn-2 transcription factor-derived peptides.
Funding support: CNPq, FAPESP and FAEP


D5
HSPB1
MEDIATES
PRL-INDUCED
CYTOPROTECTIVE
METABOLIC BENEFICIAL EFFECTS ON BETA-CELLS
D6
AND
Rosangela Aparecida Wailemann Mansano, Letcia Ferreira TerraVinicius de

Moraes GomesMaria Fernanda Pereira de Arajo Demonte ForniTalita Cristina de
OliveiraAncly Ferreira dos Santos, Giuseppe Palmisano, Leticia Labriola
University of So Paulo
Maintaining islet cell in vitro appears as an attractive strategy to increase the
pancreatic islet transplantation outcome. We have previously shown that prolactininduced pro-survival effects on pancreatic beta-cells are mediated by HSPB1. We set
out to explore the molecular mechanisms involved in HSPB1-mediated beta-cell
cytoprotection.
Lysates from cytokine and/or PRL treated Min6 cells were subjected to HSPB1
immunoprecipitation. Co-precipitated proteins were identified by SDS-PAGE
coupled to mass spectrometry. We found an enrichment of both endoplasmic
reticulum stress (ERstress) and carbohydrate metabolism related proteins. Since
recent studies have pointed that islets respiratory profile prior to transplantation may
predict their performance; we investigated whether PRL treatment could increase
beta-cell mitochondrial efficiency. We observed a cytokine-induced increase of
uncoupled mitochondrial levels, which were decreased (p<0.05-One-way ANOVA
with Tukeys post test) upon PRL treatment. HSPB1 was a key mediator of this
effect since the lack of this protein abrogated (p<0.05) PRL-induced mitochondrial
function recovery.
It has already been reported that ERstress leads to beta-cell apoptosis. Thus, we
investigated whether PRL could promote beta-cell cytoprotection upon
pharmacologically induced ERstress. We showed that ER stressorsinduced beta-cell
death was (p<0.05) inhibited by hormone-induced HSPB1 levels since its silencing
abolished PRL effects.
In conclusion, we have shown the importance of HSPB1 on PRL pro-survival
effects against ER stressors-induced beta-cell death as well as on mitochondrial
efficiency recovery. Finally, our results outline the importance of further studies on
HSPB1 functions since they could lead to beta-cell death mitigation.
Authors declared no conflicts of interest.
Support: FAPESP, CAPES, CNPq.
FROM DNA DAMAGE TO CELL DEATH: THE EFFECTS OF

AMAZONIAN BIOMASS BURNING PARTICULATE MATTER IN HUMAN
LUNG CELLS
Nilmara de Oliveira Alves (1), Alexandre Teixeira Vessoni (2), Annabel Quinet (2),
Rodrigo Soares Fortunato (3), Gustavo Satoru Kajitani (2), Milena Simes Peixoto
(4), Sandra de Souza Hacon (5), Paulo Artaxo (6), Carlos Frederico Martins Menck
(2) and Silvia Regina Batistuzzo de Medeiros (4)
1 University of So Paulo, School of Medicine, So Paulo, Brazil.
2. University of Sao Paulo, Institute of Biomedical Sciences, So Paulo, Brazil.
3. Federal University of Rio de Janeiro, Institute of Biophysics, Rio de Janeiro,
Brazil.
4. Federal University of Rio Grande do Norte, Cellular Biology and Genetics
Department, Natal, Brazil.
5. National School of Public Health at Oswaldo Cruz Foundation, Rio de Janeiro,
Brazil.
6. University of So Paulo, Institute of Physics, So Paulo, Brazil.
email:nilmaraoalves@gmail.com Fone # 55.11.3061.8531 / # 55.11.98790.9690
The Amazon Basin, located at the heart of South America, contains the largest
tropical forest world-wide. However, during dry season, the population has been
negatively affected by biomass burning in the Brazilian Amazon region. In this
study, we performed analysis of cellular and molecular effects in human lung cells
(A549 cell line) using concentration of particulate matter smaller than 10 m (PM10)
from biomass burning of Amazon region. . The PM10 samples were collected during
July - October/2011 using high-volume sampler. Subsequently, the samples were
extracted with dichloromethane and dissolved in dimethylsulfoxide to A549 cells
exposure during 24 and 72 hours. We used a combination of comet test, flow
cytometry, western blotting and fluorescence microscopy to evaluate human lung
cells exposed to PM10 concentrations. After 24 hours of exposure, there was an
increase of pro-inflammatory cytokines and in reactive oxygen species generation, in
a dose and time dependent manner. Also, the data show that PM10 can induce
autophagy in human lung cells. Besides, there was an induction of cell cycle arrest at
G1 phase, as well as an increase in the expression p53 protein and formation of DNA
strand breaks. After 72 hours, we detected a significant increase of cells in the subG1 fraction, indicating apoptosis. Additionally, we observed the phosphorylation of
H2AX (-H2AX), which correlated with the activation of caspase 3, suggesting that
the induction of -H2AX may be associated with the DNA fragmentation during
apoptosis. We also observed that necrosis is a cell demise pathway induced by PM10.
This study shows an important advance in understanding the toxic cellular and
molecular effects induced by PM10 that can be related to the increase potential of
human health impacts in the Amazon region.
Financial support: CNPq and FAPESP

D8
D7
A KUNITZ-TYPE INHIBITOR PURIFIED FROM MIMOSA REGNELLII
BENTH. SEEDS ACTIVATES KEY PROTEINS INVOLVED IN INDUCTION
OF CELL DEATH IN B16-F10 CELLS
Luciana Maria Arajo Rablo (1), Geovanna Maria de Medeiros Moura (1), Paula
Ivani Medeiros dos Santos (2), Daniela De Lucena Pedroso (1), Elizeu Antunes dos
Santos (1).
(1) Departamento de Bioqumica, Universidade Federal do Rio Grande do Norte, Rio
Grande do Norte, Brasil.
(2) Departamento de Biologia, Instituto Federal do Piau, Piau, Brasil.
Address: Departamento de Bioqumica - Centro de Biocincias - Universidade
Federal do Rio Grande do Norte - Campus Universitrio, Lagoa Nova - Caixa Postal
1524 - CEP: 59072-970 - Natal/RN - Brasil - (84)32153416 R:217 / (84)999530801
Background The progressive transformation of normal human cells to malignant
derivatives is a complex multi-step process, which requires dynamic alterations of
the genome and successful breaching of intracellular checkpoints. Protein inhibitors
are widely distributed in plant seeds and they are known to control a diversity of
events to maintain homeostasis. In subfamilies of Leguminosae the inhibitor Kunitztype are common, with one or two polypeptides chains and one active site. The
disruption in equilibrium of these molecules is the basis for disease genesis,
including tumoral events. In this work, a trypsin protease inhibitor (ITJ) was purified
from leguminosae Mimosa regnellii Benth seeds and it was evaluated due to its
capacity to induce apoptosis, describing the mechanism of action in B16-f0 cells.
Aims To purify a trypsin inhibitor from Mimosa regnellii seeds and assess its
capacity of activate key proteins in cell death events.
Methods ITJ was purified by ammonium sulphate fractionation and chromatographic
methods. The expression of death markers by ITJ incubation was evaluated with
Western Blotting, immunofluorescence and confocal microscopy assays.
Results After exposure to ITJ, tumor cells started the process of apoptosis, with
phosphatidylserine exposure on the cell membrane, cell cycle arrest, altered
mitochondrial membrane potential, p53 activation, reduction in phosphorylated ERK
expression and activation of effector caspase 3 after 72 hours of exposure, suggesting
a possible mechanism of action of the inhibitor against melanoma cells.
Conclusion ITJ induces apoptosis in melanoma cells by extrinsic pathway, behaving
like type-II cells, after 72 hours incubation.
ALKYLPHOSPHOCHOLINE HYBRID COMPOUND KILLS LEISHMANIA

AMAZONENSIS BY DIFFERENT KINDS OF CELL DEATH MECHANISMS
Joseane Lima Prado Godinho (1,3), Sara Teixeira de Macedo-Silva (1,3), Theodora
Calogeropoulou (2), Wanderley de Souza (1,3) and Juliany Cola Fernandes
Rodrigues (1,3,4).
(1) Laboratrio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofsica Carlos
Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas, 373,
CCS, Ilha do Fundo, Rio de Janeiro, Brazil, 21941-902.
(2) Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic
Research Foundation, 48 Vas. ConstantinouAvenue, 116 35 Athens, Greece.
(3) Instituto Nacional de Cincia e Tecnologia de Biologia Estrutural e Bioimagem,
Brazil
(4) Ncleo Multidisciplinar de Pesquisa UFRJ-Xerm, Diviso Biologia (NUMPEXBio), Polo Avanado de Xerm, Universidade Federal do Rio de Janeiro, Brazil.
Background: Parasites of the Leishmania genus cause leishmaniasis, a disease with
high worldwide prevalence that affects the skin, mucocutaneous, subcutaneous and
visceral tissues. The drugs used for its treatment are very toxic for humans; thus,
there are several difficulties in developing a more rational chemotherapy. Aim: The
aim of this work is to study the cell death process induced by a new molecule, TC95,
which is a hybrid compound that combines miltefosine and trifluralin in just one
scaffold. Methods: The studies of mechanisms of action and cell death were
performed in L. amazonensis. Results: Evaluating cell cycle progress in treated
promastigotes, it was observed an arrest in G2 phase. By transmission electron
microscopy the mitochondrion appeared completely disorganized, presenting a
swelling of the mitochondrial matrix with significant reduction of the mitochondrial
membrane potential, ATP production, and an increase in the ROS. By scanning
electron microscopy, TC95 altered the morphology of parasites cell body indicating
possible alterations in the cytoskeleton, which was confirmed by western blotting
analysis of the tubulin, acetylated tubulin and actin. Assays using axenic amastigotes
demonstrated the efficacy of TC95, with an IC50 value of 200 nM. In the intracellular
forms, it was possible to observe a significant reduction in the parasite burden in
macrophages after 120 h of incubation. The statics were performed using GraphPad
Prism 6 and differences were considered significant when P values were <0.05.
Conclusion: Thus, these studies suggest that TC95 is a potent and effective
compound for treatment of several species of the Leishmania genus and leading the
cell death by necrosis, autophagy and apoptosis like way.
Funding Support: CAPES

This study (protocol no. IBCCF 096/097) was approved by the Ethics Committee for
Animal Experimentation of the Health Sciences Centre, Federal University of Rio de
Janeiro (Brazil). This work was supported by CNPq, CAPES and INBEB.


D9
EFFECTS OF HUMAN KININOGENS INTERACTION WITH CELLS:
INFLUENCE OF PROTEOGLYCANS AND ZINC
Souza, D.S.P.(1), Gonalves, S.P.(1), Nascimento, F.D.(2), Justo, G.Z.(1,3),
Cavalheiro, R.P.(1), Sampaio, M.U.(1), Nader, H.B.(1), Tersariol, I.L.S.(1) and
Motta, G.(1).
(1)Departamento de Bioqumica, Escola Paulista de Medicina, UNIFESP, SP, Brasil.
(2)Programas de Biomateriais e Biotecnologia, Universidade Anhanguera de So
Paulo (UNIANSP), SP, Brasil. (3)Departamento de Cincias Biolgicas, Campus
Diadema, UNIFESP, SP, Brasil.
Contact details: si.goncalves@yahoo.com.br
Human H-kininogen (HK) endocytosis is mediated by proteoglycans but bradykininfree H-kininogen (HKa) is not internalized. The aim of the present work is to analyze
HK/HKa interactions with cell surface mediated by proteoglycans and zinc. We used
the cell lines CHO-K1 (wild type) and CHO-745 (mutant defective in
glycosaminoglycans biosynthesis) and zinc was removed from Ham-F12 medium
after treatment with Chelex-100 resin. The cell death was analyzed by flow
cytometry after annexin V and 7AAD staining. In real time experiments, using an
inverted confocal laser-scanning microscope, HK-Alexa-488 was not internalized by
CHO-K1 in the absence of zinc. The MTT assays showed a significant decrease of
CHO-K1 viability after 24 h of treatment with HK either with or without zinc
(p<0.001), and the same effect was obtained by HKa without (p<0.001) or with zinc
(p<0.01). Nevertheless, the trypan blue assays showed a significant increase in cell
death by HK only without zinc (p<0.05) and none effect was caused by HKa. The
flow cytometry indicated that under HK/HKa treatment cell death was characteristic
of necrosis and zinc increased significantly the effect of HK (p<0.05). The CHO-745
viability after 24 h of treatment with HK reduced significantly, either with or without
zinc (p<0.01) and zinc also decreased CHO-745 viability significantly either without
(p<0.01) or with (p<0.05) HK. Neither HK nor zinc caused significant death
determined by trypan blue assays. Both cell lines showed none additional cleavage of
HKa on cell surface. Our results show H-kininogen/HKa effects on cell viability
influenced by proteoglycans and zinc.
Ethics Cometee: CEP 712927
Funding Support: CAPES, CNPq and FAPESP

E CELL DIFFERENTIATION
E1
E2
EFFECTS OF THE PRODUCTS SECRETED BY TUMOR CELLS LLC IN

COCULTURE SYSTEM IN THE ADIPOGENESIS PROCESS IN VITRO
MURINE
HEMATOPOIETIC
CELLS
UNDERGO
MYELOID
DIFFERENTIATION,
ALTHOUGH
MAINTAINING
AN
UNDIFFERENTIATED POOL AFTER IL-3 AND GM-CSF STIMULI
Magno Alves Lopes, Felipe dos Santos Henriques, Felipe de Oliveira Franco, Luana
Garcia Leal, Kaltinaitis Benetton Nunes Hypolito dos Santos,Pamela Viegas Knbl,
Sidney Barnab Peres, Miguel Luiz Batista Jr.
Marina Mastelaro de Rezende1, Marcus Vincius Buri1, Christiano Marcello Vaz

Barbosa1, Edgar Julian Paredes-Gamero1,2.
1
Universidade de Mogi das Cruzes

Background: Cancer cachexia is a multifactorial syndrome with an unknown
etiology. The main symptom is the progressive reduction of the body weight.
Recently, down-regulation of adipogenic and lipogenic genes were demonstrated to
be early affected during cachexia progression in adipose tissue (AT), resulting in AT
remodeling.
Aims: Evaluate in a co-culture system the influence of the LLC tumoral cells in 3T3L1 adipogenic capacity.
Methods: Preadipocyte 3T3-L1 cells (2x104, n=3) were induced to differentiate in
coculture system with tumor cells LLC (Lewis Lung Carcinoma). The cells were
harvested at different stages of the adipocyte differentiation, i.e., days 0, 2, 4 and 8.
Afterwards, was evaluated the gene expression of classical adipocyte markers,
protein expression of apoptosis marker, cell secretion and Oil Red-O staining for
lipids accumulation performed.
Results: The co-culture of LLC tumor cells in the presence of 3T3-L1 caused a 33%
reduction in lipids accumulation, suggesting that secretory tumor cells products may
affect adipogenesis. Interestingly, a very early (day 2) down-regulation of PPAR
and C/EBP, followed by terminal genes (day 4 and 8), Adiponectin, Perilipin and
FABP4. Caspase-3 expression was increase on the last day of cell differentiation; it
occurred in the expression of proinflammatory cytokines IL-6 and TNF.
Conclusion: Overall, our results suggest that LLC secretory products impair
adipocyte differentiation in a co-culture system and increased apoptosis; seemingly,
LLC cells are able to influence the initial expression of the PPAR in 3T3-L1 cells
and consequently inhibit the whole adipogenic program.
Departamento de Bioqumica, Universidade Federal de So Paulo, So Paulo, SP,

Brazil.
2
Cruzes, Mogi das Cruzes, SP, Brazil.
Contact details:
MMR marina.mrez@gmail.com (11)9 7097-5266 (11) 5576-4868 ramal 1312
MVB marcus.buri@gmail.com (11) 5576-4868 ramal 1312
CMVB cmvbarbosa@unifesp.br (11) 5576-4868 ramal 1312
EJPG paredes.gamero@gmail.com (11) 5576-4868 ramal 1312
Background
Cytokines are proteins widely related to proliferation and differentiation of
hematopoietic stem cells, mainly IL-3 and GM-CSF, however little is known about
how it happens exactly. These effects are tied to the functional roles of its molecules
in immune and inflammatory responses, leading to leukocyte rise and activation,
although possibly affecting stem cell self-renewal and maintenance, what may be
unwanted.
Aims
The aim of this study was provide information about differentiation balance in
hematopoietic cells after IL-3 and GM-SCF stimulation.
Methods
Murine hematopoietic cells were obtained by flush of C57BL6 mice femurs and cocultured with MS-5 cells at 70% confluence. After 1 day, cells were stimulated with
IL-3 and GM-SCF (10 ngmL) separately for 3 days and incubated with antibodies in
order to use flow cytometry to analyze stem cell, progenitors and differentiated
populations. Used antibodies: Lin-PE (Gr-1, Mac-1, B220, Ter119, CD3), Sca-1PECy7, CD117-APC, CD34-FITC, FLK-2-PE, FCR- PECy7, CD117-APC, F4 80FITC, Mac-1-PE, Gr-1-PECy7, Ter119-APC.
Results
We observed an increase in myeloid progenitors (common and granulocytemonocyte, but no megakaryocyte-erythrocyte) in stimulated cells, although
presenting little effect on mature populations. Moreover, no significant alterations
were observed in stem cell pool.
Conclusion
The distinct effect of IL-3 and GM-CSF stimulation on hematopoietic populations
reinforces the possibility of specific cellular pathways involved, atop highlighting the
stem cell resistance to differentiation and its critical skills on maintenance and selfrenewal.
Ethical Approval
All procedures were approved by Institutional Ethical Committee (#1522060515).
Funding support
CAPES, FAPESP.

E4
E3
THE ROLE OF SONIC
DIFFERENTIATION
HEDHEHOG
DURING
CHICK
MUSCLE
John Teixeira, Ivone Rosa de Andrade, Jos Brito, Claudia Mermelstein.

(1)
(2)
Juliana Guimares Zulian (1), Larissa Yukari Massarenti Hosoya (1), Cruz Alberto
Mendoza Rigonati (1), Patrcia Gama (1)
(1) Laboratrio de Diferenciao Musculas e Citoesqueleto, Instituto de Cincias

Biomdicas, Universidade Federal do Rio de Janeiro.
(1)
(2) Laboratrio de Proliferao e Diferenciao Celular, Instituto de Cincias
Biomdicas, Universidade Federal do Rio de Janeiro.
Background: Myoblast fusion is a crucial step during the formation of skeletal
muscle fibers. The molecular events that control myoblast fusion are not completely
understood. Sonic hedegehog (Shh) is a morphogen that has been implicated in the
regulation of mammalian muscle differentiation.
Aims: In this study we evaluated the role of Shh during chick embryonic
myogenesis.
Methods: Myoblast cultures were obtained from 11 day-old chick embryos. Cells
were plated at 7.5 x 10<5 per dish coated with collagen. Cells are treated with
different concentrations of Cyclopamine (Shh pathway inhibitor) or with a medium
enriched in Shh, both 6h or 24h after cell plating. After 48h of treatment cells were
analyzed by immunofluorescence microscopy using anti-desmin antibodies and
DAPI. Cell cultures were quantified in relation to: number of desmin positive cells,
total number of cells, number of multinucleated myotubes, number of mononucleated
myoblasts and myotube width.
Results: Our results show that 1uM Cyclopamine induced a reduction in total cell
number, when cells were treated 6h after plating; while 4uM Cyclopamine induced
the formation of myotubes with increased width when cells were treated 6h after
plating. Shh enriched medium induced a decrease in myotube width when cells were
treated 24h after plating.
Conclusion: We can conclude from our data that Shh has different roles during early
(6h) or late (24h) chick myogenesis. Next we want to analyze the expression pattern
of Shh receptors during chick myogenesis.
Information on ethical approval and funding support: CNPq, FAPERJ. The study
was approved by CEUA-DAHEICB092-05/16.
DIFFERENTIATION OF GASTRIC SECRETORY CELLS: EFFECTS OF

EARLY WEANING AND CORTICOSTERONE

Email: zulianjg@usp.br
Av Prof Lineu Prestes 1524 ICB I 05508-000 So Paulo, SP, Brazil
Phone: 55 11 3091 7303 Mobile: 55 11 99719 4760
In gastric epithelium, mucous neck and zymogenic cells (ZC) populations are the last
to complete the maturation process, as part of mucous neck cells (MNC) differentiate
into ZC. Early weaning (EW) can induce the precocious maturation of diverse organs
and augment corticosterone levels. To evaluate the influences of EW and
corticosterone on secretory cells during stomach development, Wistar rats were
separated into groups, injected with RU486 (for corticosterone blockage, 10mg/kg)
or vehicle: suckling control (S), suckling -RU486 (SRU), early weaning (EW) and
early weaning -RU486 (EWRU) (CEUA 86/2008; 18/2015). By qPCR, we observed
that EW increased the expression of Bhlha15, Pgc, Muc5ac and Muc6 (regulatory
differentiation genes) and decreased on Pga5, which is transcribed by immature
zymogenic cells. Moreover, EW augmented Sgk1 expression (corticosterone early
response) and RU486 treatment reduced the levels, confirming efficient
corticosterone blockage. GR expression was not altered. Corticosterone antagonism
also reduced Msn, Gif, Pgc and Muc5ac expression. MNC quantification (using
histochemistry and immune-histochemistry reactions) showed that EW increased the
population, whereas RU486 treatment reduced this response. In addition, EW
augmented Mist1 and PGC immuno-stained cells. In young- adult rats, some EW
effects were still detected, as responses for Pgc and Pga5 expression persisted,
besides the reduction on Msn levels. Also, RU486 reduced MNC and PGC immunolabelled cell numbers, maintaining the effects identified immediately after treatment.
Conclusion: EW triggered and primed the precocious maturation of secretory gastric
cells and such effect was controlled by corticosterone.
Support: FAPESP JGZ: 2010/03076-0; CAPES; LYMH: 2013/18459-0; FAPESP:
2011/17415-3; 2014/21449-9.

E5
E6
ARHGAP21 PARTICIPATES IN CYTOSKELETON ORGANIZATION

DURING
MEGAKARYOPOIESIS
THROUGH
RHO
GTPASE
REGULATION
PRIMING EFFECTS OF EARLY WEANING ON H+/K+-ATPASE

EXPRESSION AND PARIETAL CELL POPULATION IN THE GASTRIC
MUCOSA
Vanessa Aline Bernusso (1), Mariana Lazarini (1,2), Joo Agostinho Machado-Neto
(1), Karin Spat Albino Barcellos (1), Sara Teresinha Olalla Saad (1)
Melissa Teles Silva (1), Gizela Agostini Zonta (1), Cruz Alberto Mendonza Rigonati
(1), Daniela Ogias (1), Juliana Guimares Zulian (1), Patrcia Gama (1)
(1) Hematology and Hemotherapy Center, University of Campinas, So Paulo, Brazil

(2) Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal
(1)Department of Cell and Developmental Biology, Institute of Biomedical Sciences,

University of So Paulo, Brazil
The authors declare that they have no competing interests

Presenting author: vabernusso@yahoo.com.br; mobile phone: 55 19 99351 2590 and
institutional phone 55 19 3521 8734
Background: Megakaryocytes are precursor cells that produce platelets via
cytoplasm extensions, requiring profound cytoskeleton rearrangements. ARHGAP21
is a RhoGAP protein involved in hematopoiesis. Aims: Investigate the role of
ARHGAP21 during megakaryocyte differentiation. Methods: Megakaryocyte
differentiation was stimulated in HEL cells with 20 nM of phorbol myristate acetate 13 -12 (PMA) and was confirmed by the expression of CD41a, CD42b and CD61
and polyploidy using flow cytometry. ARHGAP21 silencing was performed by
siRNA, after PMA treatment for 2 and 3 days and was followed by the analysis of
the expression and activity of RhoA, RhoC, Cdc42, RAC1 and their effectors.
Results: HEL cells treated with PMA presented increased cell size, polyploidy and
expression of CD41a, CD42b and CD61. Intense rearrangement of the cytoskeleton
and colocalization of ARHGAP21 and -tubulin was observed by confocal
microscopy and their interaction was confirmed by immunoprecipitation. In the days
2 and 3, ARHGAP21 upregulation was concomitant with the reduction of RhoA,
Cdc42 and RAC1 activities, but not of RhoC. ARHGAP21 silencing also increased
the phosphorylation of myosin light chain2 serine 19, DIAPH1 expression and
decreased the phosphorylation of Stathmin serine 16 and Glu-tubulin expression.
Moreover, silencing of ARHGAP21 altered the activity of proteins related to cell
adhesion, such as FAK925, p130CAS, Zyxin and expression of Vinculin and ARP3.
Conclusion: Our results suggest that the upregulation of ARHGAP21 during
megakaryocytic differentiation is important to regulate cell adhesion, actin
polymerization and microtubule stability trough the regulation of RhoA and Cdc42.
Email: melissa.teles.silva@usp.br
Av. Prof Lineu Prestes 1524 ICB I 05508-000 So Paulo, SP, Brazil
Phone: 55 11 3091-7303
The postnatal development of gastrointestinal tract depends on elements, which
include growth factors and suckling, and early weaning (EW) influences
proliferation, differentiation and maturation of gastric epithelial cells. In order to
define whether EW can affect acid secretion through expression H+/K+-ATPase and
parietal cell population and to determine the maintenance of effects, we compared
the gastric mucosa from suckling (S) and EW animals. Wistar rats were separated
from their mothers at 15 d, and stomachs were collected at 18, 30 and 60 d (CEUA
ICB/USP 18/2015). To identify parietal cells, paraffin sections were stained with
hematoxylin- eosin, where parietal cells were counted in a total of 1,000 epithelial
cells. To analyze H+/K+-ATPase gene expression, gastric samples were collected at
15, 18, 30 and 60 d for RT-qPCR. There was no significant change in parietal cell
index at 18, 30 and 60 d (S vs. EW). However, the expression of H+/K+-ATPase
pump increased after EW at 18 and 60 d (p<0.05 vs. respective S group). Our results
suggested that the number of parietal cells in gastric epithelial population is
determined early, during the third postnatal week, and whereas EW did not alter their
number, it increased the expression of proton pump in parietal membrane, which has
an essential role in differentiation and function of these cells. Moreover, as such
response was maintained until adulthood, further investigation is necessary to
determine other cellular and physiological effects on gastric mucosa. MTS is
recipient of CNPq fellowship; supported by FAPESP processes: 2011/17415-3;
2014/21449-9.
Supported by FAPESP and CNPq.

Keywords: Megakaryocyte, Cytoskeleton and ARHGAP21

E7
E8
TURNING BACK THE CLOCK ON RETINAL PROGENITORS THROUGH

MANIPULATION OF KLF4 EXPRESSION
WFDC1/ps20
DERIVED
PEPTIDE
PRONES
HUVEC
TO
DIFFERENTIATION INTO MEGAKARYOCYTES AND GRANULOCYTES
Beatriz C.Toledo (1), Maurcio Rocha-Martins (1), Rodrigo A Martins (2), Mariana
S.
Silveira (1)
Juliana Ap. Preto de Godoy (1)*, Silvia Borges Pimentel de Oliveira(1), Alexandre
F. de Oliveira(1) e Hernandes F. Carvalho(1)
1- Department of Structural and Functional Biology, Institute of Biology, State
University of Campinas (UNICAMP).
(1)Instituto de Biofsica Carlos Chagas Filho, UFRJ, Rio de Janeiro, RJ, Brasil;
(2) Instituto de Cincias Biomdicas, UFRJ, Rio de Janeiro, RJ, Brasil.
*email: ju.pgodoy@gmail.com; telephones: (19) 3521-6125/(19) 9 9614 1478

toledo.bc@gmail.com phone: 55 21 39386562/ 55 21 998212994

The mammalian retina has a limited capacity for regeneration. Retinal progenitor
cells (RPC) undergo fate restriction throughout development. Postnatal RPCs loose
retinal ganglion cell (RGC) competence and generate mainly rod photoreceptors
(RP). In this study, by interfering with the expression of a key regulator of iPS
reprogramming (KLF4), we reprogramed the competence of postnatal RPCs. First,
we tested if KLF4 is required during retinal development. Conditional knockout mice
(cKO) did not present gross visual impairment nor defects in the generation of
specific cell types, suggesting that KLF4 is not essential for transition of
competence. Although previous results showed a positive correlation between an
increase in KLF4 content and cell cycle exit, cell proliferation rate and the
expression of cell cycle regulators were unaffected in postnatal cKO retinas. Next, to
test whether KLF4 is sufficient to reprogram the postnatal RPCs, we performed gainof-function experiments. KLF4 overexpressing cells prematurely exit the cell cycle,
show an increase in cell cycle inhibitors, fail to generate RP and to express RP
specification genes. Strikingly, KLF4 overexpression induced the expression of
genes involved in RGC development. Most cells migrated to the ganglion cell layer
and projected axons into the optic nerve, which reinforces that they adopted the RGC
fate. Since post-natal RPCs are not compentent to generate RGCs, these results show
that KLF4 can reprogram post-natal RPCs into RGC-like cells. Therefore, this has
potential for the development of new glaucoma therapies. All procedures were
approved by the institutional Comitte for Animal Experimentation.
Background: WFDC1/ps20 is a member of the whey acidic protein (WAP) family

with conserved 4-disulphide core domain, which includes secreted proteins. Previous
study in our laboratory showed that ps20 protein-derived peptide 6 (p6) affected
HUVEC behavior in vitro.
Aims: To study endothelial markers in response to p6 and test cells response to
ATRA and PMA, inducers of granulocytes and megakaryocytes, respectively.
Methods: HUVECs were exposed to 0.01, 0.1 and 1.0g/mL p6 for 48 hours and
analyzed for CD34 and CD31 expression by FACS. Then, p6-treated cells were
exposed to 1 M ATRA or 20 nM PMA, inducers of differentiation into
granulocytes (CD11b+) and megakaryocytes (CD61+).
Results: Exposure to p6 decreased CD34+ (0.8% vs 1.2% in controls) and did not
affect the percentage of CD31+ cells. We considered that reduced CD34+ could mean
even less differentiated cells and tested the ability of the cells to respond to
compounds ATRA and PMA. Treatment with ATRA increased the number of
CD11b+ cells (2.39%) previously treated with 0.1g/ml p6, as compared to cells
without treatment with p6 (0.65%). Treatment with p6 resulted in marked resistance
to PMA induced cell death, promoted an increase in CD61+ cells as compared to
controls (4.47% vs 1.08%). Furthermore, platelet-like cell fragments were detected
by FACS and identified upon microscopy.
Conclusion: p6 influence cell differentiation as well as increase cell survival in
response PMA. The results so far demonstrated that p6 increases the ability of
endothelial cells to differentiate into granulocytes and megakaryocytes.
Financial support: CNPq, FAPERJ.
Funding Support: CNPq 150149/2016-6; FAPESP 2009/16150-6

E9
E10
ANALYSIS OF THE ROLE PLAYED BY THE RECK TUMOR

SUPPRESSOR GENE SPLICING VARIANTS DURING ADIPOGENESIS
AND OSTEOGENESIS
OSTEOGLYCIN
MYOGENESIS
Thais de Assis Ribas (1), Maria Fernanda Forni (2), Michelle Silberspitz Konig (2),
Gabriel Antonini (2), Sheila Maria Brochado Winnischofer (3), Mari Cleide Sogayar
(1, 2), Marina Trombetta-Lima (1)
(1) NUCEL/NETCEM, Group of Celular Terapy and Molecular, Departament of
Medical Clinic, Faculty of Medicine, University of So Paulo, Brazil
(2) Chemistry Institute, Biochemistry Department, University of So Paulo, Brazil
(3) Departament of Biochemistry and Molecular Biology, Federal University of
Paran, Brazil
e-mail: thais.assis.ribas@gmail.com
Tel: +55 (11) 2648-0236
Cel: +55 (11) 99583-9443
Rua Pangar, 100 Butant
CEP 05360-130
So Paulo SP - Brazil
Background
The extracellular matrix (ECM) and its interaction with the cells play an important
role during different processes, such as: cell growth, migration and differentiation.
The matrix metalloproteinases (MMPs) family members, associated with their
inhibitors, are crucial for ECM remodeling. The canonical RECK protein is known to
inhibit MMP-2, MMP-9 and MMP-14. Recently, our group characterized a new
RECK alternative splicing variant, namely: RECK-B, which, apparently, is unable to
inhibit MMPs and has pro-oncogenic activity in vitro.
Aim
Our aim is to analyze the role of the RECK splicing variants during osteogenic and
adipogenic differentiation protocols.
Methods
Human skin mesenchymal stem-cells (MSCs) were subjected to adipogenic and
osteogenic protocols Total RNA was extracted from these cells at three different time
periods: 7, 14 and 21 days and the mRNA expression of MMPs and their inhibitors
was analyzed by qRT-PCR. Primary cultures of MSCs overexpressing these RECK
variants or in which these RECK variants were downregulated by shRNA, are
currently being generated through transduction or nucleofection for further
functional analysis.
Results
Our data suggest that the balance between the expression of the two RECK variants
is modulated in opposite patterns during the two different differentiation protocols.
While an increase in canonical RECK, relative to RECK-B expression is observed
during the adipogenic protocol, the opposite pattern is observed during the
osteogenic protocol.
Conclusion
Our results show that RECK variants expression is differentially modulated upon
adipogenic and osteogenic protocols.
INHIBITION
BY
microRNA
miR-155
IMPAIRS
Paula Paccielli Freire (1), Sarah Santiloni Cury (1), Grasiele de Oliveira (1), Bruno
Oliveira da Silva Duran (1), Geysson Javier Fernandez (1), Leonardo Nazario de
Moraes (1), Csar Seigi Fuziwara (2), Edna Teruko Kimura (2), Maeli Dal-Pai-Silva
(1), Robson Francisco Carvalho (1).
(1) Department of Morphology, Institute of Biosciences of Botucatu, So Paulo State
University (Unesp), Botucatu, So Paulo, Brazil.
(2) University of So Paulo (ICB-USP), So Paulo, So Paulo, Brazil.
Email address: freirepp2@gmail.com
Telephone: +55 14 3880 0503
Skeletal myogenesis is a regulated process in which mononucleated cells, the
myoblasts, undergo proliferation and differentiation. Upon differentiation, the cells
align with each other, and subsequently fuse to form terminally differentiated
multinucleated myotubes. Previous reports have identified the protein osteoglycin
(Ogn) as an component of the skeletal muscle secretome differentially expressed
during muscle development. However, the posttranscriptional regulation of Ogn by
microRNAs during myogenesis is unknown. Bioinformatics analysis showed that
mir-155 potentially targeted Ogn transcript at 3-untranslated region (3 UTR). We
tested the hypothesis that miR-155 inhibits expression of the Ogn to regulate
myogenesis. C2C12 myoblast cells were cultured and miR-155 overexpression or
Ogn knockdown were induced by the transfection with miR-155 mimic, siRNA-Ogn,
and negative controls with lipofectamine. Near confluence (80-90%), myoblasts
were induced to differentiate to myotubes. Luciferase assay were used to confirm the
interaction between miR-155 and Ogn 3UTR. RT-qPCR and Western blot were
used to evaluate the mRNA and protein expression of miR-155, Ogn and the
myogenic molecular markers (Myh, MyoD, and MyoG) in myoblasts and myotubes.
Myoblasts migration and proliferation were evaluated by Wound Healing and MTT
assay, respectively. Our results show that miR-155 interact with 3UTR Ogn and
decreased the levels of Ogn in myotubes. The overexpression of miR-155 increased
MyoG expression, decreased cellular migration in myoblasts, and decreased Myh
expression in myotubes. Moreover, Ogn knockdown reduced the expression levels of
MyoD, MyoG, and Myh in myotubes. These results reveal a novel pathway in which
miR-155 inhibits Ogn expression to regulate the myogenesis process.
Information on ethical approval and funding support: This study was supported by
So Paulo Research Foundation (FAPESP 2014/13783-6).
Financial support: BNDES, CAPES, CNPq, FAPESP, FINEP, MCTI, MS-DECIT.

F CELL SIGNALING
F1
F2
METABOLIC PROFILE ANALYSIS OF MICE (C57BL/6) AFTERORAL

TREATMENT WITH THE CYTOTOXIC PEPTIDE CROTAMINE FROM
RATTLESNAKE VENOM REVEALS NOVEL ACTIVITIES FOR THIS
TOXIN THAT IS INDEPENDENT OF ITS CYTOTOXIC PROPERTY
INCREASED p53 AND DECREASED p21 ACCOMPANY APOPTOSIS

INDUCED BYULTRAVIOLET RADIATION IN THE NERVOUS SYSTEM OF
A CRUSTACEAN
Marcelo P. Marinovic1 Joana D. Campeiro1 Eduardo B. Oliveira,2 Marcelo A.

Mori3 Mirian A. Hayashi1
(1) Ps-Graduao em Fisio-Farmacologia, Setor de Produtos Naturais
(SPN),Departamento
de
Farmacologia,
Instituto
de
Farmacologia
(INFAR),Universidade Federal de So Paulo (UNIFESP/EPM). (2) Departamento
de Bioqumica, Universidade de So Paulo (USP-RP). (3) Departamento de
Gentica, Universidade de Campinas (UNICAMP).
The cytotoxic effect of crotamine was demonstrated to be dependent of its
internalization by the cells and by its actions on intracellular organelles as
lysosomes and mitochondria. As the energy balance is a process that occurs mainly
through energetic production (also called thermogenesis) in a process dependent of
3-adrenergic receptor sensibilization and activation of mitochondria uncopled
protein 1, one would predict the potential effect of crotamine in the thermogenesis,
since recent experiments confirmed the potential cytotoxic effect of crotamine and
the limited gain of weight of animals treated with this toxin. Therefore, the
objective of this study is to evaluate the action of low doses crotamine treatment in
weight gain, fatty deposits and other tissues, glucose tolerance test (GTT) and
insulin tolerance test (ITT), indirect calorimetry assessment plasma lipid profile
(triglycerides, total cholesterol, LDL, HDL), serum markers of liver function (AST,
ALT and GAMMA GT) and renal damage (uric acid and creatinine), besides the
evaluation of the intestinal microbiota (firmicutes and bacterioidetes), the gene
expression of thermogenic markers in BAT (-3 receptor, PGC1, PPAR and
UCP-1) and differentiation of white adipocytes into the subcutaneous adipose
tissue (PRDM16 and UCP-1) and adipocyte lineage (PRDM16 and UCP-1), aiming
to analyze the thermogenesis pathways in brown adipocytes and its systematic
effects. The obtained results demonstrated that treatment reduces weight gain,
white adipose depots and increased brown deposit, reduced lipid plasma markers of
liver and kidney damage, improved the response to insulin, increased resting
energy expenditure, increased expression of genes thermogenic in BAT and
differentiation in subcutaneous tissue. We can conclude that the treatment induced
a catabolic profile evidenced by the increased production of heat result of increased
systemic energy expenditure.
Gabriela Hollmann a, Rafael Lindena, Angela Giangrandeb, Silvana Allodia

a
Programa de Ps Graduao em Cincias Biolgicas-Fisiologia, Instituto de Biofsica

Carlos Chagas Filho, Universidade Federal do Rio de Janeiro-UFRJ, Riode Janeiro, RJ
21941-590, Brazil.
b
Institut de Gntique et de Biologie Molculaire et Cellulaire-IGBMC, INSERM,
Strasbourg, France.
Ultraviolet (UV) radiation can produce biological damage, leading the cell to apoptosis
by the p53 pathway. This study evaluated some molecular markers of the apoptosis
pathway induced by UVA, UVB and UVA+ UVB (Solar Simulator, SIM) in
environmental doses, during five consecutive days of exposure, in the brain of the crab
Ucides cordatus. We evaluated the central nervous system (CNS) by immunoblot-ting
the content of proteins p53, p21, phosphorylated AKT, BDNF, GDNF, activated
caspase-3 (C3) and phosphohistone H3 (PH3); and by immunohistochemical tests of
the cells labeled for PH3 and C3. After the fifth day of exposure, UVB radiation and
SIM increased the protein content of p53, increasing the content of AKT and,
somehow, blocking p21, increasing the content of activated caspase-3, which led the
cells to apoptosis. The signs of death affected the increase in neurotrophins, such as
BDNF and GDNF, stimulating the apoptotic cascade of events. Immunohistochemical
assays and immunoblotting showed that apoptosis was present in the brains of all UV
groups, while the number of mitotic cells in the same groups decreased. In conclusion,
environmental doses of UV can cause apoptosis by increasing p53 and decreasing p21,
revealing an UV-damage pathway for U. cordatus.
Financial support by FAPESP, CNPq and CAPES.

F3
CAMKII- TRANSLOCATES
STIMULATION
F4
TO
THE
NUCLEUS
UPON
EGF
CANONICAL Wnt SIGNALING IS TIME-DEPENDENTLY REGULATED BY

O-GlcNAcylation
Jerusa Arajo Quinto Arantes Faria (1); Alfredo Miranda de Goes (1); Michael H.
Nathanson (2); Dawidson Assis Gomes (1).
Miguel Clodomiro dos Santos Lucena 1, Gustavo Ventura 1, Diego Allonso 1, Adriane
Regina Todeschini 1, Gerald Hart 2, Wagner Barbosa Dias 1
1- Department of Biochemistry and Immunology, Federal University of Minas

Gerais, Minas Gerais, Brazil
2- Section of Digestive Diseases, Internal Medicine, Yale University, Connecticut,
USA.
Correspondence: jerusaquintao@gmail.com. Instituto de Cincias Biologicas.

Antnio Carlos Av. 6627, Pampulha. Cep:31270-901. Institutional number:
+5531334092631
Introduction: O-GlcNAc is a post-translational modification that regulates several

protein and cellular functions. The O-GlcNAc transferase (OGT) transfers the Nacetylglucosamine (GlcNAc) residue to target proteins and the O-GlcNAcase (OGA)
removes it. -catenin is a major player in canonical Wnt signaling by binding TCF and
promoting cell cycle progression. It was reported that O-GlcNAcylation regulates catenin subcellular localization and promotes its stability in human carcinoma.
However, it is not well established how the different subpopulations of -catenin
(nuclear or cytosolic) depends on O-GlcNAc to perform their respective functions.
Objective: This work aims to study how O-GlcNAc regulates canonical Wnt signaling.
Methodology: For monitoring O-GlcNAcylated -catenin subpopulations, cell extracts
treated with Wnt were subjected to nucleus-cytoplasm fractionation. To study the
cytoplasmic non-degraded -catenin, cells were stationed at G2/M. A confocal
microscopy was performed with marking for -catenin, OGT and OGA in various
stages of mitosis. To check whether the O-GlcNAc can interfere in nuclear -cateninTCF interaction we performed a cross-linking assay using recombinant proteins and
nuclear extracts were submitted to ELISA biding assays. Results: Our results indicate
that Wnt decreased time-dependently overall levels of O-GlcNAcylated proteins
regulating OGT and OGA protein expression and activities. O-GlcNAc also regulates
-catenin subcellular localization and its nuclear interaction with TCF. Besides, we
observed -catenin co-localization with OGT and OGA during specific stages of
mitosis. Conclusion: Our data suggest that O-GlcNAc modulates not only nuclear catenin but also the non-degraded cytoplasmic -catenin is being recruited to the
equatorial plates of the dividing cell participating in complexes in the mitotic spindle.
Keywords: O-GlcNAc, -catenin, Wnt signaling
Calcium (Ca2+) signaling within the cell nucleus regulates specific cellular events
such as cell proliferation. Nuclear and cytosolic Ca2+ can be independently
regulated, and nuclear translocation of receptor tyrosine kinases (RTKs) is one way
to locally activate signaling cascades within the nucleus. Recent data show the role
of nuclear epidermal growth factor receptor (EGFR) for tumor signaling. However,
it remains unclear which calcium machinery EGFR specifically modulates. For
that, we focused on the downstream calcium cascade, especially the Ca+2/CaM
dependent protein kinases (CaMKII), a multifunctional serine threonine kinase with
a broad range of substrates. First, we analyzed the CAMKII isoforms expressed in
SkHep-1 cells and rat hepatocytes by Real Time PCR and Western blot. Results
reveal the absence of both the alpha and beta isoforms in both cell types. A similar
pattern was observed for the gamma and delta isoforms in rat hepatocytes, although
CAMKII expression was reduced after cell plating. CAMKII isoforms present
distinct patterns regarding subcellular localization, which is cytosolic to CAMKII and nuclear to CAMKII-. The profile of Thr286 phosphorylation showed
oscillatory activation of CAMKII by EGFR signaling. CAMKII- translocates to
the nucleus upon EGF stimulation in SKHep-1 cells. The CAMKII translocation is
due to IP3/PLC-1-dependent increase in calcium levels. Also, BAPTA treatment
dramatically reduces CAMKII protein expression in SkHep-1 cells. Finally,
CAMKII- knockdown decreases BrDU uptake. Collectively, these data suggest
that CAMKII- can mediate nuclear effects as cell proliferation induced by EGFR
activation.
This work was supported by CAPES, CNPq, FAPEMIG and NIH Fogarty.
Universidade Federal do Rio de Janeiro (Instituto de Biofsica Carlos Chagas

Filho), 2Johns Hopkins University (Department of Biological Chemistry)
(Information: miguelbiof@gmail.com ;Tel : +552139386543)
Supported by: CNPq
(1)
(2)
F5
F6
EXPRESSION OF SMAD1, A CANDIDATE OF BIOMARKER FOR

DIABETIC NEPHROPATHY, IN URINE SEDIMENT OF PATIENTS
WITH TYPE 2 DIABETES
PURINERGIC RECEPTORS ARE INVOLVED IN THE STIMULATORY

ACTION OF THE CROTOXIN ON OXIDATIVE BURST PRODUCTION BY
MACROPHAGE
Aline de Jesus Souza Pereira (1), Matheus Gonalves Della Nina Raffo (1),
Matheus Moreira Perez (2), Beatriz Alves (2), Fernando Luiz Affonso Fonseca (1),
Patricia Favaro (1).
De Oliveira RBB1, Demasi M2, Sampaio SC1.
(1) Department of Biological Sciences, Federal University of So Paulo, Diadema,

So Paulo, Brazil
(2) Clinical Analysis Laboratory, ABC Medical School, Federal University of So
Contact details:
Address: R. So Nicolau, 210
Centro, Diadema SP
Email: alinejspereira@gmail.com
Telephone: (11) 3385-4137 / (11) 98076-8461 (mobile)
Diabetic nephropathy (DN) is the leading cause of renal disease, characterized by
mesangial matrix expansion. This process occurs slowly, over many years, and the
prognostic biomarkers, such as microalbuminuria, do not correlate with
glomerulosclerosis during the initial phase of the disease. Studies show that
transcription factor SMAD1, identified in the TGF- pathway, regulates the
expression of collagen type IV, an important component in glomerulosclerosis.
There are evidences that urinary SMAD1, detected by ELISA assay, could be a
predictor marker for the initial phase of this disorder. The aim of this study
included (a) the standardization of the quantitative PCR (qPCR) technique for the
detection of SMAD1 in urine sediment of patients with diabetes mellitus type 2; (b)
characterization of the expression of SMAD1 in 30 patients with diabetes without
DN and 10 healthy controls, and its correlation with markers of disease progression
and renal lesion. MannWhitney was used. Our results showed, as expected, that
there were no significant differences in the expression of SMAD1 transcripts
between the control and patient groups. But we observed, in these patients, a strong
tendency of positive Spearman correlation of SMAD1 expression and fasting
glucose levels. It is well known that blood glucose fluctuation is associated with
diabetic nephropathy, but the mechanism is not fully understood. Our results
suggest a possible relation between glucose levels and activation of SMAD1
pathway in mesangial cells. This study demonstrates that the quantification of
SMAD1 transcript level is viable by qPCR.
Pathophysiology Laboratory, 2Laboratory of Biochemistry and Biophysics. Butantan

Institute, SP, Brazil.
Crotoxin (CTX), main toxin from snake venom Crotalus durissus terrificus (CdtV)
stimulates macrophages (M) metabolism. Purinergic receptors (PRs) are crucial in the
regulation of pathophysiological processes by inducing the generation of H2O2 and NO
by macrophages and PRs can be stimulated by formyl peptide receptors (FPRs),
involved in the stimulatory activities of CTX. Aim: To investigate the possible
involvement of PRs and FPRs on H2O2 and NO production by M. Methods: M were
obtained from peritoneal cavity and adhered 96-well plate (3x105/100l) and incubated
with CTX (12.5; 25 or 50 nM) or ATP (1mM), for 2 h at 37oC. The H2O2 production
was measured using phenol red. For NO production, M were incubated with LPS
(5g/ml), for 24 h and nitrite was measured in the supernatants using Griess reagent. To
evaluate PRs and FPRs participation, M were incubated with PRs antagonists:
Suramine-10M or A43-100M and Boc-2, a selective antagonist of FPRs (100M). To
establish H2O2 production kinetic, M were placed in 96-well plate at 37oC, and CTX
or ATP were added and H2O2 production rates measured using a HRP-based detection
systems dependent oxidation of a molecular probe (Amplex UltraRed). Results and
Discussion: The results showed that CTX increased H2O2 production (12.5nM:75%;
25nM:87%) and NO production (12.5nM:5.9-fold; 25nM:17%). This stimulatory effect
induced by CTX was observed from 30 min at 120 min (65%), similar manner to ATP.
Suramine and Boc-2 antagonists blocked the stimulatory effect of CTX on the H2O2
production by M. For first, was demonstrated the involvement of the PRs in the CTX
action on M function.
Supported by CNPq Pibic

F7
F8
MOLECULAR MECHANISM OF MASPIN NUCLEOCYTOPLASMIC

SHUTTLING
CHARACTERIZATION OF MASPIN EXPRESSION, PHOSPHORYLATION

AND SUBCELLULAR LOCALIZATION DURING THE MURINE MAMMARY
GLAND DEVELOPMENT
Jeffrey Reina (1) & Nathalie Cella (1)

Magna Magalhes (1) and Nathalie Cella (1)
(1)

Sciences, University of So Paulo, Brazil.
(2)
Jeffrey Reina: Av. Professor Lineu Prestes 1524, So Paulo, Brazil. Phone: (011)
942593543, (011) 30917308. E-mail: jeffreina@usp.br,
Maspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic
biological activities, including regulation of cell proliferation, death, adhesion,
migration, gene expression and oxidative stress response. The molecular
mechanism underlying maspin function is poorly understood. Several studies
suggest that subcellular localization plays an essential role on maspin biological
function and tumor suppressor activity. Nuclear Maspin has been associated with a
good prognostic, whereas nucleocytoplasmic localization correlates with tumor
progression. The objective of this project is to investigate the mechanism
underlying maspin nucleocytoplasmic shuttling. We have identified a putative
nuclear localization signal (NLS) on maspin protein sequence. NLS deletion
reduces maspin nuclear localization in transfected cells with a maspin-EGFP
construct as observed by confocal fluorescence microscopy. However, site-directed
mutagenesis of lysine and arginine residues of the putative NLS was not enough to
abolish maspin nuclear localization. New combined site-directed mutagenesis is
necessary to identify the essential residues for maspin nuclear localization. This
data indicate that maspin nuclear translocation involves both passive and active
mechanisms. We are currently searching for maspin binding partners by mass
spectrometry to investigate how the identified molecules regulate maspin
nucleocytoplasmic shuttling.
This project is supported by FAPESP and by the Brazilian Program for Post
Graduate Students (PEC-PG) of CAPES/CNPq Brazil.
(1) Department of Cell and Development Biology, Institute of Biomedical Sciences,

magna.magalhaes@usp.br - +55-11-3091-7778 Av. Professor Lineu Prestes, 1524
ICB I Sala 428 Cidade Universitria Butant, So Paulo, So Paulo, Brasil.
ncella@usp.br - +55-11-3091-7308
Maspin is a tumor and metastasis suppressor protein. Nuclear localization of maspin in
breast cancer cells has been associated with a better prognosis, whereas maspin
cytoplasmic localization is linked to a poor prognosis. Using MCF-10A cells, our group
observed a correlation between maspin phosphorylation and its cytoplasmic
accumulation. In addition, in a tumor cell line which expresses maspin predominantly in
the cytoplasm, this protein is highly phosphorylated. These data suggest that maspin
phosphorylation correlates with cytoplasmic localization and tumorigenesis. In view of
these results and the importance of maspin subcellular localization in the prognosis of
breast cancer, the objectives of this study were to investigate maspin expression and
phosphorylation levels and subcellular localization during the murine mammary gland
development. To this aim, murine mammary glands were isolated from different
developmental stages. Maspin expression was analyzed by immunoblot and maspin
phosphorylation was detected by 2D-SDS-PAGE and Phos-tag SDS-PAGE followed by
immunoblot. Maspin subcellular distribution was determined by immunofluorescence.
Maspin is detected in late pregnancy and remains at constant levels during lactation and
involution. Maspin is phosphorylated both in lactation and involution, although
phosphorylation levels decrease in involution. Maspin is predominantly in the
cytoplasm in all luminal epithelial cells, while nuclear localization varies among these
cells. These data indicate that maspin expression and phosphorylation are
developmentally regulated in the murine mammary gland and maspin is located in the
cytoplasm of luminal epithelial cells.
This work was approved by institutional Ethical Committe on Animal Use and
supported by FAPESP (So Paulo) and CNPq (Braslia).

F9
F10
CALSARCIN-1 MAY ACT AS A NEGATIVE REGULATOR OF

CALCINEURIN-A BY STERIC HINDRANCE TO THE NFAT
TRANSCRIPTION FACTOR
LIPOTOXICITY MODULATES VESICLE TRAFFICKING-INVOLVED

PROTEINS IN HUMAN HEPATOCYTES HepG2 CELLS
Slvio Roberto Consonni* (1,2); Carlos Roberto Koscky Paier* (1); Ana Helena
Macedo Pereira (1); Alisson Campos Cardoso (1); Raphael Morales Neto (1);
Daniela Boassa (2); Mark Ellisman (2); Stephanie Lam (3); Alice Ting (3); Tatiani
Lima Brennelli (4); Fbio Csar Gozzo (4); Rodrigo Vargas Honorato (1); Paulo
Srgio Lopes de Oliveira (1); Kleber Gomes Franchini (1).
1 LNBio, Brazilian Center for Research in Energy and Materials
2 NCMIR, University of California San Diego
3 Department of Chemistry, Massachusetts Institute of Technology
4 Chemistry Institute, State University of Campinas
Julia M. Guitti de Souza1, Gabriela M. Soares1, Lucas Zangerolamo1, Sara T. Saad2,

Antonio C Boschero1, Helena C. Barbosa-Sampaio1
1: Department of Structural and Functional Biology, Biology Institute, University of
2: Haematology Centre, School of Medicine, University of Campinas, So Paulo,
Brazil.
guitti.julia@gmail.com;
+55(19)993977337
(institutional phone).
The authors declared no conict of interest.
Presenting author information: Laboratrio Nacional de Biocincias, Rua Giuseppe

Mximo Scolfaro, n 10.000, Campinas/SP, telefone: 19-3512-1187.
Background: Negative regulation of calcium-dependent phosphatase calcineurin (Cn)
by calsarcin (CS) plays key roles in regulating cardiac hypertrophy. However, the
mechanism of this regulation and the subcellular distribution of CS remain unclear.
Aims: the goals of this work were characterizing CnA-CS1 assembly and monitoring
spatial-temporal distribution of CS1 in the Z-discs in cultured neonatal rat ventricular
myocytes (MVRNs) under cyclic mechanical stretch. Methods: Recombinant protein
expression, chemical cross-linking coupled to mass spectrometry (MS/MS) and
genetic molecular probes applied to correlative microscopy and biochemical assays.
Results: The interacting surface of CnA was mapped in a pocket between the 1st and
3rd -helixes and surrounding loops, while the corresponding surface of CS1 was
mapped to the carboxyterminal loops within the Leu179-Phe185, Phe195-Ser199 and
Thr250-Leu264 regions. The assays in MVRNs under mechanical stimuli showed CS1
location change and reduced interaction with CnA. Interestingly, the cyclic stretching
did not induce modification in gene expression of CnA and CS1, as well as no
change in the phosphatase activity of CnA. Remarkably, CS1 overexpression and
silencing supported the CS1 negative regulation over CnA in stretched MVRNs.
Conclusion: The region of CnA that interacts with CS1 was found in close
proximity, but not coincident, to the -sheet 14, the main binding site for the PxIxIT
sequence of NFAT. Then, the role of CS1 in the negative regulation of CnA may
occur by steric hindrance of the NFAT transcription factor. These results could
contribute to a possible pharmacological inference.
(mobile);
+55(19)35216198
Background: Cellular vesicles are important for lipid molecules transport in

hepatocytes. Vesicles formation and transport are essential for lipids management in
the liver. ARFs are proteins involved with cytoskeleton remodeling, vesicles
formation and their fusion to the cellular membrane. Lipotoxicity and insulin
resistance - induced by palmitate treatment in vitro leading to reduced hepatocytes
cellular viability - could possibly affect cellular vesicles formation and
transportation. Aims: The aims of the study were to investigate the possible
involvement of lipids vesicles formation and transportation-involved proteins ARF1
and ARF6 in insulin resistant HepG2 cells that were treated with palmitate.
Methods: Cultured cells were treated with palmitate, 250 M or 500 M, to induce
lipotoxicity and insulin resistance in HepG2 cells. Cellular protein extracts were
loaded in polyacrylamide gel to perform Western Blotting assay to evaluate ARF1
and ARF6 protein content and insulin signaling by AKT-Ser473 phosphorylation,
using GAPDH as internal control. The groups were analyzed by Student-T test to
assess the significance of the results. Results: Here we show that 500 M palmitate
induced insulin resistance after 24h in HepG2 cells, and reduced ARF1 and ARF6
content, whilst reduced palmitate concentration (250 M) stimulated up-regulation in
ARF1 and ARF6 protein content, after 24h exposure. Conclusion: Our results show
that vesicles-associated proteins contents could be modulated in insulin resistant
hepatocytes, which suggests that lipids vesicles transport could be affected when
lipotoxicity is installed in liver cells.
Information on ethical approval and funding support: FAPESP
Ethical approval: CEUA/Unicamp 2310-1(a)

Funding support: FAPESP 2010/17086-7, 2011/01356-8

F11
F12
MOLECULAR MECHANISMS OF THE ARHGAP21 RhoGAP IN THE

VLDL VESICLES FORMATION IN HEPATOCYTES OF ARHGAP21HETEROZYGOUS C57BL/6 MICE
EXPRESSION OF GENES RELATED TO THE TGF1/SMAD SIGNALING

PATHWAY IN CHRONIC GRAFT VERSUS HOST DISEASE
Lucas Zangerolamo (1), Gabriela Moreira Soares (1), Julia M. Guitti de Souza (1),
Sara T. Saad (2), Antonio Carlos Boschero (1), Helena C. L. Barbosa-Sampaio (1)
Beatriz Corey Morini (1); Marcos Paulo Colella (1), Matheus Rodrigues Lopes (1);
Francisco Jos Penteado Aranha (1); Crmino Antonio de Souza (1); Afonso Celso
Vigorito (1); Sara Olalla Saad (1); Patricia Favaro (1,2).
(1) Department of Structural and Functional Biology, Biology Institute, University of

(2) Haematology Centre, School of Medicine, University of Campinas- UNICAMP,
Campinas, Brazil.
zangerolamo483@gmail.com; +55(19)996628671
(institutional)
The authors declared no conict of interest.
(mobile);
+55(19)35216198
(1) Hematology and Hemotherapy Center-University of Campinas/HemocentroUnicamp, Instituto Nacional de Cincia e Tecnologia do Sangue, Campinas, So
Paulo, Brazil
(2) Department of Biological Sciences, Federal University of So Paulo, Diadema,
So Paulo, Brazil
Beatriz Corey Morini: institutional phone (19) 3521-8734; mobile phone (14) 997177689; email: biatrizcorey@hotmail.com
Background
ARHGAP21 controls multiple cellular functions, such as vesicles intracellular
traffic. ARHGAP21 is a molecular partner of ARFs GTPases, controlling the
formation of vesicles. Our ARHGAP21-Heterozygous C57BL/6 mouse model (50%
of ARHGAP21) presented higher triglycerides content in the liver compared to
control when fed on a high fat diet (HFD). Then, there is a possibility that
ARHGAP21 is involved in the mechanisms associated with lipogenesis and/or liver
lipids vesicles secretion. Aims: The aims of our study were to investigate the
possible participation of ARHGAP21 and ARFs-GTPases in the formation of VLDL
vesicles and in the modulation of lipogenesis in the liver of HFD-fed ARHGAP21Het mouse. Methods: Gene expression and protein content were detected by q-PCR
and Western Blotting, respectively. Lipids accumulation was measured by Oil Red
staining. ARFs-ARHGAP21 interaction was detected by immunoprecipitation, whilst
the proteins co-localization was assessed by immunofluorescence. Results: Liver of
ARHGAP21-Het mice fed with HFD presented higher SREBP2 gene expression,
suggesting possible increase in cholesterol biosynthesis. Also, those mice presented
increased Oil Red staining, which corroborates with higher triglycerides
accumulation and hepatic steatosis. ARHGAP21 was co-localized with ARF1 and
ARF6. Immunoprecipitation showed decreased interaction of ARHGAP21-ARF in
the HFD-treated mice, which suggests that this interaction could be modulated by
liver steatosis. Conclusion: HDF induced liver steatosis in the ARHGAP21-Het
mouse and decreased ARF6-ARHGAP21 interaction, suggesting ARFs-ARHGAP21
participation in the vesicles formation, possibly interfering in the VLDL secretion in
nutritional overload, leading to increased hepatic lipids.
Chronic graft versus host disease (cGVHD) is one of the most important postallogeneic hematopoietic stem cell transplantation (HSCT) complications, due to
mortality and the impact upon quality of life. The biology of cGVHD is not yet well
understood but one hypothesis is that TGF- has a central role in the pathogenesis of
the disease. The aim of the study was to evaluate the transcript expressions of
TGF1, and its downstream target SMAD4 and SMAD5 in peripheral blood
mononuclear cells of patients submitted to allogeneic HSCT and of healthy donors.
A total of 13 samples from healthy donors and 24 patients (median-age=39 years for
both) submitted to allogeneic HSCT were included. Expression of mRNA was
detected by qPCR. The patients were classified into 3 groups as follows: 1) cGVHD,
receiving immunosuppressive treatment (IST); 2) cGVHD without IST; and 3)
tolerant, either with no history of cGVHD or after 6 months of recovering from
cGVHD. Mann-Whitney test was used. We observed a significant lower expression
of TGF1 and SMAD4 transcripts in the cGVHD without IST group when compared
with the control. A similar trend was observed for SMAD5 expression levels.
Moreover, TGF1 level was significantly increased in the tolerant group when
compared with the cGVHD without IST group. TGF is a pleiotropic cytokine with
immunoregulatory properties, implicated in the induction of alloantigen-specific
tolerance. Our results suggest an association of increased TGF expression with a
reduced risk of cGVHD. Further studies are necessary to elucidate the mechanism of
TGF regulation in the cGVHD.
Information on ethical approval and funding support: CEUA 4124-1, FAPESP

support.

F13
F14
THE SCAFFOLD PROTEIN RACK1 IS TRANSLOCATED DURING

ANTIGEN-STIMULATED MAST CELLS ACTIVATION
ERK5 ACTIVATION IN MAMMALIAN CELLS

Ahmed Alasseri (1), Michael Cross (2), Jrgen Mller (1)
Edismauro Garcia Freitas Filho (1), Constance Oliver (1), Maria Clia Jamur (1)
(1)
(1) Department of Cell and Molecular Biology and Pathogenic Bioagents, Ribeiro
Preto Medical School, University of So Paulo, Ribeiro Preto, So Paulo, Brazil.
(2)
E-mail: edismauro@usp.br
Address: Av. Bandeirantes, 3900, Monte Alegre. Ribeiro Preto, So Paulo, Brasil,
CEP: 14049-900
Phone: 55 (16) 3315-3143; 55 (16) 99621-8264.
Mast cells are immunoregulatory cells that participate in various biological events.
The action of mast cells is directly related to their activation and subsequent
mediator release. Activation via the high affinity IgE receptor, FceRI, is the best
characterized mast cell activation. Crosslinking of IgE bound to FceRI by
multivalent antigens results in the translocation of the receptor-ligand complex to
lipid rafts and the initiation of intracellular signaling events. RACK1 (Receptor for
Activated C Kinase 1) is a highly conserved intracellular adaptor protein that
interacts with a large number of proteins. As a scaffold protein, RACK1 integrates
inputs from distinct signaling pathways and is critical for fundamental cellular
activities. The present study aims to determine if RACK1 is present in mast cells and
to characterize its role in mast cell signaling. Using proteomic analyses, RACK1 was
identified in lipid rafts of RBL-2H3 mast cells. Furthermore, RACK1 was up
regulated when GD1b derived gangliosides are removed from the lipid rafts. In RBL2H3 cells and in mouse bone marrow-derived mast cells, by immunofluorescence,
RACK1 was localized throughout the cytoplasm in resting cells. After stimulation
via FceRI, the majority of RACK1 was translocated from the cytoplasm to the
nucleus. However, some RACK1 remained adjacent to the plasma membrane. The
translocation of RACK1 was confirmed by immunoblotting nuclear and cytosolic
extracts. Thus, the results suggest that RACK1 may be involved in FceRI-mediated
signaling pathways. An understanding of the role of RACK1 in mast cell activation
may lead to new therapeutic targets for allergy and inflammation.
(1) Aston Medical Research Institute, Aston Medical School, Aston University,
Birmingham, B4 7ET, United Kingdom, Tel: +44 (0) 121 204 3154, Email:
j.muller2@aston.ac.uk
(2) Department of Molecular and Clinical Pharmacology, University of Liverpool,
Liverpool, L69 3GE, United Kingdom
MAPK (mitogen-activated protein kinase) pathways are evolutionally conserved cell
signalling pathways that regulate many essential processes in eukaryotic cells,
including cell growth, differentiation, and apoptosis. ERK5 (extra-cellular regulated
kinase 5) is the most recently discovered mammalian MAPK. ERK5 expression is
ubiquitous and can be detected in the heart, placenta, lung, brain, skeletal muscle and
other tissues. Targeted deletions in mice have shown a major role for ERK5 in the
development of the cardiovascular system. Further studies have demonstrated that
the ERK5 pathway is essential to protect endothelial cells from apoptosis, implying a
cardio-protective role for ERK5. However, despite its important role, it has not been
fully clarified how ERK5 signalling is regulated in mammalian cells. A major
problem that is hindering detailed investigations is a lack of reliable reagents to
determine ERK5 activation under various conditions. We have therefore developed
assays to consistently measure ERK5 activity in cells and tissues. We will present
data showing that ERK5 is activated in response to a number of stimuli, which can
be accurately measured and quantitated. These assays will enable us to fully
investigate the regulation of the ERK5 MAPK signalling cascade in mammalian cells
and tissues to better understand this important signalling pathway.
We would like to thank King Faisal Medical City for Southern Region, Saudi Arabia
for their financial support.
F15

F16
HEPARAN SULFATE PROTEOGLYCANS INDUCE ACTIVATION OF THE

P2Y6 RECEPTOR IN CHO CELLS
EVALUATION OF ANTIANGIOGENIC EVENTS BY A KUNITZ-TYPE

INHIBITOR PURIFIED FROM MIMOSA REGNELLII BENTH SEEDS
Moura, G.E.D.D.1, Lucena, S.V.1, Lima, M.A.1, Nader, H.B.1, Paredes-Gamero,

E.J.1,2 and Tersariol, I.L.S.1,2
Luciana Maria Arajo Rablo (1), Daniela De Lucena Pedroso (1), Paula Ivani
Medeiros dos Santos (2), Geovanna Maria de Medeiros Moura (1), Elizeu Antunes
dos Santos (1).
Financial support: This work was supported by Fundao de Amparo Pesquisa do

Estado de So Paulo, FAPESP (2014/17671-8, MCJ; 2013/12861-0, EGFF).
Fundao de Apoio ao Ensino, Pesquisa e Assistncia FAEPA.
Departamento de Bioqumica, Universidade Federal de So Paulo, So Paulo,

Brazil.
2
Cruzes,
So Paulo, Brazil.
E-mail: gioconda_moura@yahoo.com.br
Address: Rua Trs de Maio, 100 4 andar, Disciplina de Biologia Molecular,
Departamento de Bioqumica, Vila Clementino, So Paulo, SP, Brazil. CEP 04044020. Institucional phone: (+55) (11) 55764442, ext. 1194 - Mobile phone: (+55) (11)
980505892
[INTRODUCTION] The purinergic P2Y6 is a G-protein coupled receptor that
modulates signaling pathways associated with nitric oxide release, bone resorption,
glucose uptake and immune responses. Among several nucleotides, UDP stimulates
P2Y6 receptor exclusively. Also, UDP can activate MAPK/ERK signaling pathways
with PKC requirement and integrins involvement. [OBJECTIVE] Here, we
investigated the role of heparan sulfate proteoglycans (HSPG) on activity, expression
and distribution of P2Y6 receptor in wild-type CHO-K1 cells and its mutant CHO745, defective in xylosyltransferase, an essential enzyme for GAGs biosynthesis.
[METHODS] P2Y6 activity was analyzed by measuring cytoplasmic Ca2+ influx,
using UDP as agonist and MRS2578 as selective antagonist. The cellular distribution
of P2Y6 receptor was measured by flow cytometry. Also, the presence of P2Y6
receptor and differences in signaling pathways in CHO cells were confirmed by
western blotting. [RESULTS] UDP activated Ca2+ influx in both CHO cells with
EC50= 3 M, these responses were blocked by MRS2578 (30 mol/L). The GAGs
enhanced the effectiveness of UDP at all concentrations tested by increasing the
maximal UDP response (CHO-K1, Emax= 351 11 URF; CHO-745, 214 4 URF),
although there were no changes in the EC50 (CHO-K1, 3.6 0.4 M; CHO-745, 2.8
0.2 M). Furthermore, it was observed that GAGs did not affect P2Y6 receptor
expression neither the cellular distribuition of this receptor in both CHO cells. In
addition, CHO-K1 cells showed 2-fold more constitutive activation of PKC than
CHO-745. [CONCLUSION] Altogether, these data suggest that the presence of
HSPG on the surface of CHO cells positively modulates P2Y6 receptor activity in
response to UDP.
Keywords: Purinergic receptors, P2Y6, CHO cells, Proteoglycans, GAGs.
Supported by: CAPES, CNPq and FAPESP.
CEP: N 3818030214
(1) Departamento de Bioqumica, Universidade Federal do Rio Grande do Norte, Rio

Grande do Norte, Brasil.
(2) Departamento de Biologia, Instituto Federal do Piau, Piau, Brasil.
Address: Departamento de Bioqumica - Centro de Biocincias - Universidade
Federal do Rio Grande do Norte - Campus Universitrio, Lagoa Nova - Caixa Postal
1524 - CEP: 59072-970 - Natal/RN - Brasil - (84)32153416 R:217 - (84)981075427
Background Angiogenesis is a highly complex physiological process, where preexisting endotelial cells, under control of several key enzymes, must break through
the basement membrane, migrate and proliferate in response to angiogenic factors to
form new vessels. To maintain homeostasis, the overexpression of these enzymes is
controled by protease inhibitors and the disruption of this equilibrium is the basis for
disease genesis, including angiogenic events. In this work, a trypsin protease
inhibitor (ITJ) was purified from the leguminosae Mimosa regnellii Benth seeds and
it was evaluated due to its ability to inhibit angiogenic events in ClPs cell line.
Aims Purify a trypsin inhibitor from Mimosa regnellii seeds and assess its ability to
inhibit angiogenic activity in ClPs cells.
Methods ITJ was purified by ammonium sulfate fractionation and chromatographic
methods. The antiangiogenic activity of ITJ was evaluated with an assay of capillarylike structures formation on Matrigel; VEGF and IL-6 expression was also assessed
by immunofluorescence and confocal microscopy assays.
Results ITJ inhibits angiogenesis in concentrations equivalent to the IC50 (0.65 mM)
and more effectively when used at half the IC50 (0.325 uM), but the concentration
equivalent to twice the IC50 was possible to observe a stimulatory effect for forming
new vessels after 72 hours of exposure. It was also observed a decrease in the
expression of VEGF and IL- 6.
Conclusion VEGF and IL-6 downrregulation coupled with the tube fomation assay
on matrigel indicates a reduction in the angiogenic process by incubation with ITJ.
Funding Support: CAPES

F17
F18
THE SMALL GTPase RhoA SIGNALS TO DNA DAMAGE REPAIR IN

TUMOR CELLS
PLASMA KALLIKREIN POTENTIATES ADP-INDUCED PLATELET

AGGREGATION
Yuli Thamires Magalhes (1), Fbio Lus Forti (1)
Tatiana F. Ottaiano1,6, Sheila S. Andrade2,4, Cleide de Oliveira1, Maria Juliano3,

Manoel J. B. C. Giro2,4, Alexander Wlodawer5, Francisco H. A. Maffei1 and Maria
L. V. Oliva1*
(1) Department of Biochemistry, Institute of Chemistry, University of Sao Paulo,

Sao Paulo, Brazil.
Mail contact: yuli.magalhaes@usp.br ; flforti@iq.usp.br
Telephone contact: Institucional: +55 11 3091-2172 - Mobile: +55 19 98898-4491
RhoA GTPases is a member Ras superfamily. It's found over expressed or activated
in many aggressive cancers and controls key regulatory proteins acting on cell cycle,
proliferation and death. Despite RhoB protects human keratinocytes from UVinduced apoptosis and RhoA acting in DNA double strand break promoted by
bacterial toxins, its roles in genomic stability needs more investigations. Here we
investigated the influence of RhoA activity in HeLa and MeWo human cancer cells
exposed to UV-radiation treatments, through the RhoA inhibitor C3 toxin and
analyzed some responses to UV-induced DNA damage. To investigate the RhoA
roles in the NER pathway, we inhibited its activity with C3 toxin in deficient cells of
XPA, XPC or XPV proteins, and analyzed survival, DNA damage and regulation of
DDR pathway after UV exposure. Clonogenic assays showed that both tumor and
NER-deficient cells inhibited by C3 have significantly reduced survival compared to
parental cells. From alkaline comet assays, the RhoA inhibition in tumor cells
exhibited increased DNA damage and a delayed DNA repair after the UV-radiation.
RhoA inhibition in tumor cells led to a decrease in the phospho-H2AX levels, while
in NER-deficient cells a strong increase was observed. Further, compatible changes
were observed in the phosphorylation of Chk1 and p53 along with RhoA inhibition
and UV treatments. These results show that RhoA affect cell survival after UVinduced DNA damage acting in the recognition and repair of DNA lesions, very
likely through the NER pathway.
Financial Support: Fapesp, CNPq, Capes
Department of 1Biochemistry, 2Gynecology and 3Biophysics, So Paulo Federal

University, SP, Brazil, 4Charitable Association of Blood Collection COLSAN So
Paulo, SP, Brazil, 5Macromolecular Crystallography Laboratory, Center for Cancer
Research, National Cancer Institute, Frederick, MD, USA 6 University Anhanguera
de So Paulo UNIAN, SP, Brazil
Background: Human plasma kallikrein (huPK) is protease that participates in the
activation of blood coagulation and fibrinolysis. HuPK has been reported to
potentiate platelet responses to subthreshold doses of ADP. Aim: Investigate the
mechanism of the potentiation of the human platelets aggregation by huPK in low
doses of the ADP. Methods: Platelets were isolated as plasma rich platelets and
human platelet aggregation was monitored by light transmission. To Immunoblot
analysis, platelets were lysed, separated and transferred onto nitrocellulose
membrane. Platelet activation was measured by lumi-aggregometer. Calcium
mobilization was measured in the confocal microscope. Results: The mechanism
finding that huPK amplifies ADP-induced platelet aggregation due to its proteolytic
activity on PARs. Such activity is mediated by the integrin IIb3 through
interactions with the KGD sequence motif present in huPK. Our data provide novel
insights into the mechanism by huPK, and suggest that integrin may act as a cofactor
focusing huPK to the platelet surface to support PAR-1 hydrolysis and consequent
activation of ADP pathway, leading to Akt, ERK and p38 MAPK phosphorylation,
and Ca2+ release. The huPK effect was blocked by a PAR-1 antagonist, by synthetic
peptides comprising KGD and KGE sequence motifs present in huPK structure, and
by the plasma kallikrein inhibitor, rBbKI. Conclusion: These new findings suggest
an alternative role for huPK in the injured vessels where the platelet concentration
increases and low amount of huPK is activated, potentiating ADP platelet
responsiveness by simultaneous binding to integrin IIb3 and modulating PAR-1
hydrolysis on the platelet surface.
Financial Support: FAPESP, CAPES and CNPq.

G CELL TRAFFICKING AND ORGANELLES
G1
G2
THE MOLECULAR MOTOR MYOSIN Va EMERGES AS A NEW

COMPONENT INTO THE PRIMARY CILIUM FUNCTION
A PHARMACOLOGIC SCREENING OF PROTEIN DISULFIDE

ISOMERASE SECRETION ROUTES IN ENDOTHELIAL CELLS DEPICTS
GOLGI INDEPENDENCE
Leandro H. P. Assis (1,2), Luciano G. Dolce (1,2), Marcos R. Alborghetti (1),

Rodrigo V. Honorato (1), Andrey F. Z. Nascimento (1,2), Talita D. Melo-Hanchuk
(3), Daniel M. Trindade (1), Celisa C. C. Tonoli (1), Paulo S. L. Oliveira (1), Jrg
Kobarg (3), Priscila O. Giuseppe (1), Mrio T. Murakami (1)
(1) Brazilian Biosciences National Laboratory, National Center for Research in
Energy and Materials, Campinas, Brazil.
(2) Graduate Program in Functional and Molecular Biology, Institute of Biology,
University of Campinas, Campinas, Brazil.
(3) Department of Biochemistry and Tissue Biology, Institute of Biology, University
of Campinas, Campinas, Brazil.
E-mail leandro.assis@lnbio.cnpem.br, Phone +55 (19) 3512-1109, Mobile +55 (19)
99191-0277 - Giuseppe Mximo Scolfaro St., n 10000, High-tech Park II, 13083100, Campinas-SP, Brazil
Myosin Va (MyoVa) is an actin-based motor that transports vesicles, organelles and
regulatory proteins. Besides its localization in actin-rich regions at the cell periphery,
MyoVa also localizes in the centrosome, an organelle that coordinates several
processes, including the formation of a sensory organelle called primary cilium. It is
known that cilium/centrosome abnormalities are associated with cancer and
ciliopathies but, despite two decades of studies, it is still unclear the MyoVa
contribution to centrosome-driven processes. By using a yeast two-hybrid (YTH)
system, we identified a cilium-related protein (CRP) as a novel MyoVa partner. The
interaction was confirmed in vitro by GST pull-down and in situ by proximity
ligation assays. Studies of microscale thermophoresis indicated that MyoVa binds to
CRP with low affinity, which is typical of transient complexes. To map the CRPbinding site, we mutated some conserved residues of the MyoVa cargo-binding
domain and evaluated these mutants using YTH assays. Our results suggest that the
CRP-binding site partially overlaps with that of Rab11 on MyoVa/b. Proximity
ligation assay and confocal microscopy data indicated that MyoVa interacts with
CRP in the centrosome during cell division and at the primary cilium base in nondividing cells. Together, our results unveil an emergent role of MyoVa into the
primary cilium function via CRP interaction.
Keywords: myosin V, cilium-related protein, centrosome, primary cilium
Funding support: So Paulo Research Foundation (FAPESP processes 2014/09720-9,
2011/20229-7 and 2009/08312-6) and National Council for Scientific and
Technological Development (CNPq processes 478059/2009-4 and 486841/2012-0).
Thas L. S. Araujo1, Julianna D. Zeidler1,2 Percllia V. S. Oliveira1,2 and

Francisco R. M. Laurindo1
1
Laboratrio de Biologia Vascular, Instituto do Corao (InCor), Faculdade de

Medicina da Universidade de So Paulo (FMUSP).
2
These authors contributed equally to this work
Thas L. S. Araujo1: Thas Larissa Araujo de Oliveira Silva.
tlarissa2006@gmail.com - Tel: (011)2661-5185 Cel: (011)98276-2710
Email:
The extracellular pool of endoplasmic reticulum (ER) redox chaperone protein

disulfide isomerase (PDI) mediates viral infection, thrombosis and vascular
remodeling. However, route(s) of PDI externalization remain unclear. We performed
a systematic screening of possible secretory pathways and underlying stimuli
affecting extracellular PDI levels in endothelial cells (EC). Results disclosed two
main extracellular PDI pools: cell surface(csPDI) and soluble non-particulated.
While a fraction of csPDI pool was decreased by inhibitors of ER-Golgi pathway,
soluble PDI secretion was largely amplified in EC at baseline or after induced
stimulation with PMA, thrombin or ATP. Also, inhibitors of Type I, II and III
unconventional routes, secretory lysosomes and recycling endosomes did not impair
basal PDI externalization. This pattern suggests predominance of Golgi-independent
Type IV unconventional secretory route, which proved to be GRASP55-independent.
In addition, these data reinforce a vesicular-type traffic for PDI. Further experiments
indicated that basal PDI traffic is ATP-independent and supported by RhoA activity,
while actin or tubulin cytoskeletal disruption markedly increased PDI secretion.
Clathrin inhibition enhanced extracellular soluble PDI, suggesting PDI dynamic
cycling through this path. Besides Golgi dependence, csPDI and soluble PDI pools
were differentially modulated by ER stress and heat shock, consistent with their
possible general independent regulation. In vascular smooth muscle cells, csPDI
translocation is Golgi-independent, while soluble PDI is undetectable, thus indicating
vascular cell-type specificity. Externalized PDI represents <2% of intracellular PDI
and showed resilience to change upon distinct challenges. Such results help clarify
signaling and homeostatic mechanisms involved in multiple extracellular PDI
functions.
(Supported by: FAPESP-Cepid Redoxoma#13/07937-8 and #2015/06210-2; F.
Zerbini)
G3

G4
THE TWO GAMMA ADAPTIN SUBUNIT ISOFORMS OF AP-1 PLAY

DISTINCT ROLES IN PROTEIN TARGETING THROUGH THE
CANONICAL DEGRADATIVE MVB PATHWAY
CD4 DOWN-REGULATION BY HIV-1 NEF REVEALS DISTINCT ROLES

FOR 1 AND 2 SUBUNITS OF AP-1 COMPLEX IN PROTEIN
TRAFFICKING
Mara Elisama da Silva-Janurio and Luis Lamberti Pinto da Silva
Lucas A. Tavares1, Eullia M. L. da Silva1, Mara E. da Silva-Janurio1, Yunan C.

Janurio1, Julianne V. de Cavalho1, Gonzalo Mardones2 and Luis L. P. daSilva1
Department of Cell and Molecular Biology, Ribeiro Preto Medical School,

University of So Paulo, Ribeiro Preto, SP, Brazil
Email contact: maraelisama@gmail.com
Telephone contact: +55 16 3315 4802
The adaptor protein (AP) complexes regulate protein trafficking in the secretory and
endocytic pathways. The AP-1 is a heterotetramer composed of composed of , , 1
and 1 subunits and its classically related with protein sorting from TGN to
endosomes. However, there are different isoforms of AP-1 subunits, suggesting the
existence of multiple AP-1 variants. In particular, there are two isoforms of the
subunit (1 and 2) but most of the studies on AP-1 are related to 1 functions,
whereas the function of 2 in protein sorting is poorly understood. Herein, we
provide evidence that the 2 subunit is involved in the targeting of transmembrane
proteins for degradation in lysosomes. To access the canonical degradative MVB
pathway, we monitor the transport of the EGF receptor (EGFR) and its ligand EGF.
Using immunofluorescence microscopy, we performed EGF uptake experiments in
control cells and in cells knockdown (KD) for 1 or 2. We observed a significant
delay in the kinetics of EGF degradation in 2 KD cells, compared to control cells,
and that delay was not observed in 1 KD cells. In addition, the degradation of EGF
receptor was analyzed after EGF stimulation in control and 2 KD cells using
surface biotinylation assays, confirmed that the degradation of endocytosed EGFR is
significantly delayed in cells KD for 2. Together, our data provide evidence that 2
is required for targeting of EGFR-EGF complexes through the canonical degradative
MVB pathway. Our data also indicates that 2 subunit has a distinct role of 1 in
protein sorting.
Financial support: FAPESP and FAEPA.
1-Department of Cell and Molecular Biology, Ribeiro Preto Medical School,

University of So Paulo, Ribeiro Preto, SP, Brazil; 2-Department of Physiology,
School of Medicine, and Center for Interdisciplinary Studies of the Nervous System
(CISNe). Universidad Austral de Chile, Valdivia, Chile.
Contact details: Department of Cell and Molecular Biology, Ribeiro Preto Medical
School, University of So Paulo, Av. Bandeirantes 3900, Ribeiro Preto, SP, 14049900, Brazil. Phone number.: +55 16 3315-3144 / +55 16 98222-7014
e-mail: lucastavares.biotec@gmail.com
HIV is the etiologic agent of AIDS and it has a worldwide distribution. The HIV
accessory protein Nef is a major determinant of viral pathogenesis as it facilitates
viral particle release, prevents viral antigen presentation and increases the infectivity
of new virus particles. All of these functions involve the ability of Nef to remove
specific host proteins from the surface of infected cells. Among the proteins downregulated by Nef is the receptor CD4. Nef binds to the adaptor protein 2 (AP-2)
complex and the cytosolic tail of CD4 in clathrin-coated pits, forcing CD4
endocytosis and directing it to lysosomes. Herein, we report that this targeting
requires a poorly characterized variant of the AP-1 complex, which comprises the
isoform 2 of -adaptin. We show by yeast three hybrid system and GST pull-down
assays that 1 and 2 are able to interact with Nef. By immunoblot assay, we found
that depletion of 2 or 1A subunits of AP-1, but not of 1, precludes Nef mediated
lysosomal degradation of CD4. In 2-depleted cells, CD4 that was internalized by
Nef accumulated in early endosomes and this alleviates CD4 removal from the cell
surface, as revealed by indirect immunofluorescence microscopy and flow cytometry
analyses, respectively. Moreover, stabilizing ESCRT-I by HRS overexpression in
NEf expressing cells, led to 2 accumulation in enlarged endosomes containing
HRS-GFP and internalized CD4. Altogether, our data provide evidence that 1 and
2 subunits compose two distinct variants of AP-1 complexes with different
functions in protein sorting.
Financial Support: FAEPA

G5
G6
THE CHARACTERIZATION OF ARC INTERACTION WITH AP-2 AND

ITS RELEVANCE FOR ARC-MEDIATED AMPA RECEPTOR DOWNREGULATION
EFFECT OF EXPOSURE TO FLUORIDE ON ENAC MEMBRANE

TRANSPORTERS AND EVALUATION OF THE VIABILITY OF KIDNEY
EPITHELIAL CELL LINE
Yunan C Janurio1, Luciana P de Almeida1, Sandrine C Wauters2, Jrgen Mller3,

Sonia AL Corra2,4 and Luis LP Da Silva1.
Mariana Rodrigues Santesso (1), Flvia Mauad Levy Abraho (1), Ligia Subitoni
Antonio (2) John Michael Edwardson (3), Marlia Afonso Rabelo Buzalaf (1),
Rodrigo Cardoso de Oliveira (1)
Department of Cell and Molecular Biology, Ribeiro Preto Medical School,

University of So Paulo, So Paulo, Brazil. 2School of Life Sciences, University of
Warwick, Coventry, UK. 3Warwick Medical School, University of Warwick,
Coventry, UK. 4School of Life Sciences, University of Bradford, Bradford, UK.
Email contact: yunan.januario@gmail.com
Telephone contact: +55 16 3315 4802 / +55 16 99765 4422
The neuronal immediate-early gene Arc (activity-regulated cytoskeleton-associated)
protein acts at excitatory synapses and regulates long-term synaptic plasticity and
memory. It is well established that Arc modulates synaptic plasticity by enhancing
AMPA receptor (AMPAR) endocytosis. We found that Arc directly interacts with
AP-2, a clathrin adaptor protein with a key role in endocytosis. The aim of this work
was to characterize Arc and AP-2 interaction. Using recombinant purified Arc and
the AP-2 complex, followed by pull-down assays, we observed a direct interaction
between these proteins. Additional pull-down assays using Arc mutants containing
successive deletions in the C-terminus revealed a motif, comprising five amino acid
residues, that is crucial for interaction with AP-2. Most importantly, further GST
pull-down analyses with an Arc mutant containing a point mutation within the
identified domain prevents Arc interaction with AP-2. Using surface protein
biotinylation and immunofluorescence microscopy, we show that mutation of the
AP-2 interacting motif in Arc compromises the capacity of Arc to reduce the levels
of AMPAR at the plasma membrane. Together, these data provide a mechanism to
explain how activity-dependent expression of Arc decisively controls the fate of
AMPAR at the cell surface and modulates synaptic strength, via the direct interaction
with the endocytic clathrin adaptor AP-2.
Financial support: FAPESP, BBSRC-UK, FAEPA and CAPES.

(2) Departament of Biochemistry and Immunology, Ribeiro Preto Medical School,
University of So Paulo, Ribeiro Preto, So Paulo, Brazil.
(3) Departament of Pharmacology, University of Cambridge, Cambridge, United
Kingdom.
Adress contact: Alameda Octvio Pinheiro Brisolla 9-75 - Cidade Universitria Bauru-So Paulo-Brasil
Email: marisantesso@usp.br
Telephone: +55 14 99661-6258
Background: It is known that fluoride interacts with mineralized tissues such as bone
and teeth, has a great ability to stimulate the formation of bone tissue and has a
recognized therapeutic action on caries. Despite its therapeutic effects when
exposure to this compound is done in an inappropriate manner can cause undesirable
reactions, moreover, the balance of fluoride into the body is modulated by ingestion,
absorption and removal, making kidney particularly vulnerable of the toxicity of
fluoride. Aims: Verify the effects of renal epithelial cells exposure to fluoride, as
well as to verify the influence of this element on the membrane transporter, through
the use of fluoride concentrations similar to those that may be found in the nephron
during chronic exposure to fluoride. Methods: M-1 cells (ATCC CRL-2023) were
treated with different concentrations of NaF and NaCl (control group for Na) (10, 40,
100, 200 and 400 M). After experimental treatment periods of 24, 48, 72, 96 hours,
viability was assessed by MTT reduction assay and Crystal Violet. To analyze the
influence of NaF in the membrane transporter (ENaC) was made
immunofluorescence test. Results: It was possible to observe significant differences
in cell viability between the highest concentrations of NaF. It was not possible to
observe differences in the expression of ENaC in cells treated with low
concentrations of NaF. Conclusion: NaF in high concentrations modulates renal cell
viability and low doses not interfering in expression of proteins that form the
epithelial sodium channel (ENaC).
Funding support: CAPES: CSF-PVEs 88881.030354/2013-01.

H CYTOSKELETON AND MIGRATION
H1
H2
DISTINCTIVE EFFECTS OF CYTOCHALASIN B IN CHICK PRIMARY

MYOBLASTS AND FIBROBLASTS
MORPHOLOGICAL ASPECTS OF SUBPELICULLAR MICROTUBULES

OF TRYPANOSOMA CRUZI
Koichi Ojima(1), Zhong-Xiang Lin (2), Ivone Rosa de Andrade (3), Manoel Luis
Costa (3) and Claudia Mermelstein (3)
(1)
(1)Animal Products Research Division, NARO Institute of Livestock and Grassland
Science, Tsukuba, Ibaraki, 3050901, Japan,
(2) Department of Cell Biology, Beijing Institute for Cancer Research, Beijing
Medical University, Beijing, 100083, China,
(3) Instituto de Cincias Biomdicas, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, RJ, 21941902, Brasil
Juliana C. Vidal (1, 2,*) and Wanderley de Souza(1,2)
Background: Actin-based structures play fundamental roles in cellular functions.

However it remains controversial how cells cope with the absence of F-actin
structures.
Aims: Short- and long-term effects of cytochalasin B (CB) on actin-complexes in
fibroblasts and myoblasts.
Methods: Myoblast cultures were obtained from 11 day-old chick embryos. Cells
were plated at 7.5 x 105 per dish and treated with CB 30 min or 72 hrs after cell
plating. Cells were analyzed by immunofluorescence microscopy using antibodies
against cytoskeletal and myofibrillar proteins.
Results: Thirty min of CB treatment dispersed subplasma actin cortices,
lamellipodia, ruffled membranes, stress fibers and adhesion plaques into actin
patches in fibroblasts and muscle cells. In contrast, 72 hrs CB treatment showed
distinct morphological effects. Fibroblasts became giant multinucleated-finger
shaped and lacked cortical actin, stress fibers, adhesion plaques and ruffled
membranes but contained immense lamelliopodia with abnormal adhesion plaque
protein complexes. Muscle cells transformed into multinucleated globular-shaped but
contained normal I-Z-I and A-bands, indicating that CB did not interfere with the
assembly of myofibrils. However, the original shape of the both cells returned within
30 min after CB removal.
Conclusion: Interesting issues concerning the role of actin compartments in
eukaryotic cells are raised since the ability to grow and assemble intermediate
filaments and microtubules occur without an actin cytoskeleton morphologically
recognizable.
Trypanosoma cruzi is a protozoan parasite and exhibits unique features, which differ
them significantly from their mammalian host. Among them, the subpelicullar
microtubules (SPMT) that follows the helical pattern along the long axis of the cell
body organized in a highly ordered array of stable microtubules placed beneath the
plasma membrane, absent in flagellar pocket and cytostome-cytopharynx complex.
The parasites life cycle involves symmetrical division and different developmental
transitional stages. The maintaining and establishment of cell shape is a fundamental
role of cytoskeleton and provides interesting models for cellular biology studies. The
morphological knowledge of SPMT during T. cruzi life cycle is limited. To analyze
this array of microtubules, tridimensional reconstruction were performed using
electron microscopy tomography and focused ion beam-scanning electron
microscopy (FIB-SEM). The observations shows that epimastigotes has
approximately 60 SPMT, among them, the microtubules next to flagellar pocket are
extremely shorter or half-length when compared to others SPMT. Besides this, the
helicoidally pattern is held. In conclusion, shorter length microtubules may represent
the first evidence that biogenesis of SPMT occurs next to flagellar pocket. At the
present time, analysis of SPMT during mitosis and metacyclogenesis are being made
and also analysis of the posterior region of cell body to elucidate how SPMT are
joined together firmly. These studies can reveal ultrastructural details about the
maintenance of cell shape even during its complex life cycle.
Information on ethical approval and funding support: CNPq, FAPERJ. The study
was approved by CEUA-DAHEICB092-05/16.

H3
(1) Instituto de Biofsica Carlos Chagas Filho, Universidade Federal do Rio de

Janeiro, Rio de Janeiro, Brazil;
(2) Instituto Nacional de Cincia e Tecnologia em Biologia Estrutural e Bioimagens,
*
vidal.ju@gmail.com, phone contact: 21 3938-6583 (Institutional) / 21 988390363

(mobile)
This work was supported by CNPq, FAPERJ, CAPES and INBEB.
H4
HIGH GLUCOSE PROMOTES APOPTOSIS OF PERIODONTAL

LIGAMENT CELLS POTENTIAL MODULATION BY microRNAs 221
AND 222
Mariana Marin Monteiro1, Cilene Rebouas de Lima1, Anna Carolina R. TempestiniHorliana2, Marinilce F. Santos1
1

2
Department of Stomatology, School of Dentistry, University of So Paulo-USP. So
Paulo/SP - Brazil. mariana.marin.monteiro@usp.br
Background: increased incidence of periodontal diseases is a common long-term
complication of diabetes mellitus (DM), a disease characterized by chronic
hyperglycemia. We have previously demonstrated that DM affects several functions
of dermal fibroblasts, such as adhesion and migration, contributing to the deficient
skin wound healing. MicroRNAs (miRs), post-transcriptional regulators of gene
expression, may be regulated by high glucose (HG), impairing different cell
functions. Aim: using human periodontal ligament cells, to evaluate the effects of
HG on migration and apoptosis, as well as its effect on the expression of miR-221
and miR-222, miRs potentially modulated in DM. Methods: Cells were obtained
from the periodontal ligament of premolar teeth of young humans extracted for
orthodontic reasons, and cultured on 1 g/ml fibronectin under low (5 mM) or high
(30 mM) glucose concentrations for 7 days. The ICB-USP Human Ethical
Committee approved this protocol. Results: High glucose reduced cell growth by
33%, mostly due to a 3x increase in cell death. High glucose also reduced cell
adhesion by 30-40%, cell spreading by 35% and cell migration by 54%. The
expression of miR-221 and miR-222 decreased nearly 40% under HG. A predicted
target for miR-221 and miR-222 is caspase 3, involved in apoptosis. Conclusion:
High glucose negatively affects migration and increases apoptosis of periodontal
ligament cells, potentially impairing healing after treatment of periodontitis,
periodontal surgeries and orthodontic movement. The reduction of miR-221 and
miR-222 may be involved in these effects, especially on cell death.
HIGH GLUCOSE AFFECTS miR-31 EXPRESSION AND IMPAIRING

MIGRATION OF DERMAL FIBROBLASTS
Cibele C. Gomes, Cilene R. de Lima, Marinilce F. Santos.
University of Sao Paulo-USP. Sao Paulo/SP - Brazil.
cibelecgomes@usp.br (55) (11) 3091-7227.
A relevant complication of diabetes mellitus is deficient wound healing. During
healing, fibroblasts play a central role proliferating, migrating and remodeling the
extracellular matrix. We have previously demonstrated that reactive oxygen species
(ROS) produced by high glucose (HG) impairs fibroblast migration, with reduced
speed and directionality, increased number of protrusions and inhibition of adhesion
maturation. MicroRNAs are non-coding RNAs, which function as posttranscriptional regulators of gene expression. MicroRNA-31 (miR-31) may be
regulated by oxidative stress and is a potential regulator of cell migration. Aim: to
evaluate the effects of HG on miR-31 expression in fibroblasts and its role on cell
migration. Dermal fibroblasts obtained from normoglycemic and hyperglycemic rats
(after induction of diabetes with streptozotocin) and NIH-3T3 fibroblasts were used.
Cells were grown under low glucose (5 mM) or HG (30 mM) for 3 days, with or
without the antioxidant N-acetylcysteine (NAC). The expression of miR-31 was
studied by real time RT-PCR and the migratory behavior was assessed by time-lapse.
The expression of miR-31 increased about 3-fold in fibroblasts from hyperglycemic
animals. In NIH-3T3, HG increased the expression of miR-31 by 50%, and NAC
treatment prevented the increase of miR-31 as well as the increase of cellular
protrusions. Exogenous expression of miR-31 in NIH-3T3 using miR mimics
increased cell protrusions and reduced cell directionality, without effects on velocity.
High glucose increases the expression of miR-31 in a ROS-dependent manner in
fibroblasts, contributing to migration impairment observed in diabetes.
Financial Support: CAPES, FAPESP, and CNPq.
Support: FAPESP, CAPES, NAPmIR/USP.

H5
H6
EVALUATION OF PROTEOLYTIC ENZYMES MONO AND CO-CULTURE

OF ENDOTHELIAL AND TUMOR CELLS TREATED WITH
AMBLYOMIN-X
SERUM STIMULATION OF QUIESCENT FIBROBLASTS INDUCES

PHOSPHORYLATION OF MYOSIN-VA AND ITS LOCALIZATION IN
FOCAL ADHESIONS
Mariana Costa Braga Schmidt1,2; Katia Luciano Pereira Morais1,2; Maira Estanislau
Soares de Almeida3, Juliana Mozer Sciani1; Ana Marisa Chudzinski-Tavassi1
1.
1
Biochemistry and Biophysics Laboratory, Butantan Institute, SP, Brazil;
2
Department of Biochemistry, Federal University of So Paulo, SP, Brazil;
3
Physiopathology Laboratory, Butantan Institute, SP, Brazil.
Rockenbach, J.A.Z1.; Pontes, C.L. 1; Espreafico, E.M. 1
Background: During the development of diseases like cancer are found high protein
levels and activity of proteolytic enzymes. In this context, Amblyomin-X is a
recombinant Kunitz-type protein, identified from a cDNA library of the salivary
glands of the Amblyomma cajennense tick that has shown to induce death of a
variety of cancer cells. In contrast, Amblyomin-X has not showed any cytotoxic
effects on normal human. Besides, Amblyomin-X promotes regression of tumor
growth and reduction of metastasis. Thus arose the hypothesis that this molecule
could be interfere in cellular migration and pericellular proteolysis. Aims:
Investigate cellular migration and release of pericellular proteases in mono and coculture of tumor (SK-MEL-28 and MIA PaCa-2) and endothelial cells (HUVEC).
Methods: Cell viability was assessed by MTT. u-PA and PAI-1 were quantified by
ELISA. Cytoskeleton was analysed through phalloidin staining and transwell
migration assay were carried out to verify cellular migration. Results: Amblyomin-X
decreased tumor cells viability, but had no cytotoxicity effect on HUVEC. Also,
Amblyomin-X induced the release of uPA and PAI-1 by HUVEC and only uPA in
SK-MEL-28 cells, no change in uPA MIA PaCa-2. Furthermore, only in tumor cells
changes were observed in the organization of F-actin and reduction of cellular
migration. Conclusion: These results suggest that antitumor action of Amblyomin-X
may be associated with the inhibition of the migration of tumor cells. However,
endothelial cells respond to Amblyomin-X treatment by modulating of different
intracellular pathways, since the increase of uPA and PAI-1 were not related to
cytoskeleton changes and cellular migration.
(1) FMRP-USP
Integrin recycling is essential for cell migration. Through this process focal
adhesions (FA) are disassembled at the cell rear and assembled at the leading edge,
allowing cell to migrate. The assembly and disassembly of FA play a key role in
various cellular processes, including cell migration, proliferation and survival. In this
work, we aim at analyzing the effect of fetal bovine serum (FBS) stimulation on the
localization and phosphorylation of myosin-Va relative to other FA proteins, in
quiescent fibroblasts. The kinetics of FA assembly was observed in a time-course
assay of fixed cells at various time points after FBS stimulation, and stained for
phosphorylated myosin-Va colocalized with FA constituent proteins. Our Results
show that p-myosin-Va localizes to FAs sites during the rapid formation of these
structures upon FBS stimulation. Therefore, this is consistent with our hypothesis
that myosin-Va participates in FA dynamics. Furthermore, our results also showed
an increase of p-myosin-Va staining throughout the cytoplasm upon serum
stimulation, and revealed that p-myosin-Va colocalizes with pFAK and vinculin in
FA sites. We demonstrated by Western blotting that FBS stimulation does not cause
change in the total amount of myosin-Va, in any of the times analyzed in relation to
the quiescent condition, but induces, after 5 and 15 minutes, an appreciable increase
of p-myosin-Va. To our knowledge, this is the first demonstration that
phosphorylation of myosin-Va increases in response to FBS and we are investigating
whether this event is connected to the dynamics of focal adhesions in fibroblasts.
Supported by CNPq, Fapes, BNDES and Unio Qumica Farmacutica

I DEVELOPMENTAL BIOLOGY
I1
I2
IDENTIFICATION AND EXPRESSION OF CPDII-PHOTOLYASE GENES

AND
APOPTOSIS-RELATED
GENES
DURING
EMBRYONIC
DEVELOPMENT USING FRESHWATER PRAWN AS A MODEL
Mir-22 CHANGES THE EXPRESSION OF OSTEOGLYCIN AND

MIOGENIC REGULATORY FACTORS DURING THE MYOGENESIS
Helosa Schramm da Silva* (1), Eliane Cristina Zeni (1), Michael Lorenz Jaramillo
Bobadilla (1), Thaline de Quadros (1), Thiciane Patrycia Gonalves Dos Santos (1),
Karla Cristina Guimares de Oliveira (1), Dib Ammar (1,2), Yara Maria Rauh
Mller (1), Evelise Maria Nazari (1).
(1) Departamento de Biologia Celular, Embriologia e Gentica, Universidade
Federal de Santa Catarina, Brazil.
(2) Universidade Catlica de Santa Catarina, Universidade Federal de Santa
Catarina, Brazil.
*helo.bio@gmail.com - Laboratrio de Reproduo e Desenvolvimento Animal - 48
37219799
Photorepair mechanism is fast and effective to remove DNA damage, which is
mediated by photolyase enzyme. However, non-repaired DNA damage could triggers
apoptosis, involving classic proteins as p53, Bcl2 family and caspases. These
processes are not necessarily conserved in all animals and not yet were elucidated in
crustaceans. Thus, the aim of this study was to identify CPDII-photolyase and
apoptosis-related genes in freshwater prawn Macrobrachium olfersi and characterize
their expression during the embryonic development and in adult tissues. tBlastn
searches against M. olfersi transcriptome and multiple alignment were conducted
using gene sequences for other invertebrates. cDNA synthesis from embryos and
adult tissues (ovary, hepatopancreas, muscle, cerebral ganglia, hemocytes) of M.
olfersi (IBAMAs permanent approval 15294/1) were used for genic expression
analysis. Six candidate genes apoptosis-related and two photolyases genes were
identified. Different profiles of transcripts of p53, TRAF6, BclX, Bax and Caspase4
were expressed in embryos and all tissues. Except Caspase3c, which was recognized
only in embryos and hepatopancreas. CPDII-photolyase genes were expressed in
embryos and also ovary, cerebral ganglia and hemocytes. Our results contribute to
the identification of important genes that can serve as targets for future molecular
studies, in attempt to better understand the cellular responses of injuries, such as
ultraviolet radiation.
Carlos Augusto Barnabe Alves, Paula Paccielli Freire, Ivan Vechetti Jr, Juarez
Henrique Ferreira, Leonardo Nazrio de Morais, Geysson Javier Fernandez Garcia,
Robson Francisco Carvalho, Maeli Dal-Pai-Silva.
Department of Morphology, Sao Paulo State University, Botucatu, So Paulo, Brazil.
Contact: karlos_augusto@aluno.ibb.unesp.br Mobile: +551438800503
Background: In mammals, myogenesis is the process of embryonic development of
muscle tissue regulated by interaction of intracellular signal transducers and nuclear
transcription factors, such as Osteoglycin (OGN) and Myogenic Regulatory Factors
(MRFs). MicroRNAs (miRs) are molecules that regulate myogenesis by targeting
mRNA and studies have shown the miR-22 regulating skeletal muscle cells
proliferation and differentiation. Aims: Evaluate the effect of overexpression of miR22 on gene and protein expression during in vitro myogenesis in skeletal muscle
cells. Methods: C2C12 cells were cultured in 6-well plates at an initial concentration
of 200,000 cells/well until they reach 80~90% of confluence. Lipofectamine was
used for overexpression through using mimetic molecules of miR-22. RT-qPCR was
performed to gene expression and Western Blot to analysis of protein levels of OGN
and MRFs. Data were analyzed by Student "t" test for independent samples and the
level of significance for all variables was 5% ( = 0.05). Results: There was a
decrease in gene expression of OGN and an increase in the MRFs (MyoD and
MyoG) in myoblasts; in myotubes there was an increase of MyoD levels. There was
a decrease in protein expression of OGN levels in myoblasts; in myotubes there was
not difference in OGN and MRFs levels. Conclusion: Our results suggest that miR22 regulates post-transcriptionally the expression of OGN also controlling the
expression of MRFs MyoD and MyoG during the during in vitro myogenesis in
skeletal muscle cells.
Funding Support: FAPESP (Process: 2014-21110-1).
Support: CAPES, CNPq, FAPESC

I3
BeWo HUMAN TROPHOBLAST CELLS SUSCEPTIBILITY
TOXOPLASMA
GONDII
INFECTION
IS
MODULATED
INTRACELLULAR LEVELS OF IRON
I4
TO
BY
Marcos Paulo Oliveira Almeida(1)*, Bellisa de Freitas Barbosa(2), Layane Alencar

Costa Nascimento(1), Mrio Czar de Oliveira(1), Eloisa Amlia Vieira Ferro(2) and
Neide Maria Silva(1)
(1) Laboratory of Immunopathology, Institute of Biomedical Sciences, Federal
University of Uberlndia, Uberlndia, Minas Gerais, Brazil.
(2) Laboratory of Immunophisiology of Reproduction, Institute of Biomedical
Sciences, Federal University of Uberlndia, Uberlndia, Minas Gerais, Brazil.
* Av. Par, 1720, Uberlndia, CEP 38400-902, Minas Gerais, Brazil; E-mail:
marcospaulooliveiraalmeida@hotmail.com; Telephone: Institutional +55 34 3225
8575; Mobile +55 34 99122 7493.
Background: Trophoblast cells represent an embryonic cell population, playing a role
in the control of Toxoplasma gondii placental crossing, and in maternal-fetal transfer
of iron, essential nutrient for pregnancy and also the survival of this parasite.
Aims: The aim of this study was to investigate the effects of the iron addition and
deprivation in T. gondii-infected BeWo human trophoblast cell line.
Methods: The cellular viability of ferrous sulfate (FeSO4) supplemented BeWo or
deprivation of iron by deferoxamine (DFO) treatment was analyzed by MTT assay.
BeWo cells treated or not with FeSO4 and/or DFO were then infected with T. gondii,
and parasitism, and Hamp (hepcidin) and TfR (transferrin receptor) mRNA
expression were evaluated by qPCR.
Results: BeWo cells showed viable when treated with FeSO4 or DFO. The addition
and deprivation of iron increased and controlled, respectively, the intracellular
parasite proliferation, and, furthermore, T. gondii infection decreased the mRNA
expression of Hamp, and drugs treatment changed the TfR gene expression in
infected BeWo cells.
Conclusion: These data suggest that the ion iron, essential to normal fetal
development, is also involved in T. gondii proliferation in the maternal-fetal
interface. Thus, the supplementation of iron in the pregnant and T. gondii-infected
female should be done with caution to avoid to get worse congenital toxoplasmosis.
IN TOTO ZEBRAFISH IMAGING REVEALS PHOTORECEPTOR CELL

DEVELOPMENT AND ITS ROLE IN RETINAL LAMINATION
Mauricio Rocha-Martins (1,2), Gauravi Deshpande (1), Jaroslav Icha (1), Caren
Norden (1)
(1) Max Planck Institute of Molecular Cell Biology and Genetics, MPI-CBG,
Dresden, Germany
(2) IBCCF, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
Correspondence: rocha@mpi-cbg.de
Phone: +493512102802/ +4917635954911
A common feature of the CNS is its laminated organization, which is established
during development. The zebrafish retina is a powerful model to investigate how
newly born neurons collectively build a layered tissue. Here, we investigate how the
most apical neurons, the photoreceptor cells (PRCs), emerge and reach their final
position. We show that PRCs in the zebrafish emerge from committed precursors
(PRCpr). These PRCpr later in development divide symmetrically within the
developing PRC layer. To determine the proliferative behavior of PRCprs, we
performed lineage-tracing and observed that all PRCprs go through one additional
round of cell division. Next, we deciphered the migratory pattern of PRCprs. Using
light sheet fluorescent microscopy, we observed that PRCprs are born from apical
progenitors and first undergo a directional and rapid basal translocation. They
subsequently move back to the apical side in a stepwise manner where they
terminally divide. To assess the relationship between migration and cell cycle, we
blocked the basal translocation by genetic interference with microtubule
cytoskeleton. We observed that interruption of the basal displacement does not affect
final division of PRCprs. So far, these results show that in order to ensure terminal
division at the developing PRC layer, PRCprs take on a stereotypic migratory
behavior, which is not functionally linked to the cell cycle. Next, we aim to explore
the molecular basis of PRCpr behavior to understand how progenitors transform into
committed precursors.
This work was performed in accordance with EU directive 2011/63/EU and
supported by the German Research Foundation (DFG).
For this work, evaluation by the ethics committee was not necessary.
Funding Support: CAPES, CNPq and FAPEMIG.


I5
I6
EGF INHIBITS FoxD3 EXPRESSION AND STIMULATES THE

PROLIFERATION OF Mitf-POSITIVE NEURAL CREST PROGENITOR
DURING DEVELOPMENT OF MELANOCYTES
HISTONE DEACETYLASE (HDAC) IS NECESSARY FOR CORRECT

EXPRESSION LEVELS OF GENES INVOLVED IN DPP SIGNAL
TRANSDUCTION DURING DROSOPHILA MELANOGASTER WING
DEVELOPMENT
Gabriel da Silva Pescador*, Bianca Luise Teixeira, Jaqueline Isoppo da Cunha,

Diego Amarante-Silva, Giordano Wosgrau Calloni, Andra Gonalves Trentin,
Ricardo Castilho Garcez.
(1) Departamento de Biologia Celular, Embriologia e Gentica, Centro de Cincias
Biolgicas, Universidade Federal de Santa Catarina, Campus Universitrio Trindade, 88040-900, Florianpolis - SC - Brazil
*Contact information: E-mail: gabrielspescador@gmail.com; Institutional phone:
(48) 3721-4582; Mobile phone: (48) 9191-1068
Pigmentation is present in all animals with many functions. Despite other kinds of
chromatophores, melanocytes are shared among all chordates and are known to arise
from the neural crest. These cells come from the neural tube closure and undergo
epithelial to mesenchymal transition to migrate and undergo differentiation.
Melanocytes arise mainly from the dorso-lateral migration pathway. When
undergoing migration, neural crest cells stop expressing Foxd3 and keep Sox10, and
later start expressing Mitf. EGF is a small protein known to be expressed in the
developing embryo and to act like a growth factor. Previous data from our group
show that EGF treatments in culture promote differentiation of neural crest cells into
neurons and melanocytes. Our aim is to assess if the growth factor EGF is important
for melanocyte differentiation, and its molecular mechanism behind it. We show that
neural crest cells, in vitro, normally express EGFR, a receptor for EGF. Also,
treatments of neural crest cells with EGF in vitro decreased the expression of Foxd3
and increased the number of Mitf positive cells. EGF seems to be crucial for neural
crest cells undergo melanocyte differentiation, as we can conclude from the
expression analyses. Our next step is to use Erbitux, a known antagonist of EGFR
used for some cancer treatments, to assess the molecular mechanism that EGF is
acting in ex vivo culture system.
This work received financial support from CNPq, CAPES, FAPESC, and has been
approved by the ethics committee from UFSC under the protocol pp00787.
Jssica Baptista1,2, Carla Marques1,2, Helena Araujo2 And Katia Carneiro1,2

1.Laboratrio De Proliferao E Diferenciao Celular, Instituto De Cincias
Biomdicas Ufrj, Rio De Janeiro Rj -Brazil;
2. Laboratrio De Biologia Molecular Do Desenvolvimento, Instituto De Cincias
Biomdicas Ufrj, Rio De Janeiro Rj -Brazil;
Background: Histone deacetylases (HDAC) are enzymes that catalyze the removal of
acetyl radical from the N-terminal tails of nucleosomal histone proteins, which then,
compacts the chromatin leading to gene expression repression. In Drosophila
melanogaster the HDAC homologue, Rpd3, plays a crucial role during
heterochromatin formation and long-term courtship memory. During Drosophila
wing imaginal disc development Rpd3 has been shown to control imaginal disc size
through suppression of apoptosis and regulation of dpp expression by a conserved
role in repressive transcriptional complexes. However, the mechanisms controlling
HDAC activity during wing imaginal disc development are not completely
understood. Aims: In this work we seek to characterize the signaling pathway that
modulates HDAC activity during wing imaginal disc development. Methods: rpd3
knockdown was generated through the UAS/GAL4 system by superexpressing rpd3
and mCherry dsRNAi in both dorsal and ventral surfaces of the wing blade during
larval development. Wing discs were then processed for qPCR, immuno staining and
phenotypical analysis by confocal microscopy. qPCR was performed for quantitative
analysis of dpp signaling gene expression levels and immuno staining of acetylated
histones H4 in four different lysine residues (K5, K8, K12, K16) and phophorylated
histone H3 for mitosis quantification. Results: rpd3 knockdown wing discs lead to
down regulation of genes involved in Dpp signaling and altered levels of histone H4
acetylation and phosphorylated histone H3. Conclusion: HDAC activity is necessary
for the correct expression levels of genes involved in Dpp signal transduction likely
as a member of a repressive transcriptional complex that targets Dpp transcriptional
regulators.

I7
I8
Smc1A IS NECESSARY FOR CELL SURVIVAL DURING OCULAR

DEVELOPMENT
HOMEOSTASIS AND ENDOCYCLES IN THE OVARIAN DEVELOPMENT

OF RHYNCHOSCIARA AMERICANA
Gabriel E. Matos-Rodrigues1, Gabriel R. Cavalheiro1, Pierre-Olivier Frappart2 and

Rodrigo A. P. Martins1
Jorge Henrique Neves1*, Julyane Batista Chaves1*, Melissa Colpani de Vitor1, Paula
Rezende Teixeira1 and Glaucia Maria Machado Santelli1
1. Institute of Biomedical Science, University of So Paulo
Programa de Biologia Celular e do Desenvolvimento, ICB, UFRJ, Brazil

Clinical Cooperation Unit Neuropathology, DKFZ, Germany.
Presenting Author: Gabriel Eduardo de Matos Rodrigues

Email: Gabriel.edu.rodrigues@gmail.com
Telephone: (21) 999910319
Adress: Av. Carlos Chagas, 373, F1-08, Labcom2, Prdio do Centro de Cincias da
Sade, Cidade Universitria, Ilha do Fundo - Rio de Janeiro - RJ, CEP 21941-902
Sister chromatids are formed during the S-phase of the cell cycle and remain
attached until the final sub phases of mitosis. This feature of proliferating cells
depends on the cohesin complex that has Smc1a, Smc3 and Rad21 as its structural
components. Mutations in the cohesin genes are correlated with few human
syndromes characterized by developmental malformations, neurodevelopmental
delay and ophthalmological problems. Although much is known about Smc1a
functions during proliferation, its role in post-mitotic cells are still to be elucidated.
To understand the role of Smc1a in vivo, we developed mice in which exons 2 and 3
of Smc1a gene were flanked by lox sequences. Different lines of Cre mice were used
to inactivate the Smc1a in specific ocular tissues: Smc1a was inactivated only in the
lens (Le-Cre), only in the retina (Pax6-Cre) or in both tissues (Nestin-Cre). Smc1a
inactivation in the surface ectoderm led to aphakia (absence of the lens) at embryonic
day 17 (E17). Loss of Smc1a in both the lens and the retina impaired eye growth
during the embryonic development. The defective eye growth was associated with
decreased the number of proliferating cells, p53 stabilization and cell death in both
tissues. Inactivation of both p53 and Smc1a decreased the cell death induced by
Smc1a loss in both tissues. Previous reports have shown that Smc1a is found in postmitotic photoreceptor cilia. Importantly, Smc1a loss specifically in retinal progenitor
cells, not only induced cell death of these proliferating cells, but also caused
malformation of the outer segment and the degeneration of post-mitotic
photoreceptors during postnatal development. We have seen for the first time that
Smc1a regulates the survival of proliferating and post-mitotic neurons during retina
development in vivo. These findings demonstrate that Smc1a is essential for ocular
development and corroborate to the understanding of the malformations caused by
cohesin complex loss of function as observed in human syndromes.
The ovary of diptera Rhynchosciara americana has unique features: ovarian follicles
develop synchronously and the last mitotic cycle of germ cells occurs early in larval
development, giving rise to two cells that hold together. One cell becomes the
oocyte-I that enters into meiosis and remains stationary while the other cell will
differentiate into nurse cell. Among the tissues that exhibit cell cycle specializations,
the ovary has three different possible scenarios: oocyte undergoes meiosis, nurse cell
has polyploid and polytene processes, and follicular cells undergoes mitosis.
This study aimed to characterize the DNA replication cycles in follicular and nurse
cells as well to check the expression levels of the innexin2 mRNA in ovarian
development of the R. americana. The innexins are proteins that form the gap
junctions connecting cell-cell and they are essential for the development and
homeostasis of multicellular organisms. The proliferative capacity of follicular and
nurse cells was measured by Click-iT EdU kit (Invitrogen) and the expression
profile of the mRNA by real-time PCR. The results showed the end of the replicative
cycle of the nurse cells, when the last peak of the ecdysone hormone occurs on the
fourth day of the pupal stage. While the expression of innexin2 occurs throughout
ovarian development, it is more expressed in the pupal stage, coinciding with the
polyploid/polytene transition of the nurse cells and increased their transcriptional
potential.
Supported by: FAPESP, CNPq and CAPES
*Both authors contributed equally
This research work was approved by the CEUA/CCS/UFRJ comitee

Financial Support: DFG, CNPq, FAPERJ, IRRF.


I9
I10
THE EFFECTS OF BIRTH WEIGHT ON MUSCLE FIBER DEVELOPMENT

IN PIGS
STUDY OF GLIOBLASTOMA MULTIFORM HETEROGENEITY AND

PLASTICITY IN CHICK EMBRYONIC ENVIRONMENT
Andria Dornas Pereira (1); Diogo Magnabosco (2); Fernando P. Bortolozzo (2);
Patrcia M. Martinelli (1); Hlio Chiarini-Garcia (1); Fernanda R.C.L. Almeida (1).
Patrcia Streit(1); Ingrid Rosenburg Cordeiro(1); Tania Cristina Leite de Sampaio e

Spoh(2); Vivaldo Moura Neto(2); Jos Marques de Brito Neto(1)
(1) Department of Morphology, Federal University of Minas Gerais, MG, Brazil.

(2) Faculty of Veterinary Medicine, Federal University of Rio Grande do Sul, RS,
Brazil.
(1) Laboratory of cell diferentiation and proliferation, Instituto de Cincias

Biomdicas, UFRJ, Rio de Janeiro, Brazil
(2) Laboratory of celular morphogenese, Instituto de Cincias Biomdicas, UFRJ,
Rio de Janeiro, Brazil
E-mail: andreiadornas@yahoo.com.br Tel: 55.37.9.9918 2772; 55.31. 3409 7800

Laboratrio de Biologia Estrutural e Reproduo, Departamento de Morfologia, ICB
UFMG.
Av. Antnio Carlos, 6627, Pampulha. CEP 31.270-901 Belo Horizonte, MG, Brasil
Background: In the last decades, the proportion of small piglets at birth has
increased. There is evidence that low birth weight piglets present worse postnatal
growth performance and poor meat quality. As the main objective of the swine
industry is meat production, knowing the effects of birth weight on skeletal muscle
fiber development seems critical.
Aim: To investigate skeletal muscle fiber development in different birth weight
piglets during three phases of the production cycle.
Methods: Female littermate piglets were selected at birth and allocated to two
treatment groups: low weight (LW: 0.8-1.0 kg) and high weight (HW: 1.4-1.7 kg).
Semitendinosus muscles samples were collected from each group at birth (n=18),
100 days (n=18) and 150 days-old (n=18) and processed for histomorphometrical
and RT-qPCR analysis.
Results: HW newborn pigs had greater muscle cross-sectional area and muscle fiber
area and diameter compared to the LW group (P<0.01). These differences were not
present in the older ages. The gene expression analysis in the newborn group,
showed a higher expression of LPL, related to lipid transport, in LW animals. At 100
days, Myf5, related to myoblast differentiation, and PAX3, related to satellite cell
activation, were highly expressed in HW and LW, respectively, while at 150 days,
there were no significant differences.
Conclusion: Even though LW piglets have smaller muscle fibers at birth, muscle
hypertrophy is not affected by birth weight during postnatal development.
Presenting author :Patrcia Streit

Adress: Macedo Sobrinho Street, 38/704, Humait, Rio de Janeiro
Institucional phone: (21) 39386481 - Mobile: (24) 992640630
E-mail patystreit@uol.com.br
Glioblastoma Multiform (GBM) is a malignant brain tumor. It is currently discussed

whether it could be originated from the malignant transformation of differentiated
cells, the astrocytes, or directly from glial or neural stem/progenitor cells.
The goal is to study whether there is presence of cancer stem cells (CSC) in GBM,
and to investigate if embryonic microenvironment is capable of controlling the
behavior of these cells. Spheroids of GBM cells were made using 3D-system culture
and they were grafted into the neural tube wall, at the anterior prosencephalic region
of chick embryos at 5-10 somites stages (ss). We use in situ hybridization for human
genomic element Alu to identify human cells in the chick embryo. After the
xenograft of GBM spheroids the development was arrested at E3(embryonic day),
E4, E6 and E8, and the forebrain was analyzed. We showed that GBM cells were
able to integrate into the neuroepithelium and they kept viable up to E8. Through
RT-PCR, we detected that GBM expressed glial and progenitors markers (GFAP,
CD133, nestin, Oct4, Nanog) and genes involved in the Hedgehog pathway (Ptc1,
Smo, Gli1, Shh), which is involved in vivo with maintenance of stem cells in the
brain. To summarize, GBM cells which express stemness markers could not only
integrate with the neuroepithelium but also they were able to recruit cells of the host
and associated with vessels. For further investigation xenografted embryos with
GBM cells will be analyzed to determine if these cells are able to give rise to neural
and/or glial derivatives.
(Funding support: CNPq and Faperj)
Key-words: pigs, birth weight, muscle fiber, meat quality.

This project was approved by CEUA/UFRGS (23732/2011).
I11
I12
ROLES OF THE ATR-ATRIP SIGNALING PATHWAY IN CENTRAL

NERVOUS SYSTEM DEVELOPMENT IN VIVO
BROMOCRIPTINE ALTERS MORPHOPHYSIOLOGY AND THE

PROLACTIN SIGNALING PATHWAY IN RAT VENTRAL PROSTATE
Gabriel E. Matos-Rodrigues1, Pedro B. Tan1, Pierre-Olivier Frappart2 and Rodrigo

A. P. Martins1
Ana Carolina L Camargo1; Flavia B Constantino1; Sergio A A Santos1; Ketlin T

Colombelli1; Suelen Franco1; Caroline N Barquilha1; Jaqueline C Rinaldi1; Bruno O
S Druran1; Maeli Dal-Pai1; Srgio L Felisbino1; Luis A Justulin Jr1.
1
2
Programa de Biologia Celular e do Desenvolvimento, ICB, UFRJ, Brazil

Clinical Cooperation Unit Neuropathology, DKFZ, Germany.
Departament of Morphology Institute of Bioscience of Botucatu, Sao Paulo State

University, Brazil.
Presenting Author: Pedro Batista Tan

Email: pedro.tan2@gmail.com
Telephone: 998899532 - Adress: Rua General Glicrio, 445/501, Laranjeiras, Rio de
Janeiro, RJ, Brazil - Institute of Biological Sciences, UFRJ, Rio de Janeiro, Brazil,
CCS, F1-08, Labcom2
Corresponding author: Ana Carolina Lima Camargo, Laboratory of Extracellular

Matrix Department, of Morphology, Institute of Biosciences of Botucatu, So Paulo
State University. Phone: (14) 38800502, email: carolgalah@gmail.com
Genomic stability is crucial for cellular and tissue homeostasis, normal development
and prevention of tumorigenesis. ATR is a PI3K-like kinase involved in the signaling
of DNA damage. ATR protein expression and stability as well as its described
functions depend on its partner ATRIP. In humans, mutations in these genes lead to
Seckel Syndrome that is associated with genomic instability, while ATR knockout
mice are embryonically lethal. Our goal is to study the roles of Atrip in the
development of the CNS in vivo. To evaluate the functions Atrip in vivo, we
generated a transgenic mice in which the first three exons of Atrip were flanked by
LoxP sequences and this was mated with different Cre lines in order to inactivate
Atrip specifically in the retina (Pax6-Cre; Atrip lox/lox) or in the brain and retina
(Nestin-Cre; Atrip lox/lox). Loss of Atrip in neural progenitor cells resulted in
developmental defects, microcephaly and postnatal lethality around P9. Atrip
inactivation also compromised retinal development, leading to DNA damage
accumulation and increased cell death in retinal progenitor cells. Atrip-deficient
adult retinas presented dysplasia and lamination defects. For the first time, we
observed that Atr-Atrip signaling pathway is essential for the survival of retinal
progenitor cells survival in vivo. The defects of Atrip-deficient adult retinas suggest a
previously undescribed role of Atr-Atrip in postmitotic neurons differentiation.
These results may contribute to a better understanding of the roles of Atr and Atrip in
human syndromes associated with genomic instability.
Introduction: Prolactin (PRL) is a polypeptide hormone synthesized by anterior

pituitary, whose most known effect is the increase in milk production by the
mammary glands. However, it has been demonstrated that prostate express prolactin
mRNA and functional prolactin receptor (PRLR), suggesting that the prostate is
target of PRL action. Although studies have demonstrated association between PRL
expression and prostate cancer, litter is known about the role of PRL during prostate
development. Aims: To investigate if PRL inhibition by Bromocriptine (PRL
secretion inhibitor) alters prostate development and growth. Methods: Sprague
Dawley rats (n=6/group) were divided into 2 groups: Control (CT) which received
sterile saline (vehicle) from postnatal day 12 (DPN12) to PND 21 (CT21) or which
received bromocriptine (BR21) (0.4 mg / kg) for the same period. After, the male
offspring were weighed, anesthetized and euthanized. Ventral prostate (VP) lobes
were collected and processed for histology, RT-qPCR and western blot (WB)
analysis. Results: The body weight did not change throughout the treatment. The BR
treatment increased VP weight. The VP from BR group presented dilated luminal
acini and reduced epithelial cell height than CT. The PRLR and STAT3 mRNA
expression was increased in the BR21 than CT group. The WB reaction
demonstrated decrease in PRL and PRLR in BR compared to CT. There is no
difference in the prostatein expression. Conclusion: The BR administration alters VP
morphology. These results suggested a posttranscriptional regulation of PRL and
PRLR protein expression. The systemic blockage of PRL also affects the ventral
prostate PRL signaling.
This research work was approved by the CEUA/CCS/UFRJ comitee

Financial Support: DFG, CNPq, FAPERJ, IRRF.
Ethical approval CEUA n 756; Funding support CAPES and FAPESP

I13
I14
LACK OF HDAC ACTIVITY IMPAIRS MESENCHYMAL REMODELING

DURING XENOPUS TAIL REGENERATION
ANDROGEN-DEPENDENT SIGNALING NETWORKS IN WOLFFIAN

DUCT MORPHOGENESIS
Nathlia Pentagna 1, Alice H. Reis 2, Fbio Mendes 2, Jos Garcia Abreu 2 and Katia
Carneiro 1
Camilla M Ribeiro1, Barry T Hinton2, Maria Christina W Avellar1.
Laboratrio de Proliferao e Diferenciao Celular, Instituto de Cincias

Biomdicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
2
Laboratrio de Embriologia de Vertebrados, Instituto de Cincias Biomdicas,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
Nathlia Pentagna M. D. Pires
Av. Carlos Chagas Filho, 373, Bloco F 2andar, sala 01 - Cidade Universitria, Ilha
do Fundo, Rio de Janeiro, Brasil - CEP:21941-902
Institutional telephone:(21) 3938-6483 - Mobile: (21) 98870-9605
e-mail: natpires87@gmail.com
Regeneration is a natural and continuous process to ensure tissue homeostasis. It
comprehends different levels of complexity ranging from the substitution of the
injured tissue until the regeneration of a whole new organism. The Xenopus laevis
presents great regeneration ability during larvae stage successfully regenerating the
entire tail upon amputation. Epigenetic mechanisms have been shown as key
modulators of Xenopus tail regeneration. In particular, our group has demonstrated
that the pharmacological inhibition of the chromatin remodeling enzyme Histone
Deacetylase (HDAC) prevented tail regeneration after amputation. Besides that
previous work from our group showed that HDAC activity inhibition gave rise to an
anti-inflammatory response enhancing wound healing and tissue repair in murine
model. This work aims to characterize a putative role for HDAC activity during cell
matrix remodeling upon Xenopus tail amputation. Stage 40 tadpoles were amputated,
under effect of anesthesia, and cultivated with DMSO (control) or HDAC inhibitor
(25nM TSA). The larvae were processed to histological procedures at 24, 48 and 72
hours post amputation. Despite of unchanged proliferating rates observed between
control and TSA treated group, TSA treated tadpoles presented increased levels of
acetylated histone H4 and diffuse collagen type I and III deposition. In contrast to the
mesenchymal tissue found in control group, TSA treated tadpoles lacked
mesenchymal cells presenting overgrowth of notochord towards the regenerative bud
likely preventing the mesenchymal remodeling necessary for full regeneration.
HDAC activity is necessary for mesenchymal remodeling during Xenopus tail
regeneration. Ethical Committee 152/13 (CEUA UFRJ).
Section of Experimental Endocrinology, Department of Pharmacology,

Universidade Federal de So Paulo, Escola Paulista de Medicina, So Paulo, Brazil.
2
Department of Cell Biology, University of Virginia School of Medicine,
Charlottesville, Virginia, USA.
Correspondent author information: Camilla M Ribeiro. Rua 03 de Maio, 100. Depto.
de Farmacologia, Unifesp-EPM, CEP: 04044-020. So Paulo, SP, Brasil. Tel: +55
1155764448. E-mail: ribeiro.cmr@gmail.com
Background: Androgens orchestrate the morphological changes the Wolffian duct
(WD) undergoes to originate the epididymis. Without an epididymis of proper shape
and size, male fertility will result. The mediators of this androgen action and their
signaling pathways are still unclear. Aim: To evaluate the involvement of the
epidermal growth factor (EGF) and MAPK/ERK signaling in mediating androgen
action during WD morphogenesis. Methods: WDs were isolated from male Wistar
rats at embryonic day 17.5 and used for RT-PCR or cultured for 72-96h on cell
inserts floating on serum-free medium, in the absence or presence of testosterone (110 nM), EGF (1.7-17 nM), PD98059 (50 M, MEK inhibitor) and AG1478 (10-20
M, selective inhibitor of EGF receptor kinase). Gross morphology was evaluated.
Results: Egf, Egfr (Egf receptor) and Etv4 (Ets Variant4; a downstream target)
transcripts were detected in WDs. A synergistic effect of testosterone and EGF was
observed on elongation and coiling of cultured WDs. The total WD area was higher
in EGF-treated WDs in comparison to ducts cultured with testosterone, an effect
blocked by co-incubation of EGF and AG1478. Furthermore, incubation of either
AG1478 or PD98059 impaired testosterone-induced WD morphogenesis.
Conclusion: Our results show that EGF and testosterone act synergistically in WD
morphogenesis. The effects of testosterone on WD elongation and coiling, in turn,
depend on activation of EGFR and MAPK/ERK signaling. These findings highlight
the relevance of androgen-dependent paracrine signaling network during WD
morphogenesis and their contribution to male fertility. Ethics Approval: CEUAUnifesp-EPM#1776201213.
Financial support: CNPq/CSF (401932/2013-3, 150066/2016-3), NIHNICHD
#069654.
Financial support CAPES, CNPq and FAPERJ.

I15
I16
DEFENSINS ARE DIFFERENTLY REGULATED AND PLAY A ROLE

DURING WOLFFIAN DUCT MORPHOGENESIS
Lucas G A Ferreira1, Camilla M Ribeiro1, Daniel S Thimoteo1, Barry T Hinton2,
Maria Christina W Avellar1.
THE DIFFERENTIATION POTENTIAL OF THE ENTERIC GLIA

Carla Pires Verssimo (1, 2, 3), Juliana de Mattos Coelho-Aguiar (1,3), Adriana
Lima (1), Fabiana Pereira Ribeiro (3), Loraine Campanati (1), Vivaldo Moura-Neto
(1,3)
Section of Experimental Endocrinology, Department of Pharmacology,

Universidade Federal de So Paulo, Escola Paulista de Medicina, So Paulo, Brazil;
2
Department of Cell Biology, University of Virginia School of Medicine,
Charlottesville, Virginia, USA.
(1) Instituto de Cincias Biomdicas, Universidade Federal do Rio de Janeiro, Brasil

(2) Programa de Ps-Graduao em Anatomia Patolgica, Faculdade de medicina da
UFRJ, Brasil
(3) Instituto Estadual do Crebro Paulo Niemeyer, Rio de Janeiro, Brasil
Correspondent author information: Lucas G A Ferreira. Rua 03 de Maio, 100. Depto.

de Farmacologia, Unifesp-EPM, CEP: 04044-020. So Paulo, SP, Brasil. Tel: +55
1155764448. E-mail: garciaii_antuerpia@hotmail.com
The Enteric Nervous System (ENS) controls the gastrointestinal functions and it is
derived from the neural crest cells. The enteric neurons and glia presented in ENS
are arranged mainly through the myenteric and the submucosal plexus. The enteric
glia plays different roles, acting, for example, in the control of intestinal motility.
Moreover, enteric glial cells are able to give rise to neurons in vivo, after stimulation
of the glial serotonin receptor 5-HT(4), or in response to chemical injury to the
enteric ganglia. The goal of this study is to investigate the differentiation potential of
enteric glia in vitro in order to establish under which conditions these cells get
committed to the neuronal phenotypes. We have already established ENS cells
cultures from adult and newborn mice under specific conditions. Both cultures
showed cells expressing simultaneously glial and neuronal markers (GFAP and IIItubulin, respectively). At lower cell plating densities, a larger number of glial cells
(GFAP-positive) acquired neuronal morphology and expressed neuronal markers.
Our results reinforce the hypothesis that the enteric glia maintains neurogenic
potential, which can be activated by tissue dissociation or injury. This work may
allow us to characterize the neuronal differentiation potential of the neural crestderived enteric glial cells, and open new perspectives for the treatment of ENS
injuries.
Morphological differentiation of Wolffian duct (WD) into epididymis is induced by

androgens. Their effects on WD epithelium are mediated via mesenchymal factors by
mechanisms poorly understood. We reported that -defensin SPAG11C acts as an
androgenregulated mesenchymal factor required for WD morphogenesis. Thus, we
investigated the expression of different defensins and their androgen modulation
during WD morphogenesis. To evaluate the effects of SPAG11C on cultured WDs,
we analyzed their gross morphology. Total RNA was extracted from male Wistar
rats WDs at embryonic days (e) e12.5-e20.5 and from adult caput epididymis (120days-old) for RT-PCR and RT-qPCR assays. WDs (e17.5) were cultured in the
absence or presence of testosterone (10 nM), flutamide (10 M; competitive
androgen receptor antagonist) and purified recombinant human SPAG11C
(hSPAG11C, 80 nM). Spag11c mRNA was detected in WDs between e12.5e20.5;
transcripts for Defb1, Defb2 and Defb22, however, were only observed in e20.5
WDs. Defb12 and Spag11e mRNAs were not detected in any age-point. Defb1 and
Spag11c mRNA levels increased and decreased, respectively, between e17.5 and
e20.5, a period when fetal plasma T increases (4,69 + 0,40 nM vs 7,15 + 0,40 nM;
n=7/age; t test *p<0.05) and the WD differentiates. Androgen modulation of these
two transcripts was confirmed by WD organotypic cultures. Incubation with
hSPAG11C impaired WD development by reducing epithelial cell proliferation. The
differential expression pattern of defensins during WD morphogenesis suggests
distinct biological roles and/or specific regulatory mechanisms driving their
expression, broadening their relevance to epididymal organogenesis and male
fertility.
Ethics committee: DAHEICB015. Financial support: INNT, CNPq, CAPES, Faperj,

Pro-Saude Assoc. Benef. Assist. Soc. e Hospitalar, Fundao Ary Frauzino.
Funding support: CNPq/CSF#401932/20133, #150066/2016-3, #101550/2016-2;

NIHNICHD#069654. Ethics approval: CEUA-Unifesp-EPM#1776201213.

I17
I18
THE BASEMENT MEMBRANE ALTERS THE SUBCELLULAR

LOCALIZATION OF YES-ASSOCIATED PROTEIN (YAP) IN A 3D
CULTURE MODEL FOR MAMMARY GLAND ACINOGENESIS
REGULATION
OF
CSCRATCH2
GENE
EXPRESSION
TRANSCRIPTION FACTORS IN NEURAL EMBRYOGENESIS
BY
Carolina Purcell Goes (1), Chao Yun Irene Yan (1)

Antonio Carlos Manucci, Ana Paula Zen Petisco Fiore, Alexandre Bruni-Cardoso.
Department of Biochemistry, Institute of Chemistry, University of So Paulo
(1)
Extracellular matrix (ECM)-signaling is crucial for determination of cell fate and
behavior during mammary gland morphogenesis and homeostasis. However, little is
known about the molecular mechanisms that regulate these processes. The Hippo
pathway, a cascade involved in the regulation of several biological processes,
including organ size, seems an important candidate as mediator of ECM cues that
change gene expression. Our preliminary data show that the basement membrane
influences the localization and concentration of YAP, an effector of the Hippo
pathway. Our main aim is to identify the partner proteins of YAP in mammary
epithelial cells grown in a tridimensional (3D) model using proximity-dependent
biotin identification (BioID) followed by mass spectrometry analysis. First, we
implemented a culture model for formation of 3D clusters of EpH4 cells (a mouse
cell line) on a non-adhesive substrate of PolyHEMA. The 3D clusters gained polarity
after addition of a reconstituted basement membrane (rBM). We also observed that
YAP localization alters from nuclear when in the absence of rBM to predominantly
cytoplasmic when the clusters were cultured with rBM. Furthermore, in the
cytoplasm of the rBM-treated cells, YAP concentrated in discrete granules. Our next
step is to transfect EpH4 cells with an expression vector containing YAP fused to a
promiscuous biotin ligase, perform cell fractionation and identify by BioID the
proteins interacting with YAP in the nuclear and cytoplasmic compartments in
response to rBM. We expect to reveal an interaction network that may bring crucial
information on the ECM-signaling mechanisms during mammary morphogenesis
and function.
(1) Department of Cell and Developmental Biology, University of So Paulo, Brazil

goes.cp@gmail.com
BACKGROUND: The neural tube ventricular zone contains a population of
proliferative cells whose transition to a post-mitotic state is regulated by proneural
genes and a molecular cascade. Both are closely linked to the neural migration
process. SCRATCH2 (Scrt2) is a transcription factor (TF) expressed in early postmitotic neural cells that plays a role in survival, differentiation and migration. cScrt2
is restricted population of cells in the inner layer of the intermediate zone of the
chick embryonic neural tubes, and this pattern is conserved in other vertebrates.
However, the mechanisms that control its transcription remain unknown. AIMS: The
absence of cScrt2 expression in the ventricular and subventricular zones suggests
that its expression could be repressed by TFs present in these regions and induced by
TFs in the postmitotic zone. METHODS: We searched the cScrt2 genomic region for
putative TFs binding sites. We evaluated the role of the TFs mAsh1, cPax6, cNgn2
and Brn in regulating cScrt2 expression through overexpression of these factors in
developing chick neural tubes. Evaluation of gene expression alterations were done
through in situ hybridization. The overexpression of mAsh1 led to a slight increase in
cScrt2 expression level. In contrast, the overexpression of cPax6 reduced cScrt2
expression and had more severe effects, such as changes in neural tubes
morphology. Likewise, the chimeric repressive construct of mBrn2-En had severe
effects, but wild type mBrn2 overexpression did not alter cScrt2 expression. cNgn2
shifted cScrt2 expression to more peripheral regions. CONCLUSION: Our data
confirms that cScrt2 is regulated by the developing neural tubes gene network.
Funding support: Fapesp and CNPq.

Ethical Committee Approval Protocol 025-04-03 (29/04/2013)
Funding Support: FAPESP and CAPES

J EDUCATION IN CELL BIOLOGY
J1
J2
USING NON-LINGUISTIC REPRESENTATIONS TO THE CELL BIOLOGY

EDUCATION: A CASE STUDY
PROFILE OF CELL BIOLOGY TEACHERS IN PUBLIC SCHOOLS OF

PARANA STATE
Gabriel Mathias Carneiro Leo (1), Paulo Roberto Custdio de Oliveira (1), Andra
Martini Ribeiro (1), Lilian Orvatti (1), Lucas Roberto Perucci (1), Marco Antonio
Ferreira Randi (2)
Gabriel Mathias Carneiro Leo (1), Marco Antonio Ferreira Randi (2)
(1) Instituto Federal do Parana, Curitiba, PR, Brazil.

(2) Department of Cell and Molecular Biology, Federal University of Parana,
Curitiba, PR, Brazil.
Graduate Program in Cellular and Molecular Biology, Federal University of Parana
(UFPR), Av. Cel. Francisco H. dos Santos, 210, Jardim das Americas, Curitiba, PR,
Brazil, CEP 81531-970, gabriel.leao@ifpr.edu.br, (41) 9909-2563.
Teaching and learning cell biology are not simple tasks, and promote the
involvement of students is a great challenge for teachers. As different people learn in
different ways, the educator must find alternatives that contribute to the development
of various learners skills. The Cell Biology is a course that involves abstract
concepts for the student, because the vast majority of the studied objects are below
the resolution limit of the human eye. The non-linguistic representations are
alternative ways to bring this information to concrete. The construction of physical
models and the preparation of drawings or posters involve materials handling,
requires three-dimensional visualization skills and can help in forming concrete
theme representations. This study aimed to analyze the use of non-linguistic
representations as a pedagogical strategy for teaching cell biology topics. The
activities involved the construction of three-dimensional models and the organization
of posters related to the proposed themes, applied to first year high school students in
a public educational institution member of the federal professional network
education. To analyze the effects of methodologies in learning, students were
evaluated before and after the classes that involved non-linguistic methods. There
was an increase in the test scores, suggesting that the methodologies can be used for
cell biology teaching, contributing to the diversification of activities, thus favoring
the relationship between students and teachers and increasing interest in cell biology.
(1) Instituto Federal do Parana, Curitiba, PR, Brazil.

(2) Department of Cell and Molecular Biology, Federal University of Parana,
Curitiba, PR, Brazil.
Graduate Program in Cellular and Molecular Biology, Federal University of Parana
(UFPR), Av. Cel. Francisco H. dos Santos, 210, Jardim das Americas, Curitiba, PR,
Brazil, CEP 81531-970, gabriel.leao@ifpr.edu.br, (41) 9909-2563.
Teachers must investigate the problems of teaching and learning resulting from its
own activity. Scientific language and the diversity of Cell Biology concepts can
accentuate the disinterest of students and decrease the motivation of teachers. In this
study the profile of Cell Biology teachers in public schools of Parana State was
analyzed in order to contribute for a better understanding of the main challenges
faced and find methodologies and strategies that enhance the teaching and learning
process. Teachers from public schools were invited to answer a questionnaire and to
participate in a semi-structured interview in which the profile and attitudes related to
the learning Cell Biology process were evaluated. In spite of being a young and
experienced group, the lecture format of class remains strongly present in
pedagogical practices, even being considered traditional by the same teachers and in
contrast to a variety of modern teaching techniques. Strategies/methodologies that
could facilitate Cell Biology teaching and learning process are little used by teachers.
Most of interviewed teachers believe there is a mismatch between what is taught and
what student learns in Cell Biology. The complexity of the subject, including the
amount of information and the extensive and difficult nomenclature in the field, and
the high abstraction level of the contents were pointed as the main problems inherent
to Cell Biology teaching and learning.
Approved by the Research Ethics Committee in Human Beings of the Hospital de
Clinicas, UFPR (CAAE: 42163015.8.0000.0102). Supported by CNPq.
Approved by the Research Ethics Committee in Human Beings of the Hospital de

Clinicas, UFPR (CAAE: 42163015.8.0000.0102). Supported by CNPq.
J3
J4
CONTINUED TEACHING TRAINING FOR CELL AND MOLECULAR

BIOLOGY
THE USE OF MODELING AND 3D PRINTING SOFTWARES TO THE

CREATION OF EDUCATIONAL TOOLS THAT CONTRIBUTE TO
ACCESSIBILITY IN CELL AND TISSUE BIOLOGY LEARNING AND
TEACHING PROCESS
Germano Carneiro da Costa (1)

(1) Health and Biological Science Institute, Federal University of Viosa, Minas
Gerais, Brazil.
Authors Contact: Rodovia LMG 818, Km 6, Campus Universitrio - Florestal, MG
CEP: 35690-000 Tel: (31) 3536-3385/ FAX: (31) 35363361. UFV-Florestal.
germanocarneiro@ufv.br; (31) 3536-3401/ 99206-6587.
Science and Technology has brought several changing in contemporary society,
which requires an improved education for providing the formation of a critical
citizen. Cell and Molecular Biology (CMB) issues are frequently found on daily
society, reported by several segments of media, which often uses misconception or
fictitious. Hence, CMB classic content is heavy and abstract, which may discourage
students. Therefore, special attention should be given for undergraduate schools
regarding new methods and models of CMB teaching. This work aimed to create a
continued teaching training for helping teachers to actualize themselves with new
concepts and new approaches which points out to contexts, interdisciplinary and
practical activities. This work was undertaken in New Talents-Capes program
along with others courses offered in UFV-Florestal Campus during 2014. The course
was conceived within three sequential modules: 1-expositive class focusing on
interdisciplinary and contextual approach for CMB teaching (4h); 2,3-didactic
practices for cell biology (2) and molecular biology (3) teaching (8h each). Science
education is currently fragmented, which makes difficult for student to understand
the collaborative evolution of knowledge. Physical and chemical concepts are crucial
for understanding molecules structures, cell dynamics and technique approaches.
Hence, experimentation is important since it has the potential to motivate students
and enhance theory learning. The course was conceived with a 94 pages booklet
which may be a helpful guide for application in schools. Therefore, teacher training
is an important strategy for science education improvement and for stimulating
students for the scientific area and to graduate education.
Financial support: Capes.
Gisele Orlandi Introni (1), Lauren Cars (2), Bianka Rauber (1), Alessandra Peres
(1), Naiane Carlesso Bassani (1), Marilda da Cruz Fernandes (1), Giovana Tavares
dos Santos (1), Claudia Giuliano Bica (1), Luiza Paul Gea (1), Mnica Fernandes
Rosa de Lima (1), Rosalva Thereza Meurer (1), Cyntia Alencar Fin (1) and Andr
Peres (3)
(1) Federal University of Health Sciences of Porto Alegre (UFCSPA)
(2) Federal University of Rio Grande do Sul (UFRGS)
(3) Federal Institute of Rio Grande do Sul (IFRS)
giseleorlandi@gmail.com
Structural details of organelles, cell morphology and tissue arrangement are the
target of scientific research seeking to understand the connection between form
(design) and function. Currently, the available technological advances to Cell and
Tissue Biology students and teachers are 3D computer graphics programs and threedimensional printing as valuable educational tools. 3D Studio Max has modeling
capabilities, a flexible plugin architecture and can be used on the Microsoft Windows
platform. We utilize a 3D printer to form successive layers of thermoplastic under
computer control to create an organelle or cell prototype. This project aims to
promote the training (a workshop) focusing on teachers, students and technicians
who are interested in creating three-dimensional images of biological structures at
various hierarchical levels and allows these models to be printed in 3D. These
prototypes will contribute to the inclusion of people with visual disabilities in
ordinary and special education with an emphasis in Cell and Tissue Biology. The
benefits of the proposed actions extend to students of primary and secondary public
schools. This project includes technical and scientific activities providing the
opportunity of a theoretical and practical learning and consequently cultural
development and social responsibility. Partnerships with (1) associations that are
concerned with people having visual impairments as well as (2) elementary and high
schools are essential to the success of these activities. Finally, it seeks to spread the
idea that the value of information and the use of technology should act
synergistically in the scenario of Brazilian Universities.
Financial support: Brazilian Ministry of Education and Culture

J5
J6
MEETING THE BRAIN: DISCUSSING NEUROSCIENCE IN BASIC

EDUCATION
UNRAVELING ORAL CANCER FOR STUDENTS OF BASIC EDUCATION
Gustavo Diniz de Mesquita Taveira, Marta Cristina da Cunha Rodrigues, Alan

Pereira da Costa, Ana Carolina Barbosa, Camila Rodrigues, Penha Cristina Barradas
Department of Pharmacology and Psychobiology. University of the State of Rio de
Janeiro.
Dissemination of science knowledge and scientific literacy favors the establishment
of a more open, inquisitive and critical society. In this sense, teaching young students
and lay people about brain development and function can bring awareness about
damages such as stroke and hypoxia-ischemia. This can also advise people about the
risks of the use of drugs, such as tobacco and alcohol, during brain development. In
this study we aim to advertise and discuss these themes with elementary students
from Rio de Janeiro schools using a customized board game named Meeting the
Brain. Our approach consisted of four stages: I) sensitization: broadcast the chosen
topics using posters placed at schools two weeks before our visit; II) presentation:
lectures and discussion about factors that affect brain development; III) game
application: students are introduced to the game that deals with themes mentioned
above; after a validation process, the game was considerate adequate for basic
students; IV) a Website, "Meeting the Brain " was used as a post-intervention tool.
We used a questionnaire to evaluate our approach. Our results show that students had
a previous knowledge about the harmful effects of alcohol and cigarettes on brain
development. Most students considered that the game helped them to understand and
consolidate the content that was covered during the lectures. Moreover, they thought
that the game was fun and easy to follow. Overall, our data demonstrate that the use
of didactic games as a scientific communication strategy is effective and has great
potential to benefit students and advisors.
Leonardo Francisco Diel(1), Natlia Angela Bortoli(1), Lisiane Bernardi(1), Carlos

Alberto Bernardes(1), Marcelo Lazzaron Lamers(1,2).
Porto Alegre, Brazil
nati-b12@hotmail.com, marcelo.lamers@ufrgs.br
The cancer process raises many questions into general society and there is a
dissemination of erroneous information about the origin/growth/treatment of tumors.
Also, the common sense links this disease to the idea of death. In order to elucidate
these basic concepts about oral cancer, we discussed issues related to cell biology and
carcinogenesis with students from Basic Education. The activity was held in 2014/02
and 2015/01 by a graduate student of Dentistry course at UFRGS, which monitored a
group of students from elementary (3) and middle (3) education from a public school.
We discussed theoretical and practical classes such as the structure and functioning of
the cell, cell proliferation and the cellular changes that lead to the development of
tumors. It was also discussed data on prevalence and incidence of oral cancer in
Brazil. The students performed oral health panels, in which it was used information
(age, gender, patients and lifestyle habits) from an epidemiological database of
patients seeking care at UFRGS dental school. Then, students were trained in basic
laboratory techniques, as well as in the use of software for data analysis and
presentation. With the collected information, the trained students prepared seminars
and supporting materials with accessible language for dissemination at school and
their community. It was noticed an evolution in the understanding of the students of
normal cellular processes, carcinogenesis and the epidemiology of oral cancer.
Funding Support: The Science Incentive Program in Mathematics, Engineering and
Letters PICMEL/FAPERGS

J8
J7
MICROSCOPY ON TRAVELING MUSEUM SCIENCES UNDER TENTS AS
PREFERRED ACTIVITY IN PUBLIC SCIENCE COMMUNICATION
Gustavo Henrique Varela Saturnino Alves (1, 2); Mariana Souza Elysio (1); Silmar
Joriatti (1); Jlia Soares Drummond (1); Ewerton Emerson Lima da Silva (1);
Lucianne Fragel-Madeira (1)
(1) Institute of Biology, Fluminense Federal University, Rio de Janeiro, Brasil.
(2) Oswaldo Cruz Institute, Fundao Oswaldo Cruz, Rio de Janeiro, Brasil.
BIOIMAGEM: AN OPEN ACCESS CENTER FOR CELLULAR AND

MOLECULAR IMAGING SHARING, EDUCATION AND RESEARCH IN
BIOMEDICAL SCIENCES
Beatriz Viana dos Santos, Izabela Caldeira, Dayble Santos, Murilo Dias da Silva,
Pamela Gabrieli, Glaucia Machado-Santelli and Jos E. Belizrio
Departamento de Farmacologia e Departamento de Biologia Celular e do
Desenvolvimento do Instituto de Cincias Biomdicas da USP. Av. Lineu Prestes,
1524, CEP 05508-000, So Paulo, SP.
Cytology is an area of biology that demands for tools and concepts about
microscopy. Microscopy depends on specific equipments that are little known by the
general population. So this study aimed to promote scientific dissemination on the
microscopy. Thus we developed two scientific popularization activities within the
traveling museum Sciences Under Tents that comprehended the observation of
neurons, retina and onion slices and a cell phone screen phone with 40x
magnification. These activities were carried out in three cities of Rio de Janeiro State
during the year of 2015. Data were obtained through public observation and through
the evaluation system in which we evaluate their preference for the exhibition
activities. The results showed that cell microscopy attracted the audience that
interacted with the equipment, its components and the representative boards of the
tissue slices in exhibition. Technological microscopy promoted discussions about the
LEDs on the mobile screen and its characteristics such as size, layout, color and
function. After analysis of the data processing system we found that, among the
exposed activities, microscopy activity took first place in the preference of the
public. The results allowed us to conclude that the microscopy activity sparked the
audience interest and promoted the dissemination of concepts and applications of
microscopy, reaching our goal.
Funding support: FAPERJ, CNPq, PROEX-UFF, CAPES, Movimento Uniforme,
Microdiz e NikitPrint
Advances in molecular imaging and computer technologies are providing scientists

and students with unprecedented possibilities to visualize internal structures of cells,
organs and organisms and to collect systematic high resolution image data under
physiological and pathological situations. Bioimagem is an integrated
interdisciplinary research program supported by several laboratories and facilities of
University of Sao Paulo that aims to apply and develop novel in situ and in vivo
imaging methodologies to study cellular and subcellular processes, disease
development and therapeutic strategies in cells and animal models. The center
provides bright field histochemical as well as multi-channel fluorescence slides
scanning services in the ZEISS Axio Scan.Z1 automated slide scanner which is able
to scan whole slide at high resolution. To make the best use of the increasingly
complex and large image data resources, we have created a digital image data bank
accessed by web-based interface named Zen browser (www.bioimagem.icb.usp.br).
This central platform provides a client-server system for accessing, analyzing and
managing of images via virtual microscope and exploring its scientific information to
research and education. We are building human and mouse histology atlases that will
serve as an essential source of image references for students and teachers in
biomedical sciences field. Throughout the sharing of virtual microscopy images
online with colleagues and students, we hope to accelerate the diffusion and
applications of new discoveries in molecular imaging for more comprehensive
understanding of cell and tissue biology at multiple levels.

K EPIGENETICS
K1
K2
HEPATOCYTE CHROMATIN ALTERATIONS PROMOTED BY VPA AND

TSA UNDER HYPERGLYCEMIA MAY BE A FUNCTION OF THE
DIFFERENT
NUCLEAR
DOMAINS
INVOLVED
AND
GENES
REGULATED RATHER THAN OF GLOBAL REMODELING
IDENTIFICATION OF REFERENCE GENES FOR miRNA EXPRESSION

ANALYSIS IN NEOPLASTIC AND NON-NEOPLASTIC GASTRIC TISSUE
SAMPLES
Marina Barreto Felisbino1, Thiago Alves da Costa1, Maria Silvia Viccari Gatti2,
Maria Luiza Silveira Mello1
1
Department of Structural and Functional Biology, Institute of Biology, Unicamp,

Campinas, Brazil and 2Department of Genetics, Evolution and Bioagents, Institute of
Biology, Unicamp, Campinas, Brazil.
Email: felisbinomb@gmail.com. Tel: (19) 3521-6124
Recent data suggest that the complexity of diabetes, characterized by chronic
hyperglycemia, cannot be entirely explained by genetic predisposition; an epigenetic
component is probably involved. Chromatin remodeling can contribute to diabetes
amelioration by differential gene transcription. Thus, epigenetic modulation could be
a novel therapeutic approach to block the progression of diabetes. The use of histone
deacetylase inhibitors (HDACi), including valproic acid (VPA) and trichostatin A
(TSA), in clinical setting is an emerging area of investigation. An increase in hepatic
glucose production is the central event in the development and progression of
diabetes. It is thus of interest to investigate whether the glucose level of the culture
medium would affect the action of VPA and TSA on hepatocytes. Under
normoglycemia, these treatments promoted chromatin remodeling, as assessed by
image analysis and H3K9ac and H3K9me2 abundance. Simultaneously, H3K9ac
marks shifted to the nuclear periphery accompanied by HP1 dissociation from the
heterochromatin and a G1 cell cycle arrest. Hyperglycemia per se induced overall
decondensation of the chromatin with loss of repressive histone modifications and
increase of activating marks. Interestingly, VPA and TSA treatments under
hyperglycemia did not intensify these results. Despite the absence of morphological
changes being promoted, HDACi treatment seems to confer a physiological meaning,
ameliorating the cellular hyperglycemic state through reduction of glucose
production. These observations suggest that the glucose level to which the
hepatocytes are subjected affects how chromatin responds to HDACi and their action
under high-glucose environment might not reflect on chromatin remodeling.
Financial support FAPESP 2012/03238-5, CNPq 304668/2014-1.
Ana Carolina Anauate Pereira1, Mariana Ferreira Leal1,2, Danielle Queiroz

Calcagno3, Fernanda Wisnieski1, Leonardo Caires Santos1, Elizabeth Suchi Chen1,
Carolina Oliveira Gigek1, Paulo Pimentel Assumpo3, Larcio Gomes Loureno4,
Carlos Haruo Arasaki4, Smia Demachki3, Ricardo Artigiani5, Rommel Rodrguez
Burbano6, Marlia Cardoso Smith1.
1
Disciplina de Gentica, Departamento de Morfologia e Gentica, Universidade

Federal de So Paulo, So Paulo, SP, Brazil
2
Departamento de Ortopedia e Traumatologia, Universidade Federal de So Paulo,
So Paulo, SP, Brazil
3
Ncleo de Pesquisa em Oncologia, Hospital Universitrio Joo de Barros Barreto,
Universidade Federal do Par, Belm, PA, Brazil
4
Disciplina de Gastroenterologia Cirrgica, Departamento de Cirurgia, Universidade
Federal de So Paulo, So Paulo, SP, Brazil
5
Departamento de Patologia, Universidade Federal de So Paulo, So Paulo, SP,
Brazil
6
Instituto de Cincias Biolgicas, Universidade Federal do Par, Belm, PA, Brazil
Correspondence to: Ana Carolina Anauate Pereira. Universidade Federal de So
Paulo (UNIFESP), Disciplina de Gentica, Rua Botucatu, 740, Edifcio Leito da
Cunha - 1 andar, CEP 04023-900, So Paulo, Brazil. Phones: (11) 5576-4848 voip
2370; (11) 99815-1021. Email: anauatte@gmail.com.
Background: Gastric cancer is the third cause of death by cancer worldwide.
MicroRNA are small non-coding RNA involving in the regulation of oncogenes or
tumor suppressor genes expression. Although reference genes are frequently used for
reverse transcription-quantitative polymerase chain reaction normalization, the
suitable reference genes for miRNA expression normalization in gastric tissue
samples was not previously determined. Aim: Identify suitable reference genes for
miRNA expression normalization in gastric tissue samples. Methods: We evaluated
the suitability of nine reference genes (U6, RNU44, RNU48, let-7a-5p, miR-28-5p,
miR-101-3p, miR-140-3p, miR-152-3p and miR-374a-3p) by using 29 pairs of gastric
tumors (T) and their matched adjacent non-neoplastic gastric tissue samples (Na)
from patients with gastric cancer and 18 non-neoplastic gastric tissue samples from
patients without gastric cancer from bariatric surgery (Nb). The stability of the
candidate reference genes was determined by using the NormFinder, geNorm,
BestKeeper, DataAssist and RefFinder software packages. Results: The best single
reference gene was miR-140-3p and the best pair was miR-140-3p+miR-152-3p for
all groups of samples. The miR-140-3p+miR-152-3p+RNU48 was the best trio for
analysis involving the T+Na+Nb and Na+Nb samples, miR-140-3p+miR-1523p+miR-101-3p for T, Na and T+Na samples, and miR-140-3p+miR-152-3p+let-7a5p for T+Nb samples. Conclusion: The miR-140-3p+miR-152-3p pair should be used
as reference genes for miRNA expression analysis in neoplastic and non-neoplastic
samples of patients with and without gastric cancer.
This study was approved by the Ethics Committee of the Universidade Federal de
So Paulo (UNIFESP), Brazil (CEP #733.152).
Supported by CNPq, CAPES and FAPESP.

K3
EPIGENETIC IMPACT OF QUERCETIN IN THE APOPTOSIS PATHWAY
Marisa C. Alvarez de Prax 1 , Victor Masso 1 , Cristiane O. Torello 1
and Sara T. Olalla Saad 1.
1
Hematology and Hemotherapy Center, University of Campinas (Hemocentro/

Unicamp) Campinas , Sao Paulo, Brazil
Epigenetic changes play a crucial role in hematological malignancies, and the protein
network that regulates these mechanisms offers potential pharmacologic regulation.
Quercetin (Qu) is one of the main flavonoids present in vegetables, fruit and
propolis, and is believed to exert antitumoral effects through several mechanisms:
acting as antioxidant, as an apoptosis inducer, as an anti-inflammatory agent and as a
chromatin remodeler. In the present study, we evaluated the effect of Qu treatment
on DNA methylation, post translational histone modifications, mRNAs and
miRNAs expression levels of genes related to apoptosis pathway. This study was
performed in vivo in two human xenograft leukemia models (approved by the
CEUA/Unicamp, number 3450-1), and in vitro using two leukemia cells lines, P39
and U937. Among highly methylated genes in P39 cell line, Qu induced
demethylation of the pro-apoptotic BCL2L11, DAPK1, dose and time dependant.
Moreover, it was observed that cells treated with Qu (50M) for 48h showed an
increase of 3 - 6 folds of histone 3(H3ac) and 4 (H4ac) acetylation in the promoter
region of BCL2L11, BAX, BNIP3, BNIP3L and APAF1. Further, the mRNA
expression levels of these genes were significantly upregulated compared to non
treated cells and/or samples from xenografts models (p<0,05). In summary, these
data suggest that the induction of apoptosis by Qu is mediated in part, not only
through changes in the methylation status, but also by enrichment of H3ac and H4ac
in the promoter regions of genes related to this pathway. Both events might
contribute favoring transcription of the genes.
Funding support: CNPq, FAPESP
CEUA: Comisso de tica no Uso de Animais

L EXTRACELLULAR MATRIX
L1
L2
FIBRINOGEC ROLE OF IL-9 IN HEPATORENAL FIBROSIS

1
Nadjania Saraiva de Lira Silva (1) , Thaise Lara Teixeira (1), Aline Alves da Silva
(1), Marlus Alves dos Santos (1), Samuel Cota Teixeira (1), Bruna Cristina Borges
(1), Francyelle Borges de Moura (2), Patrcia de Castilho (1), Claudio Vieira da Silva
(1).
(1)Laboratrio de Trypanosomatdeos da Universidade Federal de Uberlndia,
Uberlndia, Brasil.
(2)Laboratrio de Histologia da Universidade Federal de Uberlndia, Uberlndia,
Brasil.
1
nadjaniasaraiva@gmail.com. Universidade Federal de Uberlndia. Av. Amazonas Umuarama, Uberlndia - MG, Brasil. Telefone: (34)92230357.
Background: Cirrhosis is a chronic liver disease that ends up damaging the kidneys
due to the interaction between them during metabolism of toxic substances.
Aims: To evaluate the effect of IL-9 in hepatorenal cytokine fibrosis induced during
inflammation carbon tetrachloride (CCL4). Methods: We used 24 female mice
C57BL / 6, there were 4 groups (CCl4, CCl4 / IL9; IL9 and no treatment). The mice
were treated subcutaneously with carbon tetrachloride (CCl4) at a ratio 1g /g of
animal body weight mixed with olive oil, alternating two and two days for 40 days,
and in the 42th it was the day of sacrifice. During the fibrosis induction period, the
animals were treated with 100g of cytokine IL-9, alternately five and five days until
40 days of treatment. In the end of the treatment, they were euthanized according to
the American Medical Association Veterinary and the Ethics Committee in Animal
Experimentation of the Federal University of Uberlandia (CEUA/UFU-144/15). The
histological sections were stained with Picro-Sirus for collagen analysis. Collagen
was measured from the electron microscope and polarization with the help of the
program Image J. Results: The group treated with IL-9 showed an increase of
fibrosis in both the kidney and in the liver compared to control, with collagen I and
III the most abundant. Conclusion: IL-9 is a cytokine with fibrinogen role in liver
and kidney, since it increases the deposition of collagen I and III that are actively
involved in tissue fibrosis.
GENE EXPRESSION OF SMALL LEUCINE-RICH PROTEOGLYCANS

(SLRPS) IN LACRIMAL GLAND OF FEMALE MICE WITH
HYPERPROLACTINEMIA INDUCED METOCLOPRAMIDE
Ariadne Leal Araujo Stavare (1,2), Carina Verna (2), Helena Bonciani Nader (4),
Manuel de Jesus Simes (1,3), Regina Clia Teixeira Gomes (1,3).
AFFILIATION(S):
(1) Histology and Structural Biology Division of the Department of Morphology and
Genetics, Universidade Federal de So Paulo, Brazil,
(2) Department of Ophthalmology, Universidade Federal de So Paulo, Brazil,
(3) Department of Gynecology, Universidade Federal de So Paulo, Brazil, and
(4) Molecular Biology Division of the Department of Biochemistry, Universidade
Federal de So Paulo, Brazil
BACKGROUND: The SLRPs, are bioactive components of the extracellular matrix
associated to the fibrillogenesis, and cell growth and apoptosis and tissue
remodeling, and may be indicative of alterations on the functioning of the lacrimal
glands. AIMS: This report aims to assess gene expression and immunolocalization of
small leucine-rich proteoglycans, SLRPs (class I: biglycan and decorin) and (class II:
lumican and fibromodulin). Methods: 10 female/groups: control group (Ctr): 0.2 mL
of saline (vehicle) and the experimental group (HPrl): 200 g/day of
metoclopramide, dissolved in vehicle. Intervention(s): induction of
hyperprolactinemia. METHODS: After 50 consecutive days of treatment, the
animals were euthanized and the blood samples were collected for hormone
measurements. The lacrimal glands were removed and processed for gene expression
by RT-qPCR. The results were subjected to statistical test (p <0.05). RESULTS:
Gene expression alteration of the small leucine-rich proteoglycans (SLRP). Serum
prolactin levels were higher in all the animals with metoclopramide, while the levels
of estradiol and progesterone were lower compared to control group.
CONCLUSION: Our data suggest that the state of hyperprolactinemia changed
differently the gene expression of the small leucine-rich proteoglycans (SLRPs). Fact
that could explain the changes in the amount of collagen in the lacrimal gland in
female mice with HPrl reported in a previous study conducted in our laboratory.
These data suggest impairment in functioning of the lacrimal gland by elevated
serum prolactin levels and decreased estrogen and progesterone.
Report No. 8458240915
FAPESP

L4
L3
GENE EXPRESSION OF SMALL LEUCINE-RICH PROTEOGLYCANS
(SLRPS) ON THE MURINE UTERUS NON PREGNANT AND PREGNANT
WITH HYPERPROLACTINEMIA INDUCED METOCLOPRAMIDE
OLIVE OIL PROMOTES WOUND HEALING OF PRESSURE ULCERS IN

MICE
Aline Donato-Trancoso (1), Andra Monte-Alto-Costa (1), Bruna Romana-Souza (1)
Regina Clia Teixeira Gomes (1,2), Ariadne Stavare (1,3), Carina Verna (3), Helena
Bonciani Nader (4), Manuel de Jesus Simes (1,2), Jos Maria Soares Jnior (5)
AFFILIATION(S):
(1) Histology and Structural Biology Division of the Department of Morphology and
Genetics, Universidade Federal de So Paulo, Brazil,
(2) Department of Gynecology, Universidade Federal de So Paulo, Brazil,
(3) Department of Ophthalmology, Universidade Federal de So Paulo, Brazil,
(4) Molecular Biology Division of the Department of Biochemistry, Universidade
Federal de So Paulo, Brazil, and
(5) Gynecology Division of the Department of Obstetrics and Gynecology, Hospital
das Clnicas, Faculdade de Medicina da Universidade de So Paulo, Brazil
BACKGROUND: The SLRPs are involved in collagen fibrogenesis and promote
angiogenesis signal, pro-apoptotic and suppressing cell growth, yet they are capable
of inducing the signaling cascade through tyrosine kinase receptor, Toll-like and
TGF- / BMP, which are important for the embryo implantation. AIMS: This report
aims to assess gene expression and immunolocalization of SLRPs, (class I: biglycan
and decorin) and (class II: lumican and fibromodulin). METHODS: 20
female/groups: control group (non pregnant Ctr): 0.2 mL of saline (vehicle) and the
experimental group (non pregnant HPrl): 200 g/day of metoclopramide, dissolved
in vehicle. After 50 days 10 females of each group were placed for mating with
males and continued to receive treatment. The females non pregnant were euthanasia
on 50th day and the females pregnant were euthanasia on 5.5th to 6.5th post-coital day.
The uterus was processed for immunohistochemistry and gene expression by RTqPCR. The results were subjected to statistical test (p <0.05). RESULTS: In non
pregnant, the gene expression showed increase of the fibromodulin, and decrease
decorin, lumican and biglycan showed in HPrl compared to Ctr. In pregnant, the
gene expression showed increase of the decorin/lumican, and decrease
biglycan/fibromodulin in HPrl compared to Ctr. CONCLUSION: Our data suggest
that the state of hyperprolactinemia changed differently the gene expression and the
immunolocalization of SLRPs in the extracellular matrix of the endometrium of
pregnant and non pregnant. That fact could lead to a failure in embryo implantation.
(1) Department of Histology and Embryology, State University of Rio de Janeiro,

Address: Avenida Marechal Rondon, 381, So Francisco Xavier, Rio de Janeiro, RJ,
Brasil. CEP: 20.950-003
Email: alinnedonato@gmail.com
Telephone: 55 21 2334-2421
Background Pressure ulcers has an impaired wound healing due overproduction of
reactive oxygen species (ROS) and exacerbated inflammatory response. Thus, the
administration of substances with anti-inflammatory and antioxidant proprieties, as
olive oil, may be a good therapeutic strategy for promoting cutaneous wound healing
of pressure ulcers. Aim: This study investigated the effect of olive oil administration
on wound healing of pressure ulcers in mice. Methods: Male Swiss mice were daily
treated with olive oil (1.5g/kg) or water until euthanasia. One day after the beginning
of treatment, two cycles of ischemia-reperfusion by external application of two
magnetic plates were performed in skin to induced pressure ulcer formation. Mice
were killed 3, 7 and 14 days after wounding. All animal experiments were approved
(CEUA-UERJ/044/2014). Results: The olive oil administration accelerates ROS and
nitric oxide production and reduced oxidative damage when compared to water
group. The inflammatory cell infiltration was reduced by olive oil administration
when compared to water group. The olive oil administration increased reepithelialization and blood vessel number when compared to water group. The
collagen deposition, myofibroblastic differentiation and wound contraction were
accelerated by olive oil administration when compared to water group. Conclusion:
Olive oil administration improves cutaneous wound healing pressure ulcers in mice
due the acceleration of ROS and nitric oxide, decreases oxidative damage and
inflammatory response and stimulates dermal reconstruction and wound closure.
Ethical approval: CEUA-UERJ/044/2014
Support by: FAPERJ, CNPq and CAPES.
Report No. 0809/04

FAPESP 2011/12417-8

L5
L6
THE INSTILLATION OF BIODIESEL/DIESEL PARTICLES INCREASES

THE INFLAMMATORY RESPONSE OF MICE LUNG IN AN ACUTE
MODEL
HISTOMORPHOMETRIC ANALYSIS OF THE INDUCED TOOTH

MOVEMENT IN OVARIECTOMIZED RATS. (RATTUS NORVEGICUS)
Isabella Cattani-Cavalieri (1,4), Giovanna Marcella Cavalcante Carvalho (2),

Manuella Lanzetti (3), Lus Cristvo Porto (4), Andra Monte-Alto-Costa (1),
Walter A. Zin (2), Samuel Santos Valena (3), Bruna Romana-Souza (1)
1- Department of Histology and Embryology, Rio de Janeiro State University, Rio de
Janeiro, Brazil.
2- Carlos Chagas Filho Institute of Biophysics, Universidade Federal do Rio de
Janeiro, Ilha do Fundo, Rio de Janeiro, RJ, Brazil.
3- Institute of Biomedice Sciences, Federal University of Rio de Janeiro, Rio de
Janeiro, RJ, Brazil.
4- Histocompatibility and Cryopreservation Laboratory, Rio de Janeiro State
University, Rio de Janeiro, Brazil.
Presenting author adress:
Av. Marechal Rondon, 381/HLA, 20950-003. Rio de Janeiro, RJ - BRAZIL.
Telephone: +55 21 2334 2421
E-mail address: isabella.cavalieri@hotmail.com.
Background: One of the main forms of air pollution in urban centers of Brazil comes
from diesel combustion from bus and truck engines. The diesel fuel in Brazil has a
mixture of biodiesel and diesel (BS) in order to reduce the air pollution. Aim: To
investigate the effect of BS particle exposure from fuel burning by public transport
of Rio de Janeiro City in mice lung. Methods: All animal experiments were approved
(CEUA n 01200.001568/2013-87). BS particles were collected from the exhaust
pipes of buses from the public transportation fleet of Rio de Janeiro City and
dissolved in saline/dimethyl sulfoxide. Female C57BL/6 mice were intranasally
instillated during five days with 250 g of BS particles; 1000 g of BS particles and
the control with vehicle. After five days all animals were euthanized and the lungs
removed. Results: The BS particles in lung tissue were increased in 250 g group
when compared to control and increased in 1000 g group when compared to control
and to 250 g groups. The macrophage number in tissue was elevated in 250 g and
1000 g groups when compared to control. The TNF- in bronchoalveolar lavage
was increased in 250 g and 1000 g groups when compared to control. BS particles
elevated the levels of ROS in bronchoalveolar lavage in 250 g and 1000 g groups
when compared to control group. Conclusion: The acute exposure to BS particles
was capable of penetrating pulmonary parenchyma, stimulating inflammation and
increasing ROS synthesis.
Joo Paulo do Nascimento Lima, Ewerton Zaniboni, Fernanda Aparecida Sampaio

Mendona, Glucia Maria Tech dos Santos, Marcelo Augusto Marretto Esquisatto,
Maria Esmria Corezola do Amaral, Milton Santamaria Junior.
Programa de Ps-graduao em Cincias Biomdicas, Centro Universitrio Hermnio
Ometto
UNIARARAS,
ARARAS/SP,
13607-339
E-mail:
marcelosquisatto@uniararas.br Fone: +55 (19) 3543-1440.
The early phase of orthodontic tooth movement involves an acute inflammatory
response that can be influenced by systemic factors. This study aims to assess
histomorphometry during the process of bone remodeling in induced tooth
movement (OTM) in the presence and absence of the hormone estrogen. Twenty
adult female Wistar rats were used. The animals were divided into 4 groups (n = 5)
with 7 and 14 days of induced tooth movement: Control group (OTM): Tooth
movement and Experimental Group (OTM + OV): Tooth movement in
ovariectomized animals. The maxillas were isolated, macroscopically analyzed and
prepared for structural analysis using hematoxylin and eosin, Picrossiriushematoxylin and Toluidine blue techniques. The number of fibroblasts, granulocytes
and osteoclasts in traction region of distobuccal root did not differ between the
groups. However, the OTM + OV group in 14th day there was a significant increase
in the number of fibroblasts and osteoclasts and significant decrease in granulocytes.
The number of vessels was similar between the groups in both study periods. The
area of birefringent collagen fibers from mesiobuccal region of the root showed
significantly higher values at 14th day in relation the 7th day in OTM + OV groups.
The results suggest that the absence of estrogen hormone accelerated the process of
bone reabsorption during tooth movement period analyzed.
Study approved by CEUA/UNIARARAS (Parecer no 041/2015) and supported by
FHO|Uniararas and PNPD/CAPES (Process no 23038.008192/2013-01).
Support by: FAPERJ and CNPq.

L7
L8
MATERNAL
LOW-PROTEIN
DIET
AFFECTS
STRUCTURAL
ORGANIZATION OF THE KNEE JOINT IN THE RAT FETUS (RATTUS
NORVEGICUS)
Renata Moreira Acunha, Ivens Takechi Nakagawa, Rosana Catisti, Marcelo Augusto
Marretto Esquisatto.
Ometto UNIARARAS, ARARAS/SP, 13607-339
STRUCTURAL ORGANIZATION OF THE TRACHEA

SUBMITTED TO GESTATIONAL PROTEIN RESTRICTION
OF
RATS
Jaqueline Gonalves, Hamilton Ricardo Alonso, Rosana Catisti, Marcelo Augusto

Marretto Esquisatto.
E-mail: hamiltonfarma@hotmail.com Fone: +55 (19) 3543-1440.
E-mail: ivens_jp@hotmail.com Fone: +55 (19) 3543-1440.

Maternal protein food restriction affects normal fetal development. The structures of
locomotor system of the offspring can be damaged. We investigated the effect of
gestational protein restriction on the cartilage organization of epiphyses of long
bones in rat fetus on day 21 of gestation. Pregnant Wistar rats (n=10) were fed a
normal-protein (NP, 17% casein) or low-protein (LP, 6% casein) diet. The knee
joints of the fetus were removed and processed for structural and morphometric
analysis by Mallory trichrome, Picrosirius-hematoxylin and Toluidine blue
techniques. The structural organization of the tibia (T) and femur (F) showed no
alterations between the groups. The basophilia of the F and T cartilages was
significantly higher in the NP, while the acidophilia of the cartilaginous matrix is
reduced in this group in relation to LP. No differences were observed in relation to
collagen fibers distribution in F and T in both groups. The number of chondrocytes is
similar in both groups for F and T cartilages. The area of birefringent collagen fibers
in F and T cartilages was not significant among them. However, the total thickness of
the F and T articular cartilages was significantly higher in the NP group. We
conclude that the offspring of LP dams presented modifications in the extracellular
matrix deposition and reduction on the articular cartilage dimensions in F and T.
Protein restriction is the most prevalent form of nutritional disorder among children
in developing countries. This study aims to describe the histomorphometric
modifications of trachea of the offspring of rats submitted to gestational protein
restriction (GPR) on day 21 of gestation. Ten pregnant Wistar rats were fed a
normal-protein (NP, 17% casein, n=5) or low-protein (LP, 6% casein, n=5) diet. The
trachea of the fetus were removed and processed for structural and morphometrical
analysis using Mallory trichrome, Picrosirius-hematoxylin, Orcein acetic and
Toluidine blue techniques. The structural organization of the trachea showed no
alterations between the groups. However, basophilia of the tracheal cartilage was
significantly higher in the NP, while the acidophilia of the cartilaginous matrix is
reduced in this group in relation to LP. No differences were observed in relation to
elastic fibers deposition. The total thickness of the trachea was significantly higher in
the NP. The same result was found for the thickness of tracheal cartilage. On the
other hand, the mucosal measurements indicated no differences among groups. The
number of chondrocytes was higher in NP. The area of birefringent collagen fibers in
tracheal cartilage was significant higher in LP. In conclusion, our study showed that
GPR can affect the development of tracheas of Wistar rat pups and it can affect your
functional properties in adulthood.
Study approved
FHO|Uniararas.
Study approved
FHO|Uniararas.
by
CEUA/UNIARARAS
(010/2010)
and
supported
by
by
CEUA/UNIARARAS
(010/2010)
and
supported
by

L9
L10
FIBRONECTIN INDUCES A DIFFERENTIAL REGULATION OF GENES

AND MICRORNAS EXPRESSION IN LNCAP CELLS CULTIVATED IN 2D
AND 3D CELL CULTURE
THE
EFFECT
OF
CAFFEIC
ACID
PHENETHYL
ADMINISTRATION ON PRESSURE ULCER IN MICE
Bruno Martinucci*1; Brenda C. Minatel1; Maira S. Cuciello1; Alexandre L. N. dos

Santos1; Ivan J. Vechetti Jr1; Flvia K. Delella1
ESTER
Luana Graziella Bandeira (1), Andra Monte-Alto-Costa (1), Bruna Romana-Souza

(1)
(1) Department of Histology and Embryology, State University of Rio de Janeiro,

Brazil.
*martinucci.bruno@gmail.com; (14) 3880 - 0498
Presenting author: Av. Marechal Rondom, 381, 2 andar, 20950-003 Rio de Janeiro,
RJ, Brazil. E-mail address: luana.bandeira@hotmail.com. Telephone: +55 21 23342421.
Departament of Morphology; Institute of Biosciences; UNESP; Botucatu; So

Paulo; Brazil.
Fibronectin is a glycoprotein that mediates a variety of cellular interactions with the

extracellular matrix and plays important roles in cell behavior. In prostate cancer
(PCa), the expression pattern of fibronectin is disrupted and significantly reduced,
suggesting a lack of organization of the matrix. Thus, the present study was conduct
to determine if the exposure to fibronectin could alter cellular events in PCa cells.
For these, LNCaP cells were exposed to fibronectin (25g/mL) in 2D and 3D culture
models and the expression of 8 genes and 7 microRNAs involved in important
cellular processes, such as invasion, cell death and proliferation were quantified by
RT-qPCR. In 2D culture, LNCaP cells exposed to fibronectin showed more
malignant phenotype, with increased expression of BCL2 (1.52 fold), CDH12 (1.60
fold), MMP2 (2.36 fold), AKT1 (1.40 fold), miR-21 (1.46 fold), miR-125b (2.07
fold) and miR-221 (2.08 fold); and lower levels of CDH1 (0.07 fold), TP53 (0.5
fold) and PTEN (0.47 fold) compared to non-exposed cells (p<0.05). In 3D culture,
fibronectin exposure caused a less aggressive phenotype, with increased expression
of CDH1 (1.31 fold), PTEN (3.5 fold) and miR-29b (1.6 fold); and lower levels of
CDH12 (0.16 fold) and STAT3 (0.54 fold) compared to control (p<0.05). Thus,
fibronectin promotes changes in gene and microRNA expression in PCa cells and we
suggest that these changes will depend on the cell culture type and
microenvironment, since in 2D culture, fibronectin exposure showed a tumorigenic
role, whereas in 3D environment fibronectin acted as a tumor suppressor.
Financial support: FAPESP 2014/25702-0
Key words: LNCaP; prostate cancer; fibronectin, extracellular matrix, microRNAs
Background: Pressure ulcers present a chronic inflammation and oxidative damage,

which lead to impaired wound healing. Therefore, the administration of antiinflammatory and antioxidant compounds, like caffeic acid phenethyl ester (CAPE),
may have a beneficial effect on healing of these lesions.
Aim: To investigate the effects of CAPE on healing of mice pressure ulcer through
ischemia/reperfusion model (IR).
Methods: Male mice (n=20) were submitted to two cycles of IR by external
application of two magnetic plates in skin to induce pressure ulcer formation. After
the last IR cycle, a group of animals (n=10) was intraperitoneally treated daily with
CAPE (5 mol/Kg) until euthanasia. Another group of animals was treated only
vehicle. Ulcers were collected 3, 7 or 12 days after ulceration. All experiments were
approved by the Ethical Committee for Animal Use of the State University of Rio de
Janeiro (CEUA/IBRAG/026/2014).
Results: CAPE administration accelerated the wound contraction when compared to
control group 3, 7 and 12 days after ulceration. There was no difference in the reepithelialization between CAPE and control groups 12 days after ulceration.
Nonetheless, the period to necrotic skin loss was lower in the CAPE group (12 days)
than in the control group (21 days default period to necrotic skin loss). After 12
days of ulceration, the CAPE group presented a few inflammatory cells and more
organized collagenous matrix when compared to control group.
Conclusion: The administration of CAPE may improve the cutaneous wound healing
of pressure ulcers in mice.
Ethical Approval: All experiments were approved by the Ethical Committee for
Animal Use of the State University of Rio de Janeiro (CEUA/IBRAG/026/2014).
The authors declare that there are no conflicts of interest.
L11

L12
SUBCELLULAR LOCALIZATION OF YES-ASSOCIATED PROTEIN (YAP)

CORRELATES TO RESISTANCE TO GROWTH-INHIBITORY SIGNALS
FROM THE BASEMENT MEMBRANE IN MALIGNANT BREAST CELLS
STRYCHNOS
PSEUDOQUINA
EXTRACT
MODULATES
THE
EXTRACELLULAR MATRIX AND ACCELERATES SKIN WOUND
HEALING IN DIABETIC RATS
Ana Paula Zen P Fiore1, Alexandre Bruni-Cardoso1.
Reggiani Vilela Gonalves (1), Fernanda Barbosa Lopes* (1), Rmulo Dias Novaes
(2), Joo Paulo Viana Leite (3), Mariurea Matias Sarandy (4)
Department of Biochemistry, Institute of Chemistry, University of So Paulo.
Extracellular matrix (ECM) chemical and mechanical signals reprogram gene

expression leading to quiescence and differentiation of mammary gland epithelial
cells. Mechanical signals are conveyed by the Hippo-YAP pathway, which controls
organ size through cell proliferation/apoptosis. Inhibition of Hippo kinases allows
YAP translocation to the cell nucleus, were it acts as transcription co-activator. We
aimed to investigate YAP responses to the basement membrane (BM), an important
ECM compartment, in breast epithelial cells. We used the S1 (nonmalignant) and T42 (malignant) cell lines from the HMT3522, a series that reproduces breast cancer
progression in laminin rich-gels. We assessed the gene enrichment of a YAP
signature (list of YAP-regulated genes) in the expression profile of T4-2 cells and
T4-2 cells phenotypically reverted by integrin-1 signaling blockage (T4-2-R). YAP
signature was overrepresented in T4-2 expression profile in comparison to the T4-2R profile. We also evaluated whether the activation of focal adhesion kinase (FAK)
and subcellular localization of YAP were modulated in nonmalignant and malignant
cells after BM treatment. In nonmalignant cells, FAK activation decreased 2h after
the treatment, an event followed by a reduction of YAP nuclear/cytoplasmic ratio. In
malignant cells subjected to same conditions, FAK remained active and YAP
localization did not change. Additionally, in the presence of BM, the level of YAP
bound to chromatin was drastically lower in nonmalignant cells than malignant cells.
Our data suggest that nonmalignant cells may respond to BM-growth inhibitory
signaling through FAK/Hippo-YAP and that this mechanism is disrupted in
malignant cells.
Funding: FAPESP, CNPq
(1) Department of Animal Biology, Federal University of Viosa MG, Brazil. (2)
MG, Brazil. (4) Department of General Biology, Federal University of Viosa, MG,
Brazil.
Phone: (+55) 31-3899-4375. fernanda.b.lopes@ufv.br
Natural products have traditionally played an important role in drug discovery and
were the basis of most early medicines. This study aimed at evaluating whether the
ointment Strychnos pseudoquina is effective for wound healing in diabetic rats.
Thirty male rats were randomized into five treatments: Salt (0.9% saline solution),
OV (ointment vehicle), SS (sulfadiazine of silver), LE 5 (S. pseudoquina 5%) and LE
10 (S. pseudoquina 10%). Three skin wounds of 12 mm in diameter were performed
and the extract ointment was applied daily for the period of 21 days, while tissue
from different wounds was removed every 7 days. Using x20 objective lens, 10
histological fields were randomly photographed, totalizing an area of 6.21x106 m2,
which was subjected to analysis. The volumetric density of the elastic fibers (Vv)
was calculated by counting the points that occurred over type I and type III collagen,
using the ratio of Vv = PP/PT, in which PP refers to the number of points occurring
over the structure of interest, and PT to the total number of points in the test system.
Results showed that LE 5% and LE 10% groups presented a greater proportion of
type III collagen fibers in F1 and F2, when compared to controls. Thus, it is
suggested that ointment S. pseudoquina extract can stimulate skin regeneration in
rats through the production of extracellular matrix mainly fibers collagen.
For the support to such project, we would like to acknowledge the FAPEMIG
(editalAPQ 00685-14).

L13
L14
COLLAGEN FIBERS TYPE I AND III LEVELS IN WOUND HEALING OF

DIABET RATS TREATED WITH "QUINA DO CERRADO"
EFFECT OF STRYCHNOS PSEUDOQUINA EXTRACT IN THE

PRODUCTION OF ELASTIC FIBERS DURING HEALING OF SKIN
WOUNDS IN DIABETIC RATS
Mariurea Matias Sarandy* (1), Rmulo Dias Novaes (2), Joo Paulo Viana Leite
(3), Reggiani Vilela Gonalves (4)
MG, Brazil. (4) Department of Animal Biology, Federal University of Viosa, MG,
Brazil.
*Phone: (+55) 31-3899-4375. mariaureasarandy@gmail.com
Wound repair is a process that involves cellular and extracellular mechanisms
divided into three phases: inflammation, proliferation and maturation. In the final
phase of repair the type III collagen is replaced by thicker and stronger type I
collagen. The aim of this study is to analyze the effects of fractions of "Quina do
Cerrado" (Strychnos pseudoquina) on the increase of collagen type I and type III in
skin scar of diabetic rats. Three circular wounds of 12 mm of diameter were made in
the dorse of each animal. The animals were divided into 5 groups: EF5 (Extract of
"Quina do Cerrado" 5%), EF10 (Extract of "Quina do Cerrado" 10%), Sal (0.9%
saline solution), OV (ointment vehicle) and SS (sulfadiazine of silver). Products
were applied daily on the wound site for 21 days. Fragments of the skin were
removed every 7 days, processed and stained with Sirius Red to observe the levels of
collagen fibers type I and III. The groups EF5, EF10 and SS presented more
collagenous type III on day 7 but on days 14 and 21 only EF5 and EF10 showed
increase of this fiber. EF10 presented a higher production of type I collagen on day 7
compared with the other groups and on days 14 and 21 EF5 and EF10 showed a large
number of collagen type I. The fractions of "Quina do Cerrado" showed a proper
synthesis of the collagen fibers type I and III which is required for the strength and
resistance to the tissue.
Financial support: We would like to thank the Fundao de Amparo Pesquisa do
Estado de Minas Gerais (FAPEMIG) for the financial support (edital PPM00868-15).
Gabriela Diniz Pinto Coelho* (1), Mariurea Matias Sarandy (2), Rmulo Dias
(1) Department of Medicine and Nursing, Federal University of Viosa, MG, Brazil.
Brazil.
Phone: (+55) 31-3899-4375. gabriela.pinto@ufv.br
Introduction: Strychnos pseudoquina it is a Brazilian native plant, used in popular
medicine for the treatment of various lesions (1). Objective: The present study
evaluated the effect of the extract of Strychnos pseudoquina in Skin Wound in
Diabetic Rats by analyzing the number of elastic fibers. Material and Methods:
Thirty male rats were randomized into 5 treatment groups with 6 animals: LE 5
(Lyophilized Extract of S. pseudoquina 5%); LE 10 (Lyophilized Extract of S.
pseudoquina 10%); Sal (0.9% saline solution); OV (ointment vehicle) and SS
(sulfadiazine of silver). Were realized three skin wounds of 12 mm of diameter and
the extracts were applied daily over 21 days. Tissues from different wounds were
removed every 7 days. Sections mounted on histology slides were stained with
Verhoeff's method for analysis of the elastic fibers. The slides were visualized, and
the images captured using a BX-60 light microscope, the elastic fibers were
estimated using the ratio Vv [elf] =Pp[elf]/ Pt, where Pp is the number of points
that hit the structure and Pt is the total test points. Results: The groups treated with
S. pseudoquina 5% (LE 5) and 10% (LE 10), showed greater proportion of elastic
fibers in F2 and F3, when compared to controls (Sal, OV and SS). Conclusion: We
conclude that Strychnos pseudoquina in concentrations of 5 and 10% proved to have
positive effects on the morphology of the scar tissue in diabetic rats.
References:
1- F. V. Santos, I. M. S. Colus, M. A. Silva, W. Vilegas, and E. A. Varanda,
Assessment of DNA damage by extracts and fractions of Strychnos pseudoquina, a
Brazilian medicinal plant with antiulcerogenic activity, Food and Chemical
Toxicology, vol. 44, no. 9, pp. 15851589, 2006.
Financial support: We would like to thank the FAPEMIG (editalAPQ 00685-14)

L16
L15
BRASSICA OLERACEA EXTRACT INDUCES COLLAGEN FIBERS
PROLIFERATION IN CUTANEOUS WOUNDS OF WISTAR RATS
Mariurea Matias Sarandy (1), Monica Maria Lopes do Carmo* (2), Rmulo Dias
EFFECT OF FRACTIONS OF "QUINA OF THE CERRADO" ON THE

EXTRACELLULAR MATRIX IN THE MORPHOLOGY OF WOUND
Reggiani Vilela Gonalves (1), Neila Augusta Alves da Silva* (2), Rmulo Dias
Novaes (3), Joo Paulo Viana Leite (4), Mariurea Matias Sarandy (2)
Department of Medicine and Nursing, Federal University of Viosa, MG, Brazil. (3)
Brazil
(1) Department of Animal Biology, Federal University of Viosa, MG, Brazil. (2)
Department of General Biology, Federal University of Viosa, MG, Brazil. (3)
Department of Biochemistry and Molecular Biology, Federal University of Viosa,
MG, Brazil.
Phone: (+55) 31-3899-4375. monica.maria@ufv.br
Phone: (+55) 31-3899-4375. neila.silva@ufv.br
The biochemical mechanisms of the wound healing process involve mainly

disturbances in collagen production, impaired cell migration and proliferation of
keratinocytes and fibroblasts resulting in delayed re-epithelialization (1). The aim of
this study was to analyze the effects of Brassica oleracea var.captata in the increase
of collagen fibers type I and type III in cutaneous wounds of Wistar rats. Five
circular wounds of 12 mm in diameter were made by surgical incision in the skin and
the treatments applied at the wound site daily for 20 days. The animals were divided
in four groups: Balsam (B. oleracea); ointment (B. oleracea); sunflower oil
(Helianthus annuus); control (0.9% saline). The samples were processed and
embedded in paraffin. Sections of 4 m were made and subsequently stained with
Sirius Red to observe the levels of collagen fibers type I and type III. Images were
captured with polarizing microscopy and overlaid with a grid containing 300
points.Collagen fibers (type I) appear in shades of bright colors ranging from red to
yellow and thin reticular fibers (collagen type III) appear bright green. The number
of type III collagen fibers increased in groups treated with B. oleracea on days 4, 8,
12, 16 and 20. The treatment with B. oleracea showed a greater number of type I
collagen fibers compared to the other groups on days 8, 16 and 20. The application
of B. oleracea (balsam and ointment) showed great efficiency in wound healing
resulting in a strong skin rich in collagen type I.
Natural products have traditionally played an important role in new drug discovery
and were the basis of several medicines. "Quina of the Cerrado is a native plant
from South America and belongs to the family of the Loganiaceae which has
approximately 200 species. Many of them are known for their potential medicinal
secondary metabolites. This study evaluated the rate of elastic fibers in the wound
healing of the diabetic rats undergoing treatment with fractions of "Quina of the
Cerrado". 30 Rats were maintained in cages with food and water ad libitum. Diabetes
was induced by intraperitoneal injection of streptozotocin (60mg/kg). Three skin
wounds (12mm diameter) were opened on the animals' dorsolateral. The rats were
randomized into 5 groups: EF5(Extract Fraction 5%), EF10(Extract Fraction 10%),
Salt(0.9% saline solution), OV(ointment vehicle) and SS(silver sulfadiazine). The
applications were held daily for 21 days, and tissues from different wounds were
removed every 7 days. Sections mounted on histology slides were stained with
Verhoeff's method for analysis of the elastic fibers. The slides were visualized, and
the images were captured using a BX-60 light microscope, connected with a digital
camera. The elastic fibers were estimated using the ratio Vv [elf] % =Pp[elf]/
Pt[elf]. EF10 group presented a proportion of significantly higher elastic fibers
mainly on day 21, comparing to other groups. Fractions of the "Quina of the
Cerrado" proved to have positive effects on the morphology of the scar tissue in
diabetic rats.
References:
C.H. Lee, S.H. Chang, W.J. Chen, K.C. Hung, Y.H. Lin, S.J. Liu, M.J. Hsieh, J.H.
Pang, J.H.Juang, Augmentation of diabetic wound healing and enhancement of
Collagen content using nanofibrousglucophage-loaded Collagen/PLGA scaffold
membranes,
J.
Colloid Interface Sci. 439 (2015) 8897.
(We would like to thank the FAPEMIG - editalAPQ00685-14, for financial support).
Financial support: We would like to thank the FAPEMIG for the financial support
(edital PPM00868-15).

L17
L18
ANALYSIS OF THE INFLUENCE OF PHYSICAL METHODS IN THE

DECELLULARIZATION PROCESS OF CANINE PLACENTAS
ROLE OF PROGESTERONE AND PROTEASE ADAMTS 1 ON MIGRATION

AND INVASION OF OVARIAN TUMOR CELLS
Talya de Moraes Coelho (1*), Carla Maria Figueiredo de Carvalho Miranda (2),
Luciano Cesar Pereira Campos Leonel (2), Maria Anglica Miglino (2), Sonja Ellen
Lobo (2,3)
Mara A. Lima 1 and Vanessa M. Freitas 1
(1) Metodista University of So Paulo, Brazil

(2) Department of Surgery, Sector of Anatomy, Faculty of Veterinary Medicine and
Animal Science, University of So Paulo, Brazil
(3) School of Medicine, University of So Paulo, Brazil
Contact details: Rua Alfredo Mendes da Silva, 480, Jardim Jussara, So Paulo, SP;
talya_9@hotmail.com; (11) 4327-6008, (11) 96634-5586.
Tissue bioengineering makes use of biomaterials that mimic the extracellular matrix,
associated or not with stem cells and/or bioactive molecules to promote tissue
regeneration. The placenta has rich extracellular matrix. Its usually discarded after
birthday and does not require an extra surgical procedure for harvesting; thus, it
represents an interesting source of extracellular matrix to be used as decellularized
biomaterial in tissue engineering applications. The objective of this study is to
analyze the influence of physical methods on the cell removal efficiency from the
extracellular matrix of canine placentas. Placentas were harvested, washed and stored
at different temperatures (i. e., RT, 4C, -20C and -80C) before the beginning of the
decellularization process. Furthermore, some samples were processed under
agitation, whereas others, in static conditions. The presence of cellular nuclei was
analyzed using histology (samples were stained with Hematoxylin and Eosin,
Masson's Trichrome and Picrosirius); and the amount of remaining DNA in the
decellularized matrices was measured through PicoGreen assay. A greater cellularity
and increased destruction of extracellular matrix were observed in the samples
processed without agitation, as opposed to those that were prepared under agitation,
at all temperatures. No qualitative differences were noticed related to the
temperatures of storage. In conclusion, the use of agitation as physical method
associated with detergents, favors the decellularization process whereas freezing
temperatures do not optimize it.
This work has approval by the Ethics Committee on Animal Use, under the number
4195280115, and was conducted with FAPESP support.

Background: Ovarian carcinoma is the leading cause of gynecological neoplastic
death, being associated with deregulation of sex hormones. The progression of cancer
depends not only on abilities acquired by cancer cells, but also the interaction between
cells and their microenvironment. The components of the extracellular matrix (ECM)
are involved in several aspects of tumor biology. ECM components are cleaved by
proteases during physiological and pathological processes; it is essential for the
migratory and invasive ability of cells. ADAMTS (adamalysin-thrombospondin) are
secreted proteases involved in collagen processing, cleavage of the proteoglycan
matrix and angiogenesis. Our previous results demonstrated that progesterone
increases gene and protein levels of ADAMTS-1 and reduces the migratory and
invasive ability of ovarian cells compared with the cells without treatment in 24 hours.
Aims: Investigate the mechanisms by which progesterone impairs invasion and
migration of ovarian cells and if ADAMTS-1 would be involved in this process
Methods: NIH-OVCAR-3, CHO and ES-2 were submitted to wound assay or
Transwell for 24 hours in the presence of 500 nM or 1 M of progesterone with or
without RU486 (progesterone receptor inhibitor) and were compared to untreated
samples. Results: Our preliminary results showed that 500 nM and 1 M progesterone
are capable of reducing the migratory ability of the studied lines in medium without
and enriched by SFB treated with dextran, and that both these hormone concentrations
elevate levels of ADAMTS-1 protease in the lysate and conditioned medium.
Conclusion: Progesterone reduces cell migration and increases ADAMTS-1 of ovarian
cells.
Information on ethical approval: Approved (CEP-ICB N 743/15).
Funding support: CNPq 142302/2015-5

L19
L20
PROTEASE ADAMTS-1 EFFECT ON GROWTH FACTORS ACTIVITY

WITHIN THE TUMOR MICROENVIRONMENT OF FIBROSARCOMA
MACROPHAGE CULTURED OVER SCAFFOLDS OF COLLAGEN I

PRODUCED LESS NITRIC OXIDE
Heydi Noriega Guerra1, Mrio Costa Cruz2, Priscilla Ramos Lara Ribeiro1, Vanessa
Morais Freitas1
Viviane Souza de Campos, Gabriel Rabello de Abreu Cabral, Renato Augusto

DaMatta, Fernando Costa e Silva Filho.
Laboratrio de Biologia Celular e Tecidual, Centro de Biocincias e Biotecnologia,

Universidade Estadual do Norte Fluminense Darcy Ribeiro. Campos dos Goytacazes,
RJ, Brazil.

2
Center of Facilities and Support Research, Biomedical Sciences Institute,
Author(s) information
Heydi Noriega Guerra: Tel. +551130917778 E-mail hnoriegag@gmail.com; Mrio
Costa Cruz: E-mail costacruzmc@gmail.com; Priscilla Ramos Lara Ribeiro: E-mail
pmrlara@usp.br; Vanessa Morais Freitas: E-mail vfreitas@usp.br
Fibrosarcoma is a malignant tumor with the presence of immature proliferating
fibroblast. The growth and malignancy of a tumor is dictated by the surrounding
microenvironment. The extracellular matrix is a reservoir of cell binding proteins and
growth factors that affect tumor cell behavior. ADAMTS-1 (a disintegrin and
metalloproteinase with thrombospondin motifs) is a secreted protease that modifies
the extracellular matrix during malignant progression. We aim to evaluate the role of
ADAMTS-1 within the tumor microenvironment of fibrosarcoma. Here we study the
effect of ADAMTS-1 overexpression on growth factors activity. First, we transfected
HT1080 cells to overexpress ADAMTS-1, puromycin resistance gene and
fluorescent protein mCherry (HT1080-MPA). Control cells do not overexpress
ADAMTS (HT1080-MPC). Bromodeoxyuridine (BrdU) incorporation assay showed
that HT1080-MPA cells decrease proliferation when treat with HGF (10ng/mL)
compared to HT1080-MPC. Moreover, we observed that HT1080-MPA cells treated
with HGF presented a decrease in cell migration with a mean velocity of 6.120
0.416 m/hour, whereas the velocity of the HT1080-MPC treated with HGF was
13.763 1.421 m/hour. Also, Western blot analysis showed that ADAMTS-1
decreased the phosphorylation of c-Met (HGF receptor) and ERK1/2 after 30
minutes of treatment. Our results suggest that ADAMTS-1 could be sequestering
HGF, because the activation of HGF-c-Met receptor decreased. Consequently, HGFsequestered for ADAMTS-1 prevents the effects of this growth factor on
fibrosarcoma cell migration and cell proliferation, probably by decreasing activation
of ERK1/2 signaling.
This study was approved by the Ethics Committee on Animal Use - CEUA (ICBUSP). Supported by The State of So Paulo Research Foundation (FAPESP grants
2012/24108-2).
*Address: Laboratrio de Biologia Celular e Tecidual, Centro de Biocincias e

Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro. Av.
Alberto Lamego, 2000 - Parque California, Campos dos Goitacazes - RJ, 28035-200,
RJ, Brazil
*E-mail: vvncampos@gmail.com
*Phone: +55 22 2739-7310 *Cell phone: +5521 995 497 502.
Much of what is known about macrophage biology is based in the two-dimensional
culture in stiff surfaces which drastically differs from the in vivo cellular environment
because the extracellular matrix is not present. Cellular interaction with extracellular
matrix is extremely important to maintain and guide the behavior of cells. Type I
collagen (COL I) is the major and most abundant protein of extracellular matrix,
therefore, we used as a base for the three-dimensional scaffolds to culture
macrophages. Macrophages were seeded over COL I scaffolds or glass coverslips and
classically activated. Morphology, inducible nitric oxide synthase (iNOS) expression
and nitric oxide (NO) production were evaluated after culture. Macrophages cultured
on coverslips or COL I showed great spreading or rounded shape, respectively. The
NO production was statistically higher when macrophages were cultured on coverslips
compared to the cultivation on COL I. The expression of iNOS was higher in
macrophages cultured on coverslips, confirming the NO production data. Rigid and
planar surfaces such as coverslips can be stressful for the macrophage inducing high
expression of iNOS and NO production.
Information on ethical approval: This work was approved by CEUA-UENF, Protocol
301.
Funding support: CNPq, FAPERJ, UENF and CAPES.


L21
L22
MATERNAL DIABETES DISTURBS NEPHROGENESIS AND INCREASE

DEPOSITION OF EXTRACELLULAR MATRIX MOLECULES IN RENAL
CORPUSCLES OF MICE FETUSES
THE OVEREXPRESSION OF PROTEIN AHNAK INCREASES THE

NUMBER OF EXTRACELLULAR VESICLES
Mychel Raony Paiva Teixeira Morais (1), Fernanda Angela Correia Barrence (1),
Vivian Marino Mazucato (2), Rodolfo Ribeiro Favaro (1), Sebastian San-Martin (3),
Telma Maria Tenorio Zorn (1)
(1) Department of Cell and Developmental Biology, Institute of Biomedical Sciences
- University of So Paulo, So Paulo, SP, Brazil.
(2) Department of Morphology, Faculty of Medicine - University of So Paulo,
Ribeiro
Preto,
SP,
Brazil.
(3) Centro de Investigacin en Biologa de la Reproduccin- Escuela de MedicinaUniversidad de Valparaiso, Valparaiso, Chile.
Presenting authors address: Department of Cell and Developmental Biology,
Institute of Biomedical Sciences - University of So Paulo, So Paulo, Brazil.
Contact: raony@usp.br. Fone: + 55 11 30917260, 94213-0846
BACKGROUND: Maternal diabetes disturbs the embryonic and fetal development
leading the offspring to cardiovascular and renal dysfunction in adulthood as a result
of fetal reprogramming. AIMS: To investigate whether maternal diabetes disturbs
nephrogenesis and alters renal ECM in mice fetuses. METHODS: The number and
the volume of differentiated and undifferentiated renal corpuscles was evaluated by
stereological techniques on kidneys of 19 days-mice fetuses from diabetic (FDM)
and nondiabetic mothers (FNDM). Renal basement membranes and mesangial ECM
were evaluated by periodic acid-Schiff and picrosirius staining respectively. The
deposition of type IV collagen and laminin were analyzed by immunohistochemistry
and Western blot. RESULTS: Maternal diabetes significantly reduced the number of
differentiated (39.80%) and undifferentiated (56.22%) renal corpuscles, and
promoted corpuscular hypertrophy in the FDM. It was observed a thickening of renal
basement membranes and an increased deposition of collagen in the mesangial ECM
in the FDM. Additionally, it was verified an increase of type IV collagen and laminin
in the thickened basement membranes of renal corpuscles and renal tubules of the
FDM. However, Western blot data indicate decreased levels of COL4A1, COL4A3,
LAMA1 and LAMA5 chains in total kidney extracts from the FDM compared to the
FNDM. CONCLUSION: Maternal type 1 diabetes: (i) induces renal
dysmorphogenesis by restricting the number of differentiating renal corpuscles, and
by promoting a compensatory hypertrophy of differentiated corpuscles; (ii) alters
basement membranes and the mesangial matrix of the fetal kidney structures.
Priscilla Ramos Lara Ribeiro1, Heydi Noriega Guerra1, Fernanda S. Giudice2 ,

Vanessa Morais Freitas1
1

2
A.C. Camargo Cancer Center
Author(s) information
Priscilla Ramos Lara Ribeiro: Tel +55 11 97062-4640. E-mail: pmrlara@usp.br;
Heydi Noriega Guerra: E-mail: hnoriegag@gmail.com; Fernanda S. Giudice: E-mail:
fgiudice@cipe.accamargo.org.br; Vanessa Morais Freitas: E-mail vfreitas@usp.br.
Tumor cell microenvironment is a fundamental factor for the success of the
progression of a tumor. It is composed of molecules and extracellular matrix
components and stromal cells that surround the cancer cells. The communication
between these two types of cells is realized, among some mechanisms, by means of
extracellular vesicles. Possibly, vesicles regulate the proliferation, invasive behavior
and angiogenic and metastatic processes in cancer. Preliminary studies of our group
have shown the presence of protein AHNAK in the extracellular vesicles derived from
breast tumor cells MDA-MB-231. The aim of this study was to check if
overexpression of AHNAK in the well-differentiated breast adenocarcinoma cell line
(MCF-7) would increase the number of extracellular vesicles. First of all, MCF-7 cells
were transfected to overexpress AHNAK through CRISPR activating technique
followed by antibiotics selection . Then Immunoblotting technique was performed to
confirm AHNAK overexpression in MCF-7 cells. Subsequently the number of vesicles
in the MCF-7 conditioned medium was determined by NanoSight instrument. Finally,
scanning microscopy analyzed presence of cellular protrusions and vesicular structures
on cell surface. Our preliminary results have shown an increase in the vesicles number
released by MCF-7 cells with AHNAK overexpressed to an average of 13.98 0.2598
compared to control cells (average of 8.694 0.4718). We also observed by scanning
electron microscopy, an increase of cellular protusions as well as an increase of
vesicles number. Further experiments will analyze the effects of this vesicle increase
on MCF-7 cell behavior.

Funding support: FAPESP #2015/03525-2; Approved by the Ethics Committee on

Animal Use-ICB, USP, #019/2015).
L23
L24
INTERACTION DisBa-01 WITH V3/ 51/fibronectin IN CONTEXT OF

CELL ADHESION
THE IMPORTANCE OF COLLAGEN III IN IMMUNOLOGICAL SYNAPSES
Bruna Carla Casali (1), Wanessa Fernanda Altei (1), Tais Marolato Danilucci (1),
Marcela Tsuboy (1,2), Kelli Cristina Micocci (3,1), Milene Nobrega de Oliveira
Moritz (1), Ricardo Jos Soares Torquato (4), Helosa Sobreiro Selistre-de-Araujo
(1)
(1)
(2)
(1) Laboratrio de Bioqumica e Biologia Molecular, Departamento de Cincias
Fisiolgicas, (2) Departamento de Gerontologia, Laboratrio de Biologia(3) e
Envelhecimento, (3) Departamento de Engenharia de Materiais, Universidade
Federal de So Carlos, So Carlos, SP, Brasil. (4) Laboratrio de Bioqumica e
Biologia Molecular de hematfagos, Universidade Federal de So Paulo, So Paulo,
Brasil.
E-mail: brunaccasali@yahoo.com.br
Integrins 51 and V3 are cell adhesion receptors that bind to proteins of
extracellular matrix (ECM) such as fibronectin and vitronectin, key mediators of cell
adhesion and migration, and a ligand of other ECM proteins. Since integrins are also
essential for tumor cell migration, integrin inhibitors are interesting compounds
aiming the development of new drugs for metastasis prevention. In this context,
disintegrins are integrin inhibitors from snake venoms. Most disintegrins has RGD
motifs that inhibit cell adhesion and migration, however this motif is considered
promiscuous for different integrins, which could lead to unspecific and undesired
effects. DisBa- 01 is a recombinant RGD disintegrin from Bothrops alternatus
venom that blocks V3 integrin with high specificity (KD 1.6 x 107 M); it inhibits
adhesion of HMEC-1 cells to vitronectin (IC50 500nM). Here we compared the
interaction of DisBa-01 (0.1 - 40M) with 51 and V3 by Surface Plasmon
Resonance (SPR) in real time experiments. DisBa-01 showed high affinity
interaction with V3 integrin (KD 4.63 x 107M) in SPR, however, this interaction
was less specific with 51 (KD 7.62 x105M), indicating specificity to V3 integrin.
Also, we developed an adhesion assay of fluorescent stained DisBa-01 (80 mg) to
immobilized fibronectin (10 g) in 96-well plates. Fluorescence analysis showed that
DisBa-01 bound to immobilized fibronectin, thus suggesting a direct interaction with
fibronectin. Our results suggest that, despite being more specific for V3 integrin,
DisBa -01 binds directly to fibronectin and this interaction could involve 51
integrin.
Support: Fapesp, CNPq and Capes
Karen Steponavicius Cruz (1,2), Alexandre Urban Borbely (2), Vanessa Morais
Freitas (3), Alexandre Marzago Barbuto (1)
(1) Department of immunology, Institute of Biomedical Sciences, University of Sao
(2) Cell Biology Laboratory, Institute of Health and Biological Sciences, Federal
(3) Department of Cell and Developmental Biology, Institute of Biomedical Sciences,
To whom correspondence should be addressed: Karen Steponavicius Cruz, Cell
Biology Laboratory, Institute of Biological and Health Sciences, Federal University of
Alagoas, Campus A.C. Simes. Av. Lourival Melo Mota, s/n, 57072-970. Maceio,
Alagoas, Brazil. Laboratory Phone: ++558232141704. Mobile: +5582981633303. Email: kstepon@hotmail.com
Background: Although studies in two dimensional co-cultures or with matrigel and
collagen I substrates provide useful information, collagen III is the main component of
lymph node paracortical region. Aims: It was aimed to determine if collagen-III coated
three-dimensional (3D) co-cultures of dendritic cells (DCs) in different maturation
periods with CD4+ and CD8+ lymphocytes influentiate in the immunological.
Methods: T lymphocytes were separated by immunomagnetic columns. DCs at
different stages of maturation and CD4+ lymphocytes and CD8+ lymphocytes were cocultured in the Biotek 3D environment and treated or not with collagen type III with
further confocal time-lapse microscopy analyzes. Results: DCs interacted with CD4+
lymphocytes only in collagen III. DCs interaction time with CD4+ lymphocytes
decreased with maturation and mature (mDCs) preferentially interacted with a single
lymphocyte. Regarding the interaction with CD8+, mDCs interacted repeatedly with
the same lymphocyte. Migration assays showed that collagen type III or the presence
of DCs in different stages of maturation could alter the migration speed of CD8+
lymphocytes. Migratory speed of CD8+ lymphocytes after the interaction decreases
and there is a relation with DCs maturation. Mature DCs and collagen III induced IL2. Conclusion: Collagen III and mDCs are the best choice when studying
immunological synapses. High mDC expression of CD209 and PD-L1 associated with
collagen III induction of IL-2 indicate a possible tolerogenic environment which
correlates in vivo, as a cytokine was used to differentiate DCs.
Funding Support: FAPESP (2010/18139-7) and CNPq (2009/54599-5). Ethical
Approval: Ethics Committee on Human Research of ICB-USP (078.2010).


L25
L26
EFFECT OF TOPICAL OINTMENT EXTRACT ETHANOLIC MAYTENUS

ILICIFOLIA LEAVES IN WOUND SKIN TREATMENT
IDENTIFICATION AND CHARACTERIZATION OF PROTEOLYTIC

ACTIVITY IN THE VENOM OF THE SOCIAL WASP POLYBIA PAULISTA
Francyelle Borges Rosa de Moura (1), Simone Ramos Deconte (1), Bruno Antnio
Ferreira (1), Andrea Aparecida de Aro (2), Rodney Alexandre Ferreira (2), Fernanda
de Assis Arajo (1), Tatiana Carla Tomiosso (1)
Luiz Fernando Patekoski Braga (1), Gabriel Ramos de Lima (1), Marcia Helena
Appel (1), Jesiane Stefania da Silva Batista (1), Jos Rosa Gomes (1), Marcia Regina
Paes de Oliveira (1), Rafael Bertoni da Silveira (1)
(1) Institute of Biomedical Sciences, Federal University of Uberlndia, Brazil

(2) Institute of Biology, Campinas State University, Brazil.
(1) Department of Structural Biology, Molecular Biology and Genetics, State

University of Ponta Grossa, Ponta Grossa, Brazil.
*francyelle-cesm@hotmail.com; Institute of Biomedical Sciences;

Avenue Par 1720 - Block 2B Room 2 B 221 (55) 34-3225-8582 (55) 34-991980154
(luizpatekoski@yahoo.com.br; +55 42 98434005)
This study evaluates the participation of the topical use of ethanol extract of
espinheira-santa (Maytenus ilicifolia) leaves healing of skin wounds. Four dorsal
incisions were made in back with a punch of 5 mm in 32 mice BALB C. The control
group (CO) was treated daily with topical vehicle solution (vaseline / lanolin) and
other 3 groups were treated with the ointment of M.ilicilofia in the concentrations of
2%, 4% and 6%. After 7 days, skin samples were removed and submitted for
histology, stained with hematoxylin and eosin staining, toluidine blue, and Gomori
trichrome picrosirius red. Were photographed 10 areas of each blade and collagen
quantified as total area with the program Image J. Statistical analysis was performed
using nonparametric ANOVA and Tukey post test p <0.05, comparing the treated
groups with the control. Granulation tissue is essential for the repair and includes an
increased number of blood vessels and the deposition of matrix components such as
collagen I and III, proteoglycans, and glycoproteins. ES 6% showed increased
collagen deposition when compared to the control.The proportion of collagen I and
III and mast cells showed no difference. Increased collagen synthesis could indicate
a better progression in repair steps, however, the effect of M. ilicifolia should be
evaluated further by biochemical assessments for a better understanding of its effect
on skin repair.
This study was approved by the Ethics Committee on the Use of Animals of the
Federal University of Uberlndia, under the protocol 158/13.
Financial support: CAPES
Polybia paulista is a common social wasp species at urban areas in Brazil and is
involved with hundreds of medical important accidents every year. The
Hymenopteran venoms consist of a complex mixture of low molecular mass toxins, a
series of polycationic peptides and proteins/enzymes such as phospholipases (A1 and
A2), antigen-5, hyaluronidase, major royal jelly proteins and acid phosphatases.
Despite the presence of proteases are reported in the venom of P. paulista by a
proteomics-based approach, the proteolytic activity in this venom is still poorly
understood. So, this study focuses on initial biochemical characterization of
proteases present in the venom of P. paulista. The venom of P. paulista was obtained
by dissecting the inoculation apparatus and extraction of venom from glands in PBS.
Zymogram experiments using different proteins as substrates and different venom
concentrations revealed proteolytic activity on gelatin, casein and BSA. Molecular
mass estimation resulted in an apparent electrophoretic mobility of two bands at
81kDa and 220kDa respectively. By using protease inhibitors in zymographies we
concluded that activities observed in electrophoresis are due to serine proteases.
Further analysis of proteolytic activity as a function of pH demonstrated a surprising
optimal range of activity at pH 6 to 10. Although these results are still preliminary
they represent the first description of proteolytic activity in the venom of P. paulista.
The continuity of this study will certainly contribute for more comprehension about
P. paulista envenomation and opens the possibility for biotechnological use of these
proteases.
Keywords: Polybia paulista; Venom; Proteases
Supported by: CAPES, CNPq and Fundao Araucria

L27
L28
PRODUCTION
AND
CHARACTERIZATION
OF
BIOACTIVE
BIOMATERIALS OBTAINED FROM DECELLULARIZED CANINE
PLACENTAS
Luciano Csar Pereira Campos Leonel (1), Carla Maria Figueiredo de Carvalho
Miranda (1), Talya de Moraes Coelho (2)*, Guilherme Ferreira (3), Rafael Rossi (3),
(1)
Maria Anglica Miglino (1), Sonja Ellen Lobo (1, 4).
(2)
(1) Department of Surgery, Sector of Anatomy, School of Veterinary
(3)
Medicine and Animal Science, University of So Paulo.
(2) Universidade Metodista de So Paulo, Brazil.
(3) Universidade So Judas Tadeu, So Paulo, Brazil.
(4) Department of Obstetrics and Gynecology, University of So Paulo
School of Medicine.
* Rua Alfredo Mendes da Silva, 480, Jardim Jussara, So Paulo/SP Brasil, CEP
05525-000. talya_9@hotmail.com
Biological biomaterials can be produced by decellularization, which aims at
removing cells from the extracellular matrix (ECM). Placentas are organs of great
interest for tissue engineering due to the fact that they are discarded after birth and
present large amount of ECM. Our study established a method for decellularization
of canine placentas, aiming at the production of a biomaterial for clinical
applications. After ethical approval from CEUA/FMVZ-USP (protocol number
4195280115), canine placentas were harvested and subjected to different protocols
that evaluated the concentration and time of incubation in detergents, as well as the
influence of temperatures and perfusion/immersion on the process. Tissue
transparence and absence of cellular nuclei, led to selection of two protocols. SDS
was the most effective detergent for cell removal and the freezing of placentas led to
larger periods of samples incubation in detergents. Perfusion/immersion methods
were capable of removing cells. Samples from protocol I (1% SDS, 5 mM EDTA +
50 mM TRIS + 0,5% antibiotic; 1% Triton X-100) better preserved the organization
of ECM when compared to protocol II (use of 0,05% trypsin instead of 50mM
TRIS). Protocol II optimized cell removal and decreased the DNA concentration;
both protocols, retained the laminin, fibronectin and collagen type I proteins.
Collagen type III was identified only in fetal portion. In conclusion, protocol II was
more effective in the decellularization of placentas. The ability of ECM
decellularized by such method to be applied in tissue engineering strategies still need
to be evaluated in vitro and in vivo.
ROLE OF HEPARANASE ON PROSTATE CANCER PROGRESSION

Valria Ferrer (1, 2); Jerahme Martinez (2); Brian Grindel (2); Brian Engel (2);
Daniel Harrington (2); Daniel Carson (2); Mary C. Farach-Carson (2)
(1) Laboratory of Biomedicine, Paulo Niemeyer Brain State Institute, Rio de Janeiro
RJ, Brazil
(2) BioSciences, Rice University, Houston, Texas TX, USA
Perlecan (HSPG2) is a key component of extracellular matrix (ECM). In the prostate
cancer (PC) tumor microenvironment, locally produced glycosidases modify ECM
molecules to assist tumor progression. Heparanases (HPSE) degrade HS chains
attached to HSPG2 core protein releasing stored heparin binding growth factors
(HBGF) into the tumor microenvironment to favor growth and angiogenesis. To
study the role of HPSE in PC cell behavior during invasion, we used gene editing
with CRISPR-Cas9 to generate a HPSE knockout C4-2 cell line. C4-2 cells belong to
LNCaP series, which is one of best progression models available for PC. C4-2 cells
represent the predominantly osteoblastic, poorly adherent and castrate-resistant PC
disease. Genome editing mediated by nucleases has efficiently modified genes in a
variety of cell types and the frameshift knockout mutations enable identification of
gene functions. HPSE knockout C4-2 cells released lower amounts of pre-bound
FGF2 compared to parental cells in vitro. Recombinantly produced perlecan domain
I decorated with HS served as substrate. Our data suggest a role of HPSE in
facilitating growth, angiogenesis and invasion of PC cells, supporting cancer
progression and facilitating metastasis.
Supported by: CNPq, NCI, PO1 grant number CA098912.

L29
L30
URBAN AIR POLLUTION IMPAIRS THE LPS-INDUCED LESION

RECOVERY AND REPAIR
CHARACTERIZATION OF THE p63+ AND AR+ SUBPOPULATIONS OF

RWPE-1 CELLS IN CULTURE UNDER DIFFERENT STIMULUS
Natlia de Souza Xavier Costa (1), Mariana Matera Veras (1,2), Gabriel Ribeiro
Jnior (1), Luciano Belotti (1), Adair Aparecida dos Santos Alemany (1), Douglas
Hidalgo Zati (1), Paulo Hilrio Nascimento Saldiva (1), Marisa Dolhnikoff (1), Luiz
Fernando Ferraz da Silva (1).
Jlio Csar de Oliveira Santana (1), Leonardo de Oliveira Mendes (2), Hernandes F.
Carvalho (3)
(1) Department of Pathology, School of Medicine, University of Sao Paulo, Sao

Paulo, Brazil;
(2) Department of Anatomy of Domestic and Wild Animals, School of Veterinary
Medicine and Animal Sciences, University of Sao Paulo, Sao Paulo, Brazil.
Sao Paulo. Dr. Arnaldo Avenue, 455, 1st floor - room1220, Cerqueira Csar, 01246903. Sao Paulo, SP, Brazil. phone: +5511 30618531 mobile: +5511 971533233.
E-mail address: nataliasxcosta@hotmail.com.
Background: Studies have shown association between particulate matter levels and
increased mortality and morbidity rates in urban population. Acute lung injury
(ALI)/acute respiratory distress syndrome is a complex disease characterized by
intense inflammatory response, alveolo-capillary barrier damage, hypoxemia and has
high mortality rates.
Aims: Evaluate if the LPS-induced injury is altered in an individual previously
exposed to the air pollution (AP).
Methods: ALI was induced by nebulized LPS (3mg/ml). BALB/C mice with or
without ALI were exposed either to particulate matter 2.5 (PM2.5) or filtered air for 5
weeks after the ALI induction. Lungs and BALF were collected. Total BALF cell
count were obtained. Immunohistological of IL-1B, IL-6 and IL-8, collagen and
elastic fibers content analysis were performed on lung tissues.
Results: The LPS+PM2.5 group remains in a persistent inflammatory condition with
increased leucocytes (p=0.006) in BALF and pro-inflammatory cytokines, such as
IL-1(p=0.0001), IL-6(p=0.007) and IL-8(p=0.021) increased in the lung tissue,
while the LPS group shows these parameters levels closer to the control group. Both
in the parenchyma (p=0.004) and in the airways (p=0.004) the collagen content
increased only in LPS group. The elastic fibers seems to undergo more the PM2.5
effect than the LPS, since only the PM2.5 exposed groups had shown elastosis in the
parenchyma (p=0.0001) and airways (p=0.0001).
Conclusion: The exposure to AP interferes on the adequate LPS-induced lesion
recovery and repair, once the AP, specially the fine particulate matter, has a
continuous pro-inflammatory role over the lesion.
(1) Department of Structural and Functional Biology, University of Campinas, So

Paulo, Brazil. juliosantanas@yahoo.com.br, Institutional phone (19) 3521-6125, Cell
phone (14) 98149-2212
(2) Postgraduate Program in Animal Sciences, University of Western So Paulo, So
Paulo, Brazil. leobio85@gmail.com
(3) Department of Structural and Functional Biology, University of Campinas, So
Paulo, Brazil. hern@unicamp.br
RWPE-1 cells are derived from normal prostate epithelium and present two
subpopulations, one expresses p63 and the other expresses androgen receptor (AR+).
We seek to understand how the transition between the phenotypes are regulated and
how the extracellular matrix interferes in this process. RWPE-1 cells were submitted
under following experimental conditions in vitro for 48h: control (medium only), 5%
soluble matrigel (laminin-rich extracellular matrix), 10nM DHT and 5% soluble
matrigel plus 10nM DHT. Cells were submitted to immunofluorescence, flow
cytometry, PCR and qRT-PCR analyses. We characterized three RWPE-1
subpopulations, p63-only, AR-only and p63/AR double-positives. p63-only are the
smallest cells, AR-only are intermediate and large cells, and p63/AR double-positive
showed no size pattern. Matrigel alone induced an increase in cytoplasmic AR
content and decreased the number of the p63+ cells. We determined the kinetic of
AR accumulation in the cell nucleus under DHT treatment, and found a peak
activation at about 8h. Twelve hours after stimulation, AR had been relocated to the
cytosol. In addition, we characterized the subcellular localization of p63 TA and
DeltaN isoforms. p63+ cells showed nuclear localization in all treatments and colocalization with TA, but some cells showed TA in cytoplasm without nuclear p63
labeling. Matrigel and Matrigel plus DHT decreased TA labeling. DN was found in
cytoplasm, and showed no difference in subcellular localization under the different
treatments. In conclusion, matrigel induced AR protein stabilization and decreased
p63 TA isoform, showing that extracellular matrix can modulate AR and P63
expression and phenotype transition in RWPE-1 cells.
Ethical Approval: CEUA #3775-1
Funding Support: FAPESP grants 2014/22225-7 (Postdoctoral Fellowship) and
2009/16150-6 (Financial Support).
Ethical approval: CEUA 177/10

Funding support: CNPQ and FAPESP.

L31
L32
SPATIO-TEMPORAL EXPRESSION PROFILE OF MMPS MODULATORS

RECK AND SPARC DURING THE RAT OVARIAN DYNAMICS
Levin G1 ; Coelho TM1, 2 ; Nbrega NG1 ; Trombetta-Lima M1, 2 ; Sogayar MC1, 2 ;
Carreira ACO1, 2
1
NUCEL/NETCEM (Cell and Molecular Therapy Center) , Internal Medicine

2
Biochemistry Department, Chemistry Institute, University of So Paulo, So Paulo,
Brazil
To
whom
correspondence
should
be
addressed:
Gabriel
Levin
(gabriel.levin2@gmail.com), NUCEL Universidade de So Paulo, Rua Pangar,
100, Cidade Universitria, So Paulo, 05360-130 SP, Brazil., Phone: +55 11 26480230 - Mobile Phone: +55 11 99906-6477
Background: Matrix Metalloproteinases (Mmps) and their Tissue Inhibitors (Timps)
are widely recognized as crucial factors for extracellular matrix (ECM) remodeling
in the ovary, allowing for follicular growth, ovulation, luteinization and luteolysis
during the estral cycle. Recently, several genes have been linked to the modulation of
Mmps activity, including Basigin (Bsg), which induces the expression of Mmps in
rat ovaries; Sparc, a TGF- modulator which is related to increased expression of
Mmps in cancer; and Reck, which is associated with Mmps inhibition.
Aims: To better characterize the expression pattern of Mmps network members in
ovary dinamics, we have determined the expression profiles of Mmps (2,9,13,14,19),
Timps (1,2,3), Sparc, Bsg and Reck during different phases of prepuberal rat
hormone-induced folliculogenesis by qRT-PCR and Immunohistochemistry.
Results: We observed that expression peaks of the Mmps inhibitors Reck and Timp2,
were closely followed by Mmp2 and Mmp9 suppression. The opposite was also true,
higher Mmp2 and Mmp9 expressions were concomitant to lower Reck and Timp2
expressions. Also, Mmp13 expression was upregulated upon hormonal treatment.
Immunohistochemistry for Mmps 2,9,14, Reck, Sparc, revealed that gellatinases and
Sparc expression patterns were the opposites to the one displayed by Reck during a
hormone-induced estral cycle protocol of prepubescent rats.
Conclusion: Therefore, here we show, a possible correlation between Reck, Sparc
and Mmps expressions in ovary ECM remodeling throughout an induced estral cycle
model.
ALTERATIONS PRODUCED BY X RADIATION AND MMP-2 IN THE

COMPONENTS OF PERIODONTAL LIGAMENT, DENTINE AND PULP
OF THE RAT INCISOR
Leticia Sartor (1), Amanda Fischborn(1), Nadia Fayes Omar(2), Jos Koehler (3);
Maria albertina de Miranda Soares(2), Jos Rosa Gomes (2).
This study aimed to detect the alterations produced by the X radiation on the
periodontal ligament (PL), dentine and pulp of the rat incisor. Twenty one male
Wistar rats (250g) of colony of UEPG were distribuited in the groups: control (3 rats)
and irradiated (18 rats). Before the application of 1537 miligrays of X radiation
(using a nuclear accelerator from the ISPON) on the head and neck regions, rats were
anesthetized (i.p.) with xilasine and ketamine (0,1ml and 50mg/100g). The control
and irradiated rats were died at 4th day by cervical dislocation after had been
anesthetized with halotane. Rats were also died at 9th,14th and 25th day. The
hemimandibles were colected, immersed in 2% paraformaldehyde and impregnated
into paraffine. Cross sections (5m) were taken on slides and stained with
hematoxilyn/eosin, Picrosirius red and immunhostichemistry for MMP-2. Images of
sections were taken in light and polarized microscopes. The results showed that the
MMP-2 expression increased until the 9th day and a progressive morphological
changes of the fibroblast associated to the cementum, in the dentine and pulp, as well
as in the organization of the collagen fiber I and III in the PL, were observed until the
25th day. We conclude that the first action to alter the morphology of incisor
structures was the X radiation, but that changes in the extracelular matrix were
reinforced also by MMP-2 along of the times, once that they were not reversible by
the 25th day.
CEUA-27/2011.
Information on ethical approval: Ethics Committee for Animal Use of the Chemistry
Institute, University of So Paulo.
Support: BNDES, CAPES, CNPq, FAPESP, FINEP, MCTI, MS-DECIT.


L33
L34
HGF POTENTIATE EXTRACELLULAR MATRIX-DRIVEN MIGRATION

OF HUMAN MYOBLASTS: INVOLVEMENT OF MAPK/ERK PATHWAYS
EVIDENCES OF FORMATION OF CARTILAGE CANALS AT THE

RELAXATION AND RECOVERY PROCESSES OF MOUSE PUBIC
SYMPHYSIS DURING LATE PREGNANCY AND POSTPARTUM
Mariela Natacha Gonzlez (1,2), Gillian Butler-Browne (2,3), Vincent Mouly (2,3),
Wilson Savino (1,2), Ingo Riederer (1,2).
1-Laboratory on Thymus Research, IOC/FIOCRUZ, Rio de Janeiro, Brazil.
2- Fiocruz/Inserm/UPMC/International Associated Laboratory on Cell Therapy and
Immunotherapy.
3- Center of Research in Myology, Pierre and Marie Curie University, Sorbonne
Universities, Paris, France.
e-mail: marnatch@yahoo.com.ar, telephone institutional: +55 (21) 2562 1223,
Mobile: +55 (21) 972568833.
Although myoblasts transplantation has been used to correct muscle injury, this
approach still has critical limitations, including the poor cell survival and limited
migration within the recipients muscle. Herein we aimed to determine whether
hepatocyte growth factor (HGF) was able to modulate human myoblast migration on
laminin and fibronectin (proteins of extracellular matrix) in vitro. We demonstrated
by immunoblotting, flow cytometry or immunofluorescence that human myoblasts
express the HGF receptor (c-met) and the integrin chains 6 and 7 (Laminin
receptors), and 5 (fibronectin receptor). In transwell migration assays, we observed
that myoblasts migrate toward laminin and fibronectin, and migration was
synergistically increased when HGF was added. However, HGF alone did not
enhance myoblast migration. Next, we investigated the MAPK/ERK pathways
activation by the myoblasts, which is induced by HGF. We observed an increased in
ERK phosphorylated in myoblasts treated with HGF, showing the MAPK/ERK
pathways activation, as revealed by immunoblotting. Moreover, treatment with
UO126 inhibitor, specific for the MAPK/ERK pathway, decreased the HGFassociated stimulus of cell migration triggered by fibronectin or laminin. Taken
together, these data demonstrate that mechanisms involved in the migration of
human myoblasts simultaneously comprise soluble and insoluble moieties. This
notion should be taken into account for a better design of therapeutic cell
transplantation strategies in order to improve the migration of donor cells within the
host tissue.
Castelucci.B.G1., Consonni.S.R2., Joazeiro.P.P1;

1
Department of Biochemistry and Tissue Biology, Institute of Biology, University of

Campinas, Campinas, Sao Paulo, Brazil.
2
Biology of the Cardiovascular System, Brazilian Biosciences National Laboratory,
Campinas, Sao Paulo, Brazil.
Contact information:
Bianca Gazieri Castelucci

e-mail: biancastelucci@gmail.com phone: 3521-6253(Institutional)/
(011)999390452 (Mobile)
During mouse pregnancy, pubic symphysis (PS) and other pelvic structures undergo
an extensive remodeling for safe delivery and postpartum recovery to maintain the
birth canal homeostasis. The remodeling processes are finely regulated by
macrophage activity and polarization that also ensure the postpartum restore of the
pelvic support. Beside the influx of macrophages during interpubic ligament (IpL)
formation and remodeling at pregnancy, the mechanism by which these phagocytes
invade the PS is unknown. TEM and SEM analyses showed narrow canals at 18 days
of pregnancy (D18) and 3 days postpartum (3dpp) IpL in C57BL6 mice. These
canals are delimitated by fibroblastic-like cells and macrophages that can express
VEGFR2 and directly interconnect the central lacunar structure in the IpL (known as
symphyseal space) to the pubic bone marrow at 3dpp. Immunofluorescent and
enzymatic assays showed that the inner symphyseal space extracellular matrix is
like-synovia containing versican, hyaluronic acid and great amounts of active
gelatinases (MMP2 and MMP9), molecules which can modulate macrophage
activity. These characteristics suggest that during pregnancy, formation of structures
similar to cartilage canals in PS may interconnect the symphyseal space and the IpL
to the pubic bone marrow. So, these cartilage canals may play an important role not
only on PS relaxation but also on the recovery process, allowing the entrance of
cells, the removal of debris and maybe interfering in macrophage activation.
Ethical approval: CEUA/UNICAMP n 3789-1
Funding support: CAPPES/CNPq.
Finantial support: FAPERJ, CAPES, CNPq and IOC-Fiocruz.
L35

L36
TYPE V COLLAGEN IMMUNIZATION INDUCED CUTANEOUS AND

PULMONARY FIBROSIS IN C57BL/6 MICE
UNUSUAL TYPE V COLLAGEN GENES AND ISOFORMS CHAINS IN

SYSTEMIC SCLEROSISRELATED PULMONARY FIBROSIS
Las Araujo (1), Ana Paula P Velosa (1), Antonio dos Santos Filho (2), Sandra
Morais Fernezlian (3), Esmeralda M Ehr (3), Srgio Catanozi (4), Vera Luiza
Capelozzi (3), Walcy R Teodoro (1).
Begalli I (1), Velosa AP (1), Martin P (1), Carrasco S (1), AbSaber, A (2), Parra ER
(2), Capelozzi VL (2), Teodoro W (1).
(1) Rheumatology Division, Medical School from University of Sao Paulo, Sao
Paulo, SP
(2) Center Bioterial, Medical School from University of Sao Paulo, Sao Paulo, SP
(3) Pathology Department, Medical School from University of Sao Paulo, Sao Paulo,
SP
(4) Lipids Division, Medical School from University of Sao Paulo, Sao Paulo, SP
Address: Avenida Dr. Arnaldo, 455 Pacaemb So Paulo, SP; CEP: 01246-903;
e-mail: labibi_araujo@hotmail.com; Phone: (11) 3061-7211; Cellphone: (11) 9
6404-1557
Background: We discovered an experimental model of systemic sclerosis (SSc) by
immunizing healthy New Zealand rabbits with human collagen V (COLV) that
resulted in intense inflammation of the lung and skin, and progressive ECM
remodeling of the septal and bronchovascular axis.
Objective: In this study we intend evaluate the histopathological features of
pulmonary and cutaneous remodeling in C57BL/6 mice immunized by COLV.
Methods: Female C57BL/6 mice (n=10) with 7 weeks old were twice immunized
subcutaneously with an emulsion of COLV (150g) and complete Freud's adjuvant
(FA) in a thirty days interval, followed by two intramuscular boosters of COLV and
incomplete FA (IM group). The controls (n=10) were only immunized with FA in
the same way (CT group). After 75 days from the first immunization, pulmonary and
cutaneous
remodeling
was
evaluated
by
histomorphometry
and
immunohistochemistry. Ethic Comission in the Use of Animals (ECUA) 170/15.
Results: Lung from immunized mice presented a significant higher amount of type I
collagen (p<0.005), lymphocytes CD4 (p=0.002), TGF- (p<0.0025) and CTGF
(p<0.0001) when compared to control. In addition IM presented significantly
cutaneous inflammatory process (p<0.0068) and predominance of fine fibers
compared the thick collagen identified by assessment of picrosirius respectively
(p<0.0001).
Conclusions: We demonstrate that all typical pathologic manifestations of SScrelated pulmonary and cutaneous remodeling are mimicked in COL V immunized
mice, thus emerging as an appropriate preclinical model to study the mechanisms
and therapeutic approaches of lung and skin involvement in SSc.
Financial support: FAPESP, Federico Foundation.
(1) Rheumatology Division, Medical School from University of So Paulo, So

Paulo, SP
(2) Department of Pathology, Medical School from University of So Paulo, So
Paulo, SP
Address: Avenida Dr. Arnaldo, 455 Pacaemb So Paulo, SP; CEP: 01246-903;
e-mail: isa_begalli@hotmail.com; Phone: (11) 3061-7211; Cellphone: (11) 9 85619762.
Background: Type V collagen (COLV) can be a possible trigger involved in the
pathogenesis in early stages of systemic sclerosis (SSc), since an unusual
accumulation of this collagen was demonstrated in this disease. Moreover
immunogenic properties, histoarchitecture differentiated and changes in COLV
mRNA expression may suggest that such modifications, assigned to this protein may
be correlated with SSc pathogenesis.
AIMS: This study aimed to analyze morphological and molecular features of 1(V)
and 2(V) chains and the role of COL5A1, COL5A2 gene expression in
fibrillogenesis of patients with early SSc-related pulmonary fibrosis.
METHODS: Pulmonary biopsies were obtained from 7 patients with SSc-related
fibrosis and 7 healthy controls. Morphometry, immunofluorescence and qPCR were
performed to characterize microscopic changes of 1(V) and 2(V) chains and
COL5A1, COL5A2 RNA expression. Ethics Committee for Research Projects
Analysis (CAPPesq) N 0905/06.
RESULTS: The COL5A2 immunostaining showed distorted and strongly thickened
fibers in lung with irregular bundles distributed in parallel and perpendicular
arrangements. These distributions result in a dense network around the vessels in SSc
patients. In contrast COL5A1 expression was absent in lung interstitial
compartments compared with respective controls. Histomorphometric analysis of
SSc lung demonstrated COL5A2 increased expression in the bronchovascular
interstitium, compared to control (p< 0.001). The qPCR revealed increased COL5A1
and corroborate with our morphological and immunofluorescence findings in this
disease.
CONCLUSION: These results highlight major pathogenic pathways relevant to lung
in SSc-related fibrosis suggesting that a post-translational modification of 1(V)
chain and up-regulated expression of COL5A1-regulated gene can interfere with the
normal extracellular matrix lung parenchyma. Further studies on the inhibition of
1(V) chain are necessary to test if gene therapy can attenuate the effects of this
disease.
Financial support: FAPESP, Federico Foundation.


L37
L38
EXTRACELLULAR MATRIX REMODELLING BY CELLULAR STRETCH

1
Mariana Machado Leiva Ferreira , Maria Eduarda Perrud Sousa ,Thatiane Amaral
Russo1and Juliana Luporini Dreyfuss1,2
1
Disciplina de Biologia Molecular, Departamento de Bioqumica, 2Departamento de

Informtica em Sade, Grupo Interdisciplinar de Cincias Exatas em Sade,
Universidade Federal de So Paulo, So Paulo, Brazil
Address: Rua Pedro de Toledo,669,04039-901So Paulo, SP, Brazil.Phone numbers:
+55
1155764438
(1959)
/
+55
11
9975337220.
E-mail:
marianaleiva_mlf@hotmail.com.
Background: Vascular smooth muscle cells (VSMC) compose the medium layer of
vessel walls, where are subjected to cyclical mechanical deformation due to blood
stream which leads to vessel stretch and VSMC phenotype modulation. Aims:
Investigate the effects of mechanical strain on extracellular matrix synthesis and
remodeling by aortic smooth muscle cells. Methods: VSMC cells were plated on
type 1 collagen coated silicone plates and subjected to 5% and 15% of elongation
(stretch) using a frequency of 1 Hz during 4 hours or 24 hours; a static control group
subjected to no strain. After the stretch exposure several assays were performed, such
as MTT assay, metabolic labeling of glycosaminoglycans with [35S]-sulfate and
qRT-PCR to analyse gene expression of ECM molecules and growth factors.
Results: Results show that mechanical strain increased glycosaminoglycans synthesis
and cellular viability.qRT-PCR results showed an increased expression of TGF1,
TGF3, Biglycan and Glypcan; it was observed reduced expression of Connexin 43,
Syndecan4, FAK, type 3 Collagen and Fibronectin after 24 hours of exposure.
Conclusion: Mechanical stretch was able to alter VSMCs gene expression pattern
and secretion of extracellular matrix molecules, stimulating ECM remodeling and
turnover.
Financial Support: CAPES, FAPESP, CNPq.
EFFECTS
OF
MECHANICAL
FORCES
IN
REGULATING
EXTRACELLULAR
MATRIX
SYNTHESIS
BY
CULTURED
ENDOTHELIAL CELLS AND ARTERIES IN ATHEROSCLEROTIC MICE
Russo, T.A.1; Nader, H.B.1; Dreyfuss, J.L.1,2
1
Department of Biochemistry, Molecular Biology Division, Carl Peter von Dietrich

Laboratory;
2
Department of Health Informatics, Interdisciplinary Group for Science in Health
Technology, Escola Paulista de Medicina, Universidade Federal de So Paulo.
Background: Endothelial cells (ECs) respond to hemodynamic mechanical forces
such as shear stress and stretching. Under flow conditions ECs migration and
adhesion are affected and extracellular matrix (ECM) components play a role on
these events.
Aims: Investigate the expression of ECM molecules in ECs subjected to different
mechanical stimuli, evaluated on physiological and pathological conditions, and
drawing a parallel with animal model for better understanding.
Methods: ECs were submitted to shear stress using StreamerTM system. In addition,
ECs were exposed to stretch in a vacuum system FlexCellTM. Afterwards, ECs were
subjected to immunofluorescence (IF), qPCR and cell behavior assays. Ultrasound,
qPCR and IF were performed in C57BL/6 carotid arteries from apoE knocoukt
mices.
Results: Both mechanical forces induced changes in ECs morphology. Adhesion,
migration and capillary like formation where modulated positively under
physiological conditions and negatively in pathological situations. Mechanical forces
induced a higher expression in pathological conditions of the proteoglycans:
syndecan-4, perlecan, versican and decorin; adhesive proteins: as fibronectin,
collagen typeIII1, connexin-43 and growth factors: VEGFA, TGF-1 and 3.
Ultrasound measurements in carotid from apoE -/- showed an increase in the
thickness and diameter of the artery characterizing vessel dysfunction. The qPCR
and IF of arteries showed similar results of ECs.
Conclusion: The exposure of ECs to mechanical forces influences the remodeling of
the ECM and interaction of cell-matrix. These studies help to better understand how
the vascular biology is affected by mechanical forces, and how these molecules
behave in cardiovascular disease.
Financial Support: CAPES, CNPq and FAPESP.

Key Words: Mechanotransduction, endothelial cells, proteoglycans.
L39

L40
HIGH GLUCOSE INHIBITS MIGRATION AND AFFECTS THE

ORGANIZATION
OF
HUMAN
FIBROBLASTS-DERIVED
EXTRACELLULAR MATRIX
EVALUATION
CALCANEAL
TENDON
ANIMAL
SUBMITTED
DOXORRUBICIN AND TREATED WITH EXERCISES AND THYROID
HORMONE (LEVOTHYROXINE)
Gabriella Malheiros Moraes, Cilene Rebouas de Lima, Marinilce F. Santos.
Francyelle Borges Rosa de Moura 1, Jeranice Silva Barbosa1, Glenda Fernanda

Ambhl de Castro1, Fernanda Rodrigues de Souza 2 , Erica Carolina Campos Pulici 3,
Elmiro Santos Resende2, Tatiana Carla Tomiosso1

University of Sao Paulo-USP. Sao Paulo/SP - Brazil.
Background:We have previously demonstrated that fibroblasts exposed to high

glucose (HG) show impaired migration on two-dimensional substrates. The threedimensional extracellular matrix (ECM) produced by fibroblasts under HG and its
effects on migration is, however, unknown. Aim:to evaluate the effects of HG on
migration, growth, viability and the production of ECM by human foreskin
fibroblasts (HFF), as well as the effects of this cell-derived matrix (CDM) on cell
migration. Methods:HFFs were cultured under low glucose (LG, 5 mM) or HG (25
mM) for 5 or 22 days. CDM was obtained over 7 days. After decellularization, CDM
from LG and HG cells were employed for migration assays using cells cultured
under LG or HG. Time-lapse and transwell assays were used for migration analysis.
Results:After exposure to HG for 7 days, cell growth and viability were not affected,
while migration was reduced by 75%. CDM showed oriented bundles of collagen
fibers, and cells migrated preferentially overthem, which determined their
directionality. Cells exposed to HG for 5 days produced a loose CDM, compared to
cells under LG. The velocity of LG cells was about 40% higher than the other cells
on this CDM, suggesting promigratory properties. The CDM produced by cells after
22 days of HG was similar to CDM from LG cells, and there was no difference on
migration between the groups. Conclusion:HG inhibits migration and affects ECM
production and arrangement. A three-dimensional CDM stimulates migration
through composition and topography, partially compensating this effect.
Funding:FAPESP, CNPq.
1
2
Gabriella.moraes@usp.br
Institute of Biomedical Sciences (ICBIM), Federal University of Uberlndia, Brazil

Medical School (FAMED), Federal University of Uberlndia, Brazil
Physical education College, Federal University of Uberlndia, Brazil
*francyelle-cesm@hotmail.com; Institute of Biomedical Sciences;

Avenue Par 1720 Block 2B Room 2 B 221 (55) 34-3225-8582
99198-0154
(55) 34-
Doxorubicin is an antineoplastic drug that has several side activities. Achilles

tendinitis is considered a side effect and can be caused by drugs such as norfloxacin,
however there arent until then reports the effect of doxorubicin in this tissue. The
objective of this study was to evaluate the effect of doxorubicin (DX) administered
intraperitoneally (7.5 mg / kg) in the calcaneal tendon, and the effect of the following
treatments: swimming exercise with corresponding additional weight to 5% of body
weight (EX), levotiroxano hormone (TH) 10 g / 100g, DX associated with exercise
(DXEX) and associated with exercise and hormone ( DXHTEX). Statistical analysis
was evaluated by non-parametric ANOVA, Kruskal-Wallis and Dunn with p <0.05.
Histological sections were stained with hematoxylin and eosin and Sirius Red and
quantification of total protein assessed with Bradford. (EX) showed a greater number
of fibroblasts compared to (DX). Quantification of total collagen was not different
among groups, however there is a greater disturbance in the deposition of collagen
fibers in group (DX) that do not prevail in the treated groups. The hormone
associated with exercise (DXHTEX) was able to reverse type I collagen levels in
animals (DX), similar to the control group levels (CO). Exercise helps in
strengthening the calcaneal tendon structure. While the estimated drug can interfere
with the arrangement of collagen fibers may have a negative influence,
compromising the strength of this tissue.
This experiment was conducted after approval by the ethics committee CEUA- with
the protocol No. 022/14
Financial support: CAPES
(1)
(2)
(3)
(4)
L41
L42
FAK RESPONSE TO ALTERNAGIN-C IN TUMOR AND NO-TUMOR

BREAST CELLS
HEPARANASE EXPRESSION AT PHYSIOLOGICAL ABSENCE OF

ESTROGEN
Milene Nbrega de Oliveira Moritz (1); Karoline Medeiros Musquiari (2); Kelli
Cristina Micocci (3); Patty Karina dos Santos (1); Heloisa Sobreiro Selistre de
Araujo (2)
(1)
(1) Graduate Program in Evolutionary Genetics and Molecular Biology, Federal
University of So Carlos UFSCar, So Carlos,SP, Brazil.
(2)
(2) Department of Physiological Sciences, Federal University of So Carlos
UFSCar, So Carlos,SP, Brazil.
(3)
(3) Department of Materials Engineering, Federal University of So Carlos
UFSCar, So Carlos,SP, Brazil.
Juliana Mora Veridiano (1), Thrse Rachell Theodoro (1), Giuliana Petri (1), Maria
Aparecida da Silva Pinhal (1, 2), Olga Maria de Toledo Correa (1).
(1) Department of Morphology and Physiology, ABC School of Medicine, FMABC,
Santo Andr, Brazil.
(2) Department of Molecular Biology, Federal University of So Paulo, So Paulo,
Brazil.
milenemoritz@gmail.com; (16) 33518333; (16) 982237468
Av. Prncipe de Gales, 821 Prncipe de Gales, Santo Andr, SP 09060-650 (Brazil).
Tel: +55 11 4993-5475.
E-mail: olmatoledo@uol.com.br (+55) 11 98591-5198
juliana.mora@gmail.com- (+55) 11 99220-0850
Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase involved in integrin

signaling. It is associated with cell anchorage, motility and invasion, playing a key
role in metastasis. Previous studies described alternagin-C (ALT-C), an ECDdisintegrin purified from Bothrops alternatus venom, as a cell adhesion inhibitor via
21 integrin. However, the mechanisms involved in this inhibition are not fully
understood. Thus, this study aims to evaluate the adhesion effect of ALT-C in breast
tumor cells (MDA-MB-231) and non-tumoral breast cells (MCF10A), comparing its
effect on FAK response. For this purpose, adhesion and haptotactic migration assays
to type I collagen (Col I) in the presence of ALT-C (10, 100 and 1000 nM) were
carried out. In the adhesion assay, FAK protein was detected using anti-FAK
antibody and Goat Anti-Rabbit Allophycocyanin, according to the manufacturer's
instructions. For statistical analysis it was used ANOVA followed by Tukey. The
integrins content was determinate by flow cytometry and a relevant amount of 2 e 1
was confirmed for both cell lines. Only the highest concentration (1000 nM) of ALTC inhibited adhesion of tumor cells to collagen I, however in haptotactic migration
assay this effect was not observed. ALT-C increased signaling of immunostaining
FAK at 100 nM and 1000 nM in MDA-MB-231cells and only at highest
concentration (1000 nM) in MCF10A cells. Results indicate that ALT-C binds to the
collagen
receptor
(21),
and
competitively inhibits cell adhesion to collagen. However, this binding
probably activates, rather than blocks, the integrin since it enhances
FAK.
Purpose: Heparanase (HPSE) cleaves heparan sulfate (HS) side chains of

proteoglycans while the metalloproteinase-9 (MMP9) mediates shedding of syndecan
exposing the core protein of syndecan. The HPSE over expression is modulated by
estrogen. We investigated the expression levels of HPSE and MMP-9 and correlated
the expression of such molecules with the profile of sulfated glycosaminoglycans
(GAGs) in the skin of rats at different ages and periods after oophorectomy.
Methods: Skin samples from 60 days old adult females Wistar rats were randomly
distributed into four groups: 45th; 60th; 75th and 90th days after oophorectomy (OOF).
Control samples were obtained at the same period of the OOF groups. The samples
were submitted to agarose gel electrophoresis to identify and quantify GAGs. RTPCR analysis determined the mRNA expression of HPSE and MMP-9. Committee of
Ethics in Research of FMABC number 06/11.
Results: The GAGs were observed in all groups analyzed, except HS. A significant
decrease of GAGs (p<0.005) occurred in all groups (OOF and control). We observed
HS only at 90 days group (OOF and control). Furthermore, a significant
overexpression of HPSE (p=0.007) and MMP9 (p=0.007) occurred in all OOF and
the corresponding control.
Conclusion: OOF and aging promoted a decrease in the skin GAGs. This decrease
followed the rise of age and OOF period. The late appearance of HS (90 days
groups) could be related with the effect of physiological levels of estrogen in RNAm
expression of HPSE. Thus, the absence and\or decrease of estrogen with aging
promoted the increase of HPSE.
Support: FAPESP and CNPq

M GENE AND CELL THERAPY
M1
M2
MACROPHAGE-COLONY STIMULATING FACTOR GENE THERAPY

FOR LIMB ISCHEMIA IN A MOUSE MODEL
PHYSIOLOGICAL EFFECTS OF GM-CSF GENE THERAPY IN MURINE

ISCHEMIC LIMBS
Camila Congentino Gallo; Vvian Yochiko Samoto; Brbara Sampaio Dias Martins
Mansano; Gabriel Leonel Marasco; Sang Won Han.
Brbara Sampaio Dias Martins Mansano, Vvian Yochiko Samoto, Gabriel Leonel
Marasco, Camila Congentino Gallo, Sang Won Han
Research Center for Gene Therapy, Department of Biophysics, Universidade Federal

de So Paulo, So Paulo, Brazil.
Department of Biophysics, Federal University of So Paulo, So Paulo, Brazil.
Camila.congentino@gmail.com; Rua Mirassol, 207, 04044-010, So Paulo, So

Paulo, Brazil. Phones: (+5511) 98415.4125 (mobile); (+5511) 5084.7582
(institutional)
Rua Mirassol 207, Vila Clementino, So Paulo, Brazil, CEP 04044-010;

barbarasdmm@gmail.com; (mobile) +5514997546334 and (institutional)
+551150847582
Background: Peripheral artery disease is highly prevalent on the elderly, and it is

characterized by reduction of blood flow in the limbs. The macrophage-colony
stimulating factor (M-CSF) promotes proliferation, differentiation and survival of
monocytes, macrophages and bone marrow progenitor cells, which are essential
elements for vessel formation. Aims: This study aims to assess the effect of M-CSF
gene therapy on the functional restoration of ischemic limbs. Methods: Male Balb/c
10-12-weeks-old mice had surgically induced hindlimb ischemia and three days after
were electroporated with 100g of plasmid uP-MCSF. M-CSF quantification from
serum and rectus femoris muscle was performed at 0, 2, 4, 7 and 14 days by ELISA.
Weekly, the animals were assessed visually and by Laser Doppler Perfusion Imaging
(LDPI). After 30 days, muscle force and mass were determined. The animals were
euthanatized and their gastrocnemius muscles were collected for histological
analysis. Results: A peak of M-CSF expression was observed on the 2nd day
(233.250.41pg/mg) and reached background on 14th day (14.450.92pg/mg). The
ratio of muscle force/gastrocnemius weight was 3.400.56N/g for the non-ischemic
group, 0.350.12N/g for the ischemic group, 0.420.22N/g for the uP group and
2.320.99N/g for the uP-M-CSF group. LDPI analysis showed the increase of blood
perfusion in M-CSF group, but no statistical differences were found in comparison
with other groups (22.64.7(uP-MCSF); 32.812.2(ischemic) 32.68.9(uP) p=0.16).
Conclusion: M-CSF gene therapy improved muscle force.
Background: Peripheral arterial disease is characterized by the narrowing or

occlusion of blood arteries that supply the limbs, possibly evolving to chronic critical
ischemia. GM-CSF stimulates production of granulocytes and monocytes, and also
prolongs the half-life of monocytes and mobilizes endothelial progenitor cells from
bone marrow. Aims: Determine GM-CSF expression profile and assess blood flux
after transfection of murine ischemic limbs with uP-GM-CSF plasmid. Methods:
Hindlimb ischemia was induced in 10-12 weeks-old Balb/c mice by the removal of
the femoral artery. Three days later, the rectus femoris muscle was electroporated
with 100g of uP-GM-CSF plasmid in 80l PBS. Temporal expression of muscular
GM-CSF protein was obtained by ELISA. Weekly, blood flow images using Laser
Doppler and hemogram were assessed during a month. Thirty days after transfection,
gastrocnemius muscle mass and force were determined. Non-treated ischemic mice,
ischemic mice electroporated with the empty vector uP and healthy mice were used
as controls. Results: The ratio of gastrocnemius muscle force/mass (N/g) in healthy,
ischemic and treated mice was 3.400.51, 0.350.12 and 1.610.72, respectively. An
improvement of blood flux rate (%) was observed after gene therapy (30.887.82),
but it was not statistically significant when compared to the ischemic group
(32.8412.16; p=0.45). The peak of GM-CSF expression (pg/mg protein) reached
two days after transfection: 25.35.1 and 61.736.2 in healthy and ischemic muscles,
respectively. Hemogram showed no statistical significance among groups.
Conclusion: GM-CSF gene therapy accelerated recovery of muscle force and blood
perfusion of ischemic limbs.
Information on ethical approval and funding support: This project was approved by
CEUA (#9271280415) and CiBio (#2015/19), funded by FAPESP and scholarship
was provided by CAPES.
Ethical Approval: CEUA 6363240715. Funding Support: FAPESP and CNPq.
M3

M4
IN VITRO KNOCKDOWN OF ANTIAPOPTOTIC GENES IN BREAST

CANCER CELLS BY siRNA CARRIED INTO HYBRID NANOPARTICLES
EVALUATION OF DIFFERENT PROTOCOLS TO OBTAIN PLATELETRICH PLASMA
Lenidas Joo de Mello Jr(1,2), Gabriela Regina Rosa de Souza(3), Evelyn Winter(3),
Frederico Pittella(4), Tnia Beatriz Creczynski-Pasa(1,3)
Francisco Alipio de Sousa Segundo, Aldcejam Martins da Fonseca Junior, Adlio

Santos de Azevedo, Cristiana Ferreira da Silva Walter, Morgana Alves Cavalcante,
Paulo Wibiratan Lopes da Costa
(1) Graduate Program in Biochemistry, UFSC, Campus Universitrio-TrindadeFlorianpolis-SC-Brazil. CEP: 88040-900; leonidasjmj@gmail.com
(2) Daltec, Biology Dept, IFSC, Florianpolis-Brazil
(3) Graduate Program in Pharmacy, UFSC, Florianpolis-Brazil
(4) Department of Pharmaceutical Sciences, UFJF, Juiz de Fora-MG, Brazil.
The development of cancer is related with an imbalance in apoptosis process. The
BCL-2 and BCL-xL genes perform remarkable roles in the regulation of apoptosis,
promoting cell survival being relevant in tumorigenesis and drug resistance. The
knockdown of these pro-survival genes by interference RNA (siRNA) is a suitable
approach to overcome tumor progression and chemoresistance. In this study it was
developed a hybrid nanocarrier system to promote siRNAs delivery to breast cancer
cells MCF-7. The aim was to evaluate in vitro if the silencing of anti-apoptotic genes
Bcl-2 and Bcl-xL by siRNA, combined or not with chemotherapy, would succeed in
antitumor effect. The knockdown was assessed by western blot and quantitative PCR
analysis. The expression of targeted proteins by nanocarriers systems NP-siBCL-2
and NP siBCL-xL after 48h was decreased to 474% and 238% respectively. The
nanocarriers systems were also tested in MCF-7 cells with doxorubicin (DOX). The
results related to cytotoxicity expressed reduction in CC50 of DOX for both targets,
BCL-2: 2.471.1 M to 0.42 0.03 M; the BCL-xL: 46%, from 2,471.1 M to
1.150.04 M. Apoptosis was analyzed by Annexin-V assay. Compared to the
control, the increase in apoptotic cells was 20763 % when cells were incubated with
NP siBCL2 and 18818% when incubated with NP siBCL-xL. In conclusion, the
knockdown of anti-apoptotic BCL-2 and BCL-xL genes by siRNA carried by hybrid
nanoparticles showed to be effective in antitumor activity, especially for enhancing
the effects of chemotherapy for breast cancer.
Autonomous veterinarian Veterinary Medicine - Instituto Federal de Educao,

Cincia e Tecnologia da Paraba - Campus Sousa
Contact: aldcejamjunior@hotmail.com/ (+55 83)996667189
The platelet-rich plasma (PRP) is defined as a preparation having blood platelet
concentration up to eight times higher than the original sample. Its use in
regenerative medicine is mainly explained by stimulating the healing of various
tissues through the release of several growth factors. However, data on centrifugation
protocols are still scarce. Thus, it was evaluated different centrifugation protocols to
obtain PRP. Blood was obtained from equine, an aliquot was reserved for obtaining
reference values, the remaining subjected to the first centrifugation to obtain plasma,
which was subjected to second centrifugation:600/1200 rpm for 5 minutes each (T1);
600/1200 rpm for 10 minutes each (T2); 1000/2000 rpm for 5 minutes each (T3);
1800/3600 rpm for 10 minutes each (T8); Tests were performed in triplicate. Growth
was observed in platelet concentration proportional to the increase in rpm, with
218% (T1); 202% (T2); 261% (T3); 207% (T4); 275% (T5); 260% (T6); 268% (T7)
and 349% (T8) when compared with the reference value, however, in all groups,
except for T7 and T8, the protocols with same rpm lasting five minutes showed
higher concentration. It follows that increasing the rpm can lead to increased platelet
concentration, the opposite occurs with increasing spinning time duration.
This study was supported by CAPES, CNPq and IFSC.

M5
INTEGRATIVE
TOXICOGENOMIC
AND
SYSTEM
PLATFORMS TO TARGET MELANOMA GENES BY DM-1
M6
BIOLOGY
rica Aparecida de Oliveira (1), Garcia Ferreira de Souza (2), Diogenes Lima (3),
Lucas Cardozo (3), Fernanda Faio-Flores (1), Jos Agustn Pablo Quincoces Suarez
(2), Helder I. Nakaya (3), Gisele Monteiro (4), Silvya Stuchi Maria-Engler (1)
(1) Skin Biology Group, Clinical Chemistry and Toxicology Department, School of
Pharmaceutical Sciences, University of Sao Paulo, FCF/USP, Brazil
(2) Laboratory of Organic Synthesis, Anhanguera University of So Paulo, UNIAN,
Brazil
(3) Computational Systems Biology Laboratory, School of Pharmaceutical Sciences,
University of Sao Paulo, FCF/USP, Brazil
(4) Biochemical Pharmaceutical Technology Department, School of Pharmaceutical
Sciences, University of Sao Paulo, FCF/USP, Brazil
ericaoliveira@usp.br
Phone: 55 11 3091 3631
Mobile: 55 11 9 6744 0524
Melanoma is a cancer highly invasive and metastatic, with high mortality rates and
chemoresistance. The MAPK pathway is frequently overexpressed, and represents a
potent target-specific chemotherapeutic, as BRAF inhibitors. The DM-1 compound,
sodium
4-[5-(4-hydroxy-3-methoxy-phenyl)-3-oxo-penta-1,4-dienyl]-2-methoxyphenolate, a curcumin analog, has potentially useful antitumor activity in melanoma,
but the molecular pathways involved with its action remain unclear. The use of
toxicogenomic models for determining cellular responses to compounds has been
shown to be effective in the initial screening for molecular targets. In this study, we
seek to gain insights into the molecular aspects of DM-1 toxicity by the screening of
a genome-wide deletion set of individual genes in S. cerevisiae library. Systems
biology tools were applied to obtain a comprehensive view on the role of these genes
in human melanoma progression. From the primary data collected, 211 sensitive
genes were identified from which, 74 genes were selected according to human
homologs. Enrichment analysis of the genes corresponding to the sensitive mutants
reveals the key features of DM-1 toxicity, which include insulin, iron and RNA
metabolism, and oxidative phosphorylation. Pathway analysis highlighted 7 target
genes (ADK, ATP6V0B1, PEMT, TOP1, ZFP36, ZFP36L1, ZFP36L2) that are
frequently altered among normal skin, dysplastic nevus, primary and metastatic
melanomas. They are related to regulation of tumor progression, cell differentiation,
and epithelial-mesenchymal transition. Taken together, these findings provide
meaningful details into the core understanding of DM-1 toxicity regarding molecular
mechanisms and encourage further studies in the development of combinatorial
treatments for melanomas.
Keywords: melanoma; DM-1 compound, Saccharomyces cerevisiae; toxicogenomic
Conflict of interest The authors declare that they have no conflict of interest.
Funding support: Pos-Doc Fellowship grant PNPD/CAPES.

M7
Felipe Mateus Ornellas1,2, Dbora Santos Ornellas1, Sabrina Vargas Martini1, Raquel
Carvalho Castiglione1,3, Sergio A. de Souza4, Christina Maeda Takyia2, Marcelo
Marcos Morales1.
1 Laboratory of Cellular and Molecular Physiology, Institute of Biophysics Carlos
Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
2 Laboratory of Immunopathology, Institute of Biophysics Carlos Chagas Filho,
Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
3 Laboratory for Clinical and Experimental Research on Vascular Biology,
Biomedical Center, State University of Rio de Janeiro, Rio de Janeiro, Brazil
4 Department of Radiology, School of Medicine, Federal University of Rio de
Janeiro, Rio de Janeiro, Brazil.
BACKGROUD: Ischemia-reperfusion (IR) is a serious complication in renal
transplantation. Cell-based therapy by bone marrow-derived mononuclear cells
(BMMC) has already shown to improve vascularization and decrease inflammation.
AIM: Evaluate the repair capacity of BMMCs in rats submitted to IR.
METHODS: Wistar rats were divided into five groups: control (CTRL), sham-saline
(S-S), sham-BMMC (S-C), IR-saline (IR-S) and IR-BMMC (IR-C). Renal bilateral IR
was performed by clamping artery renal for 1h. BMMCs were injected i.v. 1h after
reperfusion. After 24h, renal function, histology and immunohistochemistry
(inflammation, antioxidant system, proliferation and apoptosis), RT-PCR, and ELISA
assays were carried out. BMMCs were tracked by labeled Tc99m and GFP+ BMMCs.
P<0.05 was considered significant.
RESULTS: Renal function was improved after BMMCs infusion as well as renal
structure in the IR-C group. Tc99 BMMCs tracking was visualized in the kidneys at 2
and 4h after infusion and 24h for GFP+ BMMCs infusion. The inflammatory and
biological markers: TLR-2, TLR-4, RAGE, IL-17, HMGB-1, and KIM-1 expression
were reduced, IL-10, TGF-1, Nrf2, and HO-1 expression were increased in IR-C
compared to IR-S. Apoptotic index diminished and proliferation index increased in
IR-C when compared to IR-S.
CONCLUSION: BMMCs therapy in IR model led to renal functional and structural
improvement, probably by inhibiting the inflammation, and oxidative stress of injury
besides decreasing tubular cell death and, accelerating tubular cell proliferation.
Therefore, we believe that the benefits induced by BMMCs in IR can be a substantial
promise for the development of new interventions.
ETHICAL APPROVAL: IBCCF-168
FUNDING SUPPORT: CAPES e CNPQ

M8
STRYCHNOS PSEUDOQUINA FRACTIONS INDUCES CELLULAR

PROLIFERATION AND ANGIOGENESIS IN SKIN WOUNDS OF
DIABETIC RATS
Mariurea Matias Sarandy (1), Fabiana Cristina Silveira Alves de Melo* (2),
Rmulo Dias Novaes (3), Joo Paulo Viana Leite (4), Reggiani Vilela Gonalves (2)
(1) Department of General Biology, Federal University of Viosa, MG, Brazil
(2) Department of Animal Biology, Federal University of Viosa, MG, Brazil
(3) Department Structural Biology, Federal University of Alfenas, MG, Brazil
(4) Department of Biochemistry and Molecular Biology, Federal University of
Viosa MG, Brazil.
*Phone: (+55) 31-3899-4375. facrismelo@yahoo.com.br.
The wound healing is characterized by a sequence of events that promote the repair
of the tissue after injury. The use of herbal medicine such as Strychnos pseudoquina
(Quina do Cerrado) has been widely used in traditional medicine for the treatment of
various diseases like malaria and diseases of liver, spleen and stomach. The aim of
the study was evaluate the cell proliferation and density of blood vessels in scar
tissue of diabetic rats treated with ointment-based of S. pseudoquina extracts. Thirty
rats were randomized into 5 groups: EF5 (Fraction of S. pseudoquina 5%), EF10
(Fraction of S. pseudoquina 10%), Sal (0.9% saline solution), OV (ointment vehicle),
SS (sulfadiazine of silver) (Ethics Committee Protocol 730/14 CEUA/UFV). Three
circular wounds (12 mm in diameter) were made in the skin by surgical incision and
the treatments were applied at the wound site daily for 21 days. Cuts of 4 m of
thickness were obtained and stained with hematoxylin and eosin for the analysis of
the cells and blood vessels. The groups treated with S. pseudoquina (EF5 and EF10)
showed an increase in cell numbers on days 7 and 14 when compared to the others
groups. The blood vessels proliferation increased on days 14 and 21 in EF10 group
when compared to the others groups. The use of fractions of S. pseudochina (5 and
10%) is effective in the treatment of wounds by stimulating proliferation of cells and
formation of new vessels.
Financial support: FAPEMIG (Edital PPM00868-15).
BONE MARROW DERIVED MONONUCLEAR CELLS ADMINISTRATION

ACCELERATES CELL RECOVERY AFTER BILATERAL RENAL
ISCHEMIA-REPERFUSION INJURY
EXTRACELULAR VESICULES AS POSSIBLE THERAPY FOR KIDNEY

INJURY INDUCED BY SEPSIS
Nogueira, C. M. B. 1,2, Menezes, A. L. 1 , Ornellas, F. M.

Barbosa C. M. 3, Takiya, C. M. 2, Morales, M. M. 1
1,2
, Ornellas, D. S.
1 Laboratory of Cellular and Molecular Physiology, Institute of Biophysics Carlos

Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
2 Laboratory of Immunopathology, Institute of Biophysics Carlos Chagas Filho,
Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
3 Lamoratory for Clinical and Experimental Research on Vascular Biology,
Biomedical Center, State University of Rio de Janeiro, Rio de Janeiro, Brazil.
Background: Sepsis is the major cause of death and is one of the most common
causes associated with acute kidney injury (AKI). Several studies showed the
attenuation of sepsis complications by the use of mesenchymal stromal cells from
adipose tissue (ADMSC), an possible mechanism action of this therapy could be by
extracellular microvesicles (EVs) secretion.
Aims: Characterize the model of AKI induced by cecum ligation and puncture (CLP)
and evaluate the population of EVs from ADMSC.
Methods: Wistar rats were divided in: Sham, CLP with 2 punctures (CLP-2P) and
CLP with 10 punctures (CLP-10P). After 72h, renal function was analizated. The
ADMSC was characterized by membrane markers and EVs by Western-Blotting and
by nanoparticle tracking analysis.
Results: Cells in culture adhered to the plastic dish, were positive for the expression
of MSC-positive makers and was visualized the differentiation in three cells types on
distinct conditions. Was observed large population of microvesicles in EVs and
expressed endossomal markers. There was a higher mortality in both CLP groups
compared to Sham group however in CLP-10P group it was more expressive.
Glomerular filtration rate decreased in CLP groups compared to Sham group but
more substantially in CLP-10P group. Fractional excretion (FE) of sodium increased
significantly in CLP groups compared to Sham group, FE potassium and chloride had
a decrease in CLP groups compared to Sham group.
Conclusion: Thus, CLP 10P group presented glomerular and tubular damage more
significative. The EVs that carrier endossomal markers of MSC can be used as a new
intervention for AKI.
Ethical Approval: IBCCF-044
Funding Support: CNPQ

N GENERAL CELL BIOLOGY
N1
N2
IN VITRO ASSESSMENT OF CYTOTOXIC, GENOTOXIC, APOPTOTIC

AND NECROTIC ACTIVITIES OF LATEX C-serum FROM HEVEA
BRASILIENSIS RRIM 600 IN CHO-k1 CELL LINE
METABOLIC REPROGRAMMING DURING SKIN

INSIGHTS INTO THE ROLE OF MITOCHONDRIA
Leandra Ernst Kerche-Silva1,2, Dalita Gomes Silva Moraes Cavalcante1, Eidi

Yoshihara3, Aldo Eloizo Job1
1
Department of Physics, Chemistry and Biology, Universidade Estadual Paulista

Jlio de Mesquita Filho, Presidente Prudente-SP, Brazil.
2
Medical School of Presidente Prudente, Universidade do Oeste Paulista, Presidente
Prudente-SP, Brazil.
3
Agncia Paulista de Tecnologia dos Agronegcios, Presidente Prudente-SP, Brasil.
Latex is a specific cytoplasm from laticifer cells located in the secondary phloem of
rubber tree (Hevea brasiliensis Muell. Arg.). After ultracentrifugation, latex is
separated into three major phases including a top layer of rubber particles, a clear
phase called C-serum, and a pellet known as lutoids. Some biological properties of
latex C-serum have been reported, but only a few limited research has been
conducted to explore its biological potential. Therefore, the aim of this investigation
was to assess in vitro cytotoxic, genotoxic, apoptotic and necrotic activities of Cserum latex in the CHO-k1 cell line after 24 h of exposure. Cytotoxic effects were
evaluated through MTT test, genotoxic activities through the comet assay, and
apoptotic and necrotic activities through morphological evaluation of the cells using
two fluorescent dyes Hoechst 33342 and Propidium Iodade. The MTT test showed
that C-serum latex tested did not alter the proliferative activity of the CHO-k1 cells.
Comet assay showed a lack of genotoxic effects in the cell line tested. In the
morphologic cell evaluation, C-serum latex did not raise significantly the number of
cell deaths through apoptosis or necrosis. These results demonstrate that latex Cserum did not cause any DNA damage in the tests employed. Further investigations
are being released.
The authors gratefully acknowledge Fundao de Amparo Pesquisa do Estado de
So Paulo (FAPESP) and Universidade Estadual Paulista Jlio de Mesquita FIlho
for supporting this study, and the Agncia Paulista de Tecnologia dos Agronegcios
for the use of its facilities.
Key-Words: Latex C-serum, Cytotoxicity, Genotoxicity, Apoptotic and Necrotic Cell
Death, CHO-k1.
AGING:
NEW
Julia Peloggia (1), Luis Alberto Luvano-Martnez (1), Bruno Chausse (2), Alicia
Juliana Kowaltowski (1), Maria Fernanda Forni (1)
(1) Biochemistry Department, Chemistry Institute, University of So Paulo, SP,
Brazil
(2) Faculty of Medical Sciences, University of Campinas, Campinas-SP, Brazil
Contact information:
E-mail: julia.peloggia@gmail.com
Phone: (11) 3091-8556 (institutional) and (11) 9 9136-2261 (mobile)
Background: The skin is the largest mammalian organ, and is composed by two
tissues, the epidermis and dermis. During aging, epidermal regenerative potential
declines, leading to a reduced capacity to respond to damage or stress. Maintenance
of tissue homeostasis is a high energy-demanding process and mitochondria are the
main organelle responsible for ATP production in most mammalian cells. More
importantly, mitochondrial dysfunction has recently been associated with aging in
many tissues, but has not been evaluated in skin. Aim: Therefore, the aim of this
work was to characterize bioenergetic alterations related to epidermal aging using a
murine model comprising young (2-4 months) and old (12-18 months) C57Bl/6j
male mice. Methods: Bioenergetic profile was evaluated monitoring oxygen
consumption in whole skin isolated mitochondria and in primary cultured epidermal
cells. Mitochondrial mass was also validated by citrate synthase (CS) expression and
VDAC protein levels. Gene expression was measured though RT-PCR. Results: We
observed a reduction in mitochondrial mass and function in aged epidermis
supported by lower levels of both VDAC and CS, as well as lower maximal and
spare respiratory capacity in isolated keratinocytes. Aged mice also exhibit lower
Complex I activity in isolated keratinocytes. Gene expression analysis showed a
lower expression of pentose phosphate pathway enzymes. Conclusion: Altogether,
these results suggest that epidermal aging is associated with lower mitochondrial
mass and function, as well as alterations in other metabolic pathways, indicating a
widespread metabolic reprogramming in this tissue during aging.
Supported by: FAPESP, CNPq, Guggenheim Foundation
Disclosure: The authors declare no conflict of interest
Animal Care and Use Committee Permit number: 17/2013

N4
N3
FUNCTIONAL STUDY OF RNF113A INTERACTIONS IN MAMMALIAN
CELL SPLICEOSOMES
Guilherme H Gatti da Silva1, Sidney Verissimo Filho2, Lucia Rossetti Lopes2,
Patrcia Pereira Coltri1.
MODULATION OF RANK/RANKL/OPG BY LOW-INTENSITY ELECTRIC

CURRENT HAS OPTIMIZED BONE GRAFT WITH BIO-OSS IN THE
REPAIR OF BONE DEFECTS IN THE WISTAR RAT CALVARIA
Damaris Helena Meneghetti, Leonardo Bagne, Jose Hyczy Fonseca Junior, Maria
Esmria Corezola do Amaral, Armindo Antonio Alves, Marcelo Augusto Marreto
Esquisatto, Milton Santamaria Jnior, Maira Felonato Mendes, Glucia Maria Tech
dos Santos, Thiago Antnio Moretti de Andrade, Fernanda Aparecida Sampaio
Mendona.
email: gui.h.gatti@gmail.com

Ometto UNIARARAS, ARARAS/SP, 13607-339 - E-mail:
damariis_01@hotmail.com Fone: +55 (19) 3543-1440.
Pre-mRNA splicing is the process by which introns are removed, and exons are
joined together to produce a mature mRNA for translation. It is catalyzed by the
spliceosome, a multi megadalton and dynamic ribonucleoprotein machine, composed
of 5 small nuclear ribonucleoprotein particles (U1, U2, U4, U5 and U6) and more
than 100 proteins. Spliceosome assembly is dynamic and involves several RNARNA and RNA-protein rearrangements, which are required for remodeling
spliceosome and for catalytic activation of the complex. Problems in splicing are
associated with tumor development and neurodegenerative diseases. RNF113A is the
ortholog of S. cerevisiae Cwc24p, both show similar domain, containing a RING
finger domain. The RING domain is commonly found in proteins tagged for
ubiquitination, and might regulate many cellular processes. RNF113A has been
found especially concentrated in B and Bact intermediate splicing complexes and it
also has been detected in post-catalytic complexes, indicating that this protein might
play an important role in spliceosome activation and catalysis. The role of RNF113A
in spliceossome assembly and catalysis is beginning to be explored. In this work we
analyzed RNF113A participation on spliceosome and its interactions with other
spliceosome components, proteins and snRNAs. Our results show that
overexpression of RNF113A in HEK293T cells recruit more U1, U2, U5, U6
snRNAs and PRP19 as evidenced by RT-qPCR. Interestingly, overexpression of
RNF113A is also related with an increase of PRP19 and SF3b2 protein levels. Taken
together, these results can indicate a mechanism of action for RNF113A in
spliceosomes, probably involving interaction with these proteins.
Background: The application of electric current of low intensity associated with

grafting is a strategy that aims to promote the process of osteogenesis. Aims: To
evaluate the effectiveness of electrical stimulation of low intensity (microcurrent) on
bone substitute Bio-Oss on bone healing in experimental defects induced in the
calvaria of rats. Methods: Bone defects (25mm) were induced in 96 male Wistar rats
(Rattus norvegicus), 120 days, 350g, with piezoelectric saw. These were divided into
four groups of 24 animals: C - untreated defects; E - defects filled with Bio-Oss ;
MC - defects treated with microcurrent (10A/5min/alternated days); MC+E microcurrent application in defects filled with Bio-Oss. Samples were collected
from lesion area in 10, 30, 60 and 90 days after the lesion, for
immunohistochemical analysis of RANK/RANKL/OPG and histochemistry of
TRAP; Results: Quantitative data of RANK immunostaining showed significantly
higher values in the MC+E group in all experimental times. The same was not
observed in the marking of RANKL between groups in all times. In OPG the
immunostaining increased significantly in 30, 60 and 90 day experimental in
MC+E group. The quantification of TRAP positive cells also showed significantly
higher values in the MC+E group in all experimental times. Conclusion: The analysis
of the marking for RANK/RANKL/OPG and TRAP indicated that the application of
microcurrent optimized bone graft with Bio-Oss in the repair process of defects in
the calvaria of Wistar rats.
Institute of Biomedical Sciences- University of So Paulo, Department of Cell and

Developmental Biology, So Paulo, Brazil.
2
Institute of Biomedical Sciences- University of So Paulo, Department of
Pharmacology, So Paulo, Brazil.
Funding support: CAPES and FAPESP 2013/02738-7.
Study approved
FHO|Uniararas.
by
CEUA/UNIARARAS
(052/2014)
and
supported
by

N5
N6
BENZNIDAZOLE COMBINED WITH CURCUMIN DETERMINES

IMPROVED OUTCOMES IN EXPERIMENTAL CHAGAS DISEASE
COMPARED TO BENZNIDAZOLE-BASED MONOTHERAPY
ACTIVATION OF THE OXITATIVE/NITROSATIVE STRESS IS

INVOLVED IN SKELETAL MUSCLE MYOSISTIS IN T. CRUZI-INFECTED
RATS
Rmulo Dias Novaes (1), Reggiani Vilela Gonalves* (2), Marcus Vinicius Pessoa
Sartini (3), Joo Paulo Ferreira Rodrigues (1), Eliziria Cardoso Santos (4), Raquel
Lopes Martins Souza (3), Ivo Santana Caldas (3).
Reggiani V. Gonalves (1)*, Mariurea Matias Sarandy (2), Arlete R. Penitente (3),
Maria do Carmo Q. Fialho (4), Wagner G. Gonalves (2), Izabel R.S.C. Maldonado
(2), Andr Talvani (3), Rmulo D. Novaes (5)
(1) Department of Structural Biology, Federal University of Alfenas, MG, Brazil

(3) Department of Pathology and Parasitology, Federal University of Alfenas, MG,
Brazil(4) School of Medicine, Federal University of Jequitinhonha and Mucuri
Valleys, Diamantina, MG, Brazil.
Phone: (+55) 31-3899-4375. reggysvilela@yahoo.com.br
(1) Department of Animal Biology

(3) School of Medicine, Federal university of Ouro Preto, MG, Brazil
(4) Department of Morphology, Federal University of Amazonas, AM, Brazil
(5) Department Structural Biology, Federal University of Alfenas, MG, Brazil;
Phone: (+55) 31-3899-4375. reggysvilela@yahoo.com.br
Considering the high toxicity and limited efficacy of currently available

chemotherapy, it is of utmost importance to develop new methods and therapeutic
regimens that are less hazardous and more efficient for Chagas disease (ChD)
treatment [1]. The association of curcumin to the reference chemotherapy against
malaria proved increases the effectiveness of the treatment [2]. However,
combination therapies using this molecule have never been investigated in ChD.
Thus, we evaluated the isolated and combined effect of benznidazole and curcumin
in the treatment of acute experimental ChD. Eighty-four 12-week-old Swiss mice
were equally randomized into seven groups: uninfected (NI); T. cruzi-infected and
non-treated (INF), infected and treated with 100 mg/kg Bz (B100), 100 mg/kg
curcumin (C100); 100 mg/kg Bz + 100 mg/kg curcumin (B100+C100). After
microscopic identification of blood trypomastigotes (4 days after inoculation), both
drugs were administered by gavage once a day for 20 days. Curcumin showed
limited antiparasitic, anti-inflammatory and antioxidant effects when administered
alone. When curcumin and Bz were combined, there was a drastic reduction in
parasitemia, oxidative cardiac injury, myocarditis and mortality; these results
exceeded the isolated effects of Bz. The combination of Bz and curcumin was also
effective at mitigating liver toxicity triggered by Bz, reducing circulating levels of
transaminases and liver oxidative damage. By limiting the toxic effects of Bz and
enhancing its antiparasitic efficiency, the combination of this drug with curcumin
may be a relevant therapeutic strategy that is possibly better tolerated in ChD
treatment compared with the isolated treatment with Bz.
Chagas disease caused by the protozoan Trypanosoma cruzi represents a neglected

disease worldwide [1]. Due to the lack of effective therapies, there is no cure after
the parasite install in the organs of the vertebrate host [1,2]. Through disease
evolution, oxidative and nitrosative stress has recognized implication to the
development of Chagas cardiomyopathy [2]. However, the role of reactive tissue
damage on skeletal muscle patology; especialy myositis, atrophy and necrosis is
poorly understood. Thus, this study was designed to investigate the morphological
and oxidative adaptations in skeletal muscle from mice infected by T. cruzi. Twenty
male Wistar rats were equally randomized into two groups: Control group (CG),
uninfected; and infected group (INF). Nine weeks after parasite inoculation skeletal
muscle fragments were collected for structural, ultrastructural and biochemical
analyses. By bright field microscopy was observed that infected animals presented
high tissue inflammation, connective tissue expansion, tissue fibrosis, atrophy and
fragmentation skeletal myocytes. Using transmission electron microscopy, skeletal
myocytes from infected animals presented myofibril disorganization, reduced
myofibril diameter, mitochondrial swelling and fragmentation. Furthermore, skeletal
from infected animals presented high levels of markers of oxidative (i.e.
malondialdeyde and carbonil proteins) and nitrosative stress (i.e. nitric oxide), as
well as reduced activity of antioxidant enzymes such as catalase, superoxide
dismutase and glutation-s-transferase. As all pathological changes in skeletal muscle
were more severe in animals presenting intense imbalance between
oxidative/antioxidante mechanisms, it is possible that changes the redox status is a
pivotal mechanism associated to skeletal muscle injury triggered by T. cruzi
infection.
References
[1] Santos EC, et al. Chemotherapy with benznidazole na suramin: applicability of
their concomitant treatment in mice infected with a virulent strain of Trypanosoma
cruzi. Antimicrob. Agents Chemother. 59: 5999-6006, 2015.
[2] Mimche PN, et al. The plant-based immunomodulator curcumin as a potential
candidate for the development of an adjunctive therapy for cerebral malaria. Malaria
J. 10:1-9, 2011.
Financial support: Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico
- CNPq, Brazil (449903/2014-1) and Fundao de Amparo Pesquisa do Estado de
Minas Gerais - FAPEMIG, Brazil.
N7
MODULATION OF OXIDATIVE STATUS BY EXERCISE ATTENUATES
CARDIAC DAMAGE IN EXPERIMENTAL CHAGAS CARDIOMYOPATHY
Rmulo D. Novaes (1,3), Mariurea Matias Sarandy* (2), Arlete R. Penitente (3),
Luiz Henrique M. Bozi (4), Clvis A. Neves (2), Izabel R.S.C. Maldonado (2),
Antnio J. Natali (5), Andr Talvani (3), Reggiani V. Gonalves (6)
References
[1] Rassi-Jr, A., et al. Chagas disease. Lancet 375, 1388-1402, 2010.
[2] Novaes, R. D., et al. Modulation of inflammatory and oxidative status by exercise
attenuates cardiac morphofunctional remodeling in experimental Chagas
cardiomyopathy. Life Sci. 152: 210-219, 2016.
Financial support: Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico
(CNPq) (454901/2014-3) and Fundao de Amparo Pesquisa do Estado de Minas
Gerais (FAPEMIG) (APQ-02309-14).

N8
SOD AND CAT LEVELS IN THE HEALING PROCESS OF WOUNDS OF

DIABETIC RATS TREATED WITH FRACTIONS OF STRYCHNOS
PSEUDOQUINA
Mariurea Matias Sarandy (1), Fabiana Cristina Silveira Alves de Melo* (2),
Rmulo Dias Novaes (3), Joo Paulo Viana Leite (4), Reggiani Vilela Gonalves (2)
(1) Department of Structural Biology, Federal University of Alfenas, MG, Brazil; (2)
Department of General Biology, Federal University of Viosa, MG, Brazil. (3)
Department of Biological Science and NUPEB, Federal University of Ouro Preto,
MG, Brazil; (4) School of Physical Education and Sport, University of So Paulo,
SP, Brazil; (5) Department of Physical Education, Federal University of Viosa, MG,
Brazil. (6) Department of Animal Biology, Federal University of Viosa, MG, Brazil.
Phone: (+55) 31-3899-4375. mariaureasarandy@gmail.com

Viosa MG, Brazil.
At the moment, Chagas disease has no cure after T. cruzi spreads and parasitizes
multiple organs of the vertebrate host [1]. Although the therapeutic management of
Chagas disease is almost exclusively based on antiparasitic chemotherapy (i.e.,
benznidazole and nifurtimox) [1,2], non-pharmacological strategies such as exercise
training therapy has emerged as a complementary approach for the treatment of
Chagas cardiomyopathy [3]. However, the rational basis that explains the benefits of
exercise therapy on Chagas cardiomyopathy (ChC) is poorly understood. This study
investigated the impact of an exercise program on exercise performance, heart
parasitism, immunoinflammatory response, fibrogenesis, oxidative damage, and
cardiomyocytes contractility in experimental ChC. Wistar rats were subjected to a 9week treadmill running training and challenged with Trypanosoma cruzi. Control
animals remained sedentary. Physical and metabolic performance, cardiac
morphology, oxidative tissue damage and cardiomyocyte morphology were
analyzed. Exercise training was efficient to improve physical performance (running
time, speed and workload) and anaerobic threshold (lactate production) in trained
animals. Exercise training also attenuates myocarditis, cardiac fibrosis and
cardiomyocytes hypotrophy at the same time that increased myocardial activity
catalase and superoxide dismutase, and reducing lipid and protein oxidation in
cardiac tissue. The protective adaptations to the host triggered by exercise training
contributed to reduce cardiac parasitism, inflammation, fibrosis and cardiomyocytes
atrophy. By restricting oxidative tissue damage in response to an enhanced efficiency
of endogenous antioxidant mechanisms, exercise training seem to be a beneficial
strategy to mitigate the severity of Chagas-associated cardiomyopathy. We would
like to thank the CNPq (454901/2014-3) and FAPEMIG (APQ-02309-14).
Wounds that exhibit impaired healing such as acute and chronic wounds generally
have failed to progress through the normal stages of healing. Most chronic wounds
are ulcers that are associated with ischemia, diabetes, venous disease or pressure that
result in enormous health costs. The objective of this study was to evaluate in vivo
effects in wound healing of the herbal medicine Strychnos pseudoquina fractions in
oxidative stress of diabetic rats. Thirty rats (302.2326.23g) were maintained in
cages with food and water ad libitum and the diabetes was induced by injection of
streptozotocin (60mg/kg). Three skin wounds (12mm of diameter) were created on
the animals' back which were randomized into 5 groups: EF5 (Extract Fraction of S.
pseudoquina 5%), EF10 (Extract Fraction of S. pseudoquina 10%), Sal (0.9% saline
solution), OV (ointment vehicle) and SS (sulfadiazine of silver). The applications
were made daily for 21 days and the tissues from different wounds were removed
every 7 days. Fragments of tissue were homogenized and the supernatant was used
for analysis of Superoxide Dismutase (SOD) and Catalase (CAT). On day 7 SOD
levels were higher in the EF5 and EF10 groups compared to the other groups and
EF10 showed significantly higher levels than EF5. CAT values were higher in EF10
group on day 7 compared to the other groups. S. pseudoquina fractions (5 and 10%)
had significant positive effects on antioxidant activity in wound healing in diabetic
rats.
*Phone: (+55) 31-3899-4375. facrismelo@yahoo.com.br.
Financial Support: FAPEMIG (Edital PPM00868-15).
References: [1] Nunes, M.C., et al. Ribeiro. Council on Chagas disease of the
Interamerican Society of Cardiology, Chagas disease: an overview of clinical and
epidemiological aspects. J. Am. Coll. Cardiol. 62: 767-776, 2013. [2] Bahia, M.T., et
al., Fexinidazole: a potential new drug candidate for Chagas disease, PLoS Negl.
Trop. Dis. 6: e1870, 2012. [3] Lima, M.M., et al., A randomized trial of the effects of
exercise training in Chagas cardiomyopathy, Eur. J. Heart Fail. 12: 866-873, 2010.

N9
ALCOHOL AND HIGH-FAT-DIET RETARD
CUTANEOUS WOUNDS OF WISTAR RATS
N10
ANGIOGENESIS
IN
Daiane Figueiredo Rosa* (1), Mariurea Matias Sarandy(1),Gabriela Diniz Pinto

Coelho(2), Vanessa Soares Martins(2), Srgio Lus Pinto da Matta(1), Rmulo Dias
Novaes(3),Reggiani Vilela Gonalves (4)
(2) Department of Medicine and Nursing, Federal University of Viosa, MG, Brazil
(3) Institute of Biomedical Sciences, Department of Structural Biology, Federal
University of Alfenas, MG, Brazil
(4) Department of Animal Biology, Federal University of Viosa MG, Brazil.
ALCOHOL AND HIGH-FAT-DIET INCREASE CELLULARITY AND TGF-

EXPRESSION IN CUTANEOUS WOUNDS SECONDARY INTENTION IN
RATS
Daiane Figueiredo Rosa* (1), Mariurea Matias Sarandy(1),Gabriela Diniz Pinto
Coelho(2), Vanessa Soares Martins(2), Srgio Lus Pinto da Matta (1), Rmulo Dias
Novaes(3), Reggiani Vilela Gonalves (4)
(3) Institute of Biomedical Sciences, Department of Structural Biology, Federal
University of Alfenas, MG, Brazil
(4) Department of Animal Biology, Federal University of Viosa MG, Brazil.
*Phone: (+55) 32-99100-2395. daianefigueiredo2009@hotmail.com

*Phone: (+55) 32-99100-2395. daianefigueiredo2009@hotmail.com
The new blood vessels formation is considered a crucial factor for the healing
process since it is responsible for providing oxygen to tissues and necessary nutrients
for the metabolic activity of the cells (1). Our goal was to evaluate the influence of
alcohol and high-fat-diet in the formation of new blood vessels in skin wounds of
second intention. Male Wistar rats (CEUA / UFV-213/2014) were divided into five
groups G1: Control, commercial diet and water for gavage, G2: Control, commercial
diet and water without gavage, G3: Commercial diet and alcohol (40%), G4: Highfat-diet, G5: High-fat-diet and alcohol (40%). Animals received alcohol and high-fatdiet for 61 days. For analysis of tissue vascularization, cuts of 4 m were obtained in
rotary microtome (Leica Multicut 2045, Germany), and stained with hematoxylin
and eosin. For this, a count of all structures of interest in a pattern area is performed
(AT) of 153 103 m2. We note that the groups that received alcohol (G3), high-fatdiet (G4), and alcohol and associated high-fat-diet (G5) showed less
neovascularization, it can be observed on the seventh and fourteenth day, compared
to the other groups (G1 and G2). Thus, we conclude that alcohol and high-fat-diet
can cause a delay in the healing of skin wounds of second intention, reducing
angiogenesis and consequently the nutrition in the new tissue.
Acknowledgements: The authors would like to acknowledge the Coordenao de
Aperfeioamento de Pessoal de Nvel Superior (CAPES).
1-
References
Flegg JA, Menon SM , Maini PK, McElwain DSL, et al. (2015) On the mathematical
modeling of wound healing angiogenesis in skin as a reaction-transport process.
Front Physiol 6, 262.
1-
The transforming factor beta (TGF-) has an important role in cutaneous healing
process by stimulating cell proliferation and matrix deposition, but its steady release
can lead to chronic inflammation, characterized by increased cellularity (1). Our goal
was to evaluate the influence of alcohol and high-fat-diet in the cellularity and TGF-
levels. Male Wistar rats (CEUA/UFV - 213/2014) were divided into five groups G1:
Control, commercial diet and water for gavage, G2: control, commercial diet and
water without gavage G3: Commercial diet and alcohol (40%), G4: High-fat-diet, G5:
high-fat-diet and alcohol (40%). Animals that received alcohol and high-fat-diet for
61 days. For cellularity analysis, sections of the 4 m were obtained of rotary
microtome (Leica Multicut 2045, Germany) and stained with hematoxylin and
eosin. For this, a count of all structures of interest in a pattern area is performed (AT)
of 153 103 m2. TGF- levels in the supernatant were analyzed using
immunoassay kits by ELISA (Boster Biological Technology Ltd., China). The
cellularity was increased in G3, G4 and G5 on the seventh and fourteenth day, when
compared to controls. The TGF- levels had to be elevated at day 7 and 14 in the G3,
G4 and G5 groups compared to controls. Thus, we conclude that the alcohol and
high-fat-diet can cause a delay in the healing process by increasing inflammatory
phase characterized by increased expression of growth factors and high cellularity.
Acknowledgements: The authors would like to acknowledge the Coordenao de
Aperfeioamento de Pessoal de Nvel Superior (CAPES).
References:
Ginderachte JAV (2012) The wound healing chronicles. J Blood 120, 499-500.

N11
N12
THE ROLE OF TOLL-LIKE RECEPTOR 4 IN THE DEVELOPMENT OF

CARDIOMYOCYTE HYPERTROPHY IN VITRO: PARTICIPATION OF
NF-B SIGNALLING
STRYCHNOS PSEUDOQUINA EXTRACT PROMOTES MIGRATION AND

PROLIFERATION OF MAST CELLS IN WOUND HEALING IN DIABETIC
RATS
Juliana Morais Alvim (1), Fernanda Gaisler Silva (1), Marcela Sorelli Carneiro
Ramos (1).
Mariurea Matias Sarandy (1), Gabriela Diniz Pinto Coelho* (2), Rmulo Dias
(1) Laboratory of Cellular and Molecular Biology, Federal University of ABC

(UFABC), Santo Andr, SP, Brazil

(2) Department of Medicine and Nursing, Federal University of Viosa, MG, Brazil.
(4) Department of Biochemistry and Molecular Biology, Federal University of Viosa
MG, Brazil
(5) Department of Animal Biology, Federal University of Viosa MG, Brazil
E-mail: alvim.julianam@gmail.com
Contact: (11) 3356-7479 - (11) 97520-0883
Background: Toll like receptors (TLR), which plays an important role in innate
immunity, inflammatory process and infection, activates the NF-kB pathway and
regulates inflammatory cytokines expression. Some systemic or local inflammations
are followed by cardiovascular diseases, showing that this cytokines are responsible
for modulating the cardiac tropism, decreasing the cardiac contractile. Studies using
isolated cardiomyocytes allow a specific evaluation of the effects caused by the
hypertrophic stimuli as well as the characterization of signalling pathway involved in
this process. The aim of this study is to evaluate the role of Nf-kB signalling in
cardiomyocyte tropism after TLR 4 activation.
Methods: Cardiomyocytes were isolated from wistar neonatal rats (in accordance
with the ethical committee and animal use), presenting typical morphological and
contractile characteristics that were maintained through the entire experiment. Cells
were treated for 24hs with five different concentrations of LPS (TLR4 agonist) in
order to define the dose that induces greater hypertrophic response. Alpha-actin
mRNA levels were used as cardiac hypertrophy marker. Gene expression was
evaluated by Real-time PCR. Results were expressed as mean SD and P<0.05.
Knowing the dose that promotes greater hypertrophic response, cells were treated
with small interfering RNA (siRNA) in order to silence the p65 protein and evaluate
the participation of NF-kB. Results: Alpha-actin mRNA levels has increased
approximately 50% after 100uM of LPS treatment compared with control group
(p<0.05). Conclusion: These results suggest that TLR4 was activated by their agonist
and promoted cardiomyocytes hypertrophy in vitro, allowing the siRNA
experiments.
Financial support: FAPESP and UFABC.
Phone: (+55) 31-3899-4375. gabriela.pinto@ufv.br

Introduction: The treatment of health affections using natural resources, particularly
medicinal plants and their derivatives has grown enough nowadays (1). It is a plant
popularly known in South America as Strychnos pseudoquina, and its extract is used
in popular medicine for the prevention and treatment of stomach and liver disorders
(2). Objective: Determine the effect of Strychnos pseudoquina in migration of cell
mast in repairing skin wounds in diabetic Wistar rat. Material and Methods: Thirty
male rats were randomized into 5 treatment groups with 6 animals in each group: LE
5 group (Extract of S. pseudoquina 5%), LE 10 group (Extract of S. pseudoquina
10%), Sal group (0.9% saline solution), OV group (ointment vehicle), SS group
(sulfadiazine of silver). Were realized three skin wounds of 12 mm in diameter each
and the extracts were applied daily over 21 days. Tissue from different wounds was
removed every 7 days. Scar tissue sections stained with toluidine blue were used for
the identification of mast cells. Using a 40 objective lens, 10 histological fields were
analyzed. The number of mast cells per unit histological area was calculated
according to the relation QA=mast cells/AT. Results: The groups treated with LE 5
and LE 10, showed greater proportion of inflammatory cells, mainly mast cells in F1,
F2 and F3, when compared to controls Sal, OV and SS. Conclusion: The Strychnos
pseudoquina extract demonstrated stimulate cells proliferation, and showed your
potential at the inflammatory process regulation, once this cells secretes chemical
mediators that promote cells proliferation, differentiation and tissue remodeling.
References
1- Lawrence J. and Rees G.D (2000), Microemulsion-based media as novel drug
delivery systems. Advanced Drug Delivery Reviews, 45, 89121.
2- Vasconcelos, J.M., Rodrigues, M.A., Vasconcelos Filho, S.C., Sales, J.F., Silva,
F.G., & Santana, J.G.. (2011). Dormancy break in seeds of "quina" (Strychnos
pseudoquina A. St.-Hil.). Revista Brasileira de Plantas Medicinais, 13, 507-511.
Financial support: We would like to thank the FAPEMIG (editalAPQ 00685-14)


N13
N14
TRPV1 PARTICIPATES IN THE ACTIVATION OF CLOCK MOLECULAR

MACHINERY IN THE BROWN ADIPOSE TISSUE IN RESPONSE TO
LIGHT-DARK CYCLE
QUINA DO CERRADO INDUCES CELLULAR PROLIFERATION AND

ANGIOGENESIS IN SKIN WOUNDS OF DIABETIC RATS
Maria Nathalia Moraes1, Nathana Mezzalira1, Leonardo Vinicius Monteiro de Assis,

Michael Menaker2, Ali Guler2, Ana Maria L. Castrucci1
1
Department of Physiology, Institute of Biosciences, University of So Paulo, So

Paulo, Brasil; 2 Department of Biology, University of Virginia, Charlottesville,
Virginia, USA
Reggiani Vilela Gonalves (1), Neila Augusta Alves da Silva* (2), Rmulo Dias
Novaes (3), Joo Paulo Viana Leite (4), Mariurea Matias Sarandy (2)
Viosa, MG, Brazil.
email: Nathalia.moraes@usp.br + 55 11 3091 7523.

Phone: (+55) 31-3899-4375. neila.silva@ufv.br
The clock gene machinery of some peripheral tissues is responsive to temperature
cycle. Transient receptor potential (TRPs) channels can act as thermosensors;
however, it is not fully understood how temperature is perceived by peripheral
tissues. So, we hypothesized that TRPV1 channel is involved in the synchronization
of clock genes by light and dark cycle (LD). We used wild type (WT) and TrpV1
knockout (KO) mice kept in constant darkness (DD) or LD. Interestingly, TrpV1 KO
mice displayed less total activity only when submitted to LD cycles; in addition,
TrpV1 KO showed a difference in core body temperature profile when compared to
WT mice. These data suggest that light information from the retina to the central
oscillator, and its processing are not altered in the absence of TRPV1 channel. In
brown adipose tissue (BAT) from WT mice, Ucp1 was upregulated in response to
LD. In WT mice, Bmal1 transcripts oscillates in DD, and Per1 and Per2 oscillated in
LD. Interestingly, the oscillatory profile of clock gene was abolished in TrpV1 KO
mice. We provide evidence that this channel is important in the regulation of efferent
pathways arising from the central oscillator; since TrpV1 KO mice display reduced
activity, altered body temperature profile, and loss of clock gene temporal oscillation
in BAT. Thus these data may prove useful to create a new pharmacological approach
to treat metabolic disorders having the effect of temperature and/or light through
TRPV1 activation of BAT clock gene machinery as the fundamental principle.
The wound healing is characterized by a sequence of events that promote the repair
of the tissue after injury. The use of herbal medicine such as Quina do Cerrado" has
been widely used in traditional medicine to treat various diseases, including
antimalarial effects. It has been mainly used against liver, spleen and stomach
diseases. In this way, the aim of the study was to evaluate cell proliferation and
density of blood vessels in scar tissue of diabetic rats treated with ointment-based in
fractions of the "Quina do Cerrado" extracts. Thirty rats were randomized into 5
groups: EF5(Extract Fraction of "Quina do Cerrado" 5%), EF10(Extract Fraction of
"Quina do Cerrado" 10%), Salt(0.9% saline solution), OV(ointment vehicle),
SS(silver sulfadiazine). Three circular wounds were made by surgical incision of
12mm in diameter in the skin and the treatments were daily applied at the wound for
21 days. Cuts of 4m thick were obtained and were stained with hematoxylin and
eosin for the analysis of cells and blood vessels. The groups treated with "Quina do
Cerrado" showed an increased in cell numbers on days 7 and 14 when compared to
the others groups. Neovascularization increased in EF5 and EF10 on day 14 and
EF10 showed greater number of vessels on day 21. The results showed that the use
of fractions 5 and 10% of "Quina do Cerrado" were effective in the treatment of
wounds stimulating proliferation of cells and formation of new vessels.
Funding Support: FAPESP and CNPQ
(We would like to thank the FAPEMIG editalAPQ00868-15, for financial support).
N15

N16
GUARAROBA PROMOTES PROLIFERATION OF CYTOKINES PRINFLAMMATORY IN CUTANEOUS WOUND HEALING IN DIABETIC

RATS
SUPERPARAMAGNETIC IRON-OXIDE NANOPARTICLES mPEG350

AND mPEG2000-Coated: CELL UPTAKE AND BIOCOMPATIBILITY
EVALUATION
Reggiani Vilela Gonalves (1), Laura Viera do Amaral* (2), Rmulo Dias Novaes
(3), Joo Paulo Viana Leite (4), Mariurea Matias Sarandy (5).
Adny H. Silva1, Enio Lima Jr.2, Marcelo Vasquez Mansilla2, Roberto D. Zysler2,
Horacio Troiani2, Mary Luz Mojica Piscioti2, Claudriana Locatelli3, Juan C. Benech4,
Natalia Oddone4, Vincius C. Zoldan5, Evelyn Winter1, Andr A. Pasa5, Tnia B.
Creczynski-Pasa1

Viosa MG, Brazil
Phone: (+55) 31-3899-4375. laura.amaral@ufv.br
Introduction: Guararoba, belongs to pseudoquina family. The infusion of its stem
bark is used in the traditional medicine of South America for the prevention and
treatment of stomach and liver disorders. Objective: Evaluate the effect of ointment
Guararoba in the production of TGF- pro-inflammatory cytokines in scar tissue in
diabetic rats. Material and Methods: Thirty male rats were randomized into 5
treatment groups with 6 animals in each group: GE 5 (Guararoba extract 5%), GE 10
(Guararoba extract 10%), Sal (0.9% saline solution), OV (ointment vehicle) and SS
(sulfadiazine of silver). TWere realized three skin wounds of 12 mm in diameter
each and the extracts were applied daily over 21 days. Tissue from different wounds
was removed every 7 days for TGF- analysis. TGF- levels in the supernatant were
analyzed by immunoassay kits by ELISA (Boster Biological Technology Ltd.,
China), following the manufacturers protocol. Results: The group of animals treated
with GE 5% and GE 10% showed smaller amount of TGF- at day 7, when
compared to the others groups. On the 14th there was no significant difference
between groups. Conclusion: The treatment performed with Guararoba extract were
effective to reduce the TGF- production, thereby preventing the chronic
inflammation as well as disorganized deposition of collagen fibers, since during
chronic inflammation occurs incorrect deposition of the fibers, which may promote
tissue formation in the keloid.
We would like to thank the FAPEMIG (editalAPQ 00868-15).
Departamento de Cincias Farmacuticas, Universidade Federal de Santa Catarina,

2
Div. Resonancias Magneticas, Centro Atmico Bariloche/CONICET, Bariloche,
Argentina.
3
Curso de Farmcia, Universidade do Oeste de Santa Catarina, Videira, Brazil.
4
Inst. de Investigaciones Biolgicas Clemente Estable, Montevideo, Uruguay.
5
Departamento de Fsica, Universidade Federal de Santa Catarina, Florianpolis,
Brazil.
Presenting author: Adny H. Silva Rua Euclides Lago, 112 apto 102
Florianpolis, SC, Brazil. Phone number: (48) 9942-8415 / (48) 3721-2200
adnyh@yahoo.com.br / adnyhe@gmail.com
Superparamagnetic iron oxide nanoparticles (SPIONS) were synthesized by thermal
decomposition of an organometallic precursor at high temperature and coated with a
bi-layer composed of oleic acid and methoxy-polyethylene glycol-phospholipid. The
formulations were named SPION-PEG350 and SPION-PEG2000. Transmission
electron microscopy, X-ray diffraction and magnetic measurements show that the
SPIONs are near spherical, well-crystalline, and have high saturation magnetization
and susceptibility. FTIR spectroscopy identifies the presence of oleic acid and of the
conjugates mPEG for each sample. In vitro biocompatibility of SPIONS was
investigated using three cell lines; up to 100 g/ml SPION-PEG350 showed nontoxicity, while SPION-PEG2000 showed no signal of toxicity even up to 200 g/ml.
The uptake of SPIONS was detected using magnetization measurement, confocal and
atomic force microscopy. SPION-PEG2000 presented the highest internalization
capacity, which should be correlated with the mPEG chain size. The in vivo results
suggested that SPION-PEG2000 administration in mice triggered liver and kidney
injury. This study provides data on the safety limits of SPIONS, crucial for their
future medical application.

N17
N18
ACTIVE SITE MAPPING OF LOXOSCELES PHOSPHOLIPASES D:

BIOCHEMICAL AND BIOLOGICAL FEATURES
ANTIOXIDANT EFFECT OF STRYCHNOS PSEUDOQUINA IN WOUND

Vuitikaa, L.; Chaves-Moreiraa, D.; Matsubaraa, F. H.; Justaa, H. C.; Murakamid, M.

T.; Naderc, H. B.; Senff-Ribeiroa, A; Chaim, O. Ma; Arnib, R. K.; Veigaa, S. S.
Mariurea Matias Sarandy (1), Estefanny Ruiz Garcia* (2), Rmulo Dias Novaes
(3), Joo Paulo Viana Leite (4), Reggiani Vilela Gonalves (2)

(3) Department of Structural Biology, Federal University of Alfenas, MG, Brazil
Viosa MG, Brazil
Department of Cell Biology, Federal University of Paran (UFPR), Curitiba, PR,

Brazil.
b
Multiuser Center for Biomolecular Innovation, Department of Physics, So Paulo
State University (UNESP), So Jos do Rio Preto, SP, Brazil.
c
Department of Biochemistry, Federal University of So Paulo (UNIFESP), So
Paulo, SP, Brazil.
d
Brazilian Biosciences National Laboratory (LNBio), National Center for Research
in Energy and Materials (CNPEM), Campinas, SP, Brazil.
Brown spider phospholipases D are among the most widely studied toxins in
Loxosceles venom. These toxins are very active, as they induce dermonecrosis,
exacerbated inflammatory responses, increased vascular permeability, hemolysis,
and acute renal failure. Despite their biomedical importance, the role of residues at
the catalytic interface of phospholipase D activity and other biological effects have
not yet been determined. In the present study, a comprehensive mutational
investigation was conducted using the Loxosceles intermedia phospholipase D
(LiRecDT1) as model. All variants were identified using whole venom serum
antibodies and a specific antibody to wild-type LiRecDT1 and showed native-like
conformation according to circular dichroism (CD) and differential scanning
calorimetry (DSC) analyses. The phospholipase activity of the variants H12A,
H12A-H47A, and E32A-D34A, such as vascular permeability, dermonecrosis, and
hemolytic effects were inhibited, suggesting the involvement of these residues in
catalysis. The mutant Y228A was equally detrimental to biochemical and biological
effects of phospholipase D, suggesting an essential role of this residue in substrate
recognition and binding. On the other hand, the mutant C53A-C201A (which
suppresses the hallmark disulfide bond of class II phospholipases D) did not block
phospholipase activity, but compared to the wild-type LiRecDT1, the enzyme
capacity to hydrolyze phospholipids and to promote dermonecrosis, hemolytic, and
vascular effects was diminished. The results confirm the structural features of the
biological activity of phospholipases D previously proposed by X-ray
crystallographic studies, and leave open the possibility for the design of synthetic
and specific inhibitors to Brown spider venom phospholipases D.
Keywords: brown spider, venom, phospholipase D, site-directed mutagenesis,
activity modulation.
Phone: (+55) 31-3899-4375. E-mail: tefaruga@gmail.com

The wound healing is a dynamic process that occurs after tissue injury, triggering
cellular events such as the release of molecular mediators that will protect the tissue
from damage caused by the oxidation process (1). The pharmaceutical industry has
focused on the technological development of products to improve the healing
process, mainly by identifying properties of secondary metabolites from natural
sources. This study aims to evaluate the antioxidant effect of the crude extract of
Strychnos pseudoquina, a plant native to the Brazilian, in the treatment of second
intention wounds in Wistar rats. Thirty male rats were randomized into 5 groups with
6 animals in each: QC5 group (Strychnos pseudoquina, 5% extract), QC10 group
(Strychnos pseudoquina 10% extract), Sal group (0.9% saline solution), OV group
(ointment vehicle 0.001%) and SS group (sulfadiazine of silver 0.001%). Three skin
wounds of 12 mm in diameter were realized in each animal, and the extracts were
applied topically daily over 21 days. Every 7 days, tissue samples from different
wounds were removed. The supernatant was used for the Catalase (CAT) analysis
and Superoxide dismutase (SOD). On day 14 and 21, SOD levels were significantly
higher in the QC5 and QC10 groups compared to other groups. CAT values were
higher in day 7 in the groups QC10 and QC5 compared with to other groups. QC5
and QC10% showed positive effects stimulating antioxidant defenses by increasing
the SOD and CAT activity in wound healing in diabetic rats.
We would like to thank the FAPEMIG (edital PPM00868-15).
References
1- Gonalves R.V.; Novaes R.D.; do Carmo Cupertino M.; Moraes B.; Leite J.P.; do
Carmo G.P.M.; de Mello Pinto M.V.; da Matta S.L.P. Time-dependent effects of
low-level laser therapy on the morphology and oxidative response in the skin wound
healing in rats. Lasers Med Sci. 28, 1-8, 2012.

N19

N20
EFFECTS OF SILVER NANOPARTICLES, METALS AND MIXTURE IN

MURINE MACROPHAGES (RAW 264-7)
CELL PROLIFERATION AND DIFFERENTIATION OF THE GASTRIC

EPITHELIUM: LATE EFFECTS OF THE EARLY WEANING
Andressa Glinski1*, Arandi Ginane Bezerra Junior2, Carolina Camargo de Oliveira1,

Carmen Lucia Voigt3, Francisco Filipak Neto1.
Gizela Maria Agostini Zonta (1), Juliana Guimares Zulian (1), Cruz Alberto
Mendoza Rigonati (1), Daniela Ogias (1) Patrcia Gama (1)
(1) Department of Cell and Development Biology, Institute of Biomedical Sciences,

University of the So Paulo, So Paulo, Brazil.
Departamento de Biologia Celular, Universidade Federal do Paran, C.P. 19031,

CEP 81.531-990, Curitiba, PR, Brazil.
2
Departamento Acadmico de Fsica, Universidade Tecnolgica Federal do Paran,
CEP 80230-010 Curitiba, PR, Brazil.
3
Programa Associado de Ps-Graduao em Qumica, Universidade Estadual de
Ponta Grossa, Setor de Cincias Exatas e Naturais. C.P. 992, CEP 84030-900, Ponta
Grossa, PR, Brazil
*Corresponding Author: E-mail: andressagli@yahoo.com.br , Institutional phone:
+55 41 33611680, Cell phone: +55 41 99159139
Silver nanoparticles (AgNP) are present in more than 20% of products listed in
Woodrow Wilson Database (2015). However, there is few data about toxicological
interactions of AgNP and other contaminants. The aim of the current study was,
therefore, to evaluate the effects of silver nanoparticles (AgNP), nonessential metals
(Cd, Pb ,Hg) and their associations (AgNP + metals) in murine macrophages RAW
264-7. Cells were exposed to two concentrations of AgNP (0.36 and 3.6 g ml-1), Cd
(1 and 10 M), Pb (25 and 250 M) and Hg (15 and 150 M) and the twelve
combinations of these contaminants for 4 h (DNA repair and Reactive oxygen
species ROS assays) and 24 h (cell viability, cells death and ROS assays). AgNP
had 1-3 nm diameter and zeta potential of -26.4 mv in water and -7.45 mv in culture
medium. In general, the mixtures were more toxic than individual contaminants
(lowest cell viability, and highest cell death rate and ROS levels). Cells started dying
by apoptosis before in the mixtures and increases of ROS levels occurred as soon as
4 h-exposure. ROS and DNA damage may be involved in triggering cell death. The
-H2AX-dependent DNA repair system was recruited in the cells exposed to the
highest concentrations of Hg, but not highest concentrations of Cd and mixtures of
AgNP+Cd. Toxicological interactions between AgNP and Cd, Hg and Pb occur in
RAW cells, with the mixtures being more toxic than the lone contaminants.
Funding support: Coordenao de Aperfeioamento de Pessoal de Nvel Superior
CAPES.
E-mail address: gizela.zonta@usp.br.

Contact: Tel.: (11) 3091-7303/Fax: +55-11-3091-7402.
Address: Av. Prof. Lineu Prestes, 1524 -Butant - So Paulo - SP - CEP 05508-000.
Feeding pattern regulates cell proliferation and differentiation in rat gastric
epithelium during postnatal development, and early weaning (EW) induces the
precocious maturation of gastrointestinal functions. However, few studies discussed
late effects of EW in adult stomach. Our aim was to assess the late responses of EW
on body mass and gastric cell proliferation and differentiation. Wistar rats were
divided into two groups: early-weaned at 15 days (EW) and weaned at 21 days
(suckling control, S) (CEUA/ICB USP 18/2015). The body mass, cell proliferation
(Ki-67 immunolabeling) and differentiation (pepsinogen content evaluated by
immunoblotting) were monitored at 15, 18, 30 and 60 postnatal days. We found that
body mass was lower at 18 and 30 days in EW group (vs. respective S; p<0.01).
However, at 60 days, difference was not detected, because EW females presented
higher body mass (p<0.05). Early weaning also increased proliferation index and the
thickness of mucosa at 18 days (vs. S; p<0.05), i.e. soon after the onset of treatment.
Conversely, an opposite result was observed at 30 days, when Ki-67 immunolabeling
was reduced in EW rats (p<0.05). At 60 days, there was no significant change
between the groups. As for differentiation, preliminary results indicated that EW
induced a variation in pepsinogen content, which is produced by zymogenic cells. In
summary, EW changed the pattern of body weight gain and stomach growth
immediately after the onset of solid food intake, and, whereas some effects persisted
to adulthood, others were compensated.
Support: FAPESP 2014/21449-9; GMAZ is recipient of CNPq fellowship.

N21
N22
PROFILE OF GENE EXPRESSION AND THE DEVELOPMENT OF

GASTRIC EPITHELIAL CELLS: CAN EARLY WEANING DISRUPT
GROWTH PROGRAM?
EFFECTS OF MECHANICAL CHANGES INDUCED BY MEMBRANE

CHOLESTEROL DEPLETION ON PLASMA MEMBRANE REPAIR
Kethleen Mesquita da Silva (1) Daniela Ogias (1), Ludimila Karen Cordeiro
Nogueira (1), Juliana Guimares Zulian (1), Cruz Alberto Mendoza Rigonati (1),
Patrcia Gama (1)
(1)
Natlia Fernanda do Couto (1), Luisa Rezende (1), Ana Paula Alves (2), Ubirajara
Agero (2), Oscar Nassif Mesquita (2), Luciana de Oliveira Andrade (1)

(1) Department of Morfology, Institute of Biological Sciences, Federal University of

Minas Gerais, Belo Horizonte, Brazil.
(2) Department of Physics, Federal University of Minas Gerais, Belo Horizonte,
Brazil.
Email: kethms@usp.br
AvProf Lineu Prestes 1524 ICB I 05508-000 So Paulo, SP, Brazil
Phone: 55 11 3091 7303 Mobile: 55 67 9928 6053
Address: Av. Pres. Antnio Carlos, 6627 ICB/UFMG J3 310 - Pampulha, Belo
Horizonte - MG, 31270-901. E-mail: naticouto.92@gmail.com / Phone: +55 31
3409-2791/ +55 35 99106-6152 (mobile)
The development of rat gastric mucosa is characterized by intense growth during the
first three postnatal weeks and complete differentiation is achieved on sucklingweaning transition. It is known that early weaning (EW) disturbs gastrointestinal
functional maturation, but few studies describe stomach responses. Currently, we
evaluated the profile of genes involved in growthcontrol and maturation of secretory
apparatus in mucous (MC) and zymogenic cells (ZC). Wistar rats were submitted to
EW at 15d (CEUA-ICBUSP 18/2015) and gastric samples were collected at 18, 30
and 60d for RT-qPCR. While preliminary results are obtained for genes involved in
growth, we detected that the expression of Mist1 (terminal differentiation of mucous
neck cells (MNC) into ZC) increased in EW pups at 18d. We also observed that
Mucin 6 expression (MNC) augmented in EW group at 18 and 30d. In relation genes
that are important for ZC functions, we detected that EW increased the expression of
moesin at 18 and 30d and induced GIF expression at 30 and 60d. PSGA (involved in
pepsinogen synthesis during development) declined at 18 and 30d in EW group
(p<0.05). In contrast, EW promoted an upsurge (~5x) of PSGC expression at 18d,
and such response persisted at 30 and 60d (p<0.05). Therefore, we found that EW
anticipated the expression of genes that regulate the differentiation and that some of
the changes can persist to adult life, indicating that some responses might be
imprinted throughout growth and aging.
Membrane repair after injury is an important process in retaining cell integrity,

especially for cells, which are under constant physical stress, such as endothelial
cells. However, changes in the mechanical properties of membranes could impair the
ability of cells in repairing injured membrane, and contribute to endothelial fragility.
The goal of this work was to evaluate the influence of the mechanical modulation
induced by cholesterol depletion, upon MCD treatment, on membrane repair
mechanisms. All the experiments were performed using a human umbilical vein
endothelial cell lineage. The absence of cholesterol leads to a reorganization of
cellular actin and de novo polymerization, an increase in cell rigidity, as well as an
induction of a rapid event of lysosomal exocytosis as early as 10 minutes after
MCD exposure. Additionally, during cholesterol sequestration, we observed an
increase in endocytic events between 10-20 minutes, which coincided with the peak
of exocytosis, indicating that these are most likely compensatory endocytic events. In
parallel, we observed that constitutive endocytic events, arising from pinocytic
processes, common in these cells, are blocked upon cell treatment with MCD,
suggesting that the reorganized cytoskeleton could function as a mechanical barrier
to further pinocytosis. Also, coinciding with the peak of cell rigidity induced by
MCD treatment, cells become more prone to mechanical membrane injury in
relation to non-treated cells. Together, these data show that mechanical modulation
induced by cholesterol depletion not only alters membrane traffic in cells, but also
makes cells more susceptible to mechanical injury.
Support: KMS is recipient CAPES fellowship; FAPESP processes 2011/17415-3;

2014/21449-9.
Key-words: cholesterol, membrane repair, endothelial cell.

Financial support: CAPES, CNPq, FAPEMIG.
N23
HEAT SHOCK MODULATES
MELANOCYTES
N24
GENE
EXPRESSION
IN
MURINE
MODIFICATIONS ON DNA TOPOLOGY FOUND IN REPLICATION

ORIGINS EVALUATED BY MOLECULAR DYNAMICS SIMULATIONS
Thain Rocha de Moraes, Maria Nathlia Moraes, Leonardo Vinicius Monteiro de

Assis, Ana Maria de Lauro Castrucci
Paulo Srgio Alves Bueno (1), Jos Renato Pattaro Jnior (1), Quirino Alves de
Lima Neto (2), Maria Aparecida Fernandez (2), Flavio Augusto Vicente Seixas (1).
Laboratory of Comparative Physiology of Pigmentation, Department of Physiology,

Institute of Biosciences, University of So Paulo, R. do Mato, trav. 14, no. 101, So
Paulo, 05508-900, Brazil
(1) Departmento de Bioquimica, Universidade Estadual de Maring,87020-900

Maring, Paran, Brasil.
(2) Departamento de Biotecnologia, Gentica e Biologia Celular, Universidade
Estadual de Maring,87020-900 Maring, Paran, Brasil.
email: thainarochademoraes@hotmail.com, + 55 11 3091 7523.

Background: Heat represents an important cause of DNA damage to skin cells. Clock
genes are responsible for regulating time-dependent skin traits such as DNA repair.
The role of heat shock in regulating clock gene in melanocytes is still an open
question. Aims: To evaluate the responses of normal murine melanocytes (Melan-a)
to heat shock in free-running and synchronized conditions, seeking to analyze Per1,
Hsp 90aa and Hsp 90ab expression. Methods: Melan-a cells were kept under
constant darkness at 37 C and on the 4th day divided into 2 groups:(1) no medium
change (free-running); (2) with medium change (synchronized). Then, 24 h later the
cells were exposed to heat shock (39.5 C) during 1 hour, and RNA extraction was
performed 0, 2 and 6 hours after heat shock. Results: In free-running conditions no
effect on Per1 expression was observed; however, we found a temporal increase on
Hsp 90aa six hours after the stimulus, as compared to control. Hsp 90ab was
irresponsive to the treatment. In synchronized cell population, Per1 expression
decreased immediately after the stimulus; the expression of Hsp 90aa increased at 0
and 6 h after the stimulus when compared to control cells, and no effect on Hsp 90ab
was found. Conclusion: Our data show that heat shock only affects the skin clock
gene machinery when the cell population is synchronized by medium change, thus
providing evidence that heat shock is not a synchronizing agent but it is able to
modulate clock gene expression.
Funding Support: FAPESP and CNPQ.
Conflict of interest:
None to declare
*Contact: Jos Renato Pattaro Jnior, Maria Aparecida Fernandez

E-mail: pattoze@gmail.com ; mafernandez@uem.br ;
aparecidafernandez@gmail.com
Tel.: +55 44 3011 5398
The local DNA topology in replication origins could be involved in recognition by
replication proteins. Secondary DNA structures, including intrinsically bent DNA,
can be detected, and they may indicate a specific pattern in or near mammalian
replication origins. This work aimed to simulate the topological behaviour of DNA
segments from replication origins by means of molecular dynamics, in order to
observe or not the bent formation. These evaluations were made comparing the
topology modification of DNA segments considered bent regarding others
considered not bent. Firstly, the nucleotide sequences were converted to 3D linear
DNA coordinates. Simulations were performed using gromacs-5.0 with Amber99SB
force field, PBC, SPC water, 100 mM NaCl, 1 atm, pH 7.0, and equilibration by 20
nanoseconds. The radius of gyration (Rgyr) was measured throughout the simulation.
The curvature of the segments was determined by dividing the Rgyr of the stretched
segment (initial) by the equilibrated one (final), thus providing ENDS ratio
parameter. The curvature index was obtained dividing the ENDSratio by the numbers
of base pairs from each segment. As results, all segments become bent after seven
nanoseconds and remain curved until the end of simulation. The curvature indexes
(10-2) were: 1.15 for oriGNAI3-b1, 1.26 for oriGNAI3-b2 and 1.04 for oriGNAI3nb7. These results showed that segments considered bent presented a curvature index
significantly higher compared to non-bent. The practical implication is related to the
capability of the enzymes that initiate the replication process in recognizes and bind
to these sites with higher affinity.
Funding support: CAPES, CNPQ, Fundao Araucria, Secretria de Estado da
Cincia, Tecnologia e Ensino Superior Fundo Paran and COMCAP facilities from
the Universidade Estadual de Maring-PR.


N25
N26
METABOLIC RESPONSES OF THE BRAIN OF ANTARCTIC FISH

SUBMITTED TO THERMAL STRESS
THE
EFFECTS
OF
TEMPERATURE
ON
THE
PLASMA
CONCENTRATION OF IONS AND ENERGY METABOLISM IN THE
GILLS AND KIDNEYS OF ANTARCTIC NOTOTHENIOIDS
Thaylise de Cassia Santos Przepiura (1), Tnia Zaleski (2), Cintia Machado (1),
Mariana Forgati (1), Maria Rosa Dmengeon Pedreiro de Souza (1), Priscila
Krebsbach Kandalski (1), Letcia Oliveira do Carmo Daloski (1), Luclia Donatti (1).
(1)Department of Cellular Biology, Federal University of Paran.
(2) Programa de Ps Graduao em Ecologia, Federal University of Paran.
Federal University of Paran - Centro Politcnico, s/n Jardim das Amricas CEP 81531-970 - Curitiba, PR Brasil
thay.lise@hotmail.com +55 429958-9663
Priscila Krebsbach Kandalski (1), Mariana Forgati (1), Tnia Zaleski (2), Flvia
Baduy Vaz da Silva (1), Cintia Machado (1), Luciana Badeluk Cettina (1), Maria
Rosa Dmengeon Pedreiro de Souza (1), Thaylise de Cassia Santos Przepiura (1),
Luclia Donatti (1).
(1) Department of Cellular Biology, Federal University of Paran.
(2) Programa de Ps Graduao em Ecologia, Federal University of Paran.
Federal University of Paran - Centro Politcnico, s/n Jardim das Amricas CEP 81531-970 - Curitiba, PR Brasil
The antarctic fish have your metabolism adapted to the antarctic marine
environment, making this group interesting for studies of adaptive mechanisms
involving metabolic plasticity in response to high temperatures. The study
investigates the metabolic responses of the antarctic fish's brain, Notothenia rossii
and Notothenia coriiceps, against thermal stress. Both species were exposed to
temperatures of 00.5C (control) and 80.5C (experimental) for 2, 6, 12, 24, 72
and 144 hours. This research is registered in the Ethics Committee for Animal
Experimentation of the Federal University of Paran (numbers 496 and 840). The
heat didn't affect neurotransmission by modulation of acetylcholinesterase in N.
rossii, but in N. coriiceps was modulated positively (6-12h). The energy metabolism
of N. rossii responds with negative modulation in the activity of hexokinase (12h),
lactate dehydrogenase (2-6h) and glucose-6-phosphatase (144h). N. coriiceps showed
positive modulation on glycogen phosphorylase activity (2h) and glucose 6phosphate dehydrogenase (6h) and negative modulation in glucose-6-phosphatase (2,
24-72h) and phosphofructokinase (72h). Oxidative stress in N. coriiceps was
responsible for a positive modulation of catalase (12, 44h), glutathione peroxidase
(12h) and glutathione-S-transferase (6, 72h). N. rossii negatively modulated
glutathione-S-transferase (2,12h), glutathione reductase (2h) and glutathione
peroxidase (6-12horas) and positively modulated glutathione peroxidase (72-144h).
The activities of citrate synthase, malate dehydrogenase, superoxide dismutase
activity, the level of glutathione and malondialdehyde showed not alterations in both
species. We observed that N. rossii and N. coriiceps, phylogenetically closely related
species, have different metabolic responses against thermal stress.
thay.lise@hotmail.com +55 429958-9663
Financial support: CAPES (CAPES/PNPD 2443/2011) and INCT-APA (CNPq

574.018/2008-5, FAPERJ E-26/170.023/2008).

N27
Dalita G.S.M. Cavalcante1, Andressa S. Gomes1, Renivaldo J. Santos1, Leandra
Kerche-Silva1, Caroline S. Danna1, Eidi Yoshihara2, Aldo E. Job1
(1) Department of Physics, Chemistry and Biology, Faculty of Science and
Technology, UNESP, Presidente Prudente, SP, Brazil
(2) So Paulos Agency for Agribusiness Technology (APTA), Presidente Prudente,
(1)
SP, Brazil.
dalitac@bol.com.br
To try to minimize the environmental impact of leather waste, a composite was
developed from natural rubber with leather waste tanned in chrome. To produce
materials with different colors was added iron oxide dye or titanium dioxide dye and
it was obtained a composite of more brownish (COF) or more whitish (CDT). One
possible application of these materials is in the textile industry. Thus, the aim of this
study was to evaluate the cytotoxicity of these new materials (COF and CDT) using
with CHO-K1 cell line system - test. For the cytotoxicity test using extract, liquid
extracts of the composites were obtained following the ASTM F619 (2014)
standards. The cells were exposed to COF and CDF extracts or PBS (negative
control - NC) for 24 and 48 h and the evaluation was performed by MTT assay. For
the cytotoxicity test, through direct contact, fragments of the composite were placed
on the center of the plate in contact with the cells for a period of 24 and 48 h and the
evaluation was performed by crystal violet method. The results of MTT assay
showed no significant differences in cell viability of cells exposed to COF and CDT
extracts in relation to NC, in either experimental period. In direct contact test,
composite COF and CDT were classified as two grade, which it means mild
cytotoxicity. Based on these results we can infer that the composite COF and CDT
are not cytotoxic and can be used in textile application.
Financial support: CAPES (CAPES/PNPD 2443/2011) and National Institute of

Antarctic Science and Technology of Environmental Research (INCT-APA) (CNPq
574.018/2008-5, FAPERJ E-26/170.023/2008).
N28
IN VITRO CYTOTOXICITY OF THE COMPOSITE MADE WITH

LEATHER WASTE
Funding Support: FAPESP
Notothenia rossii and Notothenia coriiceps are Antarctic notothenioids that are
ecologically and phylogenetically similar and abundant in Admiralty Bay. This study
aims to evaluate the energy metabolism and plasma concentration of ions in the gills
and kidneys of N. rossii and N. coriiceps that were exposed to temperatures of 0C
(control), 4 and 8C (experimental) for 1, 4, 15, and 30 days. This research is
registered in the Ethics Committee for Animal Experimentation of the Federal
University of Paran under the number 496. The higher temperatures resulted in the
downregulation of the activity levels of glucose-6-phosphatase in N. rossii and nonmodulation in N. coriiceps. However, the activity levels of citrate synthase, malate
dehydrogenase and lactate dehydrogenase increased in the gills and kidneys of both
species, possibly indicating an increase in metabolism. Furthermore, the energy
metabolism decreased in N. rossii after 30 days at 8C, resulting in a reduction in the
metabolic rate as a defense strategy to postpone the activation of anaerobic
metabolism. The higher metabolic rate of N. coriiceps was probably responsible for
their lower plasticity that resulted in its survival for only 4 days at 8C. The ionic
homeostasis of both species was also affected by warming, given there an increase in
Ca2+, a decrease in Cl-, and a transient increase in Mg+2 plasma concentrations that
were probably due to alterations in the membrane permeability to ions.
CYTOTOXICITY AND GENOTOXICITY ANALYSIS OF ROSMARINUS

OFFICINALIS L. (ROSEMARY) EXTRACT ON BREAST CANCER CELLS
(MCF-7)
Jonatas Rafael de Oliveira (1), Daiane de Jesus (1), Leandro Wagner Figueira (1),
Cludio Antonio Talge Carvalho (2), Samira Esteves Afonso Camargo (1), Cristina
Pacheco Soares (3), Antonio Olavo Cardoso Jorge (1), Luciane Dias de Oliveira (1)
(1) Department of Biosciences and Oral Diagnosis, Institute of Science and
Technology, Univ Estadual Paulista/UNESP. So Jos dos Campos, SP, Brazil.
(2) Department of Restorative Dentistry, Institute of Science and Technology,
Univ Estadual Paulista/UNESP. So Jos dos Campos, SP, Brazil.
(3) Development and Research Institute, Universidade do Vale do Paraba/UNIVAP.
So Jos dos Campos, SP, Brazil.
jroliveira16@hotmail.com / 55 12 98825-6374 / 55 12 3947-9334
Rosemary is an aromatic plant originated from the Mediterranean region and its
antimutagenic action has been reported on several cell lines and this DNA-protective
effect can be attributed to its biological composition. The aim of this study was to
evaluate the cytotoxicity and genotoxicity of rosemary extract on MCF-7. For this,
the cell culture was maintained in Dulbecco's modified Eagle medium (DMEM) with
10% fetal bovine serum and 1% penicillin-streptomycin under incubation (37C and
5% CO2). Posteriorly, 4 x 104 viable cells were added in microtiter plate and after 24
h adherence there was exposition for 5 min to rosemary extract (100, 50 and 25
mg/mL) or DMEM (0 mg/mL) (n=10/group). The cell viability was checked by
violet crystal (CV) assay. Regarding genotoxicity, 2 x 104 cells were added in 24well plates and after 24 h incubation there was exposition for 24 h to the different
concentrations of rosemary extract. Micronuclei (MN) were counted after addition of
flourished with DAPI by fluorescence microscopy. The results were analyzed by
ANOVA and Tukey Test (p0.05). Cell viability percentage was similar to control
group at 25 mg/mL (998%) and 50 mg/mL (9710%), however at 100 mg/mL there
was significant decrease to 838%. Additionally, the presence of MN at 25 mg/mL
was lower than the control group, however at 50 and 100 mg/mL there was no MN
formation. Thus, it can be concluded that the rosemary extract was no cytotoxic for
MCF-7 besides promote DNA-protective effect.
We do not have financial support.

N29
N30
QUALEA GRANDIFLORA EXTRACT EFFECT ON BEHAVIOUR OF THE

FIBROBLASTS AND PRE-OSTEOBLASTS CULTURES
CYTOGENETIC CHARACTERIZATION OF CELLS OF THE SALIVARY

GLANDS
OF
PECKIA
(SQUAMATODES)
INGENS
(DIPTERA,
SARCOPHAGIDAE)
Gabriela Silva Neubern de Oliveira (1), Cintia Kazuko Tokuhara (1), Flvia Amadeu
de Oliveira (1), Luiz Leonardo Saldanha (2), Anne Ligia Dokkedal (2), Rodrigo
Cardoso de Oliveira (1).
(1)
(2)
(1)Department of Biological Sciences, Biochemistry Laboratory, University of

Paulo (USP), Bauru, Brazil.
(2) Department of Biological Sciences, Natural Products Chemistry Laboratory,
Paulo State University 'Jlio de Mesquita Filho' (UNESP), Bauru, Brazil.
So
(1)
So
(2)
(3)
Adress Contact: R. Alameda Otvio Pinheiro Brisolla, 9-75 - Vila Nova Cidade
Universitria, Bauru SP, 17012-901 Brasil.
Biochemistry Laboratory
E-mail: gabriela_neubern@usp.br
Cell phone: (16)981038808
Background: Researches has shown that plant species Qualea grandiflora (QG) has
therapeutic properties, for example, anti-inflammatory and antimicrobial effects;
however, the modulatory effects of the plant are not entirely clear. The two
molecules under study: Hypoxia-Induced Factor (HIF-1) and Matrix
Metalloproteinase 14 (MMP-14) are extensively involved in physiological and
pathological processes, including repair situations. Objectives: To evaluate the effect
of QG extract in cell viability and HIF-1 and MMP-14 expression in fibroblast and
pre-osteoblasts cultures. Methods: For the cell viability test and MMP-14 and HIF1alpha activity, concentrations of: 0,0 (control group); 0,1; 1,0 and 10,0g/mL of
hydroalcoholic extract of QG were administered for periods of: 24, 48, 72 and 96h.
After each period, cell viability was assessed by MTT reduction method and the
analysis of the expression of the molecules by immunofluorescence, in both cell
types. Statistical tests were performed through GraphPadInStat and Prisma programs
being presented as mean and standard deviation. Results: QG extract did not affect
the cell viability of fibroblasts and pre-osteoblasts at concentrations until 1,0g/mL.
Immunofluorescence assays showed a modulatory effect QG extract on the
expression of HIF-1 and MMP-14, both for pre-osteoblasts and for the fibroblasts.
Conclusion: QG extract has therapeutic properties that do not alter cell viability, but
has an action on the expression of the molecules under study: MMP-14 and HIF-1,
in both cell lines.
Financial support: FAPESP #2014/05234-2 and #2014/20656-0.
Giovana Menezes Nunes (1), Kaio Cesar Chaboli Alevi (1), Ana Letcia Guerra (1),
Fernanda Fernandez Madeira (1), Patrcia Jacqueline Thyssen (2), and Maria Terclia
Vilela de Azeredo-Oliveira (1)
(1) Instituto de Biocincias, Letras e Cincias Exatas (IBILCE), Departamento de
Biologia, Universidade Estadual Paulista UNESP.
(2) Instituto de Biologia (IB), Departamento de Parasitologia, Universidade Estadual
de Campinas UNICAMP.
Listing of authors: Name: Giovana Menezes Nunes (Presenting author)

Mailing address: Instituto de Biocincias, Letras e Cincias Exatas (IBILCE),
Departamento de Biologia - Rua Cristovo Colombo, 2265 Jardim Nazareth - CEP:
15070-040 - So Jos do Rio Preto/SP - Telefone: (17) 3221-2378 - E-mail address:
giovanamn@gmail.com
Name: Kaio Cesar Chaboli Alevi ; Mailing address: Rua Cristovo Colombo, 2265
Jardim Nazareth - CEP: 15070-040 - So Jos do Rio Preto/SP - Telefone: (17)
3221-2378 - E-mail address: kaiochaboli@hotmail.com
Name: Ana Letcia Guerra ; Mailing address: Rua Cristovo Colombo, 2265
Jardim Nazareth - CEP: 15070-040 - So Jos do Rio Preto/SP - Telefone: (17)
3221-2378 - E-mail address: analebio@yahoo.com.br
Name: Fernanda Fernandez Madeira ; Mailing address: Rua Cristovo Colombo,
2265 Jardim Nazareth - CEP: 15070-040 - So Jos do Rio Preto/SP - Telefone:
(17) 3221-2378 - E-mail address: fernanda.bio56@hotmail.com
Name: Patrcia Jacqueline Thyssen; Mailing address: Rua Monteiro Lobato, 255,
Cidade Universitria - CEP: 13083862 - Campinas/SP - Telefone: (19) 33521-6299
- E-mail address: thyssenpj@yahoo.com.br
Name: Maria Terclia Vilela de Azeredo Oliveira; Mailing address: Rua Cristovo
Colombo, 2265 Jardim Nazareth - CEP: 15070-040 - So Jos do Rio Preto/SP Telefone: (17) 3221-2378 - E-mail address: tercilia@ibilce.unesop.br
The first groups of insects to colonize a corpse (two to three hours after exposure)
are the flies of the Calliphoridae and Sarcophagidae families, thus they are the most
commonly used insects in medical and criminal investigations to determine the
postmortem interval. The species of the genus Peckia hatch in the maggot form, with
their larvae placed especially during the decomposition of corpses. Peckia
(Squamatodes) ingens is a species found in the Neotropical region, present in a few
studies, mostly of morphological descriptions. Therefore, this study aimed to
cytogenetically characterize the cells of the salivary glands of P. (S.) ingens, in order
to provide new data on this important species. Five larvae of third instar provided by
the Forensic Entomology Laboratory of Campinas, Campinas, Brazil, were dissected,
the salivary glands were removed, and, through cell smashing technique, slides were
prepared and stained with Lacto-aceto-orcein and impregnation with silver ions. The
orcein technique enabled us to observe larges cells, with highly heteropycnotic nuclei
and nucleoli. The analysis with silver ions allowed us to observe the presence of a
large nucleolus in the cells of the salivary glands, possibly associated with high
metabolic activity of these cells. Thus, we characterize the cells of the salivary
glands of P. (S.) ingens, contributing thereby to the biological knowledge of this
species of high forensic importance. Financial Support: CAPES and CNPq.
N31
N32
APICOPLAST FATTY ACID SYNTHESIS IS ESSENTIAL FOR PELLICLE

FORMATION DURING THE COMPLETION OF CYTOKINESIS IN
TOXOPLASMA GONDII
ROSMARINUS OFFICINALIS L. (ROSEMARY) EXTRACT EFFECT ON

CERVICAL CARCINOMA CELLS (HELA) IN VITRO CYTOTOXICITY
AND GENOTOXICITY STUDIES
rica S. Martins-Duarte (1,2), Rossiane Vommaro (1,2), Namita Surolia (3) and
Wanderley de Souza (1,2)
Jonatas Rafael de Oliveira (1), Daiane de Jesus (1), Leandro Wagner Figueira (1),
(1) Laboratrio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofsica Carlos

Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
(1)
(2)Instituto Nacional de Cincia e Tecnologia em Biologia Estrutural e Bioimagens,
(3)Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced
Scientific Research, Bangalore, India.
(1)Department of Biosciences and Oral Diagnosis, Institute of Science and

Email: erica@biof.ufrj.br, Telephone: +55 21 3938-6580, +55 21 9887133-48

jroliveira16@hotmail.com / 55 12 98825-6374 / 55 12 3947-9334
The protozoan Toxoplasma gondii, the causative agent of toxoplasmosis, harbors a
non-photosynthetic secondary plastid (apicoplast). As in plant plastids, the fatty acids
synthesis in the apicoplast occurs by the type II fatty acid biosynthesis
(FASII) pathway. Although FASII in the apicoplast was shown to be essential for
parasite survival, the phenotypic consequences of FASII disruption for T. gondii
have not been examined in detail. Here, we combined light and electron microscopy
techniques including focused ion-beam scanning electron microscopy (FIB-SEM) to characterize the effect of FASII disruption in T. gondii, by treatment with the
FASII inhibitor triclosan or by inducible knock down of the FASII component acyl
carrier protein. Our morphological analyses show that FASII disruption prevented
cytokinesis completion in T. gondii tachyzoites, leading to the formation of large
parasites masses containing several tethered daughter cells. Quantification analysis
confirmed the division inhibition was a major phenotype of FASII disruption after
triclosan treatment and ACP knock down. Serial sections acquired with FIB-SEM of
a parasite mass showed that tethered daughter cells presented mature basal complex,
an indicative of cell maturation, but a defect in cleavage furrow establishment
prevented the assembly of a new plasma membrane between daughter cells and
complete separation at the end of cytokinesis. Addition of exogenous fatty acids to
growth medium prevented the formation of tethered daughter cells and recovered
parasite proliferation supporting the notion that FASII is essential to generate lipid
substrates required for the final step of parasite division.
Rosemary is a medicinal plant that has several biological activities. It is originated

from the Mediterranean region, however is distributed around the globe. Its
antimutagenic effect was reported in tumor and non-tumor cells. Therefore, this
study analyzed the cytotoxicity and genotoxicity of rosemary extract on HeLa. First,
the cell culture was cultivated in Dulbecco's modified Eagle medium (DMEM) plus
10% fetal bovine serum and 1% penicillin-streptomycin at 37C (5% CO2). For
cytotoxicity test, 4 x 104 viable cells were seeded in microtiter plate and after 24 h
adherence the monolayer was exposed for 5 min to different concentrations of
rosemary extract (100, 50 and 25 mg/mL) or DMEM (0 mg/mL) (n=10/group). By
crystal violet assay the cell viability was verified. For genotoxicity test, 2 x 104
viable cells were places in 24-well plates under 24 h incubation. Then, there was
exposition for 24 h to the different concentrations of plant extract. Posteriorly,
micronuclei (MN) were stained with DAPI and counted in fluorescence microscope.
The results were analyzed by ANOVA and Tukey Test (p0.05). It was verified that
only at 25 mg/mL (929%) the extract did not promote significant reduction of cell
viability, however at 50 mg/mL (85130%) and 100 mg/mL (7413%) decrease
were observed. Regarding genotoxicity, at 50 and 100 mg/mL there was no MN
formation and at 25 mg/mL it was similar to control group. Thus, it can be concluded
that the rosemary extract was no cytotoxic for HeLa and demonstrated antimutagenic
effect.
Supported by FAPERJ, CAPES and CNPq
We do not have financial support.

N33
N34
THYMUS VULGARIS L. (THYME) EXTRACT: ACTION ON CERVICAL

CARCINOMA CELLS (HeLa)
EFFECT
OF
MYRACRODRUON
URUNDEUVA
AND
QUALEA
GRANDIFLORA HYDROALCOHOLIC EXTRACTS ON CARIOGENIC
MICROCOSM BIOFILM
Leandro Wagner Figueira (1), Jonatas Rafael de Oliveira (1), Daiane de Jesus (1),
(1)

leandrowf@live.com / 55 12 99222-8920 / 55 12 3947-9334
Thyme is a medicinal plant from Mediterranean region that currently is found in all
continents. From its known effects is included the antimutagenic action. For this, the
aim of this study was to analyze the cytotoxicity and genotoxicity of thyme extract
on HeLa. This cell line was grown in Dulbecco's modified Eagle medium (DMEM)
with 10% fetal bovine serum and 1% penicillin-streptomycin (37C and 5% CO2).
After, 4 x 104 viable cells were distributed in 96-well plates, for cytotoxicity test, and
2 x 104 viable cells in 24-well plates, for genotoxicity test, both incubated for 24 h.
Then, after 5 min exposition to different concentrations of thyme extract (100, 50
and 25 mg/mL), commercially acquired at 200 mg/mL, or DMEM (0 mg/mL
control group), with n=10/group, the cell viability was quantified by crystal violet
assay. Regarding genotoxicity, after 24 h incubation with extract it was analyzed
micronuclei (MN) formation by staining with DAPI and quantification in
fluorescence microscope. The results were analyzed by ANOVA and Tukey Test
(p0.05). It was found that the cell viability decreased significantly at 50 mg/mL
(8314%), however it was similar to control group at 25 mg/mL (9413%) and 100
mg/mL (8614%). Regarding MN formation it was observed similar amount to
untreated group. Thus, it can be concluded that the thyme extract did not present
cytotoxic and genotoxic effects to HeLa.
Juliana Gonalves Pires (1), Aline Silva Braga (1), Flaviana Bombarda de Andrade
(2), Rodrigo Cardoso de Oliveira (1), Ana Carolina Magalhes (1)
Paulo, Bauru, Brazil.
(2) Department of Operative Dentistry, Endodontics and Dental Materials, Bauru
School of Dentistry, University of So Paulo, Bauru, Brazil.
Contact details: Alameda Dr. Otvio Pinheiro Brisola, 9-75 - Vila Nova Cidade
Universitria, Bauru - SP, 17012-901. Department of Biological Sciences, Bauru
School of Dentistry, University of So Paulo, Bauru, Brazil. Phone: #55 14 32358497; 55 14 98808-5557, e-mail: jupires@usp.br
Phytotherapy has been developed as an alternative to conventional antimicrobial
agents for controlling dental biofilm related to caries and periodontal disease. This
study evaluated the effect of two plants extracts (Myracrodruon urundeuva - M.u and
Qualea grandiflora - Q.g) on microorganism viability and enamel demineralization
using a cariogenic microcosm biofilm model. Human saliva (70% saliva/ 30%
glycerol) was mixed with McBain artificial saliva (1:50). Bovine enamel specimens
were exposed to human/Mc Bain saliva supplemented with 0.2% sucrose for the
biofilm formation, which was treated daily (1x60s/day) with 0.1; 1.0; 10; 100; 1000
g/ml of M.u and Q.g hydroalcoholic extracts for 14 days. Cell viability was
assessed by fluorescence using confocal microscopy (n=3, biological triplicates) and
enamel demineralization was quantified by transverse microradiography (n from 7 to
14). Samples treated with 100 g/ml M.u (62.1%), 10 g/ml M.u (74.6%) and 0.1
g/ml M.u (59.8%), 100 g/ml Q.g (67.2%) and 1.0 g/ml Q.g (64.5%) had
percentage of cell death means similar to positive control (0.12% chlorhexidine:
48.2%) and different from both negative controls (PBS: 19.0% and hydroalcoholic
solution: 33.8%) (ANOVA, p<0.0001). In respect to lesion depth, 1000 g/ml (239.3
m), 1.0 g/ml (227.5 m) and 0.1 g/ml (221.3 m) of M.u presented deeper
enamel lesion than the positive control (165.5 m) (ANOVA, p=0.0023), and no
experimental treatment differed from the negative controls (PBS: 210.0 m and
hydroalcoholic solution: 222.2 m). These results suggest that the extracts of the
plants have some antimicrobial effect without interfering in caries development.
Funding support: CAPES - Ethical approval: CAAE: 43948115.2.0000.5417
N35

N36
EFFECT OF BRAZILIAN CERRADO PLANT IN EXPRESSION AND

ACTIVITY OF MATRIX METALLOPROTEINASE IN PREOSTEOBLAST
EFFECT OF DIFFERENT COMMERCIAL MOUTHRINSES ON THE

VIABILITY AND ACTIVITY OF A CARIOGENIC MICROCOSM BIOFILM
Cintia Kazuko Tokuhara (1), Flvia Amadeu de Oliveira (1), Adriana Arruda Matos
(1), Luiz Leonardo Saldanha (2), Anne Lgia Dokkedal (2), Rodrigo Cardoso de
Oliveira (1).
Juliana Gonalves Pires (1), Aline Silva Braga (1), Ana Carolina Magalhes (1)
(1) Department of Biological Science, Biochemistry, University of So Paulo Bauru School of Dentistry, Bauru, Brazil.
(2) Department of Biological Science, So Paulo State University, UNESP Bauru,
Brazil.
Adress contact: Bauru School of Dentistry- University of So Paulo - Department of
Biological Science
Alameda Octvio Pinheiro Brisolla 9-75 - Cidade Universitria - Bauru-So PauloBrasil
Email: cintia.tokuhara@usp.br - Phone: 14 32358246; 14 996561265
Background: The Brazilian cerrado has a range of plant species still little known that
can provide many health benefits, but more scientific studies are needed to ensure the
concentrations that are not toxic and understand mechanisms. An example of plant
that has been described, with some propriety, is a Qualea grandilfora mart. Aims: So
the aim of this study was to investigate the effect of Qualea grandilfora in
expression and activity of the matrix metalloproteinase 2 and 9 (MMP-2 and 9) in
preosteoblasts. Methods: For this study, different concentrations of hydroalcoholic
extract (0.1; 1.0; 10; 100 and 1000 g/mL), remained in contact with the
preosteoblasts of mouse (MC3T3-E1 strain (ATCC)) to verify possible cytotoxic
effects of this plant. Control group was cultivated without extract. From the MTT
cell viability assay, it defined the concentrations of use, checking if it promotes
modulations in this cell type. Gene expression of metalloproteinases was evaluated
by real-time PCR and activity of matrix metalloproteinase MMP-2 and MMP-9 by
zymography assay. Results: In this study, it was found that the extract concentrations
appear to alter gene expression of MMPs in the periods evaluated, 24 and 48h. In the
enzymatic assay, activities of these molecules demonstrate not be significant
compared to the control group. Conclusion: We conclude therefore that the smaller
concentrations of Qualea promote an alteration in the expression and activity of
MMPs, however the largest concentrations of the extract, shown to be cytotoxic.

Contact details: Alameda Dr. Otvio Pinheiro Brisola, 9-75 - Vila Nova Cidade
Universitria, Bauru - SP, 17012-901. Department of Biological Sciences, Bauru
School of Dentistry, University of So Paulo, Bauru, Brazil. Phone: #55 14 32358497; 55 14 98808-5557, e-mail: jupires@usp.br
There are different oral mouthrinses containing potential antimicrobial agents in the
market, which have been indicated for patients with periodontal disease, but little is
known about their effect on the cariogenic biofilm. Therefore, this study evaluated
the effect of six commercial mouthrinses on microbial viability and lactic acid
production using a cariogenic microcosm biofilm model. Human saliva (70% saliva/
30% glycerol) was mixed with Mc Bain saliva (1:50) containing 0.2% sucrose.
Bovine enamel specimens (4 mm x 4 mm) were exposed to human saliva/Mc Bain
saliva for 14 days for the formation of biofilm, and treated daily (1x60s/day) with the
following commercial mouthrinses: Periogard, Noplak Max, Oral-B Complete,
Listerine, Malvatricin Plus and Cepacol Advanced Plus. Live and dead bacteria in
the biofilm were observed by fluorescence using confocal microscopy (n=3,
biological triplicate, ANOVA/Tukey) and lactate concentrations were quantified
enzymatically by spectrophotometry (n=3, biological duplicate, KruskalWallis/Dunn). The rinses induced cells death in a range from 50% (Periogard) to
75% (Malvatricin) of the samples; all treatments were able to significantly reduce
cells viability compared to control (p<0.0001). Listerine and Malvatricin Plus were
further more effective in reducing cell viability compared to Periogard. Samples
treated with Listerine, Periogard and Noplak Max showed lower production of lactic
acid compared to control (p<0.0001), while the other rinses were not significantly
different from control. Based on the results, Listerine seems to have the best
antimicrobial effect among the mouthrinses in this experimental model. Further
experiments need to be conducted to confirm this result.
Funding support: FAPESP
48100315.3.0000.5417
2015/11635-2;
Ethical
approval:
CAAE:
Funding support: Fapesp (#2014/05425-2 and #2014/05234-2).

N37
N38
EFFECT OF PROTEIN ANNEXIN A1 AND SERINE PROTEASE OF

BOTHROPS ATROX ON THE PROCESS OF ANGIOGENESIS: DORSAL
SKINFOLD CHAMBER EXPERIMENTAL MODEL
SMALL INTESTINAL MICROBIOTA OF PREDIABETIC MICE CHANGES

EPITHELIAL BARRIER FUNCTION IN VITRO
Rafaela B. Mols1, Marina de P. Silva1, Anwar Ullah2, Raghuvir K. Arni2, Sonia M.

Oliani1
1
Departament of Biology, So Paulo State University (IBILCE/UNESP), So Jos

do Rio Preto, So Paulo, Brazil.
2
Departament of Physics, So Paulo State University (IBILCE/UNESP), So Jos do
Rio Preto, So Paulo, Brazil.
Contact details:
Rafaela Batista Mols, rafaela.molas@gmail.com, tel. (17) 33434946, cel. (17)
981892888
Angiogenesis is the formation of new blood vessels from pre-existing vascular
network, being essential in embryogenesis, scarring, tissue transplantation and
regeneration, wound healing, and others. Recent studies have shown that the
Annexin A1 (AnxA1) protein is involved in the balance between physiological and
pathological angiogenesis, yet mechanisms and outcomes are still controversial. In
this context, several studies related snake venoms and its isolated components to
endothelial proliferation. In light of the above reports, the aim of our investigation
was to evaluate the effect of serine protease of Bothrops atrox (BaSP ) and mimetic
peptide of AnxA1 protein (Ac2-26), on angiogenesis process, by the dorsal skinfold
chamber model in female Balb/c mice, which was approved by IBILCE/UNESP
Ethical Comettee. The angiogenic potential of Ac2-26, BaSP or AC2-26 + BaSP was
assessed by quantifying the blood vessel before (day four) and after nine days of
treatments, and it showed that there was significant increase in the number of blood
vessels in all groups compared to control. Furthermore, local and systemic
inflamation was evaluated by leukocytes quantification, colorimetric analysis of the
activity of myeloperoxidase and IL-6 and TNF cytokines production. The treatments
did not induce differences in recruiting inflammatory cells and production of
cytokines. Data showed that both, Ac2-26 peptide and BaSP, improves the
angiogenesis process in vivo without inducing systemic and local immune responses
and may be useful pharmacological tools to induce angiogenesis.
Ricardo Beltrame de Oliveira (1), Leandro Pereira Canuto (1), Carla Beatriz
Collares-Buzato (1).
1. Department of Biochemistry and Tissue Biology - IB, UNICAMP, Campinas, So
Paulo, Brazil. Phone (+55) 19 3251-6331.
Gastrointestinal microbiota plays an important role in host health maintenance. Highfat diet (HF diet) induces intestinal microbiota changes and it has been hypothesized
that disturbance in microbial communities may be involved in type 2 diabetes,
obesity, hepatic steatosis and inflammatory bowel diseases. Pathogenic/commensal
bacteria may disrupt intestinal epithelial barrier which is regulated by tight junctions
(TJ) at cell level. The present study aimed to investigate the in vitro effect of small
intestinal microbiota isolated from prediabetic mice fed a HF diet (HFM) or from
those fed a chow diet (CM) in TJ-mediated paracellular barrier using the human
colon adenocarcinoma cell line Caco-2. After 6h, monolayers exposed to HFM
showed significant decrease in transepithelial electrical resistance as compared to
CM and monolayers exposed to Krebs solution without microbiota (Ctrl). Microbiota
suspension also induced increase in epithelial paracellular permeability (Papp) of the
extracelular marker, phenol red (P<0.01) when compared to Ctrl monolayers; this
increase in Papp was greater in HFM in comparison with CM (P<0.001).
Immunocytochemistry analyses showed significant decrease in the junctional content
of the TJ protein Claudin-1 (P<0.0001 HFM vs CM and Ctrl), Occludin (P<0.0001
for both CM and HFM vs Ctrl) and ZO-1 (P<0.05, for both CM and HFM vs Ctrl)
after microbiota exposure. In conclusion, these results suggest that HF diet may
disturb small intestinal microbial communities that in turn can modulate epithelial
barrier function and induce changes in TJ structure of Caco-2 monolayers.
University Ethical Committee Approval (CEUA/UNICAMP n3040-1). Financial
support: CNPq and FAPESP.
Financial Support: FAPESP and CNPQ

Ethical approval: IBILCE/UNESP Ethical Comettee (CEUA), protocolo n112/2015.

N39
N40
EFFECTS OF LONOMIA OBLIQUA VENOM ON ANGIOGENESIS
LEUKOTRIENES FROM MESENCHYMAL STEM CELLS PLAY A ROLE

IN THE SUPPRESSION OF MACROPHAGES
Alessandra Selinger Magnusson(1) Lisiane Bernardi (1) Antnio Frederico Michel

Pinto (3), Bibiana Franzen Matte(1) lvaro Macedo Laureano (4)
Marcelo
Lazzaron Lamers (1,2)
(1)
(2)
(3)
(4)
School of Dentistry, Universidade Federal do Rio Grande do Sul, Rua

Ramiro Barcelos 2492, CEP 90035-003, Porto Alegre, Brazil.
Department of Morphological Sciences, Universidade Federal do Rio Grande
do Sul, Porto Alegre, Brazil
Pontifcia Universidade Catlica do Rio Grande do Sul, Porto Alegre, Brazil
Cell Therapy Center, Hospital de Clnicas de Porto Alegre (HCPA), Porto
Alegre, Brazil
alessandra.magnusson@gmail.com; +5551 84811778; +5551 3308-5011

Lonomia obliqua is a medically important caterpillar which venom present in the
bristles leads to an envenomation syndrome characterized by ecchymosis and
hemorrhage. This suggests the presence of bioactive peptides with potential to have
pharmaceutical interest due to the ability to modulate the behavior of endothelial
cells. The aim of this study is to analyze the potential effects of Lonomia obliqua
venom on angiogenesis. An endothelial cell line (HUVEC) was exposed to different
concentrations (5g/mL, 10g/mL, 20g/mL and 50g/mL) of Lonomia obliqua
brute extract (LOBE). Using flow cytometry, it was observed that none of the doses
affected endothelial cell cycle or apoptosis after 24h of exposition. Spheroids of
HUVEC cells were plated in a 3D-collagen matrix and it was observed that LOBE
(10g/mL, 20g/mL and 50g/mL) induced an increase on cell migration consistent
with the angiogenesis process. Analysis of VE-cadherin dynamics indicates that the
immediate exposition to LOBE (10g/mL) induced a loosening of cell-cell junction,
which corroborates with the hemorrhage observed in the victims. By mass
spectroscopy, it was observed that LOBE possess several potentially bioactive
peptides. Groups of these peptides were isolated by a methanol-based fractioning of
the crude venom. The peptides present in each of the 11 fractions were characterized
by mass spectroscopy and it was analyzed the effects of each fraction on
angiogenesis. These preliminary results suggests that some of the effects of Lonomia
obliqua envenomation are due to the presence of bioactive peptides that modulate the
behavior of endothelial cells.
Funding Support: CAPES, CNPQ,UFRGS.
Talita Mendes da Silva Ventura (1), Joo Paulo Domezi (1); Flvia Amadeu de
Oliveira (1); Cintia Kazuko Tokuhara (1); Camila Peres Buzalaf (2)
(2) Centro de Cincias da Sade, Universidade do Sagrado Corao, Bauru, So
Paulo, Brazil.
Adress contact: Bauru School of Dentistry University of So Paulo - Departament
of Biological Sciences
Alameda Dr. Otvio Pinheiro Brisola, 9-75 - Cidade Universitria - Bauru - So
Paulo, CEP 17012-901.
E-mail: talitaventura@usp.br
Phone: #55 14 3235-8246, #55 14 997695390
Mesenchymal stem cells (MSCs) are multipotent progenitor cells responsible for cell
replacement of adult organisms. They stimulate tissue regeneration and have an
important role in suppressing the immune response through the synthesis of
mediators such as cytokines, indoleamine 2 3-dioxygenase and prostaglandin E2.
Besides, while MSCs synthesize and express the specific receptor for leukotrienes
(LTs), little is known about their involvement in the suppression of the immune
response induced by MSCs. Therefore, the objective was to evaluate the
underappreciated role of LTs, produced by MSCs, on the suppression of peritoneal
macrophages (PMs). For that, MSCs were obtained from 129 (wild WT) and 5-LO
KO (5-lipoxygenase knockout) mice strains. MSCs cultures were evaluated for
cellular markers by flow cytometry. MSCs were plated and treated or not with
MK886, LT inhibitor and the supernatant were collected at 6, 24 and 48h and used as
conditioned medium. After, PMs obtained from WT strain were cultured for 48h
with conditioned medium obtained from MSCs at different conditions, followed by
stimulation with LPS for 24h. PMs activity was evaluated for nitric oxide (NO)
synthesis. The results show that the cells are positive for CD44, CD90, CD73, and
CD105 and negative for CD45 and CD34. PMs cultured in presence of MSC
medium obtained at 6h presented a significant increase in NO synthesis. No
differences were observed for conditioned medium obtained at 24 and 48h. The data
suggest that LTs are produced by MSCs at early time periods and that LTs contribute
to the suppression of macrophages.
Ethical approval: 07/15


N41
N42
3,4-DIHYDROXYCINNAMIC ACID ATTENUATES THE FATIGUE AND

IMPROVES EXERCISE TOLERANCE IN RATS
PROTEOMIC ANALYSIS IN SKELETAL MUSCLE OF FISH DURING

FASTING CONDITION
Estefanny Ruiz Garcia (1)*, Reggiani V. Gonalves (1), Maria do Carmo G. Peluzio
(2), Antnio J. Natali (3), Izabel R.S.C. Maldonado (4), Rmulo D. Novaes (5)
Rafaela Nunes da Silva(1), Edson Assuno Mareco(2), Rondinelli Salomo(1), Bruno

Oliveira da Silva Duran (1), Tassiana Gutierrez de Paula(1), Letcia Gomes de
Pontes(3), Robson Francisco Carvalho(1), Lucilene Delazari dos Santos(3), Maeli DalPai-Silva(1)
(1) Department of Animal Biology, Federal University of Viosa, MG, Brazil;

(2) Department of Nutrition and Health, Federal University of Viosa, MG,Brazil;
(3) Department of Physical Education, Federal University of Viosa, MG,Brazil;
(4) Department of general biology, Federal University of Viosa, MG,Brazil;
(5) Department of Structural Biology, Federal University of Alfenas, MG, Brazil.
3,4-dihydroxycinnamic acid (3,4-DA) is a metabolite produced by the hydroxylation
of chlorogenic acid, a major phenolic phytochemical in various foods, including
fruits, honeybee propolis and coffee (1,2). It has been described that typical
consumption of coffee results in the ingestion of 0.5-1.0 g of chlorogenic acid and
250-500 mg of 3,4- DA per day, is a natural compound with high antioxidant
potential found in various foods. Animals were randomized in four groups with eight
animals per group according to the treatment administered: Vehicle (VE, negative
control): 0.5 ml of 10% ethanol solution (vehicle); DA5: 3,4-DA (5 mg/kg b.w.);
DA25:
3,4-DA
(25
mg/kg
b.w.);
VIT
C
(positive
control): Vitamin C (25 mg/kg). The fatigue test was conducted using an incremental
running protocol on a motor-driven treadmill at a constant slope of 5% with the
starting speed at 10 m/min-1.9) Treadmill velocity was increased by 1 m/min-1 every
3 min and each rat was run until fatigued. Fatigue was defined as the point at which
the animals were no longer able to keep pace with the treadmill and stayed for more
than 10 seconds in contact with the treadmill rear edge. Time to fatigue (min), speed
(m/min). This study showed that animals supplemented with 3,4-DA had higher
exercise tolerance, reduced blood lactate and markers of hepatic oxidation. Blood
glucose and antioxidant enzymes were not affected by this supplementation. 3.4-DA
may have a potential applicability to reducing the fatigue associated with the
exercise.
1)
2)
References
Kang NJ, Lee KW, Shin BJ, Jung SK., Hwang MK., Bode AM, Heo Y-S, Lee H
J, and Dong Z, Carcinogenesis, 30, 321-330 (2009).
Chung TW, Moon SK, Chang YC, Ko JH, Lee YC, Cho G, Kim SH, Kim JG, and
Kim CH, FASEB J., 18, 1670-1681 (2004).
(1) Department of Morphology, Sao Paulo State University, Botucatu, So Paulo,

Brazil.
(2) University of Oeste Paulista (UNOESTE), Presidente Prudente, So Paulo,
Brazil.
(3) CEVAP, So Paulo State University, Botucatu, So Paulo, Brazil
e-mail: rafaelanunes@ibb.unesp.br phone: (014)991751136
Proteomic tools have helped in identifying proteins as biomarkers in different
biological process in fish and it may be advantageous in improving meat quality and
food safety. Fasting/refeeding conditions promotes muscle catabolism/anabolism and
these experiments are widely used to promote compensatory growth in fish. We
globally identified and compared proteins differently expressed in white muscle of
pacu (Piaractus mesopotamicus) after fasting conditions. Two groups of fish were
used: Fasting(F) 30 days of fasting, and Control(C), continuously fed. At the end of
the experiment, muscle samples were collected (n=9) for analysis. There was a loss
of body weight and a decrease in the white muscle fiber diameters (muscle atrophy).
Sarcoplasmic proteins were extracted from white muscle and subjected to proteomic
analysis by shotgun strategy. Proteins were identified and the groups F/C were
compared using bioinformatics tools. Proteins involved in metabolism were
downregulated and proteins related to the cytoskeleton and muscle contraction were
upregulated in F group, such as the parvalbumin (Pvalb1,2 and 4). Pvalb is a
cytosolic Ca2+ buffer that promotes muscle relaxation and is highly abundant in the
white muscles of fish. Pvalb is also an important regulator of the muscle dystrophic
phenotype in mammals, to bind to Ca2+ and probably suppress proteolysis Ca2+dependent. Thus, we believe that the high levels of Pvalb in white muscle of pacu
could be a mechanism for muscle atrophy self-compensation during fasting
condition. The experimental design was approved by the Ethics Committee of the
Biosciences Institute of Botucatu (Protocol=721).
Funding Support: CNPq and CAPES.
Financial support: We would like to thank the FAPEMIG
Acknowledgment: Brazilian Biosciences National Laboratory Mass Spectrometry,

CNPEM.
N43

N44
A HIGH SELECTIVE SYNTHETIC

METALLOPEPTIDASE PHEX
SUBSTRATE
FOR
THE
THE SERUM OF DIETARY RESTRICTED MICE DOWN-REGULATES

UCP1 IN MOUSE DIFFERENTIATED BROWN PRE-ADIPOCYTES
Gabrielly M. D. Chiarantin (1,2), Raquel L. Neves (1,2), Daniela Zanatta (3), Bryan
E. Strauss (3), Maria A. Juliano (1), Adriana K. Carmona (1) and Nilana M. T.
Barros (1,2)
Raissa Guimares Ludwig (1), Beatriz Alves Guerra (1)(2), Andrea Lvia Rocha
(1)(2), Marcelo Alves da Silva Mori (1)(2).
(1) Department of Biophysics, Federal University of So Paulo, So Paulo, So

Paulo, Brazil.
(2) Department of Biology Sciences, Federal University of So Paulo, Diadema, So
Paulo, Brazil.
(3) Translational Research Center for Oncology/LIM24, Cancer Institute of the State
of So Paulo, Faculty of Medicine, University of So Paulo, So Paulo, SP, Brazil.
*Gabrielly M. D. Chiarantin
Department of Biophysics, Federal University of So Paulo.
Address: Pedro de Toledo Street, 669, So Paulo, SP 04039-032.
Institutional telephone: +55 11 5576-4450 (1977).
Mobile telephone: +55 11 99959-9535/ +55 11 97635-9535.
Email: gabydena@gmail.com
The mutated PHEX (Phosphate-regulating gene with homologies to endopeptidase
on the X chromosome) gene is responsible for the most prevalent form of rickets, the
X-linked hypophosphatemia (XLH). PHEX encodes the membrane metallopeptidase
PHEX that belongs to M13 family of peptidases. Our group characterized a selective
specificity for negative amino acids at P1position, the critical interaction of PHEX
with heparin, and identified osteopontin as the first physiological protein substrate
for PHEX. However, except for recombinant PHEX analyses in vitro, studies to
assess cellular proteolytic activity measurements have not been reported. The aim of
the present study is to develop a high selective synthetic substrate for PHEX cellular
detection. We designed and synthesized six FRET peptides based on negative
charges affinity and protein natural sequences. Among the tested peptides, AbzGFSDYEEEK(Dnp)-OH and Abz-RDDSSESK(Dnp)-NH2 showed high kcat/Km
(1100mM-1.s-1 and 734 mM-1.s-1, respectively) and selectivity for PHEX. To evaluate
measurements of endogenous cellular PHEX, osteoblast cell line MG-63 silenced for
PHEX (shRNA-PHEX) was constructed and showed normal cellular viability and
approximately 60% of decreased PHEX expression. Seeded cell lines (1,25.105) for
24h were treated for 1h or 18h with 10 M of Abz-GFSDYEEEK(Dnp)-OH or AbzRDDSSESK(Dnp)-NH2, revealing that Abz-GFSDYEEEK(Dnp)-OH shows a
significant catalytic activity distinction between MG-63 and shRNA-PHEX cells,
indicating that this substrate can monitor endogenous PHEX activity. This work
shows for the first time a substrate for cellular PHEX detection -not only for
recombinant enzymes- and could contribute to better understand the physiological
role of this enzyme.
(1) Departamento de Bioqumica e Biologia Tecidual, Universidade Estadual de

(2) Departamento de Biofsica, Universidade Federal de So Paulo, So Paulo,
Brazil.
Contact details:
Email: raissa.gludwig@gmail.com
Institutional telephone number: (19)35211167
Mobile telephone number: (19)982207272
Obesity has increased in prevalence worldwide alongside its co-morbidities. One
way to lose weight is by increasing energy expenditure, which can be achieved in
mammals by stimulation of brown/beige adipocyte recruitment and function. This
process is usually correlated with an increase in UCP1, a brown/beige adipocytespecific protein that uncouples the electron transport chain to produce heat. In this
study, we aimed to identify and characterize circulating molecules that act cell nonautonomously in adipose tissue to regulate UCP1 levels and function. We used
differentiated immortalized pre-adipocytes from mouse brown adipose tissue (BAT)
transduced with an Ucp1-promoter luciferase reporter construct (UCP1-luc BAT
cells) to screen for mouse sera that regulate Ucp1 levels. The serum samples were
collected from mice subjected to different metabolic interventions (including dietary
regimens, exercise, cold, etc) and incubated with the differentiated cells for 24h.
Serum of mice subjected to caloric restriction (CR) but not CR with dietary oil
supplementation was able to down-regulating by 44-72% the expression of Ucp1 in
vitro in differentiated UCP1-luc BAT cells. This pattern did not correlate with the
protein levels found in BAT from animals where the serum samples were collected.
These data indicate the presence of circulating molecule(s) that controls Ucp1 levels
in vitro, whereas a more complex scenario of Ucp1 regulation exists in mice
subjected to DR in vivo. This/these molecule(s) may bring insights into the
development of new drugs to combat obesity by promoting brown/beige function.
This research was made with the approval of UNIFESP ethics committee and
FAPESP support.
Support: FAPESP, CNPq, and CAPES. Ethical approval: 0317/12.


N45
N46
CYTOTOXICITY AND GENOTOXICITY ASSESSMENT OF THYMUS

VULGARIS L. (THYME) EXTRACT ON BREAST CANCER CELLS (MCF-7)
ANTI-INFLAMMATORY EFFECT OF ROSMARINUS OFFICINALIS L.

(ROSEMARY) EXTRACT ANALYZED ON LIPOPOLYSACCHARIDESTIMULATED MURINE MACROPHAGES RAW 264.7
Leandro Wagner Figueira (1), Jonatas Rafael de Oliveira (1), Daiane de Jesus (1),
(1)

Technology, Univ Estadual Paulista/UNESP. So Jos dos Campos, SP, Brazil. (1)
Fbia Lugli Sper (1), Jonatas Rafael de Oliveira (1), Daiane de Jesus (1), Leandro
Wagner Figueira (1), Cludio Antonio Talge Carvalho (2), Antonio Olavo Cardoso
Jorge (1), Luciane Dias de Oliveira (1)
(1)Department of Biosciences and Oral Diagnosis, Institute of Science and
fabiafarma@hotmail.com / 55 12 99106-8775 / 55 12 3947-9334
leandrowf@live.com / 55 12 99222-8920 / 55 12 3947-9334

Thyme is an aromatic plant originated from Mediterranean region and it has been
used by the world population as food preservative and medicinal plant, its action on
proliferation and genetic mutations has been studied. In this purpose, it was
evaluated the cytotoxicity and genotoxicity of thyme extract on MCF-7. Initially, this
cell line was cultured in Dulbecco's modified Eagle medium (DMEM) with 10%
fetal bovine serum and 1% penicillin-streptomycin (37C and 5% CO2). In 96-well
plates were added 4 x 104 viable cells/well, for cytotoxicity test, and in 24-well plates
were added 2 x 104 cells, for genotoxicity test, both incubated for 24 h. After, the
monolayer was exposed for 5 min to different concentrations of thyme extract (100,
50 and 25 mg/mL) or DMEM (0 mg/mL control group) (n=10/group). Crystal
violet assay was used to check the cell viability. The genotoxicity was analyzed after
24 h exposition to different concentrations of extract. Therefore, micronuclei (MN)
stained with DAPI were counted in fluorescence microscopy. The results were
analyzed by ANOVA and Tukey Test (p0.05). It was verified that the cell viability
was significantly decreased at 100 mg/mL (905%), however at 50 mg/mL (989%)
and 25 mg/mL (948%) there were similarity statistics to control group. Regarding
genotoxicity, the MN formation was similar to non-exposed group. Thus, it can be
concluded that the thyme extract was not cytotoxic and even caused damage to
genetic material of MCF-7.
Rosemary is a medicinal plant originated from the Mediterranean region and presents
several biological effects, including anti-inflammatory activity. The aim of this study
was to evaluate the rosemary extract action on control of the proinflammatory
cytokines production by RAW 264.7. For this end, the cells were cultivated in
Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum and 1%
penicillin-streptomycin under incubation (37C and 5% CO2). Posteriorly, in 24-well
plates was added 5 x 105 cells/well and after 24 h incubation there was exposition for
24 h to rosemary extract (100, 50 and 25 mg/mL) or DMEM (0 mg/mL)
(n=10/group). In the lipopolysaccharide (LPS)-stimulated groups, besides of the
extract, it was added 1 g/mL of LPS from Escherichia coli. Then, the supernatant
was collected in microtubes and stored at -20C for subsequent analysis of
interleukin-1 beta (IL-1) and tumor necrosis factor alpha (TNF-) by ELISA
sandwich method. The absorbance of the wells was assessed by microplate
spectrophotometer (=450 nm) and data were converted to picograms per milliliter
(pg/mL) and analyzed statistically by ANOVA and Tukey Test (p0.05). Regarding
IL-1, it was verified that the concentrations did not stimulate the cytokine
production, being similar to control group. In the LPS-stimulated groups, all
concentrations of treated groups inhibited the synthesis of this cytokine. TNF-
production was inhibited in all treated groups. On the presence of LPS, its synthesis
was significantly lower than the control group. Thus, it can be that rosemary extract
promoted anti-inflammatory effect, inhibiting significantly the proinflammatory
cytokines production.
(1)

N47
N48
THYMUS VULGARIS L. (THYME) EXTRACT SHOWS IN VITRO ANTIINFLAMMATORY EFFECT ON LIPOPOLYSACCHARIDE-STIMULATED

MURINE MACROPHAGES RAW 264.7
CHARACTERIZATION OF A TRANSGENIC MOUSE MODEL OF

PROTEIN DISULFIDE ISOMERASE A1 OVEREXPRESSION: FOCUS ON
CARDIOVASCULAR PHENOTYPE
Fbia Lugli Sper (1), Jonatas Rafael de Oliveira (1), Daiane de Jesus (1), Leandro
Wagner Figueira (1), Cludio Antonio Talge Carvalho (2), Antonio Olavo Cardoso
Jorge (1), Luciane Dias de Oliveira (1)
Carolina Gonalves Fernandes1*, Denise de Castro Fernandes1, Joo Wosniak

Jnior1, Patrcia Nolasco1, Ana Barbosa Marcondes de Mattos2, Cynthia Tararam de
Laurentys1, Victor Eidi Sakaguchi1, Francisco Rafael Martins Laurindo1.

fabiafarma@hotmail.com / 55 12 99106-8775 / 55 12 3947-9334
*Presenting author: carolbiomedica@gmail.com; (11) 2661-5185; (51) 9566-4321.
Thyme is originated from the Mediterranean region and its anti-inflammatory effect
has been related. Given the above, in this study was evaluated the thyme extract
action on proinflammatory cytokines production by RAW 264.7. Therefore, the
macrophages were cultivated in Dulbecco's modified Eagle medium (DMEM) with
10% fetal bovine serum and 1% penicillin-streptomycin under incubation (37C and
5% CO2). Then, in 24-well plates was added 5 x 105 cells/well and incubated for 24
h. After incubation, the cells were exposed to different concentrations of thyme
extract (100, 50 and 25 mg/mL) or DMEM (0 mg/mL) (n=10/group), for 24 h. In the
lipopolysaccharide (LPS)-stimulated groups, besides of the extract, it was added 1
g/mL of LPS from Escherichia coli. Posteriorly, the supernatant was collected in
microtubes and stored at -20C for subsequent analysis of interleukin-1 beta (IL-1)
and tumor necrosis factor alpha (TNF-) by ELISA sandwich method. The test was
read in microplate spectrophotometer (=450 nm) and optical densities were
converted to picograms per milliliter (pg/mL) and analyzed statistically by ANOVA
and Tukey Test (p0.05). It was verified that the IL-1 concentration was
significantly increased only at 100 mg/mL. On the presence of LPS there was
synthesis inhibition this cytokine in the treated groups. Regarding TNF-, all treated
group concentrations inhibited the synthesis of this cytokine, both in the absence as
in the presence of LPS. Thus, it can be that thyme extract presented antiinflammatory effect with significant inhibition of the proinflammatory cytokines
production.
Protein disulfide isomerase(PDIA1) is a thioredoxin superfamily chaperone from

endoplasmic reticulum and the prototype of a family involved in redox protein
folding catalysis. Our previous studies indicate that PDIA1 associates with Nox
complex and is required for growth factor-triggered Nox1 activation and migration in
vascular smooth muscle cells. PDIA1 overexpression promoted Nox1 overactivation.
However, implications of PDIA1 effects in cardiovascular pathophysiology are
unclear. Here, we investigated the cardiovascular phenotype of a transgenic mouse
model of PDIA1(TgPDI) developed by our group, depicting constitutive ubiquitous
PDIA1 overexpression. In TgPDI mice, we evaluated histology of aorta/ blood
vessels (cellularity, elastic fiber content, vessel area, wall thickness), blood pressure,
heart rate (plethysmography) at baseline and after Angiotensin(Ang)-II continuous
infusion (1000ng/kg/day, 28 days). TgPDI mice appeared normally fertile and
exhibited no gross macro or microscopic alterations. TgPDI demonstrated small but
frequent changes in aortic histology, with hypotrophy, elastic fiber disorganization
and reduced cellular content. Importantly, blood pressure measurements showed no
statistically significant differences at baseline, but after Ang-II infusion TgPDI mice
exhibited an increase in early hypertensive response by average 10%(p<0.01 to
p<0.05 at distinct points). Also, heart rate was reduced in TgPDI mice early after
Ang-II infusion (p<0.01). Oxidant generation (lucigenin reductase activity) was
enhanced in TgPDI aortae. Thus, TgPDI model enables exploration of cardiovascular
implications of PDIA1 redox effects. The enhanced responses to Ang-II infusion in
these mice indicate (patho)physiological relevance of PDIA1 overexpression.
Vascular Biology Laboratory, Heart Institute (InCor) - University of So Paulo, So

2
Genetics and Molecular Cardiology Laboratory, Heart Institute (InCor) - University
of So Paulo, So Paulo, So Paulo, Brazil.
Work approved by Scientific/Ethics Commitee from InCor/FMUSP and supported

by FAPESP(Cepid-Redoxoma) and CAPES.

N49
GALECTIN-3
CONTRIBUTES
ENDOMETRIOSIS
N50
TO
THE
DEVELOPMENT
OF
Rmulo Medina de Mattos1, Manuelly Da Conceicao Gomes1, Nathalia de Oliveira

Meireles da Costa2, Eliane Gouva de Oliveira Barros1, Julianna Henriques da Silva1,
Celia Yelimar Palmero1, Felipe Leite de Oliveira1, Luiz Eurico Nasciutti1
1 Institute of Biomedical Science, Federal University of Rio de Janeiro, Rio de
Janeiro, Brazil
2 National Cancer Institute, Rio de Janeiro, Brazil
Contact: romulo_medina@hotmail.com
(21)98082-5885
Backgound: Angiogenesis is essential for endometriotic lesions development and
maintenance. Galectin-3 is related with angiogenesis promotion, and It has been
shown that the expression of galectin-3 is higher in endometriosis than in eutopic
endometrium.
Aim: To evaluate the differences between the endometric lesions developed in wild
type mice (WT) and mice deficient in galectin-3 (Gal-3 -/-).
Methods: Endometriosis experimental model was developed in both animals and the
morphology; angiogenesis and inflammation processes were analyzed.
Results: The developed endometriotic lesions were bigger in WT when compared
with Gal-3 -/- animals, showing the presence of glandular structures and regular
endometrial stroma. In addition, a higher expression of vascular endothelial growth
factor (VEGF) and its receptor Flk-1, and a reduced expression of cyclooxygenase-2
were also observed in WT endometriotic lesions. The analysis of peritoneal
macrophages revealed a greater presence of these cells in WT mices. corroborating
with the above results and the literature data that indicate an important role of
macrophages in the development of endometriosis. The macrophage
immunohistochemical distribution in WT endometriotic lesions seems to be
increased, but it needs to be quantified. Furthermore, arginase activity was higher in
the peritoneal fluid of WT than in Gal-3 -/- animals; nitric oxide (NO) expression
was not observed.
Conclusion: These results indicate that galectin-3 is an important modulator of
endometrosis development, controlling macrophage recruitment and angiogenesis
process.
The experimental procedure used in this study was approved by the Ethics
Committee of Animal Use (CEUA) of Centro de Cincias da Sade (CCS)
Reference number 156/13.
Financial Support: CNPq, Capes, Faperj
IN VITRO EFFECTS OF TWO NANOEMULSIONS CONTAINING THE

ORBGNYA SPECIOSA OIL AND COPAIFERA LANGSDORFFII OIL-RESIN
ON
HUMAN
STROMAL
CELL
CULTURES
OF
EUTOPIC
ENDOMETRIUM AND ENDOMETRIOTIC LESIONS
Henriques da Silva J1, Furtado PS2, Pereira LCB3, Ferrari R4, Barros EG1, Palmero
CY1, de Mattos RM1, Fernandes PD5, Cabral LM2, Nasciutti LE1.
1
Research Program in Cell and Developmental Biology, Institute of Biomedical

Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
2
Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, Federal University
of Rio de Janeiro, Rio de Janeiro, Brazil.
3
Laboratory of Tissue Bioengineering, Applied Metrology Directorate of Life
Sciences, National Institute of Metrology, Quality and Technology - Inmetro, Rio de
Janeiro, Brazil.
4
Institute of Gynecology, Moncorvo Filhos University Hospital, Federal University
of Rio de Janeiro, Rio de Janeiro, Brazil.
5
Pharmacology Laboratory of Inflammation and Nitric Oxide, Cellular and
Molecular Pharmacology Programme, Institute of Biomedical Sciences, Federal
University of Rio de Janeiro, Rio de Janeiro, Brazil.
Contact: henriques.julianna@gmail.com
(21) 987517672
BACKGROUND: The hormonal treatment for endometriosis frequently fails to
eradicate endometriotic implants and a new therapeutic treatment is needed.
AIM: To investigate the in vitro effect of two nanoformulations containing Orbgnya
speciosa babassu oil (SNEDDS 18) and Copaifera langsdorffii copaiba oil-resin
(SNEDDS 18 + COPA) on human eutopic endometrium and endometriotic lesions
stromal cells. METHODS: SNEDDS 18 and SNEDDS 18 + COPA were developed,
not showing toxicity. Eutopic endometrium and ectopic endometrium stromal cells
(EuESCs and EctESCs) were isolated and cell viability, morphology and necrosis
induction were analyzed after 48h of treatment with 50 g/mL of SNEDDS 18 or
SNEDDS 18 + COPA. RESULTS: SNEDDS 18 and SNEDDS 18 + COPA treatment
of EctESCs reduced the cell viability and changed the cell morphology, with actin
microfilaments disorganized, disassembled and disrupted. These treatments also
increased the lactate dehydrogenase release into the extracellular medium of
EctESCs. These effects were not observed in the EuESCs. The nanoemulsion
SNEDDS 18 + COPA exherted a greater effect in EctESCs than in EuESCs.
CONCLUSIONS: Our data indicate that SNEDDS 18 and SNEDDS 18 + COPA
have a greater impact on the behaviour of human endometriotic than on the eutopic
endometrium stromal cells. The better result of the composed nanoemulsion
SNEDDS 18 + COPA in EctESCs suggests the synergistic effect of the copaiba oilresin and babassu oil. These preliminary results supports the idea that these
nanosystems should be further investigated as a novel and valuable alternatives to
treat endometriosis.
These studies were approved by the Research Ethics Committee from Clementino
Fraga Filho Teaching Hospital of the Federal University of Rio de Janeiro (UFRJ),
Rio de Janeiro, Brazil, Protocol No. 23002513.7.0000.5257, 2014,and informed
consent form was obtained from the donors.
Financial support: CNPq, CAPES, FAPERJ
N51
N52
AC2-26 MIMETIC PEPTIDE OF ANNEXIN A1 IS ABLE TO INDUCE AN

ANTI-PARASITIC EFFECT IN HUMAN PLACENTAL EXPLANTS
INFECTED BY TOXOPLASMA GONDII
ANTHELMINTIC
EFFECTS
IN
CAENORHABDITIS
ELEGANS
DETERMINED BY A CYTOTOXIC CATIONIC PEPTIDE FROM A SOUTH
AMERICAN RATTLESNAKE VENOM
Marystela Fvero de Oliveira Cardoso (1), Anglica de Oliveira Gomes (2), Juscile
Brogin Moreli (1), Caroline de Freitas Zanon (3), Ana Elizabete Silva (3), Luana
Ricci Paulesu (4), Francesca Ietta (4) , Eloisa Amlia Vieira Ferro (5), Sonia Oliani
(3)
Dal Mas, C.1; Moreira, J.T.1; Pinto, S.2; Monte, G.G.1; Nering, M.B.1; Oliveira,
E.B.3;
Gazarini, M.L.4; Mori, M.A.2; Hayashi, M.A.F.1
(1) From the Post-graduation in Structural and Functional Biology, Federal

University of So Paulo (UNIFESP), Paulista School of Medicine (EPM), So Paulo,
SP, Brazil. (2) Departament of Structural Biology, Institute of Biological and Natural
Sciences, Federal University of Tringulo Mineiro (UFTM), Uberaba, MG, Brazil.
(3) Department of Biology, Instituto de Biocincias, Letras e Cincias Exatas; So
Paulo State University (UNESP), So Jos do Rio Preto, SP, Brazil. (4) University of
Siena, Italy. (5) Laboratory of Immunophysiology of Reproduction, Federal
University of Uberlndia (UFU), Uberlndia, MG, Brazil.
Corresponding author: Juscile Brogin Moreli, Rua Cristovo Colombo, 2265, So
Jos do Rio Preto, SP, Brazil, 15054-000. Phone.: +55 17 3221 2381 +55 17
996727002. juscielemoreli@gmail.com.
Background: The trophoblast has been considered as an important component to
prevent vertical transmission of parasites. Aims: Considering the studies regarding
the anti-parasitic role of annexin A1 protein (ANXA1) on T. gondii transmission,
this study was conduct to investigated a potential protective effect of ANXA1 in
human placental explants from first and third trimester of gestation. Methods: We
used immunohistochemical and densitometric analyses to verify the expressions of
ANXA1, formyl peptide receptors (FPR1 and FPR2) and COX-2 in the first and
third trimester placentas infected or not with T. gondii. Pharmacological treatments
using mimetic peptide Ac2-26were carried in the placentas from third trimester. The
parasitism rate was performed by -galactosidase assay and prostaglandin E2 (PGE2)
levels in supernatants by ELISA. This study was approved by Research Ethics
Committee from Federal University of So Paulo, Federal University of Uberlndia
and University of Siena. Results: Expressions of ANXA1 are coincident only with
the FPR1 receptor in trophoblastic cells of uninfected and infected groups, with
significant increase in cytotrophoblast of first trimester samples. After peptideAc2-26
treatment, the intracellular parasites decrease and ANXA1 expression increase in
syncytiotrophoblast. The expression of COX-2 and PGE2 levels were lower in the
infected group compared with respective uninfected group without treatment.
Conclusion: Together, our data highlight that Ac2-26 peptide is able to induce an antiparasitic effect in placental explants of the advanced gestational age. In this context,
the major indication is that FPR1 receptor, COX-2 and PGE2 levels may be involved
in the control mechanism of T. gondii infections. Funding support: FAPESP.
Departamento de Farmacologia, Universidade Federal de So Paulo

(UNIFESP/EPM),
So Paulo, Brazil; 2 Departamento de Biofsica, Universidade Federal de So Paulo
(UNIFESP/EPM), So Paulo, Brazil; 3 Departamento de Bioqumica e Imunologia,
Universidade de So Paulo (USP-RP), Ribeiro Preto, Brazil; 4 Departamento de
Biocincias, Universidade Federal de So Paulo (UNIFESP), Santos, SP, Brazil
Crotamine is a positively charged peptide composed by 42 amino acid residues from
the venom of a South American rattlesnake Crotalus durissus terrificus. We have
characterized several biological activities of crotamine in the last few years,
including the antimicrobial, antifungal, antitumoral, antimalarial activities and cellpenetration properties. Crotamine also shows cytotoxic effects, which is mostly
dependent on its ability to target acidic vesicles such as lysosomes, promoting the
disruption of these vesicles and release of their content that triggers the cell death.
The use of Caenorhabditis elegans as an in vivo infection model for evaluation of the
antifungal activity of crotamine, allowed us to observe the lethality of worms at
higher concentrations of this toxin. Then, aiming to investigate the potential
anthelmintic activity of crotamine, we confirmed the concentration and time
dependent nematicide effect of this peptide. Considering the lysosomotropic property
of crotamine observed in tumoral cells, we have also investigated the uptake and
accumulation of this toxin in the acidic vesicles of worms by confocal microscopy
and by fluorimetry. The use of a metachromatic dye acridine orange, which
accumulates mainly in the acidic vesicles, allowed us to confirm the disruption of
these acidic vesicles in the presence of increasing concentrations of crotamine. These
findings provide insights into a new potential application for crotamine, as its
lysosomotropic properties may represent a novel class of anthelmintics with potential
to overcome the problems of resistance of these parasites to the currently available
drugs.
Ethical approval: 171905/13.
Financial support by FAPESP, CNPq and CAPES.

N53
N54
PHARMACOLOGICAL
BONE
MODULATION
THROUGH
OSTEOCLASTS INHIBITION ARRESTS TOOTH MOVEMENT
ACTIN AND ENDOCYTOSIS IN TRYPANOSOMA CRUZI EPIMASTIGOTES

Aline Araujo Alves; Narcisa Leal da Cunha e Silva
Gabriel Schmidt Dolci (1), Natalia Koerich Laureano (1), Anna Christina Medeiros
Fossati (2).
Instituto de Biofsica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brazil
nataliakoerich@hotmail.com
Contact: alinealves@biof.ufr.br, +55 21 3938 6580, +55 21 98827 2060

Trypanosoma cruzi epimastigotes present high endocytic activity through two
specific portals on their anterior region: the flagellar pocket and the cytostomecytopharynx complex. In these forms, actin is concentrated in the anterior region,
near the endocytic portals (Cevallos et al. Exp. Parasitol. 127:249, 2011).
Nevertheless, the participation of actin cytoskeleton on endocytosis is unclear.
Corra and coworkers (Exp. Parasitol. 119:58, 2008) have shown a drastic reduction
in transferrin uptake and alterations in the cytopharynx structure after treatment with
Cytochalasin, a drug that destabilizes actin filaments. However, Cytochalasin target
in epimastigotes is not determined yet. Here we show by cytometry analyses that
both Cytochalasin D and Latrunculin B, a drug that blocks actin polymerization,
were able to reduce tracer uptake, reinforcing actin as the target of these drugs.
Latrunculin B had a stronger effect, suggesting that endocytosis requires actin
polymerization. Electron microscopy confirmed tracer retention in the cytostomecytopharynx of treated cells. Three-dimensional models showed that both drugs
reduced cytopharynx length, leading to its disappearance in some cells. Using the
antibody produced by Cevallos and coworkers, we localized actin around flagellar
pocket and along cytopharynx in epimastigote ultrathin sections. Our results indicate
a relationship between actin and the epimastigote endocytic portals, indicating actin
participation on the initial stages of endocytosis.
(51) 3308-5011 - (51)8302-8988
Introduction: In addition to the cholesterol-lowering, a class of drugs namely statins

seems to enhance osteogenesis and suppress bone resorption, which could represent a
clinical concern during orthodontic treatment. This study aimed to determine
whether atorvastatin (ATV) might affect the orthodontic tooth movement (OTM)
through osteoclasts inhibition. Furthermore, we analyzed potential adverse effects of
ATV on long bone turnover and endochondral ossification. Methods: Rats started to
be administered with ATV (15mg/Kg) or saline, via gavage (n=12, per group), two
weeks prior to the initial OTM. The tooth displacement was measured after 7, 14,
and 21 days, while the maxillary and femur histologic sections were obtained after
14 and 21 days of OTM. The sections were prepared to H&E and TRAP staining,
then histomorphometric analysis was performed Results: Atorvastatin resulted in a
significant decrease of tooth movement (p<0.05), and osteoclasts number (p<0.05).
Independently of drug administration, the OTM increased number of osteoclasts and
reduced bone volume rate, when compared to the control maxillae, without OTM.
Furthermore, it seems that long-term statins administration did not affect femur bone
turnover and endochondral ossification. Conclusions: Considering the methodology
used in this study, we found that atorvastatin is able to minimize orthodontic tooth
movement through osteoclasts inhibition, which could represent a clinical concern.
These results shed light on osteoclasts as a cellular target modulating maxillary bone
metabolism and orthodontic tooth movement.
Support: CAPES, FAPERJ, CNPq.
The experimental design of this study was approved by COMPESQ UFRGS and
CEUA/UFRGS.
Funding Support: CAPES, CNPQ, FAPERGS.

N55
N56
INVESTIGATION OF THE MACROPHAGES ADHESION ON DIAMONDLIKE CARBON FILMS CONTAINING TiO2 NANOPARTICLES
EXPRESSION AND CHARACTERIZATION OF LEISHMANIA MAJOR

TELOMERASE REVERSE TRANSCRIPTASE CATALYTIC DOMAIN RT
C.C. Wachesk1, J.A. Portes2, S.H. Seabra2. P.G. Theofilo2, P., V.J. Trava-Airoldi3,
A.O. Lobo1, F.R. Marciano1
Vitor Luiz da Silva, Maria Alejandra Viviescas1, Maria Isabel Nogueira Cano1
Genetics Dept.1, Institute of Biosciences of Botucatu, Universidade Estadual
Paulista, UNESP, Botucatu, So Paulo, Brazil.
Laboratory of Biomedical Nanotechnology - IP&D / UNIVAP So Jos dos

Campos, SP, Brazil.
2
Laboratory Technology in Cell Culture UEZO - Rio de Janeiro, RJ, Brazil,
3
Laboratory Associated of Sensors and Materials - INPE - So Jos dos Campos, SP,
Brazil.
Leishmania telomeres are composed of hexonucleotide TTAGGG repeats maintained

by the action of telomerase. The parasite telomerase ribonucleoproteic complex is
minimally composed of a protein component TERT (telomerase reverse
transcriptase) that catalyses the addition of telomeric repeats and an RNA component
(TER) that associates with the protein component and serves as template for telomere
addition by telomerase. The TERT component varies considerably among different
species, but keeps some structurally conserved domains that serve different
functions. The catalytic domain is named RT (retrotranscriptase) and contains the
motifs 1, 2 and A-E, being responsible for telomerase activity. The aim of this work
is characterize the RT domain of Leishmania major TERT and to establish a
purification protocol for the heterologous protein expressed in bacterial cells
(Escherichia coli, Bl21). Bioinformatics studies show structural conservation among
L. major TERT RT domain and other retrotrancriptases described so far. L. major
TERT RT domain was cloned into the pET28a+ expression vector to allow the
recombinant protein to be expressed with a N-terminal 6xHis-tag in order to facilitate
protein purification. Preliminar mini-induction and expression tests showed that the
recombinant protein is expressed in the soluble fraction of the bacterial extract. We
are currently standardizing the purification protocol. Purification of L. major TERT
RT domain will make possible to perform biochemical assays that might give
insights about the catalytic mechanism of telomeric repeat in L. major.
*Cristiane da Costa Wachesk NANOBIO, IP&D, UNIVAP, Av. Shishima Hifumi

2911 Urbanova CEP 12240-000, SJCampos, SP, Brazil. cris_cw@hotmail.com
(12) 981247203
DLC coatings are useful for the designing of the biocompatible surfaces for
biomedical implants [1]. In the last recent years, various authors have reported the
production and characterization of TiO2-DLC films for biological applications [2-3].
The interaction of cells with the surface of materials has an extreme importance for
the effective use of these medical implants [4]. The purpose of the current
manuscript is to investigate the cell adhesion in direct contact with DLC and TiO2DLC coatings. The films were deposited on stainless steel disks using plasma
enhanced chemical vapor deposition technique. Raman scattering spectroscopy
characterized the film structure. The contact angle of the samples was measured with
two different liquids in order to calculated the surface free energy and its work of
adhesion. The samples were surgically inserted into the peritoneal cavity of CF-1
mice and maintained for 7, 15 and 30 days. As the concentration of TiO2 increased,
the films increased the macrophage viability, becoming more thermodynamically
favorable to cell adhesion. The increasing number of macrophages indicate a higher
adhesion between the cells and the films. Adherent macrophages fixed on the
samples were quantified on scanning electron micrographs.
Keywords: Diamond-like carbon, titanium dioxide, cell adhesion, macrophages

REFERNCIAS:
[1] Ahmed M. H., et al. Characteristic of silicon doped diamond like carbon thin
films on surface properties and human serum albumin adsorption. Diamond and
Related Materials (2015) Volume 55, , Pages 108116
[2] Goodman, S. B. et al. The future of biologic coatings for orthopaedic
implants. Biomaterials, (2013) v. 34, n. 13, p. 3174-3183..
[3] Tsai P. C., et al. Effects of nanotube size and roof-layer coating on viscoelastic
properties of hybrid diamond-like carbon and carbon nanotube composites. C A R B
O N 8 6 ( 2 0 1 5 ) 1 6 3 1 7 3
[4] Wachesk C. C. Estudo da interao de clulas L929 com superfcies recobertas
por filmes de carbono tipo-Diamante (2011). Dissertao apresentada ao Programa
de Ps Graduao em Engenharia Biomdica da Universidade do Vale do Paraba.

O HOST-PATHOGEN INTERACTION
O1
O2
THE IMPACT OF TRYPANOSOMA CRUZI P21 ON HOST TISSUE

FIBROSIS
THE ROLE OF NEUTROPHILS IN CONTROLLING THE PARASITE

BURDEN OF TOXOPLASMA GONDII ON MURINE MACROPHAGES
Nadjania Saraiva de Lira Silva (1) , Thaise Lara Teixeira (1), Aline Alves da Silva
(1), Patrcia de Castilho (1), Claudio Vieira da Silva (1).
Iasmim P Santos, Helene S Barbosa, Rafael M Mariante
(1)Laboratrio de Trypanosomatdeos da Universidade Federal de Uberlndia,

Uberlndia, Brasil.
(2)Laboratrio de Histologia da Universidade Federal de Uberlndia, Uberlndia,
Brasil.
nadjaniasaraiva@gmail.com. Universidade Federal de Uberlndia. Av. Amazonas Umuarama, Uberlndia - MG, Brasil. Telefone: (34)92230357.
Background: Chagas disease is a serious public health problem, caused by
intracellular protozoan Trypanosoma cruzi. This disease is characterized by acute
and chronic phase, and the last, in cases majority, results in fibrosis in the heart
muscle tissue. Aims: Evaluate fibrogenic activity Trypanosoma cruzi P21 from
chronic inflammation model in polyester sponge. Methods: To analyze the tissue
fibrosis formation were used rP21 protein (obtained by transfecting bacterium
Escherichia coli strain BL21 with plasmid pET-28th (+) with the gene rP21, bacterial
extract (B.E.), and PBS for control. We used 18 male C57BL / 6 mice in total, six per
group, for polyester sponge incision of 1.2 cm of diameter in the inter-scapular
region. After surgery the animals were treated for 9 days every 72 hours with PBS,
40ug/mL of rP21 or B.E. In the end of the treatment, they were euthanized according
to the American Medical Association Veterinary and the Ethics Committee in
Animal Experimentation of the Federal University of Uberlandia (CEUA/UFU059/08). The sponges histological sections were stained with Picro-Sirus for collagen
analysis. Collagen was measured from the electron microscope and polarization (to
see collagen I and III) with the help of the program Image J. Results: The rP21
treatment induced higher collagen deposition in the extracellular matrix compared to
the control group and to the B.E treated group. Type I collagen was more abundant
than type III. Conclusion: we proposed that Trypanosoma cruzi P21presents profibrogenic activity. This activity can be an important factor that intensify cardiac
fibrosis during Chagas disease.
(1)
(2)
(3)
(4)
Laboratory of Structural Biology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro,

Brazil.
Phone: +55(21)25621018 +55(11)976477443
iasmimpolido@gmail.com
Toxoplasma gondii is an obligate intracellular parasite that infects several warmblooded species. The interaction between parasite and immune cells is determinant
for T. gondii survival and dissemination. Macrophages act as Trojan horses and, once
infected, can migrate to tissues, therefore disseminating infection. Neutrophils act in
the control of parasite load in macrophages infected with trypanosomatids. Its
implication in T. gondii infection is, however, unclear. Here, we investigate the role
of neutrophil in controlling the parasite burden in macrophages infected with T.
gondii. Peritoneal macrophages from either C57BL/6 or Swiss mice were infected
for 4-h with tachyzoites from either ME49 or EGS strains, respectively. Unbound
parasites were washed away and cells were cultivated for 48-h in the presence or
absence of neutrophils. The rate of infected macrophages was about 45% for ME49
and 68% for EGS, irrespective the presence of neutrophils. The number of
intracellular parasites, however, varied substantially [% infected cells without x with
neutrophils (parasite burden) ME49: 23.8% x 35.9% (<10), 17% x 11.1% (10-30),
4.1% x 0.25% (>30); EGS: 45.5% x 51.8% (<10), 17.9% x 16.4% (10-30), 4.4% x
0.6% (>30)], indicating that neutrophils act inhibiting T. gondii proliferation. This
inhibitory effect was partially prevented by an inhibitor of neutrophil elastase (an
important neutrophil microbicide agent). Therefore, we have evidences that
neutrophils control parasite burden in T. gondii infected macrophages, where
neutrophil elastase may play a role. This study can bring valuable information on the
mechanisms that orchestrate parasite growth inside host cells.
Supported by Oswaldo Cruz Institute/Fiocruz.

O3
O4
P. GRANATUM L. INCORPORATION INFLUENCE ON RESILIENT

MATERIALS USED IN DENTURE MANUFACTURING: ACTION
AGAINST C. ALBICANS
EFFECTS OF ETHANOLIC EXTRACTS OF ZINGIBER OFFICINALE

ROSCOE (GINGER) IN THE CONTROL OF IN VITRO AND IN VIVO
TOXOPLASMA GONDII INFECTION
Rafaela A. S. Alavarce (1), Marcelo M. R. Lopes (2), Paula S. B. H. Karam (3),

Eliane F. da Costa (1), Nara L. M. Almeida (1), Karin H. Neppelenbroek (4), Carla
A. Damante (3), Vinicius C. Porto (4), Vanessa S. Lara (1).
Natlia Carnevalli Miranda* (1), Layane Alencar Costa Nascimento (1), Neire
Moura de Gouveia (2), Foued Espindola Salmen (2), Neide Maria Silva (1).
(1) Department of Stomatology (Pathology), Bauru School of Dentistry, University

of So Paulo, Bauru, So Paulo, Brazil.
(2) Bauru School of Dentistry, University of So Paulo, Bauru, So Paulo, Brazil.
(3) Department of Prosthodontics (Periodontics), Bauru School of Dentistry,
University of So Paulo, Bauru, So Paulo, Brazil.
(4) Department of Prosthodontics, Bauru School of Dentistry, University of So
(5)
Alameda Dr. Otvio Pinheiro Brizola, n 9-75, 17012-901 Bauru, So Paulo, Brazil.
Tel.: +55 14 32358378; +55 14 982300106
E-mail: rafaela@fob.usp.br
Background: Even though there has been therapeutical advances, there has been a
higher increase of infectious diseases by opportunistic fungi. Denture Stomatitis
(DS) is the most prevailing and permanent problem for denture wearers, strongly
associated with the presence of C. albicans. Antifungal agents incorporated to
temporary soft lining materials may provide a slow and continuous release of drugs.
Within this context, phytotherapeutical drugs, such as P. granatum L., may play an
important role in DS treatment.
Aims: This study aims to evaluate in vitro if a resilient denture liner and a tissue
conditioner, incorporated with phytotherapeutical P. granatum L., show
antimicrobial activity against C. albicans.
Methods: The antimicrobial action was assessed on biofilms made by C. albicans
ATCC 90028 and SC 5314 strains, trough XTT assay. Such biofilms were induced
and kept in aerobic incubation conditions or microaerophilic ones, at 37C for 12h
and 24h, under conditioned medium, obtained by indirect contact with the materials
either included or not with the phytotherapeutical drug, in concentrations of 100, 50
and 25mg/mL.
Results: From the XTT assay, in all the phytotherapeutical concentrations, there has
been a considerable inhibition of the fungal metabolism, varying from 35 to 98% in
relation to the materials without the phytotherapeutical drug (CS and CC). Most of
the cases presented a MIC (Minimum Inhibitory Concentration) of 25mg/mL.
Conclusion: The incorporation of P. granatum extract into the resilient materials
maybe a promising alternative therapy in the DS prevention and treatment.
(1) Laboratory of Immunophatology of the Institute of Biomedical Sciences, Federal

University of Uberlndia, Uberlndia, MG, Brazil.
(2) Laboratory of biomolecules of the Institute of Genetics and Biochemistry,
Federal University of Uberlndia, Uberlndia, MG, Brazil.
* Corresponding
+5534992114433
author:
natty_vet@hotmail.com
Telephone
contact:
Background: Toxoplasmosis is an infection caused by the obligate intracellular

parasite Toxoplasma gondii, which affects all warm-blood animals, including
humans. In Brazil, most of the adult population (50-80%) is seropositive for
infection. Medicinal plants have been studied to control some parasitic infections.
The extract of Zingiber officinale Roscoe is known to have antiinflammatory,
anticancer, antioxidant and antimicrobial effects.
Aims: Evaluate the in vitro and in vivo effect of the crude ethanolic extract of
Zingiber officinale Roscoe (EExtZO) in experimental toxoplasmosis.
Methods: For in vitro experiments, cell viability and parasitism were evaluated. For
parasitism analysis, NIH/3T3 fibroblasts were infected with RH-2F1 strain and
treated with 30 - 200g/ml of EExtZO. In vivo, C57BL/6 mice were orally infected
with 20 ME-49 cysts and treated with 2.5mg/day of EExtZO by gavage during 7
days after infection. The clinical parameters and histological analysis were evaluated.
Results: In vitro, the treatment with 120g/ml and higher concentrations controlled
the parasite proliferation in NIH/3T3 cells. In vivo, C57BL/6 mice infected and
treated with EExtZO presented similar body weight loss and morbidity score to nontreated and infected animals (control). The EExtZO decreased the tissue parasitism in
the small intestine and lung of infected mice, despite not statistically significant. In
addition, the EExtZO induced an increase of the intestinal Paneth and goblet cells in
infected mice.
Conclusion: This study suggests that EExtZO presents a potential anti-Toxoplasma
activity.
Ethical approval and funding support: CAPES, CNPq and FAPEMIG. Approved by
CEUA, number 006/15.
Conflict of interest statement: None declared.

Information on ethical approval: This work was approved by ethics committee,
CAAE: 44951715.6.0000.5417
Funding support: Fapesp

O5
O6
THE ROLE OF THE CYTOSKELETON OF ENDOTHELIAL CELLS

AGAINST PLASMODIUM BERGHEI
MYCOBACTERIUM SMEGMATIS AFTER CHOLESTEROL CONSUMPTION

DOES CHANGES IN BIOACTIVE MOLECULES OF CELL WALL
Daniela Debone1, Luana dos Santos Ortolan2, Michelle Klein Sercundes1, Flvia
Afonso Lima2, Claudio Romero Farias Marinho2, Sabrina Epiphanio1
Ana Cristina Doria dos Santos (1), Victor Hugo de Souza Marinho (1), Edilene
Oliveira da Silva (2), Jos Luiz Martins do Nascimento (2), Chubert Bernardo Castro
de Sena (1).
1
2
Faculty of Pharmaceutical Sciences University of So Paulo

Institute of Biomedical Sciences University of So Paulo
(1)
Background. Infections by Plasmodium spp. can lead to a serious respiratory

(2)
condition with pulmonary complications, named acute lung injury and acute
respiratory distress syndrome (ALI/ARDS). Acute inflammation, dysfunction and
(3)
increased permeability of the pulmonary alveolar-capillary barrier and consequent
formation of edema characterize this syndrome. Stimuli such as VEGF and TNF
can directly increase endothelial permeability through actin rearrangement, via RhoGTPases signaling, leading to dysfunction of the endothelial barrier. DBA/2 mice
infected with Plasmodium berghei ANKA develop ALI/ARDS similar to that
observed in humans. Aims. The purpose of this research was to assess cytoskeletal
changes in DBA/2 mice primary microvascular lung endothelial cells (PMLEC) and
verify the signaling pathways of the Rho-GTPases in the presence of P. berghei
ANKA-infected red blood cells (iRBC). Method. PMLEC were stimulated by TNF,
VEGF or IFN followed by incubation with iRBC. Immunofluorescence assays were
performed to analyze actin rearrangement and microtubules destabilization. Western
blot for RhoA and Cdc42 proteins were conducted to assess alterations in signaling
pathways of Rho-GTPases. Results. P. berghei ANKA, VEGF, TNF and IFN
stimuli, in association or not, caused morphological disturbances in actin
microfilaments of PMLEC and an increase of intercellular spaces. The transwell
permeability assay showed that iRBC induced an increase of microvascular
permeability in PMLEC. Moreover, immunofluorescence images showed probable
microtubules destabilization, in these cells, caused by iRBC. Conclusion. The present
data demonstrate the involvement of P. berghei ANKA and other stimuli in
cytoskeleton disorganization and alveolar-capillary barrier dysfunction, which may
contribute to ALI/ARDS pathogenesis. Ethics Statement. Research approved by the
Animal Health Committee of the Biomedical Sciences Institute of the University of
So Paulo (CEUA n051 page 32 book 03).
Funding Support. CNPq and FAPESP.
(1) Laboratory of Structural Biology, Institute of Biological Sciences, Federal

University of Par, Belm, Par, Brazil.
(2) Laboratory of Protozoology, Institute of Biological Sciences, Federal University
of Par, Belm, Par, Brazil.
(3) Laboratory of Molecular and Cellular Neurochemistry, Institute of Biological
Sciences, Federal University of Par, Belm, Par, Brazil.
Ana Cristina Doria dos Santos- Federal University of Par, Institute of Biological
Siences, Laboratory of Structural Biology, Augusto Corra Av., 01, Guam, 66075110, Belm, Par, Brazil. Phone/Fax/institutional: 0559132018232; Phone/mobile:
05591980230323; Email address: anadoria@ufpa.br ; tina.biomed@gmail.com .
The cell wall is the hallmark of Mycobacterium species due to be rich in bioactivity
lipids and glycoconjugates, such as phosphatidylinositol mannoside (PIMs),
lipomannan (LM) and lipoarabinomannan (LAM). Much of these components are
essential during infection to immunemodulate the infected host cells: lung
macrophage. The infected cells become foamy macrophages due to accumulation of
cholesterol, known as alternative source of energy and carbon to maintain the bacilli
able to grow. The present work investigated the possible modulations of cell wall
using the unpathogenic Mycobacterium smegmatis after cholesterol consumption in
minimal media, mimicking the intracellular environment of infection. Filipin staining
showed that M. smegmatis was able to accumulate the cholesterol, what induced slow
growth in minimal medium. The analysis of lipid fractions from cell wall in thin layer
chromatography (TLC) shows that cholesterol consumption induced less
accumulation of PIMs during growth at stationary phase. The fraction of
glycoconjugates after separation by electrophoresis and staining for glycans, showed
that LAM after cholesterol consumption in minimal medium becomes bigger than the
ones from control group in 7H9 medium (25-30 kDa to 30-50 kDa), resembling the
ManLAM from pathogenic species. Thus, our results show that unpathogenic species
of mycobacteria also can accumulate and catabolize the cholesterol, changing its
physiology and cell wall architecture, suggesting possible essential adaptation for the
new environment during infection.
Funding support: CAPES, Universal-CNPq

O7
O8
NEW ARCHITECTURE OF MYCOBACTERIAL CELL WALL AFTER

CHOLESTEROL CONSUMPTION INDUCES BACILLUS RESISTANCE
Pedro Henrique de Aviz Silva (1); Ana Cristina Doria dos Santos (1); Jos Luiz
Martins do Nascimento (2); Chubert Bernardo Castro de Sena (1).
(1)
(2)
(1) Laboratory of Structural Biology, Institute of Biological Sciences, Federal

University of Par, Belm, Par, Brazil
Sciences, Federal University of Par, Belm, Par, Brazil
Pedro Henrique de Aviz Silva, Federal University of Par, Institute of Biological
05591983624459; Email adress: pedro.aviz@icb.ufpa.br
Mycobacterium tuberculosis overcomes the mechanisms mediated immune response
and are able to respond and survive under different conditions of oxidative stress
developed within pulmonary macrophages. For this, the accumulation of cholesterol
in the host cell provides an alternative source of energy and carbon for keeping their
physiological functions. An important key factor for the success of the bacillus in the
spread of infection is unusual structure of the cell wall. The aim of this work is to
evaluate the role of cholesterol consumption by mycobacteria to it becomes
resistance to hydrogen peroxide stress. We used the nonpathogenic Mycobacterium
smegmatis grown in 7H9 broth supplemented with glycerol (control group) and
minimal medium supplemented with cholesterol. Cells in early stationary-phase were
treated with 20 mM H2O2 for three hours to evaluate the cell viability and cell wall
components. We found that cholesterol consumption provided the bacillus resistance
to chemical stress, not affecting the growth. The glycolipids and glycoconjugate
fractions were analyzed by thin layer chromatography (TLC) and SDS-PAGE. The
data showed that PIMs, GPL and TDM were not affected by the H2O2 treatment.
However the amount of LM and LAM were reduced after H2O2 treatment only when
the cholesterol was used as carbon and energy source. Thus, the consumption of
cholesterol allowed the biosynthesis of molecules resistant to action of H2O2, and
suggests the development of strains resistant to stress of the natural immune
response, it propose that similar changes can occur within granulomas, contributing
to the evolution of tuberculosis.
Supported by: CAPES; Universal-CNPq
DETECTION OF CIRCOVIRUS IN CRIMSON-BELLIED PARAKEET

(PYRRHURA PERLATA), BY NEGATIVE STAINING TECHNIQUE FOR
TRANSMISSION ELECTRON MICROSCOPY
Fernanda Falco de Queirz; Gilberto Pereira de Oliveira Jnior; Ana Maria Cristina
Rebello Pinto da Fonseca Martins; Marcia Helena Braga Catroxo
Electron Microscopy Laboratory, Biological Institute of So Paulo, SP, Brazil.
The Avian circovirus belongs to Circoviridae family and Circovirus genus. The
circovirosis is one of the major diseases that affect psittacines, causing heavy losses
and damage to creators. The main symptoms described are characterized by loss and
feather dystrophy and defects in its beak, in addition to severe anemia and leukopenia,
immune deficiency. Transmission may occur via inhalation or excretions of viral
particles expelled from the powder of feathers. Pyrrhura perlata species are
distributed from the Centre and southwest of the Amazon to the north-eastern Bolivia.
Although not classified in the threatened category, this population has suffered a
significant decline due to habitat loss. In July 2015, were sent to the laboratory of
electron microscopy of Biological Institute of So Paulo, SP, Brazil, stool samples of
a Crimson-bellied parakeet (Pyrrhura perlata), with clinical suspicion of circovirose.
The bird was suffering from gastrointestinal problems, changes in the skin and
mucous membranes and in coloring and structure of feathers. The sample was
processed by negative staining technique, being suspended in 0,1M phosphate buffer
pH 7.0, placed in contact with metal grids, previously coated with collodion film and
carbon, drained with filter paper, negatively contrasted with ammonium molybdate
2% and pH 5,0. By the transmission electron microscope Philips EM 208, particles
were viewed with similar morphology to circovirus, non-enveloped, spherical,
isometric and characterized as "complete" and "empty", measuring between 17 and 20
nm of diameter. This is the first occurrence of circovirus in Crimson-bellied Parakeet
(Pyrrhura perlata).

O9
O10
HERPESVIRUS DETECTION IN EXOTIC BIRDS PSITTACIFORMES BY

ESSENTIAL BIOACTIVE COMPONENTS OF MYCOBACTERIAL CELL

WALL ARE CHANGES AFTER CHOLESTEROL CONSUMPTION
Marcia Helena Braga Catroxo; Fernanda Falco de Queirz; Gilberto Pereira de

Oliveira Jnior; Ana Maria Cristina Rebello Pinto da Fonseca Martins
Jaqueline Batista de Lima (1), Victor Hugo de Souza Marinho (1), Ana Cristina
Doria dos Santos (1), Jos Luiz Martins do Nascimento (2), Chubert Bernardo Castro
de Sena (1)
Electron Microscopy Laboratory, Research and Development Center for Animal

Health, Biological Institute of So Paulo, SP, Brazil.
Exotic birds psittaciformes occupy wide part of the avian species in the world. Viral
diseases such as Pacheco's disease, however, are observed both in captive birds as in
wild putting at risk the conservation of some endangered species. The etiologic agent
(PsHV-1) is a member of the Herpesviridae family and lltovirus genus. The main
clinical signs are represented by anorexia, somnolence, lethargy, ruffled feathers,
yellowish diarrhea, regurgitation and neurological signals with progression to death
within 48 hours. The transmission occurs via contact with contamined food, water,
feces or from an ill bird as well as from carriers, who appear asymptomatic. From
January 2014 to April 2016, were sent to Biological Institute of So Paulo, SP,
Brazil, stool and organ fragments samples of 22 birds psittaciformes exotic. The
samples were processed for transmission electron microscopy utilizing negative
negative staining (rapid preparation), immunoelectron microscopy and
immunocytochemistry (immunollabeling with colloidal gold particles) techniques.
On the transmission electron microscopy isometric herpesvirus particles, exhibits
enveloped and non-enveloped particles and showing nucleocapsids stain penetrated
and non-stain penetrated measuring 120-200 nm of diameter were visualized in
samples of 10 birds ((1 Anodorhynchus hyacinthinus, 1 Ecletus rorattus, 1 Pionites
leucogaster, 2 Deroptyus acciptrinus, 1 Poicephalus senegalus, 1 Psittacula krameri,
1 Aratinga jandaya, 1 Ara chlropterus and 1 Ara ararauna). The presence of
aggregates formed by antigen-antibody interaction, characterized the positive result
obtained at the immunoelectron microscopy technique for herpesvirus. In the
immunocytochemistry technique, the antigen-antibody reaction was strongly
enhanced by the dense colloidal gold particles.
(1) Laboratory of Structural Biology, Institute of Biological Science, Federal

University of Par-PA, Brazil
Science, Federal University of Par-PA, Brazil.
Jaqueline Batista de Lima- Federal University of Par, Institute of Biological
05591983388347;
Email
address:
Jaqueline_batista17@yahoo.com.br
;
jaquelinebiotecnologia@outlook.com.
The development of tuberculosis is related to the accumulation of host cholesterol
and bioactive lipids and glycoconjugates of mycobacterial cell wall. These features
are know to confer the immunomodulatory abilities to shutdown the immune
response of the host cell (macrophage) and develops the foamy macrophages, a
hallmark of tuberculosis disease. The aim of this work is to evaluate the role of
cholesterol during the biosynthesis of bioactives cell wall components. We used the
nonpathogenic Mycobacterium smegmatis, grown in Middlebrook 7H9 broth (control
group) or minimal medium containing glycerol or cholesterol as carbon and energy
source, mimicking the nutritional conditions found inside the macrophages. Lipid
fractions from extraction of cell wall were analyzed by thin layer chromatography
(TLC). Our data showed that cholesterol consumption maintained the biosynthesis
and accumulation of the essential cell wall components: phosphatidylinositol
manosides (PIMs) and trehalose dimicolate (TDM). Differently, we observed that
cholesterol consumption decreased the accumulation of mycolic acids and increased
the accumulation of glycopeptidolipids (GPLs). The accumulation of mycolic acids
was confirmed by auramine fluorescence staining. Hexadecane partitioning showed
that these modulations in cell wall induced less cell surface hydrophobicity. All data
were compared with control group. These results clearly show the essential role of
cholesterol to modulate the cell wall biosynthesis during nutritional shortage. It
suggests that such modulations might also changes the bioactivity of cell wall during
the host infection, maintaining the bacilli available to develop the tuberculosis
diseases.
Funding support: CAPES, Universal-CNPq
O11

O12
EFFECTS
OF
ETHANOLIC
EXTRACT
OF
TRICHODERMA
STROMATICUM ON EXPERIMENTAL CEREBRAL MALARIA IN C57BL/6
MICE
EFFECT OF MK886 TREATMENT, AN INHIBITOR OF 5-LIPOXYGENASE

PATHWAY, IN PARASITE BURDEN DURING ACUTE EXPERIMENTAL
TOXOPLASMOSIS
Yusmaris Josefina Cariaco Sifontes (1), Wnia Rezende Lima (1), Layane Alencar
Costa Nascimento (1), Romulo Oliveira de Sousa (1)*, Jane Lima dos Santos (2),
Neide Maria Silva (1)
Ester Cristina Borges Araujo(1), Mrio Czar de Oliveira(1), Marcos Paulo Oliveira
Almeida(1)* and Neide Maria Silva(1)
(1) Laboratory of Immunophatology of the Institute of Biomedical Sciences, Federal

University of Uberlndia, Uberlndia, MG, Brazil.
(2) State University of Santa Cruz, Ilhus, BA, Brazil.
Corresponding author: yusmaris_c@hotmail.com

Telephone contact: +5534991630872 (mobile); +55 34 3225 8575 (institutional)
Background: Cerebral malaria is the most fatal consequence of malaria. Currently,
with the rapid spread of drug-resistant strains is necessary to develop new
alternatives of treatment. The fungal extracts can be a potential source of antimalarial
compounds. In this regard, a fungal genus Trichoderma has gained relevance in the
last decades from which have been characterized volatile and nonvolatile compounds
with antimicrobial properties.
Aims: Evaluate the in vivo effect of crude ethanolic extract of Trichoderma
stromaticum in experimental malaria of C57BL/6 mice infected with Plasmodium
berghei ANKA strain.
Methods: We inoculated C57BL/6 mice with red blood cells infected with
Plasmodium berghei and treated them with 2,5mg/day of crude ethanolic extract of
the fungus T. stromaticum during 18 days. Parasitaemia, weight loss, morbidity and
survival rate of the animals were evaluated.
Results: C57BL/6 treated with crude ethanolic extracts of T. stromaticum exhibited
greater weight loss in the first five days of infection that untreated and infected mice.
Infected mice treated with fungus extract showed higher survival rate, lower
parasitaemia and morbidity score compared with untreated group.
Conclusion: Administration of crude ethanolic extract of T. stromaticum is able to
diminish the parasitaemia and clinical signs associated with malaria at different time
points and protect mice against mortality on experimental cerebral malaria.
Ethical approval: This work was approved by Comisso de tica no Uso de Animais
(CEUA) of the Federal University of Uberlndia.
Funding support: CAPES, CNPq and FAPEMIG.
(1) Laboratory of Immunopathology, Institute of Biomedical Sciences, Federal

University of Uberlndia, Uberlndia, Minas Gerais, Brazil.
* Av. Par, 1720, Uberlndia, CEP 38400-902, Minas Gerais, Brazil; E-mail:
marcospaulooliveiraalmeida@hotmail.com; Telephone: Institutional +55 34 3225
8575; Mobile +55 34 99122 7493.
Background: Toxoplasma gondii induces type 1 immune response in infected hosts
and it can lead to serious consequences mainly in immunocompromised individuals
and pregnant women. 5-Lipoxygenase (5-LO) is an enzyme required for production
of lipid mediators leukotrienes and lipoxins and interferes in others parasitic
infections.
Aims: The aim of this study was investigate the participation of 5-LO during
experimental toxoplasmosis.
Methods: C57BL/6 mice were daily treated with MK886 (inhibitor of 5-LO
pathway) or vehicle and infected with 20 ME49 T. gondii cysts. Clinical signs and
mortality were observed daily. At day 7 post-infection, small intestine and liver
were collected for histological analysis. In order to verify liver damages, glutamatepyruvate transaminase (GPT) was measured in serum samples.
Results: MK886 treatment did not alter morbidity or body weight of infected mice.
Additionally, MK886 and vehicle treated mice presented similar inflammatory scores
and goblet cell numbers in small intestine. However, MK886 treatment increased
inducible nitric oxide synthase (iNOS) positive cells and parasite burden in small
intestine. Regarding to liver, MK886 treatment decreased parasitism and enhanced
iNOS positive cell numbers. Furthermore, T. gondii infection increased GPT levels,
especially in MK886 treated mice.
Conclusion: These data suggest that although 5-LO pathway inhibition increases
iNOS positive cells in small intestine and liver of infected mice, different
mechanisms are involved in parasite elimination in these infection sites.
Ethical Approval: This work was approved by Ethics Commission on Use of
Animals (CEUA) Federal University of Uberlndia with protocol number 078/14.
Funding Support: CAPES, CNPq, FAPEMIG.

O13
O14
TLR7/TLR8 ACTIVATION EXPRESSED BY TROPHOBLAST CELLS

MODULE CYTOKINE PRODUCTION BY MYELOID DENDRITIC CELLS
AND PLASMACYTOID DENDRITIC CELLS IN THE ANTI VIRAL
RESPONSE
IDENTIFICATION OF AVIAN PARAMYXOVIRUS IN PSITTACINES

BIRDS BY TRANSMISSION ELECTRON MICROSCOPY
Elaine Cristina Cardoso(1), Luanda Mara da Silva Oliveira(3), Flavia Afonso

Lima(2), Caroline Guedes Borgato(1), Simone Correa-Silva(1), Maria Notomi
Sato(3) and Estela Bevilacqua(1)
(1) Departament of Cell and Developmental Biology, Institute of Biomedical
Sciences, University of Sao Paulo
(2) Departament of Parasitology, Institute of Biomedical Sciences, University of Sao
Paulo
(3) Laboratory of Dermatology and Immunodeficiencies, LIM-56, Department of
Dermatology, Medical School, Tropical Medicine Institute of So Paulo, University
of So Paulo, So Paulo, So Paulo, Brazil
Corresponding author Estela Bevilacqua bevilacq@usp.br Elaine Cristina Cardoso
enialeccardoso@gmail.com Mailing address: Avenida Professor LineuPrestes,
1524, Butantan 05508-000 Phone: +55 11 30917307/3091-8050
Background: Mother-to-child transmission (MTCT) of HIV-1 has been significantly
reduced with the use of antiretroviral therapies, resulting in an increased number of
HIV-exposed uninfected infants. The consequences of HIV infection on the innate
immune system of both mother and newborn are not well understood. Elegant studies
recently showed that neonates exposed to HIV-1 have impaired IFN-a and TNF-a
production by pDCs and mDCs. Our previous studies, however, demonstrated that
the adjuvant effect of natural or synthetic TLR7/8 ligands, such as CL097 in
neonates exposed to HIV-1. This signaling pathway showed to be preserved in the
newborns, suggesting a role as protective factor related to HIV-1 vertical
transmission. In addition, evidences also indicate that expression of Toll-like
receptors (TLR) by trophoblast cells might regulate the differentiation and activation
of immune cells, likely a key modulation for successful gestation and fetal
protection. Aims: Herein, we analyzed the production and secretion of cytokines by
trophoblast cells involved in an antiviral response. Methods: Extravillous trophoblast
(EVT) cells isolated from placentas of healthy mothers and trophoblast cell lines
(BeWo, HTR-8 and Swan71) were infected with HIV-1 and activated with agonists
for intracellular TLRs. Results: Our results showed a potential modulatory effect of
CL097 (TLR7/8), Gardimiquimod (TLR7) and CPGA (TLR9), which may reflect the
improved anti-viral response during pregnancy. Conclusion: These findings raise
new possibilities in the search for better tools in the development of vaccine
adjuvants as well as antiretrovirals with better efficiency.
Gilberto Pereira de Oliveira Jnior; Fernanda Falco de Queirz; Ana Maria Cristina
Rebello Pinto da Fonseca Martins; Marcia Helena Braga Catroxo
Electron Microscopy Laboratory, Biological Institute of So Paulo, SP, Brazil.
Diseases caused by the paramyxovirus occur worldwide in both free-living species as
captive, causing significant losses to the nature and damage to breeding. Avian
paramyxovirus belong to Paramyxoviridae family and Avulavirus genus. Avian
paramyxovirus that make up the Avulavirus genus are divided into 10 different
serotypes (APMV 1-10). The serotype 2 (APMV-2) and 3 (APMV-3) are those that
affect the Psittaciformes, and many species are sensitive. The main clinical signs and
symptoms are represented by anorexia, pneumonia, weight loss, diarrhea, ruffled
feathers, conjunctivitis, nerve signals and high mortality. From January 2014 to April
2016, were sent to the Biological Institute of So Paulo, SP, Brazil, stool and organ
fragments samples of the 43 psittacines birds for the diagnosis of viral agents. The
samples were processed by negative staining techniques, being suspended in 0.1M
pH 7.0 phosphate buffer, placed in contact with metal grids, previously coated with
collodion film and carbon, drained with filter paper, negatively contrasted with
ammonium molybdate 2% and pH 5 0. After examining the transmission electron
microscope, paramyxovirus particles, pleomorphic, measuring between 100 and 300
nm in diameter, containing an envelope covered with spikes and characteristic helical
herring-bone-like nucleocapsid, were observed in 28 bird samples (12 Nymphicus
hollandicus, 8 Amazona aestiva, 3 Agapornis sp, 1 Anodorhynchus hyacinthinus, 1
Aratinga Jandaya, 1 Aratinga solstitialis, 1 Melopsittacus undulatus, 1 Eupsittula
cactorum). The technique was efficient for the rapid diagnosis of paramyxovirus.

Conflict of interests: The present data has no conflict of interests

O15
O16
EFEITO DO TRATAMENTO DAS CULTURAS PRIMRIAS DE FD COM

BLOQUEADORES E AGONISTAS DE TLR-2 E TLR-6 NA INFECO POR
L. (L.) AMAZONENSIS
Camila Guerra Silva, Roger Magno Macedo-Silva, Paolla Roberta de Paula Pereira
Pinto, Stella Oliveira da Silva, Suzana Crte-Real Faria
Instituto Oswaldo Cruz/Fiocruz
Marisol Pallete Briceo1+, Layane Alencar Costa Nascimento1, Nathalia Pires

Nogueira, Paulo Victor Czarnewski Barenco1, Eloisa Amlia Vieira Ferro2, Karine
Rezende-Oliveira3, Luiz Ricardo Goulart4, Patrcia Terra Alves4, Bellisa de Freitas
Barbosa1, Wnia Rezende Lima1, Neide Maria Silva1
1
In the infection by protozoa Leishmania genus, the early stages of infection are
crucial for the evolution of disease, being involved several factors, including the
interaction of molecules at the parasites surface in the initial response of the host
cells. To elucidate the involvement of TLR-2/1 and TLR-2/6 were performed
activation assays and blocking the receptors in the infection by L. amazonensis in
dermal fibroblasts (DF) obtained from primary cultures of skin of C57BL/6 mice
embryos. DF were plated in DMEM at 37C with 5% CO2/24 h. After, the cells were
incubated for 30 min/37C with 10 g/mL anti-TLR-2 or 10 g/mL anti-TLR-6 in
DMEM with 2% BSA. For activation with agonists or blocking of dimers TLR-2
cells were incubated with 0,1g/mL Pam3Cys-Ser- (Lys) 4 (Alexis) or 0,1g/mL
MALP-2 (Alexis) for 12 h/37 C. After, the cells were infected with L. amazonensis
promastigotes by 2, 6, 24 and 48 hours of interaction. For morphological and
phenotypic analysis used light microscopy and electronics, immunofluorescence and
flow cytometry. Ethics committee for animal, FIOCRUZ N LW-2/12 were used.
Cultures treatment with agonists and blockers revealed that modulation of TLR-2 can
reverse the susceptibility of FD, which may be a determining factor for the
development of an immune response more effective in controlling infection by L.
amazonensis, and a way in the search for alternatives in the development of new
therapies for the leishmaniasis treatment.
Supported by IOC/FIOCRUZ/ CNPq/ FAPERJ.
TOXOPLASMA GONDII INFECTION PROMOTES EPITHELIAL BARRIER

DYSFUNCTION OF CACO- 2 CELLS
Laboratory of Immunopathology, Institute of Biomedical Sciences, Federal

University of Uberlndia, MG, Brazil.
2
Laboratory of Immunophysiology of Reproduction, Institute of Biomedical
Sciences, Federal University of Uberlndia, MG, Brazil.
3
Laboratory of Biomedical Sciences, Federal University of Uberlndia, MG, Brazil.
4
Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal
University of Uberlndia, MG, Brazil.
+
Corresponding Author: Marisol Pallete Briceo; Email: marislpb@gmail.com

Immunopathology Laboratory, Institute of Biomedical Sciences, Universidade
Federal de Uberlndia, Av. Par, 1720, Bloco 2B, Uberlndia, Minas Gerais, Brazil,
38 400-902. Telephone contact: +55 34 99309 5513 (mobile), +55 34 3225 8481
(institutional)
Background: After oral infection, T. gondii invades intestinal cells, induces
breakdown of intestinal physiology and barrier functions and causes intestinal
pathology in some animal species. Although parasites invasion into host cells is a
known phenomenon, the effects of T. gondii infection in the intestinal barrier are still
not well established. Aims: Evaluate the morphological and physiological
modifications on the colorectal adenocarcinoma-derived Caco-2 cell line during T.
gondii infection. Methods: Caco-2 cells were maintained for 20 days in culture and
infected with T. gondii tachyzoites for 24 hours. To analyze paracellular permeability
in Caco-2 cells, transwell system was used to assess transepithelial electrical
resistance (TEER) and FITC-Dextran endocytosis. For analysis of tight junctions
integrity and microvilli structure, occludin, ZO-1, claudin-1 and actin filaments were
studied by immunofluorescence and immunoblotting assays. Results: In infected
Caco-2 cells, the dextran uptake (endocytosis) and distribution was smaller. The
infection leads to the partial loss of microvilli at the cell surface. Claudin-1, ZO-1
and occludin expressions were co-localized by immunofluorescence and presented
discontinuous net patterns in infected cells. Immunoblotting analysis at 24 hours
post-infection revealed decreasing expression of occludin and ZO-1 proteins,
whereas claudin-1 expression level was not altered. T. gondii decreased TEER in
Caco-2 cells 24 hours after infection. Conclusion: Our results suggest that T. gondii
infection may lead to the loss of integrity of intestinal mucosa, resulting in impaired
barrier function.
Ethical approval: For this work, evaluation for ethical approval was not necessary.
Funding support: CAPES, CNPq and FAPEMIG.

O17
O18
INFECTION OF MOUSE NEUROSPHERES BY TOXOPLASMA GONDII

INCREASES
PANNEXIN1
EXPRESSION
AND
HOST
CELL
PROLIFERATION
IMUNNOMODULATION OF MACROPHAGE BY C-TYPE LECTIN AND

ITS EFFECTS ON LEISHMANIA BRAZILIENSIS
Aranda-Souza, M.A.1; Correia, M. T. S.2; Figueiredo, R.C.B.Q.1
Thalyta Priswa de Souza Andrade, Pedro Henrique G. Victorino, Paloma Vieira dos
Santos, Luiza Bendia Pires, Helene Santos Barbosa, Daniel Adesse
Departamento de Microbiologia, Centro de Pesquisas Aggeu Magalhes, Fundao

Oswaldo Cruz, Recife, Brazil
2
Departamento de Bioqumica, Centro de Cincias Biolgicas, Universidade Federal
de Pernambuco, Recife, Brazil
Laboratrio de Biologia Estrutural, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro,

Brazil
Congenital toxoplasmosis is one of the leading causes of microcephaly within the
TORCH syndrome (that includes Rubella, Syphilis, CMV and Herpes).
Toxoplasma gondii is an obligate, intracellular parasite belonging to the phylum
Apicomplexa and affects up to a third of the worlds population. However little is
known about the cellular and molecular mechanisms by which the parasite alters
cortical neurogenesis. Gap junction proteins are involved in neuronogenesis in mice
and studies have shown that T. gondii alters cell-cell coupling in astrocytes and
leptomeningeal cells. We used primary neural progenitor cells (NPCs) isolated from
Swiss Webster mice to infect with tachyzoite forms of T. gondii (ME49 strain).
Cultures were analyzed for parasite load and specific genes expression at different
times using qRT-PCR and Confocal and Electron Microscopy. RT-PCR indicated the
occurance of spontaneous tachyzoite-bradyzoite conversion at 96 hpi. qPCR revealed
that infected NPCs have unaltered levels of gap junction gene Gja1 (Cx43). Levels
of Pannexin1, which is involved with proliferative state of NPCs, were increased.
qPCR analyses revealed that Nestin expression was also increased at 96 hpi,
suggesting that the infection lead to augmented proliferation of host cells. This was
further assessed by the quantification of the size of the neurospheres, revealing that
infected cultures had bigger spheres, indicating increased proliferation. This model
has proven to be a valuable tool to investigate in vitro the mechanisms involved in
the pathogenesis of congenital toxoplasmosis and elucidate how this infection alters
the correct development of the CNS.
American tegumentary leishmaniasis is an endemic disease in Brazil and the main

causative agent is Leishmania braziliensis. These parasites replicate intracellularly in
macrophages and the mechanisms underlying host resistance involves the immune
response. Thus, pro-inflammatory cytokines and macrophage activation are
important in the infection inhibiton. The aim of this study was to investigate the
effect of a C-type lectin, BLL, in the immune response to L. braziliensis infection.
Peritoneal macrophages were plated at 106cells/mL (37C/5%CO2) and infected with
promastigotes forms of L. braziliensis. The culture was incubated for 24h at different
concentrations of lectin BLL and after, stained using Giemsa and the results were
expressed as Infection Index and percentage of macrophage infection. The
supernatants were incubated with Griess reagent for the quantification of the nitrite
concentration and also stained utilizing BD CBA Mouse Th1/Th2/Th17 Cytokine
Kit. Additionally, the cells were submitted to transmission electronic microscope
processing for evaluation of ultrastructural alterations. BLL significantly inhibited
the survival of amastigote (86%7.8) and the percentage of infected macrophages
was decreased. The lectin also induced enhancement of nitrite production. Similarly,
the release of proinflammatory cytokines, but not of Th2 cytokines, was significantly
induced. On ultrastructure analysis, cells treated with lectin presented alterations on
parasitophorous vacuole and on the parasites. Overall, our results suggest that the
immunomodulatory effects of BLL on macrophages is a possible mechanism
responsible for the inhibition of Leishmania infection in these cells and could supply
the research of new therapeutic targets for leishmaniasis.
Use of animals was approved by the Ethics Committee in the Use of Laboratory
Animals (CEUA-IOC), protocol number L-048/2015.
Acknowledgements: CAPES, FIOCRUZ

P METHODS IN CELL BIOLOGY
P1
P2
NANOMATERIALS TO EFFICIENTLY DELIVER GENES TO DIFFICULTTO-TRANSFECT CELLS
SUCCESSFUL TRANSFECTION
CULTURE OF FISH
Fernanda Maria Policarpo Tonelli (1), Samyra Maria dos Santos Nassif Lacerda (2),
Luiz Orlando Ladeira (3), Rodrigo Ribeiro Resende (1).
Bruno Oliveira da Silva Duran (1), Guilherme Alcars de Ges (1), Paula Paccielli
Freire (1), Edson Assuno Mareco (2), Rondinelle Artur Simes Salomo (3),
Robson Francisco Carvalho (1), Maeli Dal-Pai-Silva (1).
(1) Department of Biochemistry and Immunology, Federal University of Minas

Gerais, Belo Horizonte, Brazil. Av. Antnio Carlos 6627, Pampulha, Caixa Postal
486, 31270-901, Belo Horizonte, MG.
(2) Department of Morphology, Federal University of Minas Gerais, Belo Horizonte,
Brazil.
(3) Department of Physics, Federal University of Minas Gerais, Belo Horizonte,
Brazil.
IN
PRIMARY
MYOBLAST
CELL
(1) Department of Morphology, Institute of Biosciences of Botucatu, So Paulo State

University (Unesp), Botucatu, So Paulo, Brazil.
(2) University of West So Paulo (Unoeste), Presidente Prudente, So Paulo, Brazil.
(3) Aquaculture Center, So Paulo State University (Unesp), Jaboticabal, So Paulo,
Brazil.
Email address: duran@ibb.unesp.br
Telephone: +55 14 3880 0503
(31) 3409-2780/(31)84019831. tonellinanda@gmail.com

Difficult-to-transfect cells are a challenge when it comes to trying to develop new
gene delivery reagents. So, nanomaterials emerge as an important tool as they can
suffer a wide range of modifications on their structure, by functionalization, to
efficiently deliver genes. This study aimed to investigate the ability of nanographene
oxide (NGO), carbon nanotube (CNT), nanodiamond (ND), gold nanorod (NR) and
phosphate nanoparticle (NPC), as well as Lipofectamine 2000, in promoting the
delivery of a commercial plasmid (containing the gene of cyan fluorescent protein) to
the difficult-to-transfect cells: rat cardiomyocytes, rat DRGs, U373 and C6.
Nanomaterials were synthesized, functionalized, and complexed to pAmCyan1-N1.
After transfection, cells viability was determined; protein expression was observed
by fluorescence microscopy 24 hours after exposure. The synthesis of the mRNA of
interest was detected through qRT-PCR. All gene delivery vehicles were able to
transfect cells. However, CNTs overcome the delivery capacity of the commercial
reagent in cardiomyocytes and induced lower cell death rate; when it comes to C6
cells, NRs and CNTs induced significantly higher levels of transcription than
Lipofectamine. In U373, the commercial reagent proved to be the best gene delivery
strategy. In DRGs, the NRs were the best delivery vehicles, overcoming the capacity
of the commercial reagent and also inducing lower rate of cell death. Thus,
functionalized nanomaterials can promote the delivery of genes to difficult-totransfect cells; combining low toxicity and high efficiency, CNTs, NDs and NRs are
interesting alternatives. However, gene delivery efficiency and death rate induction
vary with target cell type.
Financial support: CNPq.
Background: Skeletal muscle in fish constitutes around 60% of body mass and the
primary myoblast cell culture, an in vitro model, is becoming a useful tool to
understand the regulation of muscle growth. Aim: The aim of our work was to
perform a transfection technique in primary myoblast cell culture of pacu (Piaractus
mesopotamicus), which is a powerful method to improve our knowledge of the
regulatory pathways involved in fish muscle growth. Methods: Fast-twitch muscles
were collected from juvenile pacus to establish the primary myoblast cell cultures.
The myoblasts were studied in three phases: commitment and early proliferation (2-4
days), late proliferation and early fusion (5-8 days), and late fusion and myotube
formation (9-12 days). Initially we performed a transfection of plasmids conjugated
with the green fluorescent protein (pEGFP-N1 Vector) and, to confirm the
establishment of this technique, we performed another transfection using inhibitors
of miR-1 (anti-miR-1), which is a microRNA involved in muscle growth. Results:
When the myoblasts reached a 60-70% confluence, we added 4 L of plasmid-GFP
(1 g/L) and 7.5 L of Lipofectamine during 15 hours. The amounts of 4 L of
anti-miR-1 (10 M) and 7.5 L of Lipofectamine during 15 hours were sufficient to
promote the miR-1 inhibition in the primary myoblast cell cultures of pacu.
Conclusion: Our results are very promising and represent a major advance in
research that involves fish muscle growth, since transfection of myoblasts allows the
examination of isolated regulatory molecules under precisely controlled conditions.
Information on ethical approval and funding support: This study was supported by
So Paulo Research Foundation (FAPESP 2013/10196-0) and approved by Ethics
Committee on Animal Use (CEUA 506) of Biosciences Institute, UNESP,
Botucatu, So Paulo Brazil.
P3
P4
COMPARATIVE STUDY OF PREPARATION TECHNIQUES FOR SEM

ANALYSIS OF STABILIZED MURINE FIBROBLASTS (NIH 3T3) CELLS
CHARACTERIZATION OF THE CYTOPLASMIC CONTENT TO SEA

URCHIN COELOMOCYTES USING CYTOLOGY AND ENERGYDISPERSIVE X-RAY SPECTROSCOPY (EDS)
Susane Lopes (1,2), Eliana de Medeiros Oliveira (2), Ana Paula Voytena (1), Deise
Rebelo Consoni (2), Marcelo Maraschin (1)
(1) Laboratrio de Morfognese e Biqumica Vegetal, da Universidade Federal de
Santa Catarina, Santa Catarina, Brasil.
(2) Laboratrio Central de Microscopia Eletrnica, da Universidade Federal de Santa
Catarina, Santa Catarina, Brasil.
susane.lopes@ufsc.br, (48) 3271-8727 / (48) 9144-9162, Universidade Federal de
Santa Catarina, Rua: Eng. Agronmico Andrei Cristian Ferreira, s/n, - 88040-900,
Florianpolis SC.
BACKGROUND: Several methods for scanning electron microscopy (SEM) sample
preparation have been described in literature, showing profit images and results for
adhered cells culture. The sample must be representative to avoid misleading
conclusions.
AIMS: To evaluate sample preparative techniques for SEM analysis of stabilized
murine fibroblasts (NIH 3T3) cells.
METHODS: NIH 3T3 fibroblasts were inoculated in previously poly-L-lysine
treated glass slides. The cells were incubated on appropriate culture medium. Upon
reaching confluence, cells were fixed with Karnovsky solution during 1 h, post-fixed
in 1% (m/v) osmium tetroxide during 1 h, followed by drying processes: critical
point (CP), hexamethyldisilazane (HMDS), 40C oven (24 h), silica gel desiccator at
room temperature during 24 h. The CP and HMDS dried samples were also analyzed
with and without the cell post-fixation step. Samples were dehydrated in ethanol in
increasing concentrations (70%-100%, v/v). The glass slides were sputter-coated
with gold and analyzed in SEM.
RESULTS: All the samples showed good cell integrity, except the non-post fixed
and CP dried ones. The non-post fixed and HMDS samples, as well the post fixed
and HMDS, oven and desiccated dried ones showed similar SEM morphology. The
post fixed and CP dried samples revealed and superior pseudopodes and vesicles
integrity.
CONCLUSION: These observations indicate that post-fixed and CP dried techniques
allowed to preserve better cellular morphologic integrity.
Vinicius Queiroz (1,2), Enrique Eduardo Rozas Sanchez (3) and Mrcio Reis
Custdio (1,2).
(1) General Physiology Department, University of So Paulo, So Paulo, Brazil.
(2) Center for Marine Biology (CEBIMar), University of So Paulo, So Paulo,
Brazil.
(3) Chemical Engineering Department, Semi-Industrial, University of So Paulo, So
Paulo, Brazil.
Correspondence: Vinicius Queiroz vinicius_ufba@yahoo.com.br. Phone number:
+55 11 3091-7522, Cel Phone: +55 11 98301-4102
In Echinoidea three major categories of coelomocytes are recognized: phagocytes,
spherulocytes and vibratile cells. However, only the red spherulocyte had its content
properly characterized. In all other the evidences are speculative, based on mostly in
some stain characteristics. Thus, this work evaluated if the energy-dispersive X-ray
spectroscopy (EDS) can provide complementary information about the chemical
nature of the echinoid coelomocytes. Cells from Eucidaris tribuloides were fixed in
glutaraldehyde 2.5% (4-6h/4C) and analyzed by EDS system coupled to a scanning
electron microscope and by Mallorys Trichrome (MT) or Toluidine Blue (TB)
stains. Phagocytes, vibratile cells and two spherulocytes (colorless and granular)
were identified and formed two contrasting groups. The first two showed a similar
pattern, with low concentration of carbon and nitrogen (<5%) and very low levels of
phosphorus and sulfur (<1%). Vibratile cells are metachromatic with TB indicating
polysaccharides, and the low level of nitrogen corroborates this evidence. For both
spherulocytes, the EDS indicate higher concentration of carbon and nitrogen (~10%)
and a little more phosphorus (1-1.5%) and sulfur (1-3%). With MT, the colorless
spherulocyte showed affinity to the methyl blue, suggesting also polysaccharides,
while the granular spherulocytes stained with acid fuchsine, indicating a peptide-rich
moiety. The EDS analyses corroborated this pattern, showing almost 2-fold nitrogen
and 5-fold cytoplasmic sulfur in granular spherulocytes (possibly methionine and
cysteine, involved with protein synthesis). We believe that the EDS approach,
coupled with the standard cytochemical methods, can be useful and provide an
elemental signature to each cell subpopulation.
KNOWLEDGMENTS AND FINNACIAL SUPPORT: CAPES e UFSC.

Financial support: CAPES Foundation and FAPESP (proc. 15/21460-5).

P5
P6
STANDARDIZATION OF AN IMMUNOFLUORESCENCE TECHNIQUE

FOR THE DETECTION OF PROTEINS OF THE RENIN-ANGIOTENSIN
SYSTEM IN THE MICE KIDNEY
ESTABLISHMENT OF A PROTOCOL FOR SKIN BIOPSIES

CAPUCHIN MONKEY (SAPAJUS APELLA) VITRIFICATION
Brito, P. L. (1); (1); Silva, J. A. F. (1); Ferreira Jr, W.A. (1), Chweih, H. (1);
Silveira, A. A.(1); Almeida, C. B. (1); Fabris, F. C. Z (1); Costa, F. F. (1); Conran,
N. (1)
(1) Vascular Inflammation Laboratory, Center of Hematology and Hemotherapy,
School of Medical Sciences, University of Campinas (UNICAMP), Brazil.
(1)
Corresponding author: Pmela Lara de Brito
Telephone: +55 (19) 3521-8611
(2)
e-mail: lara06brito@gmail.com
Vascular Inflammation Laboratory, Center of Hematology and Hemotherapy, School
(3)
of Medical Sciences, University of Campinas (UNICAMP), Brazil.
Background: Kidneys are responsible for the homeostasis of body fluids and
excretion of metabolic and xenobiotic products. Hemodynamic changes in the kidney
lead to the activation of the renin-angiotensin system (RAS), a complex hormonal
aimed at electrolyte homeostasis and blood pressure control. Aims: Standardization
of an immunofluorescence technique for the assessment of the expression of proteins
of the Rennin-Angiotensin System (RAS) and macrophages in the mouse kidney.
Methods: Kidneys from C57BL/6 mice were dissected and immersed in OCT
mounting medium and cryostat sections (10 m thick) were xed with
paraformaldehyde and incubated with methanol/hydrogen peroxide solution and 3%
bovine serum albumin (BSA) solution. Tissue sections were incubated with primary
antibodies goat anti: renin, angiotensin and F4/80; rabbit anti: nephrin, AT2, ACE
and CD68 (diluted 1:100, 1:200 and 1:500). Sections were then incubated with antigoat and anti-rabbit secondary antibodies Donkey anti-Goat IgG Alexa Fluor 488
conjugate, Donkey anti-Rabbit IgG Alexa Fluor 555 conjugate and the nuclei were
stained with DAPI. Results: Nephrin, ACE, Ang and AT2 were observed in the renal
tubules of C57BL/6 mice, with scarce presence of macrophages. Renin was found
localized in the glomerulus and within the renal tubules. Conclusion: Using an
immunofluorescence technique, it was possible to identify the expression of proteins
of the RAS in different locations of the mouse kidney, as well as the presence of
macrophages. Thus, this technique may be employed for evaluating kidneys of mice
with renal damage (as occurs in sickle cell disease) to identify changes in elements of
the RAS.
Financial support: FAPESP.
Ethics Committee: Protocol n 3810-1
OF
Gabriela Jaques Rodrigues (1), Carla Maria Figueiredo de Carvalho Miranda (3),
Sheyla Farhayldes Souza Domingues (2), Cinthia Tvora de Albuquerque (1), Mayra
Pauline Ribeiro Costa (1), Marcela da Silva Cordeiro (1), Adriel Behn de Brito (2),
Danielle Cristina Calado de Brito (2), Julyne Vivian Guimares de Carvalho (2),
Silmara Leticia Gonalves Lima (2), Cleideanny Cancela Galvo (2), Juliana
Gonalves Lima (2).
(1) Veterinary Stem Cell Laboratory. Institute of Veterinary Medicine. Federal
University of Par. Castanhal-PA-Brazil.
(2) Laboratory of Biotechnology and Reproduction of Amazonian Animals. Institute
of Veterinary Medicine. Federal University of Par. Castanhal-PA-Brazil.
(3) Anatomy department of domestic and wild animals. University of So Paulo.
Carla Maria Figueiredo de Carvalho Miranda; Endereo: Av. Orlando de Marques
Paiva, 87. Departamento de anatomia dos animais domsticos e silvestres.
FMVZ/usp/ SP; E-mail: carlacarvalhovet@gmail.com; Telefone: (11) 95131-7895.
Vitrification is a quick, easy and less expensive method than slow cryopreservation.
Vitrifying skin samples is relevant as it allows studies with stem cells isolated from
this tissue. The aim of this study was to develop a vitrification protocol for skin
biopsies of capuchin monkey (Sapajus apella), evaluating two different
concentrations (0.1 and 0.5 M) of trehalose and sucrose as extracellular
crioprotectors, and RPMI and DMEM as base medium. A skin fragment from
Sapajus apella was divided into a vitrification and control group. For the
vitrification, the following treatments were employed: T1 (DMEM + 40% EG + 0.1
M SUC); T2 (DMEM + 40% EG + 0.5 M SUC); T3 (DMEM + 40% EG + 0.1 M
TRE); T4 (DMEM + 40% EG + 0.5 M TRE); T5 (RPMI + 40% EG + 0.1 M TRE).
For thawing, the sample went through three washing solutions. The evaluation of the
treatments was carried out by cellular culture and histology. After thawing, cellular
growth was present only in the T5 treatment. T5 histological analysis showed
fibroblast integrity and citoplasmatic vacuoles, which denote tissue damage, though
the fibroblasts remained intact. In conclusion, the protocol using RPMI associated
with trehalose was an efficient protocol for skin biopsy vitrification.
Ethical terms: This project was authorized by the ethical committee of experimental
animal research number 245-14 (CEPAE-UFPA), and by SISBIO number 46990-1.
P7
P8
NEONATAL CARDIOMYOCYTES ROS PRODUCTION, METABOLISM

AND FUNCTION PERFORMANCE IN VITRO ARE INFLUENCED BY
EXTRACTION METHOD
AN EFFECTIVE METHOD FOR THE EVALUATION OF SPHEROID CELL

MIGRATION
Leonardo Jensen (1), Elida Neri (1), Nathalia Oliveira (1), Vinicius Bassaneze (1),
Rafael Dariolli (1), Dbora Levy (2), Douglas Veronez (3), Sergio Bydlowski (2),
Idagene Cestari (3), Jos Eduardo Krieger (1)
1 - Laboratory of Genetics and Molecular Cardiology/LIM 13, University of So
Paulo School of Medicine Heart Institute (InCor), University of So Paulo Medical
School
2 - Laboratory of Genetics and Molecular Hematology/LIM31, University of So
Paulo School of Medicine Heart Institute (InCor), University of So Paulo Medical
School
3 - Bioengineering Division, University of So Paulo School of Medicine Heart
Institute (InCor), University of So Paulo Medical School
Elida Neri: e-mail: elidaneri@gmail.com
telephone institutional: (11) 2661-5521
mobile: (11) 96723-8711
Neonatal cardiomyocytes from several different species are widely used since late
1960's for developmental, toxicological, pharmacological and physiological studies.
The objective of the study was to show that different methods of the cardiac cell
extraction can alter the basal state of the cells in vitro. Cells of neonatal rat heart
(were done according the ethical committee 285/12) 1-2 days were obtained by the
following extraction methods: Preplating (PP) and Percoll (PER) and maintained in
vitro for eight days. Cell types, nitrotyrosine and troponin 1 were measured in
fluorescence assays for High-Throughput Screening (HCS). Analyses of
mitochondrial stress and intracellular influx of calcium were performed with living
cells in HCS. The dosages of hydroxy-and ethidium and ethidium were performed in
HPLC. Production of nitrite and LDH activity were quantified by
chemiluminescence. Cardiomyocytes obtained from pre-plating procedure had a
higher lactate production (Per versus PP; meanSEM; 6.130.09 mol/cell versus
7.500.42 mol/cell). This correlated with an increased reactive oxygen species
(ROS) production, indirectly measured by ethidium (0.62250.059 pmol/cell versus
1.3810.042 pmol/cell) and hydroxy-ethidium production (0.031 0.003 pmol/cell
versus 0.0520.004 pmol/cell). Also protein nitration could be detected on cells
under (HCS) (1.9x1054817 versus 3.9x10526980; P<0.0001), as well as
nitrotyrosine accumulation in the cell medium (0.00720.0011 versus
0.00190.0003) indicating nitrosactive stress on these cells. In conclusion cells
extracted using the PP protocol presented higher metabolic activity, oxidative stress,
apoptosis and contractile activity under basal conditions or under isoproterenol
challenge compared with cells extracted using the protocol of Per.
Adam A. Martens*, Flvia S. Belarmino*, Lais I. Fukase*, Marcelo M. de Souza*,

Marcel Shiniti Urabayashi*, Glucia M.M. Santelli*
Departamento de Biologia Celular e do Desenvolvimento, Instituto de Cincias
Biomdicas, Universidade de So Paulo.
presenter, email:marcelo.msouza@usp.br,
(11)3091-7229 mobile:(11) 99368-8892
telephone
contact:
institutional:
Background: 3D or spheroid cell culture represents a model closer to in vivo tumors

than monolayer cell cultures. They have been proposed for the study of different
aspect of drug effects. The evaluation of cell migration in this model requires
modifications of the usual techniques.
Aims: The aim of this study was to establish a protocol for the evaluation of cell
migration from spheroids.
Methods: Cells lines SK-MEL 147, HT-47D (melanoma cells), MCF-7 and Hs-578T
(breast cancer cells) were plated in non-adherent Petri dishes to form self-assembled
spheroids. After 7 and 14 days, stable spheroids were transferred to adherent cell
culture dishes, and the area of spheroid adhesion and spreading was measured at
different time points. Cell migration was inhibited interfering in actin cytoskeleton
dynamics with sponge-derived compound, Jasplakinolide and Geodiamolide H, to
induce actin cytoskeleton impairment. Cells were stained with phalloidin-FITC and
propidium iodide and images were obtained with light microscope, fluorescence
microscopy and confocal laser scanning microscope. Image analysis was performed
with Zen Lite from Zeiss microscopy.
Results: The measurement of spheroid adhesion and spreading was able to accurately
determine the cell migration from spheroids. Cells from different origins displayed
different migration behavior, with breast cancer cells showing a more circular
spreading, whereas melanoma cells were more heterogeneous. Fluorescent staining
confirmed cytoskeletal disorganisation of cells treated with the drugs.
Conclusion: The present method is an effective tool to evaluate cell migration from
spheroids and actin cytoskeleton dynamics.
*authors contributed equally.
Financial Suport: CAPES, CNPq and FAPESP.

Q MORPHOLOGY AND CELL BIOLOGY
Q1
Q2
ACUTE TOXOPLASMA GONDII INFECTION ALTERS THE GOBLET

CELLS SUBPOPULATIONS IN THE ILEUM OF RATS
HOMOCYSTEINE INDUCES IMPAIRMENTS ON MITOCHONDRIAL

DYNAMICS IN MESENCHYMAL CELLS DURING LIMB DEVELOPMENT
Larissa Carla Lauer Schneider (1), Joquebede Caroline Pessoa Nascimento (1),
Evandro Jos Beraldi (1), Stephanie Carvalho Borges (1), Dbora de Mello Gonales
SantAna (1), Nilza Cristina Buttow (1).
Gilian Fernando Bourckhardt*, Manuela Sozo Cecchini, Maria Lusa da Silveira

Hahmeyer, Yara Maria Rauh Mller, Evelise Maria Nazari
Universidade Federal de Santa Catarina (UFSC), Brazil
(1) Department of Morphological Sciences, State University of Maring (UEM),

Maring, Brazil.
E-mail: lari_uem@yahoo.com.br
Phone: 55 44 3011 4928
Mobile: 55 44 9101 4332
Goblet cells (GCs) produce the mucus layer that coats the intestinal surface, acting as
the first line of innate defense in the intestine. The disruption of this barrier is
required for the dissemination of parasites, as in the case of Toxoplasma gondii
infection. We evaluated in this study the action of T. gondii on the population of
goblet cells in the ileum of rats infected in the acute phase. Male Wistar rats (60 days
old) were randomly distributed in 9 groups (n = 5): not infected (control group) and
infected by the inoculation of 5000 sporulated T. gondii oocysts (ME-49 strain,
genotype II). Infected animals were further distributed in the following groups: 6, 12,
24, 48 and 72 hours, 7, 10 and 15 days after infection. The histological stainings of
periodic acid-Schiff (PAS), alcian-blue (AB) pH 1.0 and 2.5 were used to observe
the mucins modulation by the quantification of GCs. All animal procedures were
approved by the Ethics Committee for Animal Experimentation of UEM (Protocol n.
079/2013). No significant change was observed in the number of AB 1.0+ GCs
during the infection. However, there was a significant decrease in the subpopulation
of AB 2.5+ GCs in all groups, except in group 12 hours. PAS+ GCs also had their
density decreased up to 24 hours, being similar to control values 15 days after
infection. The acute T. gondii infection changed the density of the GCs
subpopulations producing acidic and neutral mucins, resulting in a more fluid mucus.
*e-mail: gilian.bourckhardt@posgrad.ufsc.br
Mitochondria are energy powerhouse performing multiple metabolic functions in
normal/abnormal cellular conditions. However, the mitochondrial dynamics in
hyperhomocysteinemia is unclear. Thus, the aim of this study was to investigate
whether homocysteine (Hcy) affects the fission/fusion mitochondrial dynamics in
mesenchymal cells during limb development. Chicken embryos were treated with
20mol D-L Hcy at E2 and return to incubator chamber until E6, when were
analyzed. Untreated embryos received exclusively 50L saline solution (Ethics
Committee 175/CEUA/UFSC/2014). First, ultrastructural analysis allows recognize
subcellular damage, as dilations in the mitochondrial crests, presence of lysosomes
and autophagic vacuoles, after Hcy-treatment. Then, by flow cytometry, proteins
involved in mitochondrial fission/fusion were analyzed. We observed that Hcy
promotes mitochondrial fission recognized by increased Drp1-positive cells (551.00
50.45; 340.67 27.76, p<0.01). In contrast, Hcy induces a decrease in the
mitochondrial fusion, identified by Mfn1-positive cells (121.67 2.78; 276.00
24.46, p<0.0001). Moreover, an increase in autophagic vacuoles after Hcy-treatment
(6,419.67 1164.26; 2,112.00 576.24 p<0.0001) was recognized by acid dye
acridine orange. Additionally, mitochondrial integrity and mitophagy related proteins
were analyzed. Hcy promotes an increase in mitochondria damages, recognize by
PRK8-positive cells (2,582.00 146.76; 1,419.00 152.58 p<0.001). Also, the Hcy
induced an increase in the autophagosome-marker LC3II (2,787.33 62.38; 1,286.83
75.13 p<0.001). Finally, an increase in the autophagic flux, identified by P62
positive-cells (1,553.33 95.46; 367.00 100.00 p<0.0001) were observed after
Hcy-treatment. Our results contribute to better understand the multifaceted toxicity
of Hcy on mesenchymal cells during limb development.
Support: CAPES, FAPESC

Q4
Q3
EVALUATION OF HISTOPATHOLOGY AND GENOTOXICITY OF
SKELETAL MUSCLE AFTER SCALD BURN INJURY IN THE YOUNG
WISTAR RATS
SHORT TERM ADMINISTRATION OF A HIGH-FAT DIET IMPAIRS

WOUND REPAIR IN MICE
Fernanda Seabra Schanuel (1), Andra Monte-Alto-Costa (1)
Hananiah Tardivo Quintana (1), Jeferson Andr Bortolin (1), Daniel Araki Ribeiro
(1), Flavia de Oliveira (1).
(1) Department of Bioscience, Federal University of So Paulo, Santos-SP, Brazil.
(1) Tissue Repair Laboratory; Rio de Janeiro State University, Rio de Janeiro, Brazil.
E-mail: hananiah@hotmail.com
Address: Silva Jardim Street, 136, 11050-300. Federal University of So Paulo,
Campus Baixada Santista, Lab 328, Santos-SP.
Phone: (13) 37783844 / (13) 996540970
Corresponding authors address:

PhD. Student Fernanda Seabra Schanuel
Rio de Janeiro State University (UERJ) - Tissue Repair Laboratory - Rua Marechal
Rondon, 381/HLA - 20950-003. Rio de Janeiro, RJ - BRAZIL.
Telephone: +55 21 2334 2421
Mobile: +55 21 985304545
E-mail address: nandasschanuel@gmail.com
Background: Burn injury (BI) in children is associated with a pronounced persistence

of catabolism, resulting in muscle wasting, tissue loss and inflammation, which
contribute to burn mortality. The major frequency of this trauma occurs by scald hot
liquids and lesions covering more than 40% of the body, resulting increased
metabolism and other systemic alterations that persist months after injury. Aims:
Evaluate temporal of histopathology and genotoxicity in the skeletal muscle after BI
extensive in the young rats. Methods: This research was approved by the Ethics
Committee on Animal Use of Federal University of So Paulo (CEUA-UNIFESP:
4857080514). Therefore, we used 60 male 21-day-old Wistar rats, divided in two
groups: Control (C) and subjected to scald burn injury (SBI). The burn area was 45%
of the total body surface area. The animals were euthanized after 1, 4 or 14 days
post-burn injury or simulation (C1, C4, C14, SBI1, SBI4 and SBI14) and the
gastrocnemius muscle was dissected for the following analysis: histopathological,
hematoxylin-eosin and sirius red and analysis of genotoxicity by comet assay.
Results: Histopathological results showed morphological changes in muscle fiber
and type collagen fiber in the groups SBI in comparison to C. The analysis of the
comet assay presented no significant difference. Conclusion: During experimental
days, the BI of great extension was sufficient to cause changes in histopathology,
increase of collagen and alteration in the collagen type in the skeletal muscle distant
of injury. However, DNA damage was not found in this tissue.
Background: Wound healing is a physiologic process that involves sequential and

overlapping stages. The consumption of food that has a great quantity of saturated fat
has increased is western diet, affecting the metabolism and human health. So the
importance of diet composition on wound healing process should be considered.
Aims: This study investigated the effect of short term administration of a high fat
diet on cutaneous wound repair in mice. Methods: This study was approved by
CEUA/036/2014. Male mice were randomly divided into control (fed control chow:
76% of calories from carbohydrates, 14% from protein and 10% from fat) and diet
(fed high-fat diet: 26% calories from carbohydrates, 14% from protein and 60% from
fat) groups and fed different diets during 20 days. Body weight was measured at the
beginning of experiment and 7, 14, and 20 days later. Oral glucose tolerance and
intraperitoneal insulin tolerance tests was performed. After 10 days of diet
administration, an excisional lesion was made and lesion was collected 10 days after
wounding for histological analysis. Wound contraction and re-epithelialization was
measured on the day of lesion and 3, 7 or 10 days after wounding. Results: There
was no difference between the average body weight gain neither fasting blood
glucose between groups. The high fat diet group presented delayed wound
contraction and re-epithelialization. Ten days after wounding high-fat diet group
presented increased inflammatory cell amount and cellular density. Conclusion:
Short term administration of high fat diet impairs cutaneous wound healing in mice.

Q5
Q6
MODERATE EXERCISE CONCOMITANT TO STRESS POTENTIATES

THE DELAY ON WOUND HEALING
EFFECTS OF RESVERATROL ON SKIN REPAIR IN WISTAR RATS

SUBJECTED TO CALORIE-RESTRICTED DIET
Bianca de Oliveira Saguie1, Bruna Romana-Souza1, Andra Monte-Alto-Costa1
Viviane Theodoro, Ana Cristina Christovan, Fernanda Aparecida Sampaio

Mendona, Maria Esmria Corezola do Amaral, Marcelo Augusto Marretto
Esquisatto, Glucia Maria Tech dos Santos.
Tissue Repair Laboratory; - State University of Rio de Janeiro, Rio de Janeiro,

Brazil.
Corresponding authors address:
Msc. student Bianca de Oliveira Saguie
State University of Rio de Janeiro (UERJ) - Tissue Repair Laboratory - Rua
Marechal Rondon, 381/HLA - 20950-003. Rio de Janeiro, RJ - BRAZIL.
Telephone: +55 21 2334 2421
Mobile: +55 21 979688626
E-mail address: bsaguie@live.com
Background: Wound healing is a complex process involving cells and mediators. It is
divided in three overlapping phases: inflammation, proliferation and remodeling.
Stress-induced prolonged inflammation impairs cutaneous wound healing. Moderate
physical training may inhibit the adverse effects of stress on skin healing via an antiinflammatory mechanism. Aims: Investigate the effect of moderate exercise on
cutaneous wound healing of chronically stressed mice. Methods: Mice were trained
five times per week for 8 weeks using a treadmill at moderate intensity or received
no training. Animals were submitted to daily rotational stress from the 6th week until
euthanasia. Exercise was performed simultaneously with the stress model for two
weeks. Two excisional cutaneous lesions were created in the animals dorsum after
two weeks of rotational stress and collected 10 days after wounding to histological
analysis. Wound contraction and re-epithelialization was measured 3, 7 or 10 days
after wound. Results: Exercise did not change blood lactate. Plasma normetanephrine
levels were increased in the animals of the stressed+exercised group (ST+EX)
compared to those of the stressed+sedentary group (ST+SE). The ST+EX group
presented an impairment in the wound contraction and re-epithelialization.
Additionally, the neo-epidermis of ST+EX group was thinner than that of the ST+SE
group. The ST+EX group demonstrated a reduction in the neutrophil and
macrophage number, but an increase in the amount of blood vessels and
myofibroblast density when compared to ST+SE group. Conclusion: Moderate
exercise in association with stress potentiates the stress effect, delaying wound
healing.

E-mail: marcelosquisatto@uniararas.br Fone: +55 (19) 3543-1440.
A considerable number of studies have demonstrated that resveratrol (a polyphenolic
SIRT1 activator, mimics the anti-ageing effects of calorie restriction) has potent antiinflammatory and antioxidative properties. Therefore, the aim in this study was to
evaluate the effects of resveratrol in the skin repair process of rats under calorierestricted diet (CR). Fourty male animals with 120 days old were anesthetized and a
10mm-punch experimental lesion was made on their back. After, they were
randomly divided into four groups: without treatment (C), topic-treated resveratrol
(R), under calorie-restricted diet (CR), and under calorie-restricted diet + topictreated resveratrol (CR+R). Tissue samples were taken after 3 and 10 days of lesion
and were subjected to structural and morphometric analysis using Mallory
Trichrome, Dominici, Picrossirius-hematoxylin and Toluidine blue techniques. No
significant changes were observed in the sequence of structural events in repair
process among the groups. The number of fibroblasts was similar in 3 days for all
groups, but at 10 days CR+R presented the higher values compared to C. The
amount of granulocytes did not differ between the groups and periods. The number
of vessels was higher in CR+R at 3 days and in 10 days was in R and CR+R versus
C. The content of collagen was similar in all groups and experimental times. The
area of birefringent collagen fibers of the R and CR+R groups presented the higher
values in relation to the others at 3 and 10 days. In conclusion, our findings show that
resveratrol may have a beneficial effect on skin wound healing, especially in the
animals submitted to calorie-restricted diet.
Study approved
FHO|Uniararas.
by
CEUA/UNIARARAS
(023/2014)
and
supported
by
Ethical approval: CEUA/009/2013

Support by: FAPERJ, CNPq and CAPES.

Q7
Q8
THERAPY BY ELECTROACUPUNCTURE AND LASERPUNCTURE

STIMULATES REPAIR PROCESS IN EXPERIMENTAL WOUNDS IN
ADULT FEMALES OF WISTAR RATS (RATTUS NORVEGICUS)
EFFECTS OF ACUPUNCTURE AND MOXIBUSTION THERAPIES IN THE

REPAIR PROCESS OF EXPERIMENTAL WOUNDS IN ADULT FEMALES
OF WISTAR RATS (RATTUS NORVEGICUS)
Ivens Takechi Nakagawa, Hamilton Ricardo Alonso, Juan Guzman Quispe

Cabanillas, Fabrcio Campos Kuroda, Fernanda Aparecida Sampaio Mendona,
Glucia Maria Tech dos Santos, Andrea Aparecida Aro, Maria Esmria Corezola do
Amaral, Marcelo Augusto Marretto Esquisatto.
Hamilton Ricardo Alonso, Juan Guzman Quispe Cabanillas, Fabrcio Campos

Kuroda, Jos Roberto Passarini Junior, Fernanda Aparecida Sampaio Mendona,
Glucia Maria Tech dos Santos, Andrea Aparecida Aro, Maria Esmria Corezola do
Amaral, Marcelo Augusto Marretto Esquisatto.


E-mail: ivens_jp@hotmail.com Phone: +55 (19) 3543-1440.
E-mail: hamiltonfarma@hotmail.com Fone: +55 (19) 3543-1440.
The effects of electroacupuncture (E) and laseracupuncture (L) in the soft tissue
healing processes is still controversial. Our objective was to describe the effects of
these therapies on the wound healing in the adult female Wistar rats. Ninety animals
were divided in control (C), E and L groups. The animals were anesthetized and a
10mm-punch experimental lesion was made. After 24h, the animals were treated in
alternated days with needles associated to electrical current (2mA/4Hz/15min) or
laser pen (660nm/100mW/21s) on the B13-Feishu, B17-Geshu and E36-Zusanli
acupoints. Animals were sacrificed at 7, 14 and 21 days after lesion for structural and
biochemical analysis. The quantitative data were compared statistically. The number
of fibroblasts in E and L was greater than C at 14 and 21 days. The amount of
granulocytes was lower in E and L at 21 days. The number of vessels was
significantly higher in E and L at 21 days. The amount of glycosaminoglycans and
hydroxiproline was similar in all groups and times. Zymogram for MMP2 and MMP9 does not presented differences among groups. The amount of type I collagen was
significantly higher in E and L in all experimental times. In relation to type III, the
amount was significantly higher in E and L at 21 days. The amount of TGF-1 and
VEGF was significantly higher in E and L at 7 days. Electro and laseracupuncture
showed to have a positive influence on wound healing and tissue regeneration.
Acupuncture (A) and Moxibustion (M) are procedures used in Chinese Traditional
Medicine in the treatment of systemic pathologies. The aim of this study was to
describe the effects of these therapies on the wound healing in the adult female
Wistar rats. Ninety animals were divided in control (C), A and M groups. The
animals were anesthetized and a 10mm-punch experimental lesion was made. After
24h, the animals were treated in alternated days with needles or Artemisia's burning
rolls on the B13-Feishu, B17-Geshu and E36-Zusanli acupoints. Animals were
sacrificed at 7, 14 and 21 days after lesion for structural and biochemical analysis.
The quantitative data were compared statistically.The number of fibroblasts in A and
M was greater than C at 21 days. The amount of granulocytes was similar among the
groups in all experimental periods. The number of vessels was significantly higher in
A and M in relation to C at 21 days. The amount of glycosaminoglycans and
hydroxiproline was similar in all groups and times. Zymogram for MMP2 and MMP9 isoforms does not presented differences along all experimental times and groups.
The amount of type I collagen was significantly higher in A and M at seven days. In
relation to type III, the amount was significantly higher in A and M at 21 days. No
differences were noted in TGF1 and VEGF analysis. The therapy of A and M
affects positively the tecidual reorganization after experimental lesion in female adult
rats.
Study approved
FHO|Uniararas.
by
CEUA/UNIARARAS
(014/2014)
and
supported
by
Study approved
FHO|Uniararas.
by
CEUA/UNIARARAS
(014/2014)
and
supported
by

Q9
Q10
COMPARATIVE STUDY OF THE ORGANIZATION OF THE DIGESTIVE

SYSTEM OF SPECIES BELONGING TO THE SUPERORDER
CONDYLOGNATHA
DAMAGES CAUSED BY SUBLETHAL DOSES OF MALATHION IN

MELIPONA QUADRIFASCIATA L. MIDGUT EPITHELIAL CELLS
Camila Silva Leal (1), Alberto de Freitas Ribeiro (1), Alexandre Hiroshi Utiyama (1)
(1)
(2)
(1) Departamento de Gentica e Biologia Evolutiva - Universidade de So Paulo

camila.leal@usp.br - phone numbers: (55 11) 30917579 (55 11) 998858800
Address: Rua do Mato, 277 - CEP 05508-090 - So Paulo, SP, Brasil
Unlike most insects, members of the group Condylognatha (Thysanoptera and
Hemiptera) present a luminal membrane complex associated to enterocyte microvilli
in the form of perimicrovillar membranes (PMM). The PMM are observed in
members of the Heteroptera suborder and they seem to have evolved in the
Condylognatha ancestor as an adaptation to a sap-sucking habit. However, further
studies described two different organizations of the luminal membrane complexes in
Hemiptera: the modified perimicrovillar membranes (Sternorrhyncha) and the flamelike luminal membranes (FLM) (Auchenorrhyncha), suggesting that the luminal
membrane complex is more diverse than previously expected. Thus, this work
presents a morphofunctional study of the digestive system of Dalbulus maidis
(Hemiptera: Cicadellidae), Mahanarva fimbriolata (Hemiptera: Cercopidae),
including two representative species of Thysanoptera: Frankliniella schultzei
(Thripidae) and Liothrips sp. (Thripidae). Both adult male and female specimens
were used in this study. For transmission and scanning electron microscopy the
material was fixed in a glutaraldehyde/osmium tetroxide in cacodilate buffer,
according to routine procedures as detailed in Caldeira et al. Insect Biochem. Physiol,
64:1 (2007). Ultrastructural observations revealed the existence of FLM associated
with the enterocyte microvilli of D. maidis and M. fimbriolata. Nevertheless, in the
two thysanopteran species, the luminal membrane complex shows a different
organization with the presence of intermicrovillar trabeculae and fewer luminal
membranes. These findings indicate the occurrence of a rather diverse organization
in the luminal membrane complex among the Condylognatha species.
This study is supported by FAPESP and CAPES
Keywords: digestive system, Hemiptera, Thysanoptera, morphology
(1) Department of Histology, Embriology and Cell Biology, Federal University of

Gois, Gois, Brazil. Instituto de Cincias Biolgicas, Av. Esperana, s/n - Setor
Itatiaia, Goinia - GO, CEP: 74001-970
(2) Federal Institute of Education, Science and Technology of Gois, Gois city,
Gois, Brazil.
(3) Department of Botany, Federal University of Gois, Gois, Brazil.
Phone: 55 62 83033664, e-mail: pedrovalebrito@yahoo.com.br
Bees are responsible for pollinating native plant species, as well as plant species
grown for food production. When exploring flower resources in cultivated fields, as
tomatoes fields, they may consume pollen contaminated with pesticides. Even
sublethal doses of some pesticides can cause cellular damage reducing workers
fitness, contributing to reduce bee populations. This study investigated the effect of
sublethal doses of Malathion (organophosphate), in the midgut cells of Melipona
quadrifasciata. Adult workers of M. quadrifasciata were kept in laboratory
conditions and fed for three days with 50% sucrose syrup added with 2,5x10-6% of
Malathion. The control group was fed only with 50% sucrose syrup. After this period
their midgut were processed for TEM. The midgut epithelial cells are columnar with
basal extracellular labyrinth, characterized by mitochondrion richness. The apical
portion of these cells has a dome shape with microvilli. This portion of the cell does
not have many organelles, except by some electron lucid vesicles. The median
portion of the cells is rich of RER, mitochondrion and some electron lucid vesicles.
Midgut cells from workers fed with contaminated syrup have large electron lucid
portions, with no organelles, except few mitochondria. The basal cellular membrane
labyrinth and the apical microvilli are preserved, as well as the cellular shape.
However, most of the cytoplasmic organelles are disorganized and the cytoplasm is
filled with granular material. It is probable that even very small dosage of Malathion
may affect the physiological functions of the M. quadrifasciata midgut, although the
cellular shape is preserved.
Acknowledgements:
FAPEG
Laboratory of High Resolution Microscopy LabMic - UFG

Q12
Q11
MORPHOLOGICAL
ANALYSIS
BY
HISTOCHEMICAL
AND
ULTRASTRUCTURAL TECHNICS OF HUMAN SUBLINGUAL SALIVARY
GLAND AND STUDY OF IMMUNOHISTOCHEMICAL EXPRESSION OF
EGF; EGFR; FGFR-2
Sergio Mestieri Chammas* (1), Tamires Albuquerque Gomes (2), Marcelo
Cavenaghi Pereira da Silva (2), Adriana da Costa Neves (1).
(1)
(2)
Pedro Brito (1), Iago Schardong (1), Frederico Dutra (1), Walquiria Arruda (1),
Carlos Melo (2), Edivani Franceschinelli (3)
ANALYSIS OF DENERVATION EFFECTS ON THE REPULSIVE

GUIDANCE MOLECULE A (RGMA) EXPRESSION IN SKELETAL
MUSCLE CELLS
Samira Incia Santos e Silva (1*), Bruno Machado Bertassoli (1), Gerluza Aparecida
Borges Silva (1), Erika Cristina Jorge (1)
(1) Department of Morphology, Federal University of Minas Gerais, Minas Gerais,
Brazil
(1)Laboratrio de Gentica, Instituto Butantan, So Paulo, Brasil

(2) Universidade Federal de So Paulo, So Paulo, Brasil
(*) samira.inacia@hotmail.com, +553134093033; +5531996182828

* Telephone number : +55 11 2627-9701
Cell phone: +55 11 98177-1987
e-mail: sergio.chammas@butantan.gov.br
Background: The salivary glands secrete proteins called growth factors, during aging
the parenchyma of the secretory portions tends to be replaced by connective and
adipose tissue, decreasing the functional activity of these glands, causing dry mouth.
Aims: Study by immunohistochemistry, the pattern of EGF, EGFR and FGFR-2
expression in sublingual salivary glands (SLG) and compare imunohistochemical
expression of these protein between adults (30- to 60 years old) and elderly people
(over 60 year old). Methods: Morphological analysis was performed by
histochemical techniques and by transmission electron microscope.
Immunohistochemistry was used to verify the expression of EGF, EGFR and FGFR2. Quantification of immunohistochemistry was made by Image J software. Results:
Analyzing SLG of elderly people we found that the glandular parenchyma
replacement by fat appears to be at the expense of mucous cells than the serous ones.
In both groups, EGF expression was positive in serous cells with cytoplasmic
granular staining. The mucous cells presented weak membrane staining. Duct cells
exhibited mainly nuclear staining. FGFR-2 showed mainly granular cytoplasm
staining in serous and duct cells. Mucous cells were negative for FGFR2. The
expression of EGFR was remarkable in serous cells with granular cytoplasmic
stained but the mucous cells were negative. Ducts cells presented granular and
cytoplasmic staining. Analyzing the quantitative data of immunohistochemical
expression of EGF, EGFR and FGFR2, it was not observed differences between
adults and elderly people. Conclusions: EGF, EGFR and FGFR-2 are not the main
factors to maintain glandular parenchyma as they are similarly expressed during
aging.
This project was supported by FAPESP
This project was approved by the
26517914.3.1001.5505
ethics
committee
of
RGMa is a molecule described playing role as a repulsive clue for the axon
formation. Recent data, however, have described RGMa in skeletal muscle
precursors in the somites; and later, in a striated pattern in the sarcoplasma, and also
in the sarcolemma of adult cells. These data suggested an association of RGMa with
skeletal muscle development and maintenance. In this work, we proposed to
investigate RGMa expression in atrophied skeletal muscle cells, induced by
denervation. Adult swiss mice had their left sciatic nerve axotomized to induce
gastrocnemius atrophy. The right side was used as control. After 14 days, the
atrophic condition was confirmed by RT-qPCR for Atrogin-1 and MuRF-1; by
measuring muscle fibers area; and by weighting muscles mass. RGMa expression in
atrophied muscles was evaluated by RT-qPCR and immnunofluorescence.
Gastrocnemius mass and fibers area reduced approximately 65% and 74%,
respectively. Relative expression analysis revealed the upregulation of both
Antrogin-1 and MuRF-1 in gastrocnemius muscle, confirming atrophy induction.
Relative expression revealed a 2-fold upregulation of RGMA in the
atrophic/denervated skeletal muscle. RGMa expression pattern has revealed an
increase of the protein expressed on the sarcolemma. Our data suggested an
association of RGMa with the atrophic signaling pathway.
All animal procedures were conducted following the instructions of the Ethics
Commission on Animal Use (CEUA-UFMG).
This work was supported by CNPq and Fapemig.
UNIFESP:

Q13
Q14
COULD AN INTEGRATIVE APPROACH BRING NEW INFORMATION

ABOUT ECHINODERM COELOMOCYTES? A STUDY USING THE SEA
URCHIN EUCIDARIS TRIBULOIDES AS MODEL
HISTOLOGY OF THE SMALL INTESTINE OF AMPHISBAENA ALBA

(SQUAMATA, AMPHISBAENIDAE)
Cnthia Kazue Arizono, talo Augusto da Costa Soares, Fernanda Barbosa Lopes,
Renato Neves Feio, Sirlene Souza Rodrigues Sartori
Vinicius Queiroz, Mrcio Reis Custdio

Department of General Physiology, University of So Paulo, So Paulo, Brazil.
Center for Marine Biology (CEBIMar), University of So Paulo, So Paulo, Brazil.
Correspondence: Vinicius Queiroz vinicius_ufba@yahoo.com.br.
Phone number: +55 11 3091-7522, Cel Phone: +55 11 98301-4102
Like others invertebrates, echinoids has cellular immunity as their first line of
defense and three categories of coelomocytes are registered: phagocytes,
spherulocytes (red and colorless) and vibratile cells. Evidences from various
techniques (i.e. an integrated approach) can bring very useful information about
coelomocytes however this kind of study is scarce yet. Thus, this work demonstrates
that an integrative approach (live cells, cytochemistry and electron microscopy) can
brings advantages in echinoderm coelomocytes studies. The coelomic fluid of
Eucidaris tribuloides was withdrawn through a syringe preloaded with anticoagulant
solution and living cells and cytospun slides were observed. These slides were
prepared using a cytospin and finally stained with Toluidine Blue (TB), Hematoxylin
and Eosin (H&E) or Mallory Trichrome (MT). For electron microscopy, cells were
fixed in glutaraldehyde 2.5% (2-4h) and followed usual TEM and SEM protocols.
Seven subpopulations were observed: four previously documented and three new
(progenitor cell and granular and irregular spherulocytes). For spherulocytes (except
the irregular) and vibratile cells, a maturation process was observed. Early cells
showed large nucleus and reticulated cytoplasm (spherulocytes) or large cytoplasm
with low affinity to TB (vibratile cell). In the final stage, they presented small
nucleus and a spherulous cytoplasm or small and spherulous cytoplasm with high
affinity to TB, respectively. The detection of new cell-subpopulations and maturation
process shows how this approach can be useful. Consequently, in the absence of
specific markers to echinoderm cells, and to other invertebrates, more attention
should be given to this kind of study.
Departamento de Biologia Animal, Universidade Federal de Viosa, Viosa, MG
This work describes the histology of the small intestine of Amphisbaena alba,
fossorial animal that feeds on small invertebrates. Three animals were euthanized for
collection of intestinal fragments, that were processed according to the routine
procedure for inclusion in resin. Sections of 3m thickness were stained with
toluidine blue and Periodic Acid-Schiff (PAS) conjugate with Alcian Blue (AB). The
small intestine is a relatively long tube and it is thickened at its cranial portion,
becoming narrower caudally. In its inner lining there are numerous tall, slender folds.
The mucosa is lined by simple prismatic epithelium with brush border cells and
goblet cells PAS-AB positive. The lamina propria, formed by loose connective
tissue, is mixed with the submucosa because of the absence or poor development of
the muscularis mucosae. The muscular tunic is formed by two smooth muscle layers,
an inner circular and outer longitudinal. The serosa is formed by a connective tissue
lined by mesothelium. The small intestine of A. alba resembles the other reptilian
species, but differ from those of mammals, especially the absence of histological
distinction between intestinal segments, although the initial portion is anatomically
distinct; and the absence of villi and crypts, although the tall folds resemble villi and
may enlarge the absorptive surface. As in the small intestine of other vertebrates, the
brush border cells are certainly responsible for the final digestion and absorption of
nutrients while goblet cells are responsible for lubrication and protection to secrete
neutral mucins (PAS-positive) and acid (AB-positive).
Financial support: Fapemig.
Financial support: CAPES Foundation and FAPESP (proc. 15/21460-5).

Q15
Q16
STRYCHNOS PSEUDOQUINA EXTRACT ENHANCES CELLULAR

PROLIFERATION IN WOUND HEALING IN DIABETIC RATS
EFFECT OF GUARAROBA EXTRACT FOR THE NEOANGIOGENESIS

DURING THE WOUND HEALING IN DIABETIC RATS
Reggiani Vilela Gonalves (1), Fernanda Barbosa Lopes* (1), Rmulo Dias Novaes
(2), Joo Paulo Viana Leite (3), Mariurea Matias Sarandy (4)
Reggiani Vilela Gonalves (1), Laura Viera do Amaral* (2), Rmulo Dias Novaes
(3), Joo Paulo Viana Leite (4), Mariurea Matias Sarandy (5).

Viosa MG, Brazil
(4) Department of General Biology, Federal University of Viosa, MG, Brazil.

Viosa MG, Brazil
(5) Department of General Biology, Federal University of Viosa, MG, Brazil.
Phone: (+55) 31-3899-4375. fernanda.b.lopes@ufv.br

Phone: (+55) 31-3899-4375. laura.amaral@ufv.br
Plants are an unlimited source of potentially active substances and many of them are
used to help promote wound healing. This study aimed at describing the action of
Strychnos pseudoquina extract in the cellular proliferation during wound healing of
diabetic rats. Thirty male rats were randomized into five treatment groups with six
animals each. The procedure included the use of Salt (0.9% saline solution), OV
(ointment vehicle), SS (sulfadiazine of silver), LE 5 (S. pseudoquina 5%) and LE 10
(S. pseudoquina 10%). Three skin wounds of 12 mm in diameter were performed and
the ointment extracts were applied daily over the period of 21 days, while tissue from
different wounds was removed every 7 days. The sections were stained with
hematoxylin and eosin (H&E). Stereological parameters of cells volumetric density
(Vv) were calculated by counting the points a superimposed grid over fibroblasts,
using the ratio of Vv = PP/PT, in which PP refers to the number of points occurring
over the structure of interest, while PT represents the total number. Regarding the
cellularity, groups treated with S. pseudoquina (5% and 10%) have shown a
decrease in the number of cells on the 7th , 14th and 21st days (F1, F2 e F3
respectively), when compared to the other groups. Thus, these results have shown
that S. pseudoquina extract has an anti-inflammatory effect, reducing the number of
cells and consequently improving wound healing.
Such project was only possible due to the support from FAPEMIG (edital APQ
00685-14).
Introduction: Neovascularization are striking processes in wound healing, mainly

during the inflammatory phase. It assures a suitable environment for cell metabolic
activities and provides a better skin recovery. Objective: Investigate the effect of the
guararoba extract during wound healing in diabetic rats. Material and Methods:
Thirty male rats were randomized into 5 groups with 6 animals in each group: GE 5
(Guararoba extract 5%), GE 10 (Guararoba extract 10%), Sal (0.9% saline solution),
OV (ointment vehicle) and SS (sulfadiazine of silver). Three skin wounds of 12 mm
in diameter each were realized and the extracts were applied daily over 21 days.
Tissue from different wounds was removed every 7 days. Cuts of 4 m thick, were
obtained using a 40 objective lens and 10 histological fields were analyzed. The
sections were stained with hematoxylin and eosin (H&E) for analysis of blood
vessels. Images were captured for digital camera and Stereological parameters of
volumetric density (Vv) were calculated by counting the points over blood vessels
using the ratio: Vv = PP/PT. Results: The groups treated with Guararoba 5% (GE 5)
and 10% (GE 10), showed larger proportion of blood vessels during of the days 14
and 21 when compared to controls (Sal, OV and SS). Conclusion: The treatments
performed with Guararoba shown to be effective in the healing process by providing
oxygen and nutrients for cell proliferation.
We would like to thank the FAPEMIG (editalAPQ 00868-15).

Q17
Q18
EVALUATION OF HISTOMORPHOMETRIC CHANGES IN TOOTH

MOVEMENT ASSOCIATED WITH PERIODONTAL DISEASE IN THE
PRESENCE OF DIABETES MELLITUS
HISTOLOGY OF THE ESOPHAGUS

(SQUAMATA, AMPHISBAENIDAE)
Ewerton Zaniboni (1), Leonardo Bagne (1), Rodrigo Dalia (1), Mara Felonato
Mendes (1), Maria Esmria Corezola do Amaral (1), Mauro Pedrine
Santamaria (3), Marcelo Augusto Marretto Esquisatto (1), Fernanda Aparecida
Sampaio Mendona (1), Glucia Maria Tech dos Santos (1), Milton Santamaria
Junior (1,2).
(1) Graduate Program of Biomedical Sciences, Heminio Ometto University
Center, UNIARARAS, Sao Paulo, Brazil, 13607-339
(2) Graduate Program of Orthodontics, Heminio Ometto University Center,
UNIARARAS, Sao Paulo, Brazil.
(3) Division of Periodontics, College of Dentistry, State University of So
Paulo, UNESP, Sao Jose dos Campos, Sao Paulo, Brazil.
E-mail: taozaniboni@hotmail.com Fone: +55 (19) 3543-1440.
Background: Diabetes Mellitus and periodontal disease can produce disorders in
periodontal repair during orthodontic therapy. Aims: To evaluate the histological
changes during tooth movement (OTM) in the Diabetes mellitus and periodontal
disease. Methods: Fifty male Wistar rats, 90 days, 300g were divided into 5 groups
(n=10): OTM-Healthy submitted to OTM (force application of 40g for 7 days on first
molar); OTM+L-Healthy submitted to OTM and induced with periodontal disease
(ligature on first molar); OTM+D-diabetic induced with Alloxan and submitted to
OTM; OTM+D+L-diabetic, submitted to OTM and induced periodontal disease;
D+L-diabetic and induced periodontal disease. The samples were collected on the
7th day of OTM and stained with hematoxylin-eosin, picrossirius-hematoxylin and
toluidine blue, for histomorphometric analysis of fibroblasts, osteoclasts,
granulocytes, blood vessels and birefringent collagen fibers. The data were analyzed
by ANOVA and Tukey's test at 5% significance level. Results: Histomorphometric
analysis revealed a significant increase of granulocytes in OTM+D+L when
compared to other groups (OTM=19.82.6, OTM+D=22.81.8, OTM+L=21.82.1,
OTM+D+L=24.62.1, D+L=23.62.9). There was a significant decrease in blood
vessels in diabetic animals in different groups (OTM=5.20.7, OTM+D=4.80.8,
OTM+L=5.10.5, OTM+D+L=4.40.8, D+L=4.60.6). The number of fibroblasts
(OTM=30.92.8, OTM+D=24.82.4, OTM+L=27.23.8, OTM+D+L=26.52.2,
D+L=25.23.5), osteoclasts (OTM=5.90.8, OTM+D=4.80.7, OTM+L=4.40.6,
OTM+D+L=3.90.8, D+L=3.80.4) and percentage of birefringent fibers
(OTM=51.17.3, OTM+D=42.16.6, OTM+L=44.66.8, OTM+D+L=39.78.1,
D+L=41.97.2) were higher in OTM. Conclusion: The presence of Diabetes Mellitus
interfered in the neovasculogenesis and aggravated the inflammatory process in
OTM when associated with periodontal disease.
OF
AMPHISBAENA
ALBA
Cnthia Kazue Arizono, talo Augusto da Costa Soares, Fernanda Barbosa Lopes,
Renato Neves Feio, Sirlene Souza Rodrigues Sartori
Departamento de Biologia Animal, Universidade Federal de Viosa, Viosa, MG
Morphological studies are scarce, thus, this work sought to describe the histology of
the esophagus of Amphisbaena alba, reptil squamata known as two-headed snake.
Three animals were euthanized for collection of esophageal fragments, that were
processed according to the routine procedure for inclusion in resin. Sections of 3m
thickness were stained with toluidine blue and Periodic Acid-Schiff (PAS) conjugate
with Alcian Blue (AB). The esophagus is extensive (measuring 12cm on average)
and narrow, and its wall consists of four layers: mucosa (epithelium, lamina propria
and muscularis mucosae), submucosa, muscular and adventitia. The epithelium is
pseudostratified with ciliated cells, basal cells and mucus-secreting goblet cells PASAB-positive. The lamina propria, formed by loose connective tissue, and muscularis
mucosae, composed of a smooth muscle band, are well developed. The submucosa is
composed of loose connective tissue with blood and lymph vessels; the muscular is
composed of a thick layer of smooth muscle arranged circularly and other thinner
longitudinal. In the transition between esophagus and stomach there is not an
anatomical sphincter and the boundary between the two is difficult to distinguish,
with a smooth increase in tube caliber and wall thickness. Histologically the
transition is evident, with the most significant change observed in the mucosa, with
the emergence of the gastric glands and the change in the epithelium, which becomes
simple prismatic composed solely of mucus-secreting cells. Esophageal cilia comply
role in cleaning and possibly aid in swallowing. Mucus is important for lubrication
and protection, even against gastric acid.
Study approved by CEUA/UNIARARAS (Parecer no 022/2014) and supported by

FHO|Uniararas and PNPD/CAPES (Process no 23038.008192/2013-01).

Q19
Q20
PROTECTIVE
EFFECT
OF
LASER
THERAPY
(LT)
ON
ACETYLCHOLINE RECEPTORS(nAChrs) AFTER LOCAL ANESTHESIA
Cristiane Neves Alessi Pissulin (1), Paula Aiello Tome de Souza (2), Ivan Jos
Vechetti Jr(3), Selma Maria Michelin Matheus(4)
(1) Graduation Program on the General Bases of Surgery, FMB, Unesp, Botucatu,
So Paulo, Brasil; Department of Anatomy, Oeste Paulista University (UNOESTE),
Presidente Prudente, SP, Brasil.
(2) PhD So Carlos Federal University , So Carlos, SP, Brasil.
(3) PhD in General and Applied Biology , IB/Unesp, Botucatu, So Paulo, Brasil.
(4) Department of Anatomy, IB/Unesp, Botucatu, So Paulo, Brasil.
Local anesthetics act both in the voltage-dependent channels and in the nAChRs of
neuromuscular junctions(NMJ). Bupivacaine is one of the LA widely used, but it is
considered very aggressive. LT is an option for injury treatment. This study is about
the effects of LT in sternomastoid NMJ and nAChr after Bupivacaine. Fifteen adult
male Wistar rats (CEUA/IBB n509) were used. The muscles received Bupivacaine
0.5 % on the right antimere and sodium chloride 0.9 % on the left. After 24 hours,
the animals were divided into: Control and LT groups that received one daily
application of GaAs 904 nm on both antimeres for 5 consecutive days. After the
muscles were removed and the morphology and morphometry of NMJ were
evaluated by confocal laser scanning microscopy and the nAChR protein levels by
Western Blotting.The NMJ and nAChrs with sodium chloride didn't show
alterations. Confocal microscopy analysis showed a decrease in the number of
synaptic elements and fluorescence intensity in NMJs after Bupivacaine. After LT
the branches of the NMJs increased and became more elongated. The morphometric
analysis revealed that the values of area (76.30), perimeter (74.69) and relative
planar area(8.75) were larger after LT. These data showed that the NMJ complexity
increased after LT. All the groups had the same 1 protein expression. There was a
positive correlation between 1 and subunits after LT and an increased of subunit
expression. These results indicate that LT can be used after LA, due to its protective
effect on nAChrs and NMJs.
Financial support: Fapesp: 2013/26649-3
EFFECTS OF A CEREBRAL PALSY EXPERIMENTAL MODEL ON THE

NUMBER OF NUCLEIS AND CAPILLARIES OF EXTENSOR DIGITORUM
LONGUS (EDL) MUSCLE
Caroline Covatti (1); Pmela Buratti (1); Bruna Hart Ulsenheimer (1); Lucinia
Chasko Ribeiro (2); Lgia Aline Centenaro (2); Mrcia Miranda Torrejais (2).
(1) Discente do programa de Ps Graduao - Nvel Mestrado em Biocincias e
Sade, Universidade Estadual do Oeste do Paran, Cascavel, Brasil.
(2) Docente da disciplina de Anatomia Humana, Universidade Estadual do Oeste do
Paran, Cascavel, Brasil
address: Rua Universitria, 1619 - Jardim Universitrio, Cascavel - PR, Brasil
e-mail: pamela_buratti@hotmail.com
instuticional: (45) 3220 - 3194
mobile: (45) 8818-7275
Cerebral palsy (CP) is a chronic encephalopathy, which causes motor disturbances.
Several animal models have been developed to reproduce the clinical features found
in patients with CP. This study aimed to quantify the number of muscle fibers, nuclei
and capillary in the extensordigitorumlongus (EDL) muscle of rat pups subjected to
a experimental model of CP. Ethics Committee on Animal Use of UNIOESTE
approved this study. Wistar male pups were divided in to groups: Control (CTL) pups of mothers injected with saline during pregnancy and PC - pups of mothers
injected with lipopolysaccharide during pregnancy, submitted to neonatal anoxia and
sensorimotor restriction. On the day of birth, the animals of CP group were placed in
a closed chamber with nitrogen flow (100%) to perform the anoxia. During 30 days
PC group were also submitted to an immobilization of hindlimbs, named as
sensorimotor restriction. At 48 days of age, the animals were euthanized, the EDL
muscle was collected and stained with hematoxylin-eosin. The ratio of nuclei/muscle
fiber and of capillary/muscle fiber was higher in PC group (21% and 18%,
respectively). The increase in the capillary density and in the nuclei number
associated with hypertrophy of muscle fibers are characteristics of spastic muscles, a
finding frequently observed in patients with CP.
Thus, this animal model seems to reproduce part of the signs observed in patients
with this disease.

Q21
Q22
HISTOENZIMOLOGIC AND MORPHOMETRIC ANALYSIS OF THE

EXTENSOR DIGITORUM LONGUS (EDL) MUSCLE OF RATS
SUBJECTED TO AN EXPERIMENTAL MODEL OF CEREBRAL PALSY
EFFECTS OF ROACUTAN (ISOTRETINOIN)

INTESTINE OF ADULT WISTAR RATS
Caroline Covatti (1); Pmela Buratti (1); Ana Tereza Bittencourt Guimares (3);
Lgia Aline Centenaro (2); Mrcia Miranda Torrejais (2).
(1) Discente do programa de Ps Graduao - Nvel Mestrado em Biocincias e
Sade, Universidade Estadual do Oeste do Paran, Cascavel, Brasil.
(2) Docente da disciplina de Anatomia Humana, Universidade Estadual do Oeste do
Paran, Cascavel, Brasil
e-mail: pamela_buratti@hotmail.com
mobile: (45) 8818-7275
Cerebral palsy (CP) refers to a posture and movement disorder, attributed to a nonprogressive injury to the brain. The objective of this study was to quantify the
number and area of different muscle fibers in extensor digitorum (EDL) muscle of
rats subjected to a CP model. This study was approved by the Ethics Committee on
Animal Use of UNIOESTE. Wistar male pups were divided in to groups: Control
(CTL) - pups of mothers injected with saline during pregnancy and PC - pups of
mothers injected with lipopolysaccharide during pregnancy, submitted to neonatal
anoxia and sensorimotor restriction. On the day of birth, animals of CP group were
placed in a closed chamber with nitrogen flow (100%) to perform the anoxia. From
the first postnatal day (P1) to P30, PC group were submitted to a sensorimotor
restriction with the immobilization of hindlimbs. At 48 days of age, the animals were
euthanized, the EDL muscle was collected and stained with the NADH-TR reaction.
This histoenzimologic analysis allows the characterization of the muscle fibers in
type I, IIA and IIB. No significant differences where found between the experimental
groups in relation the number of each type of muscle fiber, but there was an increase
of 16% in the cross-sectional area of type IIB fibers in the CP group. It is known that
patients with CP have muscle hypertrophy as a result of spasticity.
ON
THE
SMALL
Adlia Contiliano Expedito, Susana Puga Ribeiro, Andra Costa Goulart, Camila
Gonalves Oliveira Chagas, Srgio Luis Pinto da Matta, Sirlene Souza Rodrigues
Sartori
Departamento de Biologia Animal, Universidade Federal de Viosa, Viosa, MG.
Isotretinoin is widely used to treat severe acne. However, as participates in the

regulation of the rate of growth and differentiation of many cell types, its use
generates multiple collateral effects. This study sought to determine the effects of
isotretinoin in the digestive morphophysiology. The experiment was carried out for
60 days with 29 adult Wistar rats divided into 4 groups: control group 1 (n = 6)
received 1.0 ml of water/day, control group 2 (n = 5) received 1.0 ml of soybean
oil/day, treated group 3 (n = 9) received 0.2 mg Roacutan in 1.0 ml of soybean
oil/day, treated group 4 (n = 9) received 0.4 mg Roacutan in 1.0 ml of soybean
oil/day. Subsequently the animals were euthanized to collect fragments of the
duodenum, jejunum and ileum, that were fixed in Carson formalin solution for 24
hours, dehydrated in increasing concentrations of ethanol and embedded in
methacrylate. They were obtained 3 thick sections that were stained with toluidine
blue and periodic acid-Schiff (PAS). It used analysis of variance followed by Student
Newman-Keuls test (a = 0.05). There were no significant differences in the evaluated
parameters that were thickness of layers, height and width of villi, crypt depth,
thickness of the brush border and number of PAS-positive goblet cells, except for the
villus height in duodenum, which was higher in group 4. This reflects the potential of
isotretinoin for cell proliferation and may have implications on the digestive
physiology by increasing the intestinal surface.
Thus, this CP animal model seems to reproduce this clinical finding of CP.

Q23
Q24
CHANGES IN CHROMATIN STRUCTURE MAY ELICIT SENESCENCE IN

PRIMARY CULTURES OF MOUSE CORTEX ASTROCYTES
GROWTH HORMONE
FORMATION
Trika Gonalves do Carmo Oliveira, Vinicius Freitas Fernandes, Renata Graciele

Zanon, Alberto da Silva Moraes
Ccero Fagner Messias de Lima (1), Maria Danielma dos Santos Reis (1), Fernando
Wagner da Silva Ramos (1), Silvana Ayres-Martins (1), Salete Smaniotto (1).
Undergraduation Program in Biomedicine, Institute of Biomedical Sciences,

Federal University of Uberlandia.
Department of Anatomy, Institute of Biomedical Sciences, Federal University of
Uberlandia
Department of Cell Biology, Histology, and Embryology, Institute of Biomedical
Sciences, Federal University of Uberlandia
(1) Laboratory of Cell Biology, Institute of Biology and Health Science, Federal
University of Alagoas, Alagoas, Brazil.
E-mail: tarika.goliveira@gmail.com. Telephone contact: +55 34 99123-4235
Background: Studies have indicated that the growth hormone (GH) may act as
proangiogenic factor, inducing endothelial cell function and angiogenesis in vivo.
Aims: We aimed the GH in vitro effects on endothelial cells, especially on their
migratory capacity and in the formation of capillary-like structures.
Methods: The study was carried out with the endothelial cell line tEnd.1. These cells
were treated with GH (10-9, 10-8 and 10-7 M) at different time points and further
analysed for cell proliferation; deposition of extracellular proteins and expression of
integrins; cell migration on a transwell system and formation of capillary-like
structures on a three dimensional cell culture model with matrigel.
Results: We could demonstrate an increase on tEnd.1 proliferation after GH
treatment at concentration of 10-8 M for both 8 and 24 hours. This same
concentration, at 24 hours, induced cellular morphological changes on tEnd.1,
increased laminin and fibronectin (FN) deposition, and augmented the expression of
FN receptors VLA-4 and VLA-5. The GH also modulated the tEnd.1 migration on
transwell chambers, with increased number of migrating cells, on both 10-8 and 10-7
M concentrations, compared with non-treated cells. The treatment with GH at 10-8
and 10-7 M concentrations significantly stimulated capillary-like tube formation at 6,
12 e 24 hours compared to tEnd.1 without treatment.
Conclusion: These findings can shed lights on therapeutic angiogenesis, and
particular those pathologies where the cardiovascular system has been compromised.
Background: Primary cell cultures cannot be indefinitely maintained because

successive mitotic events along cultivation contribute to telomere shortening, thus
triggering replicative senescence and cell cycle arrest. Cell aging is associated with
changes in chromatin structure, but it is unclear if these changes take place before or
after the senescence events, being, respectively, a cause or a consequence of
senescence.
Aims: To evaluate if changes in chromatin are an ongoing event along cell
cultivation or a latter event of senescence.
Methods: Primary cultures of astrocytes obtained from newborn Swiss cerebral
cortexes were used. Every time cells reached confluency, they were counted for
estimation of Population Doublings (PDL), and recultured, from where some cells
were separated, spread and fixed in histological slides, followed by cytochemical
staining of nuclei by Feulgen Reaction. Images of stained nuclei were captured and
analyzed in ImageJ. Mann-Whitney test was used with significance for p<0.05.
Results: Cultured astrocytes had a total of 13 PDLs, with an evident decreased
proliferation capacity after PDL 9. However, even in lower PDLs, there was
chromatin unpackaging with a more homogeneous distribution, which were more
evident in higher PDLs. Chromatin unpackaging occurred mainly in heterochromatic
areas which is an indication of a senescence associated loss of heterochromatic areas
and increased genome instability.
Conclusion: Senescent associated changes in chromatin structure are an early event
of cell senescence, and take place even before cells acquire senescent phenotype,
being a trigger of senescence.
Ethical Approval: all procedures were approved by the Local Ethical Committee
(protocol 085/11).
INDUCES
IN
VITRO
BLOOD
VESSEL
Disclosure: Authors declare that they have no competing interests.

danielma.reis@icbs.ufal.br; 82 3214-1704/ 82 9 9938-4827
Funding support: Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico

(CNPq) and Fundao de Amparo Pesquisa do Estado de Alagoas (FAPEAL).

Q25
Q26
AEROBIC TRAINING PROTOCOL IN CHRONIC HEART FAILURE

ALTERS MUSK/RAPSYN SIGNALING PATHWAYS IN THE SYNAPTIC
TRANSMISSION BY NICOTINIC ACETYLCHOLINE RECEPTORS
(nAChR)
CONFOCAL MICROSCOPY SHOWS THAT CHRONIC HEART FAILURE

CHANGES STRUCTURAL ASPECTS OF NEUROMUSCULAR JUNCTIONS
IN DIAPHRAGM MUSCLE IN RATS
Paula Aiello Tom de Souza Castro (1), Luana Campos Soares (2), Rodrigo Wagner
de Souza (3), Antonio Carlos Cicogna (4), Dijon Campos Soares (4), Katashi Okoshi
(4) Marcia Gallaci (5), Walter Garrido (5), Selma Maria Michelin Matheus (6),
Maeli Dal Pai Silva (7)
(1) post-doctorated in Physical Therapy, So Carlos Federal University, So Carlos,
SP, Brasil.
(2) Master Science in So Paulo Universiy, So Paulo, Brasil.
(3) post-doctorated in Sao Paulo University, So Paulo, Brasil.
(4) Department of Medical Clinic, FMB/UNESP/Botucatu, So Paulo, Brasil.
5) Department of Pharmacology, IB/Unesp, Botucatu, So Paulo, Brasil.
(6) Department of Morphology, IB/Unesp, Botucatu, So Paulo, Brasil.
Heart failure (HF) is a myopathy that causes muscle weakness, fatigue, labored
respiration, metabolic and neuromuscular activity changes to sustain the increased
workload. Physical exercise is a safe and effective therapeutic intervention to
improve clinical status, functional capacity and quality of life during heart failure.
The present study aimed to evaluate the aerobic training protocol about molecular
and functional characteristics of synaptic transmission in diaphragm muscle in rats
with HF. In order to do that, 40 rats were used in this experiment. They were divided
in Sham group (10 rats without aortic stenosis), AS group (10 rats with aortic
stenosis), ShamTR group (10 rats with training and without aortic stenosis) and
ASTR group (10 rats with aortic stenosis and with aerobic training). At 18 weeks
after surgery, when the AS animals presented a cardiac dysfunction as measured by
echocardiography, both groups (Sham and AS) were randomly redistributed in either
sedentary untrained groups (Sham and AS) or groups that underwent 10 weeks of
aerobic training protocol (ShamTR and ASTR). At 28 weeks, the AS group
presented cardiac dysfunction and HF induced an increase in the nAChR1 and
MusK synaptic regulators and a decrease in Rapsyn expression that ancored nAChR.
The functional properties as safety margin and fatigue resistance showed an increase
in the diaphragm muscle. Aerobic training protocol attenuated the deterioration of
cardiac function and prevented molecular and functional changes, in nAChR1 and
MusK synaptic regulator expression. . Thus, the study has important implications for
better understanding of HF pathophysiology indicating the potential therapeutic
effects of aerobic exercise to improve diaphragm muscle function in this condition.
This study was approved by the Ethical Comitee (#412) and received support from
Fapesp proc. 2012/02850-9.

Q27
Paula Aiello Tom de Souza Castro (1), Luana Campos Soares (2), Rodrigo Wagner
de Souza (3), Antonio Carlos Cicogna (4), Dijon Campos Soares (4), Katashi Okoshi
(4) Selma Maria Michelin Matheus (5), Maeli Dal Pai Silva (6)
(1) post-doctorated in Physical Therapy, So Carlos Federal University, So Carlos,
SP, Brasil.
(2) Master Science in So Paulo Universiy, So Paulo, Brasil.
(3) post-doctorated in Sao Paulo University, So Paulo, Brasil.
(4) Department of Medical Clinic, FMB/UNESP/Botucatu, So Paulo, Brasil.
(6) Department of Morphology, IB/Unesp, Botucatu, So Paulo, Brasil.
Heart failure (HF) is a myopathy that causes tachypnea and limited physical
capacity. Diaphragm is an inspiratory muscle most affected during HF and can
promote fibers type modulation, alterations in the contractile properties and synaptic
transmission activity. Physical exercise is an effective therapeutic intervention to
improve quality of life during heart failure and may partially attenuate some of the
cardiac and skeletal muscle abnormalities. The present study aimed to evaluate
structural characteristics of neuromuscular junctions (NMJs) in diaphragm muscle in
rats with HF submitted to aerobic training. In order to do that, 40 rats were used in
this experiment. They were divided in Sham group (10 rats without aortic stenosis),
AS group (10 rats with aortic stenosis), ShamTR group (10 rats with training and
without aortic stenosis) and ASTR group (10 rats with aortic stenosis and with
aerobic training). At 18 weeks after surgery, when the AS animals presented a
cardiac dysfunction as measured by echocardiography, both groups (Sham and AS)
were randomly redistributed in either sedentary untrained groups (Sham and AS) or
group that underwent 10 weeks of aerobic training (ShamTR and ASTR). Area,
density and dispersion of 30 motor endplates were measured in all animals in order
to determine fiber proportions for each group using laser scanning confocal
microscopy. At 28 weeks, AS group presented cardiac dysfunction, changes in the
NMJs structure with an increase in area, density and dispersion motor endplate for
each fiber type. The aerobic training attenuated the deterioration of cardiac function,
but did not reverse the NMJs structural changes. Therefore, the study demonstrated
that chronic heart failure induced by aortic stenosis promoted changes in diaphragm
NMJ structure, but aerobic training did not modify this condition in the synaptic
transmission.
This study was approved by the Ethical Comitee (#412) and received support from
Fapesp proc. 2012/02850-9.
Q28
IN VITRO STUDY USING OSTEOGENIC CELLS FOR EVALUATION OF

CYTOTOXICITY OF Ti-20Mo ALLOYS DOPED WITH DIFFERENT
OXYGEN CONTENTS
Anne A. Capra (1,2); Tatiani A.G. Donato (1,2); Renata A. Nogueira (1,2); Raul O.
Arajo (1,2); Carlos R. Grandini (1,2)
OLMESARTAN DECREASES THE INFLAMMATORY PROCESS AND

OXIDATIVE STRESS IN AN INTESTINAL MUCOSITIS MODEL
Raimundo Fernandes de Arajo Jnior, Ana Luiza Cabral S Oliveira, Vincius
Barreto Garcia, Pedro Brito Borba, Aurigena Antunes de Araujo
UFRN
(1) UNESP Univ Estadual Paulista, Laboratrio de Anelasticidade e Biomateriais,

Bauru, Brazil
(2) IBTN Institute of Biomaterials, Tribocorrosion and Nanomedicine Brazilian
Branch, Bauru, Brazil
The ideal biomaterial should induce predicable, controlled, guided and rapid healing
of host tissues. Titanium (Ti) is the consensual biomaterial employed in implants for
allowing a well osseointegration. Recently, promising alloys that added niobium
(Nb), tantalum (Ta), zirconium (Zr) and molybdenum (Mo) to Ti are being
investigated. These alloys are a new class of Ti -based alloys, which avoid Al and V,
while exhibiting low values of Youngs modulus, quite attractive as a biomaterial.
The in vitro cytotoxicity tests represent the initial step of the process, and could be
considered as a pre-selection of those materials. To investigate the biocompatibility
as well as the differentiation of osteoblastic cells cultived on a Ti-20Mo alloy after
annealing heat treatment comparing with Ti-20Mo alloy doped with different
oxygen, in vitro cell viability, immunofluorescense and alkaline phosphatase were
used. For this, MC3T3-E1 line cells were cultured on Ti-20Mo alloys with base
medium [MEM supplemented with 10% FBS and 1% gentamicina]. These cells
were exposed on Ti-20Mo alloys for 48 hours to assay MTT and indirect
immunofluorescence. The alkaline phosphatase assay was analyzed with 7 days. The
present results demonstrated that all studied alloys presented no cytotoxic effects on
the osteogenic cells. In addition, a high activity of alkaline phosphatase was
observed. All of them, independently from the treatment, showed a central and
flattened cell body and numerous and long processes.
The authors thank Brazilian agencies CNPq (grants #481.313/2012-5,
#307.279/2013-8 and 400.705/2015-0)and FAPESP (grant #2015/25.562-7) for
financial support.
Methotrexate (MTX) is a pro-oxidant compound that depletes dihydrofolate pools

and is widely used in the treatment of leukaemia and other malignancies. The
efficacy of methotrexate is often limited by mucositis and intestinal injury, which are
major causes of morbidity in children and adults. The aim of this study was to
evaluate the effect of olmesartan (OLM), an angiotensin II receptor antagonist, on an
Intestinal Mucositis Model (IMM) induced by MTX in Wistar rats. IMM was
induced via intraperitoneal (i.p.) administration of MTX (7 mg/kg) for three
consecutive days. The animals were pre-treated with oral OLM at 0.5, 1 or 5 mg/kg
or with vehicle 30 min prior to exposure to MTX. Small intestinal homogenates were
assayed for levels of the IL-1b, IL-10 and TNF-a cytokines, malondialdehyde and
myeloperoxidase activity. Additionally, immunohistochemical analyses of MMP-2,
MMP-9, COX-2, RANK/RANKL and SOCS-1 and confocal microscopy analysis of
SOCS-1 expression were performed. Treatment with MTX + OLM (5 mg/kg)
resulted in a reduction of mucosal inflammatory infiltration, ulcerations,
vasodilatation and haemorrhagic areas (p<0.05) as well as reduced concentrations of
MPO (p,0.001) and the pro-inflammatory cytokines IL-1b (p<0.001) and TNF-a
(p<0.01), and increase anti-inflammatory cytocine IL-10 (p<0.05). Additionally, the
combined treatment reduced expression of MMP-2, MMP-9, COX-2, RANK,
RANKL(p<0.05) and increased cytoplasmic expression of SOCS-1 (p<0.05). Our
findings confirm the involvement of OLM in reducing the inflammatory response
through increased immunosuppressive signalling in an IMM. We also suggest that
the beneficial effect of olmesartan treatment is specifically exerted during the
damage through blocking inflammatory cytocines.

Q29
Q30
EFFECTS OF THE X RADIATION ON THE RAT SKIN MORPHOLOGY
MORPHOLOGICAL CHANGES IN THE LUNGS OF MARIJUANA

SMOKING MOTHERS: STUDY IN MICE
Matheus da Silva Santin (1); Jos Koehler (2); Jos Rosa Gomes (3).
(1) Acadmico do 3 ano do curso de Medicina da UEPG
(2) Mestre em Ps Graduao em Cirurgia pela PUC-PR- rea Medicina, sub-rea
Clnica Mdica - Professor Assistente da Universidade Estadual de Ponta Grossa-PR
Diretor mdico e diretor tcnico do Instituto Sul Paranaense de Oncologia
(3) Doutor em Biologia Buco-Dental pela FOP/UNICAMP-rea Histologia e
Embriologia - Mestre em Cincias-ICB/USP-rea de Biologia Celular e do
Desenvolvimento - Professor Adjunto da Universidade Estadual de Ponta Grossa-PR
Coordenador do Mestrado em Cincias Biomdicas UEPG-PR
Matheus da Silva Santin. mssantin@hotmail.com (42)99359133
Abstract: The presente study shows the effect of X radiation on the morphology of
the skin rat. Twenty one male Wistar rats (250g) of colony of UEPG were
distribuited in the groups: control (3 rats) and irradiated (18 rats). Before the
application of 1537 miligrays of X radiation (using a nuclear accelerator from the
ISPON) on the head and neck regions, all rats were injected with xilasine and
ketamine (0,1ml and 50mg/100g). After that, rats were died at 4th, 9th,14th and 25th
days and were anesthetized with halotane, and died by cervical dislocation. One hour
before died, all rats were injected with BrdU i.p. (1ml/kg). The skin of head was
collected and immersed in 2% of paraformaldehyde, after dehydrated and included in
paraffine. Sections of 5m were taken on slides and stained with Masson trichome,
hematoxilyn/eosin, Picrosirius and immunohistochemistry. Images of sections were
taken and analysed.The results showed progressive alterations, produced by
radiation, in the morphology of the dermal and epidermis components like, collagen
fibers degeneration, disappearance of sebaceous glands and hair follicles. Changes in
the proliferative process in the epidermal and glandular cells, mainly on 4th and 9th
days was observed. On 25th day, epithelial structures have already exhibit a slow
regeneration process when comparated with the control group. We conclude that the
degenerative effects of radiation in the epithelial structures are immediatly and in the
conective tissue they are progressive, but may be reversible due the cell proliferation
process.
CEUA-27/2011 process.
Janine Braz (1), Vilessa Gomes (2), Srgio Matta (3), Naianne Clebis (4), Moacir
Oliveira (2), Danielle Morais (4), Carlos Moura (2).
(3)
(4)

Sao Paulo. Dr. Arnaldo Avenue, 455, 1st floor - room1220, Cerqueira Csar, 01246903. Sao Paulo, SP, Brazil. phone: +5511 30618531 mobile: +5511 979609281. Email address: lubelotti@usp.br.
Background: During pregnancy, the respiratory system undergoes significant
morphofunctional changes. Marijuana is the most widely used illicit drug during
pregnancy and its effects on the respiratory system of expectant mothers are not well
understood and requires clarification.
Aims: Assess marijuana smoke exposure effects on pregnant mice lung.
Methods: Pregnant Balb/c mice (n=10) were daily exposed (nose-only) either to
marijuana smoke [0.2g of Marijuana] (Group MA) or filtered air (Group FA) during
5 minutes, from 5.5 dpc (days post coitus) to 17.5 dpc and euthanized on 18.5 dpc.
Lungs were collected and total and differential BALF cell count were obtained. We
estimated total and volume densities, surfaces and surface densities of lung
compartments and septum arithmetic mean thickness by stereological methods.
Results: Body weight on 18.5 dpc of MA group was slightly reduced but without
statistical difference from FA group. Lung total volume (p=0.009) was increased in
MA groups. Parenchyma evaluation revealed that septum (p=0.028) and alveolar
space (p=0.010) volume were increased in MA group without air-blood barrier
thickening. Lung total volume per body weight ratio was increased in MA group
(p=0.008). BALF cell count were not statistically different between groups.
Conclusions: Exposure to marijuana smoke can alter pulmonary architecture by
increasing total lung, septum and alveolar air space volume, most likely not related
to lung inflammation or edema. The apparent lack of inflammatory response could
be a consequence of the 9-tetrahydrocannabinol role in suppressing cell function
and cell-mediated immunity.

Q32
EFFECT OF VITAMIN C IN THE SPERMIOGENESIS OF DYSTROPHIC

MICE
(2)

Paulo, Brazil;
(2) Department of Anatomy of Domestic and Wild Animals, School of Veterinary
Medicine and Animal Sciences, University of Sao Paulo, Sao Paulo, Brazil.
Ethics Committee approval CEUA n043/15.

Q31
(1)
Luciano Belotti (1), Natlia de Souza Xavier Costa (1), Sarah Gomes de Menezes
Benevenuto (2), Gabriel Ribeiro Junior (1), Paulo Hilrio Nascimento Saldiva (1),
Marisa Dolhnikoff (1), Mariana Matera Veras (1,2)
(1) Department of Surgery, School of Veterinary Medicine and Animal Science,

(2) Department of Animal Science, Federal University Rural of Semi-rido,
1Mossor, Brazil
(3) Department of Biology, Federal University of Viosa, Viosa, Brazil.
2(4) Department of Morphology, Federal University of Rio Grande do Norte, Natal,
Brazil.
3-
EFFECT OF DIFFERENT GLYCOSAMINOGLYCANS IN ARTERIAL

THROMBOSIS TIME AND RE-ENDOTHELIALIZATION OF THE
CAROTID ARTERY OF MICE AFTER INJURY INDUCED BY FERRIC
CHLORIDE
Andressa Raysa da Costa e Silva, Claudio Chrysostomo Werneck e Cristina Pontes
Vicente.
(1) Departament of Structural and Functional Biology, State University of Campinas,
Campinas, Brazil.
(2) Departament of Biochemistry e Tecidual Biology, State University of Campinas,
Campinas, Brazil.
E-mail: costaandressa51@gmail.com. Address: Rua Charles Darwin s/n, bloco N, 3
andar. Phone: 55 (19) 3342-9548. Mobile: 55 (19) 99843-2578
Address - Prof. Orlando Marqus Paiva Avenue, 87, Butant, So Paulo.

Contact: +55 19 3565-6882 / +55 19 98164-6600.
E-mail jani_karla@yahoo.com.br
Vitamin C or ascorbic acid (AA) is an antioxidant that protects DNA from free
radicals. Low levels of seminal AA can damage the spermatic DNA, its synthesis
and cause abnormal sperm infertility in men. However, there are no reports of its
therapeutic use for Duchenne Muscular Dystrophy (DMD). Therefore, this study
aimed to evaluate the effect of ascorbic acid on spermiogenesis of dystrophic mice
(mdx). Eight (8) adult (60 days old), male mice were used in this experiment, being 4
C57BL/10 mice (control group supplemented - CS60) and 4 C57BL/10Mdx mice
(group dystrophic supplemented - DS60), approved by Ethics Committee (Permit
number: 064/2013 CEUA / UFRN). Supplementation was performed with 0,005g/Kg
of ascorbic acid diluted in water and given by oral gavage for 30 days. The animals
were then euthanized, and the testicles sampled, weighted, cross-sectioned and fixed
in Karnovsky solution (24h) for inclusion in histological resin for spermatogenic
yield analysis and methacrylate for transmission electron microscopy (TEM). The
results were evaluated by statistical test of Kruskal-Wallis followed by Mann
Whitney with Bonferroni correction, with 5% significance level. Vitamin C
increased the spermatogenic yield DS60 (29.87 1.11) compared to the CS60 group
(23.56 5.57) (P0.05). In addition, ultrastructural analysis showed the spermatids
and sperm dystrophic were normal morphology, as well as acrosome has presence of
axial filament axonema, fibrous sheath. These findings compared to CS60
demonstrated absence of morphological changes in DS60 animals.
Thus, the ascorbic acid did not change the morphological patterns of
spermatogenesis in DMD.
The dermatan sulfate (DS), heparin and low molecular weight heparin (LMWH) are
glycosaminoglycans (GAG) that have antithrombotic, anticoagulant and antiinflammatory activity. We analyzed the influence of these agents in thrombosis and
neointimal formation after arterial injury in mice, using the injury model induced by
ferric chloride. The injured carotid arteries were examined histologically in order to
identify the magnitude of thrombus and the presence of neointima. The analyzes
were performed respectively immediately after injury time to 0 days and 14 days
after the injury being treated with different glycosaminoglycan. At time zero was
observed 20% of thrombosis in animals treated with 1 mg/kg LMWH, 10% on DS
treated with 10 mg/kg and about 3% in animals treated with heparin 1mg/kg. There
was a significant response in preventing neointimal formation when the animals were
treated with heparin compared to untreated group of animals (p<0.05). The analysis
of partial thromboplastin time activated (APTT) showed an increase of
approximately 3000% of the APTT in the group treated with heparin within 24
hours; in the animals treated with LMWH 1.086% 24 hours after arterial injury and
treated with DS 1.025% at 24 hours; after 72 hours APTT normalizes all groups. TT
no significant difference in animals treated with heparin, LMWH and DS. These
partial analyzes indicate that heparin was more effective in inhibiting thrombotic
response in the vessel and preventing neointimal by model proposed by ferric
chloride injury.
This project was approved for the ethics committee from UNICAMP (CEUA)
(protocol number 3892-1).
Funding Support: Capes, FAPESP.


Q33
Q34
CAFETERIA DIET EFFECTS ON THE GOBLET CELLS POPULATION OF

THE JEJUNUM OF OBESE WISTAR RATS
THE ORIGIN OF PROSTATE-SECRETED IgA AND IgG
Mylena de Campos Oliveira (1), Pmela Buratti (2), Evandro Jos Beraldi (3), Rose
Meire Costa Brancalho (3), Sandra Lucinei Balbo (3), Allan Cezar Faria Araujo (4),
Marcia Miranda Torrejais (4), Angelica Soares (4)
(1)
(1) Cincias Biolgicas, Universidade Estadual do Oeste do Paran, Paran, Brasil(2)
(2) Mestrado em Biocincias e Sade, Universidade Estadual do Oeste do Paran,
Paran, Brasil
(3)
(3) Centro de Cincias Biolgicas e da Sade, Universidade Estadual do Oeste do
Paran, Paran, Brasil
(4) Centro de Cincias Mdicas e Farmacuticas, Universidade Estadual do Oeste do
e-mail: mylenac.oliveira@hotmail.com
mobile: (45) 9802-2041
(4)
(5)
Obesity is both a disease and one of the most important risk factors for comorbidities such as diabetes and cardiovascular diseases. The cafeteria diet has been
widely used for the study of obesity as a model that simulates the high energy
density being consumed by society. Due to the intestinal ability to adapt in response
to the type of food ingested, the aim of this study was to evaluate the effects of
cafeteria diet on the proportion of goblet cells in the jejunum of obese Wistar rats.
All of the procedures were approved by the Ethics Committee on Animal
Experiments at the State University of the West of Paran. Male rats were fed
standard chow (CON group) or cafeteria diet (CAF group) for 38 weeks, and visceral
fat and the small intestine were collected at the end of this period. Samples of the
jejunum were subjected to histological processing and the Periodic Acid Schiff
staining for the quantification of goblet cells. Data were analyzed by Student's t test
or nonparametric equivalent (p<0.05). The cafeteria diet induced obesity in animals,
confirmed by higher body weight and weight of the retroperitoneal and
periepididymal fats. Quantitative analysis of goblet cells of the intestinal epithelium
showed a decrease in the density of these cells in the CAF group compared to the
CON group. Given that the function of goblet cells is related to mucus secretion,
changes induced by the cafeteria diet may have influenced the protective function of
the intestinal barrier.
Juliete A. F. Silva (1), Manoel F. Biancardi (1,2), Osvaldo A. B. E. SantAnna (3),

Hernandes F. Carvalho (1)
(1)Departamento de Biologia Estrutural e Funcional, Instituto de Biologia,
UNICAMP, Campinas - SP, Brasil.
(2) Departamento de Histologia, Embriologia e Biologia Celular, Instituto de
Cincias Biolgicas, UFG, Goinia - GO, Brasil.
(3) Departamento de Imunoqumica, Instituto Butantan, So Paulo - SP, Brasil.
Corresponding author: Juliete Aparecida Francisco da Silva
e-mail: juliete@gmail.com
Fone: 55(19) 3521-6125
Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, UNICAMP,
Instituto de Biologia, UNICAMP.
Rua: Charles Darwin, s/n Bloco N.
CEP: 13083-863 Campinas/SP.
Background: the prostate secretes part of the IgA and IgG found in the semen, but is
unknown how these immunoglobulins (Igs) reach the prostate fluid through the gland
epithelium. Aims: quantify (and locate) immune system cells and immunoglobulins
IgA and IgG in the prostate and investigate how the Igs reach the prostatic secretion
in Wistar rats. Methods: rats were immunized with two doses of ovalbumin (OVA)
or PBS in conjunction with SBA-15 adjuvant via oral (by gavage) with 180 days
interval. The labeling of IgA, IgG, and immune cells was made by
immunofluorescence. Tetramethylrhodamine-labeled monomeric IgA (TRITC-IgA)
was injected i.v. Results and Conclusions: (i) Prostate immune cells did not organize
clusters or niches, which would characterize a mucosal lymphoid tissue. (ii) We
found IgA, but not IgG, both in putative plasma cell in the ventral prostate (VP)
stroma and inside the prostate epithelial cells, in non-immunized rats. In addition, we
did not find neither CD45RA nor IgA staining in the other prostate lobes. Therefore,
PV have an active mechanism to concentrate IgA, but not IgG, via transcytosis
toward the gland lumen, which involves interaction with polymeric immunoglobulin
receptor (pIgR). (iii) Oral immunization resulted in increased number of IgAsecreting plasma cells in the stroma and, surprisingly, in the presence of IgG+ (and
rare IgA+) CD45RA+ B cells in the prostate epithelium. Intraepithelial IgG+ plasma
cells are in direct contact with the prostate lumen, while most of IgA+ plasma cells
are in the gland stroma in immunized animals.
Comit de tica em Experimentao Animal (CEEA): 2337-1.
Financial support: FAPESP, CNPq.

Q35
STORE-OPERATED
CALCIUM
LARYNGEAL MUSCLES
Q36
ENTRY
IN
MDX
INTRINSIC
SODIUM ARSENITE SUBACUTE EXPOSITION REDUCES LEYDIG CELL

POPULATION IN WISTAR RATS
Veridiana Carvalho dos Santos(1)*, Ananda Barbosa Muniz(1), Robson Francisco

Carvalho(2), Maeli Dal-Pai-Silva(2), Cintia Yuri Matsumura(1), Renato Ferretti(1)
Niumaique Gonalves da Silva (1), Ianny Brum Reis (2), Luciana Schulthais Alto
(1), Marcos de Lucca Moreira Gomes (3), Juliana Castro Monteiro Pirovani (1)*
(1) Departament of Anatomy, Institute of Biosciences of Botucatu, So Paulo State

University, Botucatu, SP, Brazil.
(2) Departament of Morphology, Institute of Biosciences of Botucatu, So Paulo
State University, Botucatu, SP, Brazil.
(1)Department of Agrarial and Biological Sciences, UFES, So Mateus, ES, Brazil,

(2)Department of Structural and Functional Biology, UNICAMP, Campinas, SP,
Brazil
(3) Departmento of Strutural Biology, UFTM, Uberaba, MG, Brazil
*e-mail: veridianacs@live.com. Adress: Rua Professor Doutor Antonio Celso

Wagner Zanin, s/n, Botucatu, SP CEP 18618-689. Phone: (14) 3880-0012.
E-mail: niumaique@hotmail.com
Address: DCAB/CEUNES/UFES, So Mateus, ES, Brazil
Telephone contact: +55 (27) 9 9961-9776
The intrinsic laryngeal muscles (ILM) are involved in vital functions that include
respiration and phonation, due to their unique superfast contraction and fatigue
resistance. During contraction, over time the sarcoplasmic reticulum (SR) is depleted
of calcium (Ca2+). Store operated Ca2+ entry (SOCE), performed by channels such as
Orai1, Stim1 and Trpc1 enables Ca2+ entry from the extracellular space to replenish
Ca2+ in the SR for subsequent contractions. The ILM are protected from myonecrosis
in the absence of dystrophin in Duchenne Muscular Dystrophy (DMD). ILM differ
from affected muscles by their ability to handle Ca2+. Imbalance in SOCE
homeostasis could be related to muscle protection or degeneration in the DMD. We
investigated if SOCE could be related to ILM protection in mdx mice. We analyzed
genes and protein expression of SOCE using quantitative PCR and Western Blot in
ILM and diaphragm (DIA) of C57/BL10 (Ctrl) and dystrophic (mdx) mice. The mdx
DIA showed Stim1 and Trpc1 lower expressed and Orai1 higher expressed compared
with control. SOCE were not differentially expressed in ILM (mdx vs. Ctrl).
Although the protein levels were not consistent with gene expression, mdx ILM
showed decreased protein levels of STIM1 and TRPC1 in relation to its respective
control. These results demonstrate SOCE plays a critical part to replenish Ca2+ for
contractions, which prevent excess of intracellular Ca2+ and it protects mdx ILM
from myonecrosis.
Experiments were approved by the Animal Care and Use Committee (CEUA #461).
Acknowledgements: FAPESP (2013/10633-0), PROPe (2052/002/14) and CAPES.
Sodium arsenite remais at the global biogeochemical cycles due to its

biodegradability. Besides being a water contaminant it is used in industrial and agroindustrial processes causing potential human health risks. The intoxication by
arsenite results in toxic effects that trigger pathologies amongst them changes in
gametogenesis. The present study evaluated the effect of sodium arsenite in Wistar
rats testis. Ten animals were divided into two groups : control (water) and sodium
arsenite (5mg/Kg/day). Water and sodium arsenite were administered daily by
gavage (0,5mL/animal) during twenty-one days. Twenty-four hours after treatment
animals were weighed and euthanized overdose of anesthetic. The testis were
removed, weighted and processed for light microscopy. Diameter and length of
seminiferous tubules , height of germinal epithelium, volumetric proportion and
volume of tubular and interstitial compartments were not altered. Already the
proportion and volume of Leydig cell were reduced with treatment. The individual
volume of these cells were not altered but the number of the Leydig cell in the testis
and per gramme of the testis were lower after treatment. Therefore the sodium
arsenite administered twenty-one over days reduced Leydig cell population
corroborating literature data.
The experimental design was approved by Committee on Ethics in Research with
Experimental Animals of Universidade Estadual de Campinas (protocol number
2294-1).
Financial support: CNPq (PIBIC)

Q37
Q38
THE EFFECT OF PHYSICAL EXERCISES ON THE LEVELS OF

ENDOTHELIAL PROGENITOR CELLS AND ON ARTERIAL THROMBUS
FORMATION AND RESOLUTION
ANNUAL CHANGES IN THE EXPRESSION OF ANDROGEN RECEPTOR

(AR) IN THE PROSTATIC COMPLEX OF PALLASS MASTIFF BAT
MOLOSSUS MOLOSSUS (CHIROPTERA:MAMMALIA)
Maiara Ferreira Terra1, Denise Gonalves Pedrosa1, Maria Jlia Marques1, Claudio
Chrysostomo Werneck2, Cristina Pontes Vicente1
Manuela Tosi Comelis*1; Mateus Rodrigues Beguelini2; Sebastio Roberto Taboga3,

Rejane Maira Ges3, Eliana Morielle Versute1.
Department of Structural and Functional Biology, Institute of Biology, University

of Campinas UNICAMP, SP, BRAZIL
2
Department of Biochemistry and Tecidual Biology, University of Campinas
UNICAMP, SP, BRAZIL
1: Department of Zoology and Botany, UNESP-IBILCE, So Jos do Rio Preto,

Brazil;
2: Center of Biological and Health Science, UFOB, Barreiras, Brazil;
3: Department of Biology, UNESP-IBILCE, So Jos do Rio Preto, Brazil
Email: maiara6_terra@hotmail.com
Address: Charles Darwin Street, w/n, Block N, Institute of Biology, State University
of Campinas, SP, Brazil.
Institutional phone: +55 (19) 35216126
Mobile: +55 (19) 992710024
Correspondent author: manuela1213@gmail.com telephone contact: (17) 32212369 mobile: (17) 98114-3870
Physical exercises can reduce blood pressure by promoting vasodilatation and

improving blood circulation. It can also increase the release of hematopoietic and
endothelial progenitor cells to peripheral blood helping in the vascular remodeling
after injury. The aim of this study is to analyze the influence of physical exercises on
the arterial thrombosis time, size of the thrombus and its re-absortion. Mice C57Bl/6
of 8 weeks were exercised during 21 days on treadmill at 12m/min and arterial lesion
was induced by ferric chloride (FeCl3) on right carotid artery. Using histological
analysis we observed a 21% reduction of thrombus area in exercised animal and an
increase of 83% occlusion time after injury in exercised animals when compared to
controls (p0,05). We quantified the number EPCs by flow cytometry in peripheral
blood and observed an increase in 300% CD34+ cells and 100% CD34/CD31
positive cells in exercised mice and a decrease after arterial injury. Exercised and
injured mice presented an increase in CD34/CD31 positive cell when compared to
injure non-exercised mice. In conclusion, our study demonstrates that physical
exercise exerts favorable effect on the vascular response, increasing thrombus
formation time, reducing the area of thrombus and increasing the number of
progenitor cells in peripheral blood in C56Bl/6 mice.
This project was approved by Ethics Committee of UNICAMP, protocol n 3550-1.
Financial support: Capes and FAPESP
Bats undergo considerable influence from abiotic factors on their reproductive

strategies, detected through behavioural or functional assessment of the gonads and
accessory glands. Molossus molossus is a member of the family Molossidae known
to be polyestric, with two annual reproductive breeding seasons in Brazil, males
present spermatogenesis activity throughout the entire year, one in MarchApril and
another in November. Our aim was to evaluate seasonal variations in Androgen
receptor expression in the prostatic complex of Molossus molossus (Molossidae).
The prostatic complex from specimens collected in March, October, December and
January was removed, fixed in Methacarn, sectioned after histological processing
and submitted to Immunocytochemistry of AR. We observed variations in the AR
expression between the months, with a higher AR expression in October, showing an
intense activity in the gland during this month. On December/January there is
a lower AR expression, and with the analysis of the general morphology, we can
infer that the gland is regressing. Finally, in March, there was more significant levels
of AR expression than the observed in December/January. This data shows the
variation of AR expression in the prostatic complex during the year, which were
consistent with the two reproductive peaks previously described for this bat species.
IBAMA Processo: 02027.001957/2006-02
CEUA IBILCE: 126/2015.

Q39
Q40
EVALUATION OF ALTERATIONS IN THE BRAIN OF BEES Apis mellifera

EXPOSED TO IMIDACLOPRID THROUGH THE MALDI-IMAGING
TECHNIQUE
ALTERATIONS OF THE COLLAGEN TYPE I AND III IN THE TENDON

REGENERATION TREATED WITH F1 PROTEIN AND INFLUENCE OF
SUTURE TECHNIQUE
Aline Fernanda Catae, Thaisa Cristina Roat, Marcel Pratavieira, Anally Ribeiro da
Silva Menegasso, Mario Sergio Palma, Osmar Malaspina.
Diego Pulzatto Cury (1), Alice Cristina Rodrigues (2), Ii-sei Watanabe (1)
Center of Social Insects Studies (CEIS), University of So Paulo State (UNESP). Av.
24A, 1515, CEP: 13506-900, Rio Claro, So Paulo, Brazil
E-mail: aline_catae@hotmail.com
Telephone: (55) (19) 3523-5027
Mobile: (55) (19) 98115-7606
A serious problem about the use of pesticides in the agriculture is the fact that these
products act not only on the pests, but also on non-target insects. Imidacloprid, a
neonicotinoid, is extensively used in Brazil. Based on the increasingly use of
insecticides and considering its effects on bees, this study aimed to analyze the
alterations on the distribution of proteins in the brains of bees exposed to
imidacloprid through the use of MALDI-Imaging. For this, forager bees were
exposed to a diet containing a sublethal concentration of imidacloprid (LC50/100:
0.0146 ng/L diet). Individuals were collected 1 and 8 days after the beginning of
food supply and their brains were sectioned by criostate. The chemical printer ChIP1000 was used for trypsin and matrix deposition, and the MALDI-MS spectra were
acquired by MALDI ToF-ToF. The spectra were converted into images by
MSiReader v0.05 software, creating density maps for proteins. Results showed that
imidacloprid caused an increase in the relative expression of glutamine synthetase.
This intensification may result from an attempt to detoxification front of an
accumulation of glutamate and ammonia. The thioredoxin peroxidase was also more
expressed after exposure to the insecticide. It is related to the protection against
oxidative stress and with the inhibition of apoptosis. The alterations in the relative
expression of these proteins demonstrate that imidacloprid could cause biochemical
changes that can imbalance the neuronal functions. The MALDI-Imaging is an
eficiente techinique to show the structures that are affect by the insecticide.
(FAPESP: 2014/14070-3; 2012/13370-8; 2012/50197-2).
(1) Department of Anatomy. Institute of Biomedical Sciences, University of So

(2) Department of Pharmacology. Institute of Biomedical Sciences, University of
So Paulo, So Paulo, Brazil.
2415, Prof Lineu Prestes av, ICB III.
Laboratory of Cell and Tissue Ultrastructure. - Zip Code: 05508-000 - So Paulo-SP,
Brazil
diegocury@usp.br ; +55 11 3091-7386
Background: Calcaneal tendon is one of the most frequently injured since the
incidence of rupture in North America is 5.5 to 9.9/100,000 habitants and 6.8 to
18/100,000 in Europe. It occurs mostly in men and is more frequent between the
third and fourth decade of life. There are many suture methods, however, KesslerTajima (KT) suture with nodes between the stumps is widely used to avoid the
bottleneck of microcirculation. After surgery, the patients return to their activity on
approximately 85-270 days. Aim: Apply F1 protein in the injured tendon with the
intention of decrease repair time, even as analyze to influence of the suture
techniques in the regeneration, as well as ultrastructural alterations of the collagen
that make up tissue. Methods: SD rats with 3 months-old were organized into
groups: C (control), K (KT), S (simple suture), KP (KT plus F1), SP (simple suture
plus F1). All groups were analyzed 2 and 4 weeks after surgery. To ultrastructural
analysis was used transmission electron microscopy (TEM), WB technique was used
to analyze collagen I and III, MMP-2, -9, TIMP-1, -2 and GAPDH. Results: TEM
revealed that group SP after 4 weeks of surgery presented a better ultrastructural
characteristic. WB showed that group SP 2 weeks has a higher amount of collagen I,
and group K 2 weeks more collagen III and MMP-2. Group KP 2 weeks has more
GAPDH, TIMP-1 and -2, control group presented more MMP-9.
Experiments were approved by the ethical committee of the ICB/USP (no. 016/14).
Fapesp 2015/24087-3.

Q41
Q42
BURNS HEALING IS FACILITATED BY TREATMENT OF ALOE VERA

EXTRACT ASSOCIATED WITH ELECTROSTIMULATION
LAMINA PROPRIA T-LYMPHOCYTE, NATURAL KILLER AND

NATURAL KILLER T CELL POPULATIONS DURING THE PEAK OF THE
5-FLUOROURACIL-INDUCED INTESTINAL MUCOSITIS IN C57BL6/J
MICE
Sofia Poletti, Letcia L. Lucke, Fernanda O. Bortolazzo, Narianny Armbrust, Renata

M. Acunha, Marcos G. Mattos, Fernanda O. G. Gaspi, Andra A. Aro, Edson R.
Pimentel, Marcelo A. M. Esquisatto, Fernanda A. S. Mendona, Glucia M. T.
Santos.
Ometto, UNIARARAS, ARARAS/SP, Brazil.
Departamento de Biologia Estrutural e Funcional - Instituto de Biologia, UNICAMP,
(1)
CAMPINAS/SP, Brazil.
E-mail: poletti.sofia@gmail.com Fone: +55 (19) 3543-1440.
(2)
Alternative therapies are less invasive and inexpensive presenting good results (3)
in
healing wounds. This study investigated the action of Aloe vera extract associated
with the application of magnetic electrostimulation (EEM) in the repair of the 2nddegree burns in rats. Sixty male Wistar rats with 120 days were anesthetized and the
lesion was induced on the dorsal skin using metal plate. Then, they were divided into
groups C, treated with carbopol gel; F-treated with A.vera extract in gel of carbopol
(AV); H-treated with EEM (Haihu CD9) and carbopol gel; H+F-treated with EEM
and AV. Samples were collected from lesions on the 7th, 14th and 21st days after
injury for structural and morphometric analysis evaluating the number of
granulocytes, fibroblasts, blood vessels and area of birefringent collagen fibers.
Besides, it was quantified the MMP-2 and MMP-9 isoforms. The number of
granulocytes and blood vessels showed no significant differences in the different
groups and experimental periods. In F, H and H+F presented significant higher
content of MMP-9 in 14th experimental days. There was a gradual increase in the
number of fibroblasts and birefringent collagen fibers in the samples of all groups
with special emphasis on the H and H+F. Samples from the H and H+F presented
increase in the content of MMP-2 throughout the trial period. A.vera is beneficial in
the modulation of the inflammatory process while EEM alone and associated with
A.vera promote positive effects on collagen fibers deposition in this experimental
model.
Study approved
FHO|Uniararas.
by
CEUA/UNIARARAS
(075/2014)
and
supported
by
Cristhyane da Costa Aquino (1), Camila Fernandes (2), Jardlon Costa Albino (1),
Ramon da Silva Raposo (3), Ana Carolina Bencio Alves (1), Antonione Santos
Bezerra Pinto (1), Dulce Maria Nascimento Coelho (1), Lilia Maria Carneiro Cmara
(2), Reinaldo Barreto Ori (1)*
Federal University of Ceara
(2) Laboratory of Medical Immunology, Department of Pathology, Institute of
Biomedicine, School of Medicine, Federal University of Ceara
(3) Experimental Biology Core, University of Fortaleza (UNIFOR).
* e-mail: oria@ufc.br, telephone: + 55 85 33668239
5-fluorouracil (5-FU) is one of the most used anti-neoplastic drugs for colon cancer.
5-FU may cause intestinal mucositis in some patients. Bone marrow-derived natural
killer (NK), NKT and T cells may be involved in the immunoinflammatory process
during intestinal mucositis. The objective of this study was to quantify those
subpopulations of lymphocytes in the small intestinal lamina propria at the peak of
the 5-FU-induced intestinal mucositis. Male C57BL6J mice weighing 19-25g were
either challenged by 5-FU injection (150 mg/Kg, single dose, i.p) (n=6) or receiving
saline (n= 6) and euthanized after 72h post-5FU. The overall small intestine was
collected for histological processing and myeloperoxidase (MPO) activity. In another
subset of mice, the lamina propria lymphocytes were extracted from the overall small
intestine for flow cytometry analyses. Blood was drawn from the retroorbital plexus
for leukometry analyses. The animals were weighed daily prior to 5-FU
administration until the end point. Protocols were approved by the UNIFORs
Ethical Committee. The 5-FU challenge caused significant weight loss and
leukopenia. Villous blunting, increased crypt depth, decreased villus/crypt ratio, and
higher MPO activity were found in all small intestinal segments. Flow cytometry
data showed reduced NK CD3-CD16+ and CD3+CD4+ T helper cells, following 5FU-challenge. In addition, there was a significant increase in the percentage of NKT
cells (CD3+CD16+) and CD3+CD8+ T cytotoxic cells (p<0.05). Altogether our
findings suggest an important effect of 5-FU on T and NK cell-populations at the
peak of inflammation of the intestinal mucositis.

Q43
ASTROCYTIC CHANGES
TRAUMATIC STRESS
Q44
INDUCED
BY
EXPERIMENTAL
Lucas Athaydes Martins1, Lisiani Saur1, Pedro Baptista1,

Laura Neves1, Rgis Mestriner1, Lder Xavier1
POST-
Pamela Bagatini1,
1. Cell and Tissue Laboratory, Biosciences College. Pontifical Catholic University of

Rio Grande do Sul (PUCRS).
Corresponding Author:
Lucas Athaydes Martins
Nursing, Nutrition and Physical Therapy College - Pontifical Catholic University of
Rio Grande do Sul - Avenida Ipiranga, 6681 - Prdio 12 / 8. andar - Porto Alegre,
RS, Brazil - CEP: 90619-900
e-mail: lucas.athaydes18@gmail.com
Phone: (55) (51) 33203646 or (55) (51) 82978648
Exposure to traumatic events can result in a psychiatric condition known as posttraumatic stress disorder (PTSD).The neuropathology of PTSD and its
symptomatology have been attributed to the hippocampus and amygdala, regions that
are involved in explicit memory processes. The aim of our study was to investigate
whether PTSD is able to change morphology, density and expression of GFAP in
astrocytes from the stratum radiatum of the hippocampus and the medial amygdala
and correlate the data obtained with the orientation index of the polarity of
astrocytes. Thirty male rats were divided in two groups: control (n = 15) and PTSD
(n = 15). The inescapable shock protocol, in which the animals are exposed to a
single episode of footshock, was used to induce PTSD. Our results show that, in the
hippocampus, PTSD is capable of decreasing the density of GFAP+ astrocytes as
well as altering astrocytic morphology, as shown by the reductions observed in the
total number of primary processes, in the number of primary processes in the lateral
quadrants, and the degree of branching in the lateral quadrants.The analysis of the
orientation index indicates that PTSD alters the polarity of hippocampal astrocytes.
No alterations were observed in the amygdala astrocytes. The main findings of our
study are that event alters the morphology, changes the polarity and decreases
hippocampal astrocytes, suporting the concept that these cells play an important role
in PTSD symptomatology.
All procedures were in accordance with the universitys ethical committee, (CEUA
13/350-PUCRS).Support: CNPq,CAPES,FAPERGS.
PROLIFERATIVE ACTIVITY AND HORMONAL RECEPTORS IN THE

GERBIL MAMMARY GLAND DURING GESTATION, LACTATION AND
INVOLUTION
Ellen Cristina Rivas Leonel (1), Silvana Gisele Pegorin de Campos (1), Douglas
Alexandre Leonel (1), Luiz Roberto Falleiros Junior (1), Sebastio Roberto Taboga
(1)
(1) Department of Biology, Institute of Biosciences, Humanities and Exact Sciences
(IBILCE) So Paulo State University (UNESP). Rua Cristvo Colombo, 2265,
Jardim Nazareth, So Jos do Rio Preto, So Paulo, Brazil, 15054-000.
Telephone: 55 17 3221-2200.
ellenleonel@yahoo.com.br
Mobile:
55
17
99635-8364.
E-mail:
The mammary gland has been considerably studied. Despite that, its knowledge is
limited in gerbils, an interesting species applicable for cancer studies. This work
aimed to describe the morphology, the proliferation pattern and hormonal receptors
expressed in the female gerbil mammary gland epithelium during gestation, lactation
and involution. Animals were euthanized at 14 and 21 gestational days, 7 and 14
lactation days and 3 and 5 days post-weaning. The mammary glands were extracted,
fixed, histologically processed and sectioned. The sections were stained with
hematoxylin and eosin, Gomoris reticulin and periodic acid & Schiff (PAS)
reaction. The meanSD epithelium height and acini diameter (m) were evaluated.
Immunohistochemistry was performed for -actin and to determine the percentages
of PCNA, PR, ESR-1 and ESR-2 positive cells (meanSD). Besides replacement of
adipose tissue by epithelium, reticular fibers replace collagen and PAS intensity
varies throughout the gland. Epithelium height rises during gestation (32,44,6) and
lactation (25,44,9). Acini diameter changes considerably in gestation (69,915,0),
lactation (139,742,0) and involution (70,724,4). Mioepithelial cells are thicker
during lactation and involution. The proliferation indexes were higher during
gestation (60,26,1) and lactation (65,08,9). Percentages of cells showing PR were
higher in the mid-pregnancy (42,319,5) and the lactation beginning (59,88,3).
Higher percentages of ESR-1 were present during gestation (58,019,3) and ESR-2,
otherwise, was higher during lactation (68,27,1). The mammary gland
modifications during preparation for milk secretion and involution involve
substantial morphological changes besides alterations in the pattern of hormone
receptors and proliferation of glandular epithelium.
Ethics commission approval: 099/2014.
Acknowledgements: FAPESP(2015/01548-5), CAPES.

Q45
Q46
EFFECT OF THE SODIUM BUTYRATE ON METABOLIC PARAMETERS

AND INTESTINAL EPITHELIAL BARRIER OF PREDIABETIC MICE
ULTRASTRUCTURAL CHARACTERIZATION OF THE FLAGELLAR

REGION OF SPERMATIDS IN FOUR SPECIES OF PHYTOPHAGOUS
BUGS (HETEROPTERA: PENTATOMIDAE)
Valquiria Aparecida Matheus (1), Letcia Cristina Scarappichia Monteiro (1),

Ricardo Beltrame de Oliveira (1), Carla Beatriz Collares-Buzato (1)
(1) Department of Biochemistry and Tissue Biology, Institute of Biology,
UNICAMP, Campinas, SP, Brazil
e-mail: valviski@gmail.com - Telephone institutional: (19) 3342-2597
Recent studies suggest that the peripheral insulin resistance associated with type 2
diabetes may result from systemic inflammation triggered by an intestinal epithelial
barrier dysfunction. Sodium butyrate has been shown to strengthen this barrier in
animal models of intestinal diseases. In this study, we investigated the effect of diet
supplementation with butyrate (5% w/w) on metabolic parameters and on the tight
junction (TJ)-mediated intestinal barrier function in both normal and
obese/prediabetic C57 mice fed a regular (control) or high-fat diet (HFD) for 60
days, respectively. Butyrate treatment significantly inhibited all the HFD-induced
metabolic dysfunctions evaluated, i.e. significantly reduced the weight gain and
adiposity as well as the insulin resistant state, the fasting and postprandial
hyperglycemia and hyperinsulinemia. In addition, HFD-fed mice treated with this
fatty acid did not display compensatory hyperplasia of pancreatic beta cells as seen
in prediabetic mice after HFD only. Butyrate enhanced the intestinal epithelial
barrier, as revealed by the FITC-dextran permeability assay, which was accompanied
by a significant increase in junctional content of the TJ-associated claudin 1 in the
intestinal epithelia of jejunum, ileum and colon of both control and HFD mice. In
conclusion, our results suggest that supplementation with butyrate inhibits the
deleterious effects of HFD intake on metabolic parameters and induces an increased
expression of the tight juncional protein, claudin 1, resulting in an increase of
intestinal epithelial barrier in mice.
This research was approved by the Ethics Committee of Experimental Animal
IB/UNICAMP (#3439-1) and financially supported by CNPq, CAPES and FAPESP.
Fernando Cesar Silva Junior (1), Emi Rosane Silistino de Souza (1), Tatiani Seni de
Souza Firmino (1), Luis Lenin Vicente Pereira (1), Mary Massumi Itoyama (1)
(1) Departamento de Biologia, Instituto de Biocincias, Letras e Cincias Exatas
UNESP - Universidade Estadual Paulista, Campus de So Jos do Rio Preto, So
Paulo, Brasil.
Contacts: Fernando Cesar Silva Junior
Instituto de Biocincias, Letras e Cincias Exatas, IBILCE UNESP - Rua Cristvo
Colombo, 2265 - Jardim Nazareth - 15054-000 So Jos do Rio Preto, SP Brasil
Telefone: + 55 (17) 3221-2200/cel: + 55 (17) 98180-4807
e-mail: ju_fcsj@hotmail.com
Heteroptera is a suborder of the Hemiptera order, which has about 40,000 species
distributed in eighty families, among them is the Pentatomidae family that stands out
for being one of the largest families with about 4,100 species, representing
approximately 11% of total species Heteroptera. Pentatomidae species of Edessa
meditabunda, E. collaris, Chinavia impicticornis and Thyanta perditor were studied
in this work due to its economic importance, considering that are phytophagous
species that causes major losses in several crops, besides being spermiogenesis
species not previously described in the literature ultrastructurally. The species were
collected in the region of So Jos do Rio Preto and male specimens had their
testicles extracted, fixed and then processed for analysis by transmission electron
microscopy. The specimens showed structural and ultrastructural characteristics
similar to those found in many specimens of different orders, among these features
are the presence of two symmetrical mitochondrial derivatives, presence of
paracrystalline structures inside, and the microtubule axonema standard 9 + 9 + 2,
these being typical characteristics of insects. Morphologically, the mitochondrial
derivatives are different in E. meditabunda, E. collaris, C. impicticornis e T. perditor
specimens which may be a very interesting feature for future phylogenetic and
taxonomic studies for the group Heteroptera.
Keywords: ultrastructures, mitochondrial derivatives, axoneme.
Financial Support: FAPESP (Process:2013/19864-5), CAPES.
Q47

Q48
STEM CELLS TRANSCRIPTION FACTORS EXPRESSION IN AGING

SUBMANDIBULAR GLAND
COMPARATIVE ANALYSIS OF MITOCHONDRIAL

MORPHOLOGY OF FOUR SPECIES OF COREIDAE
Tamires Albuquerque Gomes (1) Sergio Mestieri Chammas (2) Tiago Nicoliche
Maria (1) Adriana da Costa Neves (2) Marcelo Cavenaghi Pereira da Silva (1)
Emi Rosane Silistino de Souza (1), Fernando Cesar Silva Junior (1), Tatiani Seni de
Souza Firmino (1), Luis Lenin Vicente Pereira (1), Mary Massumi Itoyama (1)
(1) Department of morphology and genetics, Universidade Federal de So Paulo, So

Paulo, Brazil
(2) Laboratory of genetics, Instituto Butantan, So Paulo, Brazil

UNESP - Universidade Estadual Paulista, Cmpus de So Jos do Rio Preto, So
Paulo, Brasil.
The main function of the major salivary glands is saliva production, process that the
submandibular gland, composed by different types of acini and ducts, is responsible
for 60% of the total volume produced. The saliva provides buffering and protection
for structures of the oral cavity and beginning of digestion, however it is known that
aging decrease saliva flow. This study aims to characterize the morphological
modifications caused by the aging process in human submandibular glands of adult
and senile. A morphological analysis was performed with different types of
coloration as hematoxilin/eosin (HE), periodic acid of Schieff (PAS) and
Picrosirius, and immunohistochemistry for the expression of transcription factors
Sox-2, Oct-4, Nanog, Stat-3, C-Kit and Musashi-1. It was observed reduction of
acini and increase of adipose tissue in senile submandibular gland and through
immunohistochemistry analysis, we observed weak immunostaining for the
transcription factors Sox-2, Oct-4, Nanog and Stat, while a strong cytoplasmic
immunostaining for C-kit and Musashi-1 were seen in adult and senile glands. It can
be concluded that aging causes alterations on the morphology of the submandibular
salivary gland, and it can be in part explained by weak immunostaining of
transcription factors that influence in the salivary flow.
Contacts: emi.rosane@hotmail.com + 55 (17) 98136-9084
FAPESP 2015/03967-5
CEP/UNIFESP 618131
DERIVATIVES
The Coreidae family (Heteroptera) can be found in all zoogeographic regions of the
world. The individuals belonging to this family are all phytophagous and some have
economic importance, such as Corecoris fuscus, Leptoglossus gonagra, L. zonatus
and Sphictyrtus fasciatus, for being agricultural pests. The aim of this study was to
compare the morphological differences of mitochondrial derivatives during
spermiogenesis of four species of Coreidae. Adult males of these species were
collected and testicles were extracted. Then, they were processed and analyzed by
transmission electron microscopy, which is an important tool to be used in
phylogenetic and taxonomic analysis, due to the ultrastructures of germ cells being
highly conserved. We observed that the mitochondrial derivatives have a similar
morphology to the same order, being symmetrical, periformes, positioned bilaterally
to the axoneme. The face, when turned to the axoneme, is concave and, when turned
to the plasmatic membrane, convex. But the mitochondrial derivatives morphology
has a slight difference when compared between species of the same order, possibly
being more rounded, tapered, narrowed or reniformed. We conclude that the
morphology of mitochondrial derivatives is species-specific, however it resembles
more of the mitochondrial derivatives of species belonging to the same order. The
data obtained are important tools to assist in phylogenetic and systematic analysis.
New studies of the stages of spermiogenesis will reveal new synapomorphies and
deepen the knowledge related to Heteroptera group in an ultrastructural level, whose
studies are still scarce.
Keywords: ultrastructures, mitochondrial derivatives,spermiogenesis.
Financial Support: FAPESP (Process:2013/19864-5), CAPES

Q49
Q50
OBSERVATIONS ABOUT THE TONGUE MUCOSA OF WHITE-EAREDOPOSSUM (DIDELPHIS ALBIVENTRIS), EMPLOYING SCANNING AND
ULTRASTRUCTURAL ANALYSIS OF SPERMATOGENESIS OF

MARTAREGA MEMBRANACEA (HETEROPTERA, NOTONECTIDAE)
Tatiani Seni de Souza Firmino, Luis Lenin Vicente Pereira, Fernando Cesar Silva
Junior, Emi Rosane Silistino de Souza, Mary Massumi Itoyama
Brbara Tavares Schfer (1), Althen Teixeira Filho (2), Ii-Sei Watanabe (3)
(1) Department of Surgery, Faculty of Veterinary Medicine and Animal Science,
(2) Department of Morphology, Institute of Biology, Federal University of Pelotas,
Pelotas, Brazil.
(3) Department of Anatomy, Institute of Biomedical Sciences, University of So
2415, Professor Lineu Prestes Av., ICB III.
Laboratory of Cell and Tissue Ultrastructure - Zip code: 05508-000 - So Paulo/ SP,
Brazil
barbaraschafer@gmail.com ; +55 11 3091-7386 ; +55 11 95497-6955
The White-Eared-Opossum (Didelphis albiventris) is a marsupial that occupies a
large variety of habitats, spreading through several Brazilian biomes, with an
important role in seed dissemination. The aim of this research is the study of its
lingual morphology. The tongues were collected from animals found dead in roads in
the state of Rio Grande do Sul, Brazil, and submitted to processing for scanning
(SEM) and transmission (TEM) electron microscopy. Through TEM the stratum
corneum demonstrate, to adults and fetuses, the epithelial renewal process through
the release of the most superficial cells; the granular layer is more tenuous in fetuses
than in adults, where tonofilaments and keratohyaline granules were observed; the
spinous layer was similar in both ages and marked by the large number of
desmossomes. Through SEM the mucosa analysis revealed filiform papillae with
different morphologies according to the region. The fungiform papillae are scattered
in the apex and body, and partially covered by an elevation of mucosa. Circumvallate
papillae are 3 in number and form a triangle on the lingual root. Foliate papillae are
small and thin elevations in the caudolateral border of the tongue, ranging from 12 to
16. The fungiform, circumvallate and foliate papillae subepithelial tissue present an
irregular surface with some dorsal depressions. The characteristics observed in the
tongue of D. albiventris are similar to those observed in other marsupial species.
UNESP/IBILCE
The suborder Heteroptera has seven infraorders with about 80 families. The familes
aquatic and semi-aquatic are widely distributed and surprised by his ability to inhabit
an extraordinary variety of ecosystems, being found in freshwater and marine
habitats, with varied range of altitude from 0 to 4,700 m. Ultrastructural studies on
aspects of spermatogenesis and specifically of the structure of sperm in Heteroptera
are still scarce, therefore the objective of this study was to analyze the ultrastructures
of spermatogenesis, through ultra-thin sections, contrasted with uranyl acetate [2%]
and lead citrate [2%], analyzed by transmission electron microscopy, using testis of
adult male of Martarega membranacea. The images obtained from ultra-thin
sections show the two mitochondrial derivatives (MD), of large size positioned
bilaterally with respect to axonema (Ax). With respect to the organizational pattern
of Ax, was observed which is of type 9 + 9 + 2. We also note that the core and the
acrosome continue compacting in the training process of the spermatozoid, was
possible to identify the nucleus region with lacto-acetic orcein staining, and in
longitudinal sections was possible to identify the nucleus (N), the acrosome (Ac) the
centriole adjunct. After the analysis we can conclude that the ultrastructural features
of spermatogenesis Martarega membranacea Heteroptera are similar to others
described in literature.
Keywords: spermiogenesis, mitochondrial complex derived mitochondrial
This research is supported by the So Paulo Research Foundation (FAPESP.

Proc.:2015/05065-9).
Experimental procedures were approved by the Ethic Committee on Animal Use
(CEUA/FMVZ/USP).

Q51
Q52
CAFETERIA DIET PROMOTES MORPHOMETRIC CHANGES IN THE

INTESTINAL WALL OF OBESE WISTAR RATS
Bruna Hart Ulsenheimer (1), Amanda Pugens (2), Matheus Felipe Zazula (2),
Mylena de Campos Oliveira (2), Maria Lucia Bonfleur (3), Evandro Jos Beraldi (3),
Allan Cezar Faria Araujo (4), Marcia Miranda Torrejais (4), Angelica Soares (4)
(1) Mestrado em Biocincias e Sade, Universidade Estadual do Oeste do Paran,
Paran, Brasil
(2) Cincias Biolgicas, Universidade Estadual do Oeste do Paran, Paran, Brasil
(3) Centro de Cincias Biolgicas e da Sade, Universidade Estadual do Oeste do
(4) Centro de Cincias Mdicas e Farmacuticas, Universidade Estadual do Oeste do
Iran Augusto Neves da Silva (1), Sarah Gomes de Menezes Benevenuto (1), Adair
Alemany (2), Marlise Di Domenico (2), Elia Tamaso Espin Garcia Caldini (2), Maria
Anglica Miglino (1), Marco Antonio Garcia Martins (2), Mariana matera Veras
(1,2)
(1) Departamento of Surgery, School of Veterinary Medicine and Animal Sciences,
University of So Paulo, So Paulo, Brazil;
(2) Departamento f pathology, School of Medicine, University of So Paulo, So
Paulo, Brazil.
Corresponding author: Departamento f Pathology, School of Medicine University of
So Paulo. Dr. Arnaldo Avenue, 455, 1st floor room 1220, Cerqueira Csar, 01246903. So Paulo, SP. Brazil phone: +5511 30618531 mobile: +5511 953162511. Email adress: irnaugusto@usp.br.

e-mail: matheuszazula@gmail.com
mobile: (45) 9945-2148
Obesity is a chronic and epidemic disease, represents a major challenge to global
public health. Studies in animals show that the consumption of hypercaloric or highfat diets induces obesity and associated disorders such as insulin resistance and type
2 diabetes. Considering the importance of the small intestine in the context of
obesity, the aim of this work was to evaluate the effects of cafeteria diet on the
morphometric parameters of the wall of the duodenum and jejunum of obese rats. All
of the procedures were approved by Ethics Committee on Animal Experiments at the
State University of the West of Paran. Male Wistar rats were fed standard chow
(group CON) or cafeteria diet (CAF group) ad libitum for 38 weeks, and visceral fat
and the small intestine were collected at the end of the experimental period. Samples
of the duodenum and jejunum were subjected to histological processing and
hematoxylin-eosin staining for the morphometric analysis of the intestinal wall. Data
were analyzed by Student's t test (p<0.05).The cafeteria diet caused increases in body
weight and in retroperitoneal and periepididymal fat weight, confirming the
installation of obesity. The morphometric analysis of the intestinal tunics showed an
increase in villiheight, crypt depth, thickness of the muscular layer and total wall in
the duodenum of the CAF group, but no significant changes in the jejunum. The
results lead to infer that the high caloric and fat content of the cafeteria diet may have
influenced the intestinal absorptive capacity, favoring weight gain and visceral
adiposity.
STRUCTURAL CHANGES IN PLACENTAL OF MARIJUANA SMOKING

MOTHERS
IS
ASSOCIATED
WITH
LOW
BIRTH
BABIES:
EXPERIMENTAL STUDY IN MICE
Background: Prevalence of marijuana use during pregnancy ranges from 3-30% and
the legalization of recreational use this percentage may increase. Human studies of
its effects on the placenta are scarce and existing experimental studies use nonrealistic exposure routes and dose. To better understand the impacts of recreational
use during pregnancy we developed a mouse model of realistic exposure that mimic
humans use under the aspects of dose and route of exposure.
Aim: Asses the impacts of maternal marijuana low dose exposure via inhalation on
fetal and placental outcomes.
Methods: We exposed pregnant BALB/c mice (n = 12) daily (nose-only) to either
marijuana smoke [0.2g of Marijuana- 0.3% THC] (Group MA) or filtered air (FA
Group) during 5 minutes from 5.5 to 17.5dpc. On 18.5dpc pregnancy was
terminated, fetus and placenta macroscopically examined, weighted and fixed. We
used stereological methods to assess placental structure.
Results: We did not observe any gross abnormalities in placenta or fetus. MA
placentas presented greater total volumes (p <0.004) that were consequence of
increased labyrinth and decidua compartments. Besides this compensatory growth of
the placenta, MA fetus were smaller (CR length) and lighter (p <0.01, 8% reduction
in weight). When we analyzed the fractional contribution of each placental
compartment to its total volume (chorionic plate, labyrinth, junctional zone and
decidua) no significant change was seen.
Conclusions: Low dose exposure to marijuana smoke during pregnancy affects
placental structure and impairs fetal growth. Increases in placental specific
compartments suggest compensatory mechanisms that fails to attend fetal demand.
Bioethics Committee FMVZ-USP N 1455120116
Funding support: CNPq and CAPES


Q53
Q54
LONG TERM EFFECTS OF CHRONIC LOW DOSE MARIJUANA

SMOKING
HABITS
E
ON
CARDIOVASCULAR
FUNCTION:
PRELIMINARY STUDY IN MICE
ANALYSIS OF MORPHOMETRY INSTESTINAL OF RATS SUBMITTED

TO NUTRITION CONTAINING TRANSGENIC GOAT MILK EXPRESSING
HUMAN LYSOZYME
Janaina Iannicelli Torres (1), Rafael Dariolli (3), Giovana M. Di Constanzo (1),
Adriana M. Fonoff-Oliveira (1), Marco Antonio Garcia Martins (1), Nilmara de
Oliveira Alves (1), Mariana Matera Veras (1,2);
Julyana Almeida Maia (1), Ana Valesca Pinto de Lima (1), Katiane Queiroz da
Silva* (1), Marcelo Berlolini (2), Reinaldo Barreto Ori (3), Luciana Relly Berlolini
(4)

(1)
Paulo, Brazil;
(2) Department of Surgery, School of Veterinary Medicine and Animal Sciences,
University of Sao Paulo, Sao Paulo, Brazil.
(2)
(3) Lab of Genetics and Molecular Cardiology, InCor, School of Medicine,
(3)
(1) Laboratory of Molecular Biology and Development, University of Fortaleza,

Avenida Washington Soares, 1321, Edson Queiroz, 60.811-905, Fortaleza, Cear,
Brazil.
(2)School of Veterinary Medicine, Federal University of Rio Grande do Sul, Porto
Alegre, RS, Brazil.
(3)Laboratory of the Biology of Tissue Healing, Ontogeny and Nutrition,
Federal University of Cear, Rua Cel. Nunes de Melo 1315, 60430-270, Fortaleza,
CE, Brazil.
(4) Pontifical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre, RS,
Brazil.

Sao Paulo. Dr. Arnaldo Avenue, 455, 1st floor - room1220, Cerqueira Csar, 01246903. Sao Paulo, SP, Brazil. phone: +5511 30618531 mobile: +5511 986299385. (4)
Email address: janainaiannicelli@hotmail.com.
Background: Marijuana use in adolescence or early adulthood is increasing
worldwide, however, there are many gaps in our knowledge about the impacts of the
smoking habits on different health endpoint such as cardiovascular system in young
people. Thus, we developed a mice model to mimic human exposure to study these
effects.
Aims: Investigate by echocardiography the effects of chronic marijuana smoke on
cardiac function of juvenile and young adult mice
Methods: Balb/c mice (n=8) were daily exposed (nose-only) to either marijuana
smoke [0.2g of Marijuana] (Group MA) or filtered air (Group FA) during 5 minutes,
from 21 to 90 post natal day. Non invasive echocardiography evaluations were
performed on days 21th, 60th and 90th day post exposure. Systolic and diastolic left
ventricular functions were evaluated.
Results: Our data, although not statistically significant due to small number of
animals per group, suggest that chronic low dose marijuana use can adversely affect
cardiovascular function even in young individuals. We observed that as time
progress there is a decrease in the diastolic diameter and this could be an early
indicator of future impairments of systolic function
Conclusions: Our data suggest that chronic exposure to marijuana smoke during
young ages may predispose individuals to later life cardiovascular problems
Ethics Committee approval CEUA n 039/16.
* email: katiane1002@yahoo.com.br, telephone: (85) 34773030

Genetically Modified Organisms (GMOs) can be used to produce food with high
nutritional value. Transgenic goats expressing human lysozyme (hLZ) developed
significant changes in properties and functionality of their milk. In this study, we
evaluated the protective role of goats milk expressing recombinant human lysozyme
(TGM) in intestinal morphology of rats subjected to malnutrition. Protocols were
approved by the UNIFORs Ethical Committee. This study were done with, forty
Wistar rats, twenty-one days of age and initial body weight of thirty to thirty-three
grams. The animals were divided into nourished group (Nut) which were not
submitted to malnutrition protocol and received on demand the commercial feed
(Purina) and saline solution at 0.9% by gavage. Four groups (Des) of the animals
were subjected to malnutrition protocol by reducing of 70% of daily feed. The
groups composed by undernourished rats were divided into the following groups:
group (i) Desn-Saline, which receiving saline solution at 0.9%, group (ii) Desn-rhLZ,
which receiving commercial lysozyme solution at 300 mg/mL (Sigma) diluted in
0.9% saline, group (iii) Desn-nGTM, which received goats milk from non-transgenic
animals and group (iv) Desn-GTM, which receiving goats milk expressing human
lysozyme. After twenty two days of the experiment, fragments of the jejunum were
collected for morphometric analysis. The malnourished group which received
transgenic goats milk expressing human lysozyme (Desn-GTM) showed greater
height of the intestinal villi (p <0.0001). This suggests that the goats milk expressing
recombinant human lysozyme showed a protective effect on the intestinal barrier of
malnourished submitted to food restriction.

Q55
PHARMACOLOGICAL INDUCTION
REDUCES ORTHODONTIC RELAPSE
Q56
OF
OPG
OVEREXPRESSION
Gabriel Schmidt Dolci(1), Leonardo Francisco Diel(1), Anna Christina Medeiros

Fossati(2).
leocvr@bol.com.br
Introduction: The statin class of drugs enhances osteogenesis and suppresses bone
resorption, which could represent a plausible biological mechanism for mitigation of
orthodontic relapse. We aimed to determine whether atorvastatin (ATV) might affect
orthodontic relapse and osteoclastogenesis by modulating expression of RANKL and
OPG, crucial molecules involved in bone turnover. Furthermore, we analyzed the
adverse effects of ATV on femur turnover and endochondral ossification. Methods:
Wistar rats were subjected to orthodontic tooth movement (OTM) for 21 days,
followed by removal of the appliance and start of ATV or saline administration. Up
to 7, 14, and 21 days of ATV administration, tooth relapse was measured and
maxillary and femur sections were obtained and prepared for H&E, TRAP, and
immunohistochemical (RANKL and OPG) staining. Results: ATV decreased tooth
relapse (p=0.03) and osteoclast count (p=0.04), which were positively correlated
(p=0.006). Statin administration increased periodontal expression of OPG (p=0.008),
but not of RANKL protein. ATV administration also enhanced growth plate cartilage
thickness. Conclusions: Statin-induced OPG overexpression reduces relapse after
OTM, in a phenomenon correlated with decreased osteoclast counts. This
phenomenon sheds light on OPG as a molecular target that modulates maxillary bone
metabolism and orthodontic relapse.
The experimental design of this study was approved by COMPESQ UFRGS and
CEUA/UFRGS
Funding Support: CAPES, CNPQ, FAPERGS.
EXPOSURE TO THE FLAVONOID CHRYSIN INCREASES THE

ESTROGEN RECEPTOR ALPHA IMMUNOSTAINING IN THE PROSTATE
OF ADULT FEMALE GERBILS (MERIONES UNGUICULATUS)
Mnica Sousa Campos (1), Rodrigo Fernandes de Lima (2), Naiara Cristina Souza
Ribeiro (2), Glucia Maria Cavasin (2), Lus Octvio Regasini (3), Fernanda Cristina
Alcantara dos Santos (2), Sebastio Roberto Taboga (1)
(1) Department of Biology, Laboratory of Microscopy and Microanalysis, University
Estadual Paulista UNESP, Rua Cristvo Colombo, 2265, So Jos do Rio Preto,
So Paulo, 15054000, Brazil
(2) Department of Histology, Embryology and Cell Biology, Federal University of
Gois, Goinia-GO, 74001970, Brazil
(3) Department of Chemistry and Environmental Sciences, Institute of Biosciences,
Letters and Exact Sciences, University Estadual Paulista UNESP, So Jos do Rio
Preto, Brazil
monicabioufg@yahoo.com.br; Phone +556299598756; +556235211765
rodrigolima_biologia@hotmail.com; phone +556235211765, +556281535797
Chrysin (5,7-dihydroxyflavone) is a natural biologically active compound extracted
from honey, plants and propolis. This chemical interacts with sexual hormones
receptors, modulating its functions, through inhibition or activation of the hormonal
pathways. The aim of this work was to evaluate the morphophysiology of female
gerbil prostates exposed to chrysin. For this, 90-days-old female gerbils received,
through gavage, daily doses of chrysin (50mg/Kg diluted in 0.1 ml mineral oil) for
21 days. Another group received only the mineral oil (0.1 ml/animal). The animals
were divided into 3 subgroups and euthanized after 3, 7 and 21 days of treatment.
The prostatic complexes were removed, fixed in metacarn solution and embedded in
Paraplast. Haematoxylin-Eosin and immunostaining for estrogen receptor alpha
(Anti-ER for quantitative analysis) were applied. The morphological analysis of the
groups demonstrated structural epithelium disorder with hyperplasic prostatic foci.
The ER-positive stromal cells showed an immunostaining increase in the stromal
compartment of all treated groups. These data suggest that chrysin can act as an
estrogenic agonist leading to an increased number of ER-positive stromal cells.
Thus, the presence of hyperplasic foci is a result of a proliferative action of ER
through epithelium-stroma interaction, resulting, in harmful lesions due to
continuous exposure of chrysin on the prostate.
Ethical committee approval: CEUA/UFG: N 024/13. Financial Support: CAPES,
CNPq and FAPEG.

Q57
Q58
MATERNAL PROTEIN RESTRICTION IMPAIRS ANGIOGENESIS

THROUGH DOWN REGULATION OF AQUAPORIN-1 AND VEGF
PATHWAY IN VENTRAL PROSTATE OF RAT OFFSPRING
MORPHOLOGICAL
ANALYSIS
BY
HISTOCHEMICAL
AND
ULTRASTRUCTURAL TECHNICS OF HUMAN SUBLINGUAL SALIVARY
GLAND AND STUDY OF IMMUNOHISTOCHEMICAL EXPRESSION OF
EGF; EGFR; FGFR-2
Ketlin Thassiani Colombelli 1, Srgio A.A. dos Santos 1, Flvia B. Constantino 1,

Ana C.L. Camargo1, Caroline N. Barquilha1, Suelen Franco1, Luis M. Frediane 1,
Jaqueline C. Rinaldi1, Luis A. Justulin Jr 1
1
Sergio Mestieri Chammas* (1); Tamires Albuquerque Gomes (2)

Cavenaghi Pereira da Silva (2); Adriana da Costa Neves (1);
Laboratory of Extracellular Matrix, Department of Morphology, Institute of

Biosciences of Botucatu, So Paulo State University, Botucatu, Brazil.
Email: ketlin.colombelli@gmail.com, Phone: (14) 38800502, (14) 98109 1652.
* Telephone number : +55 11 2627-9701

Cell phone: +55 11 98177-1987
e-mail: sergio.chammas@butantan.gov.br
Background: The impairment of offspring prostate development and growth has been
demonstrated in a model of perinatal protein restriction (PPR). Some studies
demonstrated reduction of angiogenesis in brain and lung in offspring from protein
restricted mothers. Aim: To verify if PPR (gestational and lactational periods) alters
angiogenesis in the ventral prostate (VP) of offspring males. Methods: Male Sprague
Dawley rats born from mothers that received normal diet (17% protein) (Control
Group-CTR) or hypoprotein diet (6% protein) during the gestational period (GLP
group), or during the gestational and lactational periods (GLLP group) were
weighted and euthanized at post-natal day (PND) 10 and 21. The urogential complex
(UGC) and VP were collected for morphological, immunohistochemical and Western
Blotting analyses. Results: The body, UGC and VP weights were reduced in
restricted groups compared to age-matched CTR. Luminal and epithelial
compartment of VP decreased in restricted groups compared to CTR. The
immunostaining for cell proliferation (Ki67), androgen receptor (AR) and basal cell
marker (p63) and -actin immunoreactivity were reduced in restricted groups. The
microvascular density decreased in restricted groups and this result was associated
with reduction in immunostaining of aquaporin-1 (AQP-1), VEGF and its receptor
(VEGF-R) in the VP. These results were confirmed by western blotting
quantification of PCNA, AQP-1, VEGF and VEGFR expression. Conclusion: The
PPR induced impairment in VP microvascular angiogenesis through downregulation
of VEGF signaling. This result can be associated with delay in cellular
differentiation and prostate growth in male offspring.
Ethical approval: CEUA (Protocol 670).
Funding support: CAPES and FAPESP.
; Marcelo
Laboratrio de Gentica, Instituto Butantan, So Paulo, Brasil

Universidade Federal de So Paulo, So Paulo, Brasil
Introduction: The salivary glands secrete proteins called growth factors, during aging
the parenchyma of the secretory portions tends to be replaced by connective and
adipose tissue, decreasing the functional activity of these glands, causing dry mouth.
Objectives: Study by immunohistochemistry, the pattern of EGF, EGFR and FGFR-2
expression in sublingual salivary glands (SLG) and compare imunohistochemical
expression of these protein between adults (30- to 60 years old) and elderly people
(over 60 year old). Methods: Morphological analysis was performed by
histochemical techniques and by transmission electron microscope.
Immunohistochemistry was used to verify the expression of EGF, EGFR and FGFR2. Quantification of immunohistochemistry was made by Image J software. Results:
Analyzing SLG of elderly people we found that the glandular parenchyma
replacement by fat appears to be at the expense of mucous cells than the serous ones.
The EGF expression was positive in serous cells with cytoplasmic granular staining.
The mucous cells presented weak membrane staining. Duct cells exhibited mainly
nuclear staining. FGFR-2 showed mainly granular cytoplasm staining in serous and
duct cells. Mucous cells were negative for FGFR2. The expression of EGFR was
remarkable in serous cells with granular cytoplasmic stained but the mucous cells
were negative. Ducts cells presented granular and cytoplasmic staining. Analyzing
the quantitative data of immunohistochemical expression of EGF, EGFR and
FGFR2, it was not observed differences between aldults and elderly people.
Conclusions: EGF, EGFR and FGFR-2 are important to maintain glandular
parenchyma and act differently in glandular cellular types during aging.
This project is being funded by FAPESP
This project is approved by the ethics committee of UNIFESP:
26517914.3.1001.5505

Q59

Q60
POSSIBLE ANGIOGENIC EFFECT IN THE MESENTERIC BLOOD

VESSELS FROM YOUNG WISTAR RATS SUBMITTED TO HIGH-FAT
AND LOW-CARB DIET
Patrcia Andressa de Almeida Buranello (2), Isabella Rodrigues Ramires (1), Rayane
Bernardes Estevam (1), Lucas Fernandes Rezende (1) and Marcelo Rodrigues Pinto
(1)*.
(1) Uberaba University (UNIUBE), Uberaba, Minas Gerais, Brazil.
(2) Federal University of Tringulo Mineiro (UFTM), Uberaba, Minas Gerais,
Brazil.
*Corresponding author: mrodriguespinto@gmail.com
Presenting: pathburanello@gmail.com
Address correspondence to: Marcelo Rodrigues Pinto
Laboratory of Biopathology and Molecular Biology - Uberaba University (UNIUBE)
1801 Nen Sabino Av Uberaba 38055-500 - Fone:+55 34 33258035 - Uberaba,
Minas Gerais, Brazil - E-mail: mrodriguespinto@gmail.com
The increased consumption of diets high in saturated fats is a major public health
concern in human societies. The adipose tissue expansion can lead to increased
adipocyte hypoxia, which stimulates angiogenesis, a process of new blood vessel
formation by the sprouting of the preexisting vascular network. In present work, we
evaluate the possible effect of high-fat diet in the mesenteric blood vessels of Wistar
rats. Male rats (N= 20) were equally separated into two groups. The control group
received a balanced diet, while the experimental group received a high-lipid diet.
Diets plus water were offered ad libitum, for eleven weeks. After sacrificing the rats,
the mesentery was removed and stained with toluidine blue. Ten fields for each
sample/rat were acquired in 100x magnification and vessels counted using ImageJ
software. Macroscopically, was observed a higher fatty deposit around the larger
mesenteric vessels in rats fed with high-fat diet. In addition, histological analysis
showed that 60% of these rats presented increase in number and diameter in
mesenteric blood vessels. Our data showed that high-fat diets can contribute to
angiogenic effect in mesenteric vessels. This increased vascularity in principle
suggests an improvement in the absorptive capacity of nutrients. However
angiogenesis can be related to tumoral maintenance, and is a reflection of chronic
inflammation. Currently, more efforts are being applied to investigate this effect in
the murine model.
This study was approved by the Ethics Committee for Animal Experimentation of
the Uberaba University (n036/2014). Financial support: FAPEMIG.
HISTOLOGICAL ANALYSIS OF THE SPERMATOGENESIS

OLOLYGON LUIZOTAVIOI (ANURA, HYLIDAE)
OF
Rafael Siqueira Chagas (1), Alessika Moura de Souza (2), Maria Rita Silvrio Pires
(2,3), Katiane de Oliveira Pinto Coelho Nogueira (1,4).
(1) Laboratrio de Biomateriais e Patologia Experimental, Universidade Federal de
Ouro Preto, Minas Gerais, Brazil.
(2) Laboratrio de Zoologia de Vertebrados, Universidade Federal de Ouro Preto,
(3) Departamento de Biodiversidade, Evoluo e Meio Ambiente; Universidade
Federal de Ouro Preto, Minas Gerais, Brazil.
(4) Departamento de Cincias Biolgicas, Universidade Federal de Ouro Preto,
rsiqueirachagas@gmail.com 03135591215 031991006349
Descriptions for gametogenesis are important for a better understanding of
reproductive biology, especially of neotropical anurans, which data are still scarce in
literature. Similar studies were made on different anuran species, although there is no
such kind of description for Ololygon luizotavioi, which is endemic in Brazil and can
be found in Serra do Espinhao-MG. This study aims to describe the histological
organization of the seminiferous elements of O. luizotavioi. Five samples of the
species were used. The specimens came from Zoological Museum of Universidade
Federal de Ouro Preto and its use was approved by the Ethics Committee of the same
institution.The tests were submitted to histological routine for microscopic analysis.
Anatomically, the tests measured 3.40.66 mm. Microscopically, it was observed
that in O. luizotavioi, as well as another anuran species, spermatogenesis also occurs
in the seminiferous locule where elements of the germ epithelium are organized in
spermatogenetic cysts, each cyst containing cells in the same differentiation stage.
Characterization of each cellular type were made turning possible the identification
and differentiation of germ lineage cells. In the basis of the epithelium there are
spermatogonia I, biggest cells and associated with Sertoli cell, that suffer mitotic
proliferation and originate cysts conteining spermatogonia II. After growth and
differentiation, the spermatocytes I are originated, and through meiotic division
originate the spermatocytes II, which suffer the second meiotic division and become
haploid cells, the spermatids I. After proeminent differentiation, these cells originate
the spermatids II, and with the spermiogenesis process the spermatozoa appear.

Q61
Q62
HYPERBARIC OXYGEN (HBO) TREATMENT IMPROVES ARTERIAL

THROMBUS RESOLUTION IN MICE
SUGAR CANE BIOPOLYMER IN TREATING CALCANEAL TENDON

INJURY: HISTOMORPHOLOGICAL AND IMMUNOHISTOCHEMISTRY
EVALUATION
Denise Gonalves Pedrosa1, Maiara Ferreira Terra1, Claudio Chrysostomo Werneck2,

Selma Giorgio3, Cristina Pontes Vicente1.
Department of Structural and Functional Biology, State University of Campinas,
So Paulo, Brazil.
2
Department of Biochemistry and Tecidual Biology, State University of Campinas,
1.
So Paulo, Brazil.
3
Department of Animal Biology, State University of Campinas, So Paulo, Brazil.2.
3.
E-mail: denise-pedrosa@hotmail.com
Adress: Charles Darwin street, w/n, Block N, Institute of Biology, State University
of Campinas, SP, Brazil.
Phone: +55 (19) 3521-6126 Mobile: +55 (19) 98364-0634
Hyperbaric oxygen (HBO) therapyinvolves exposing the body to 100 percent oxygen
at a pressure that is greater than oneatmosphere. The combination of high pressure
and pure oxygen increases the concentration of oxygen in bloodstream, which
permeates into body tissues helping to promote neovascularization, healing process,
stem cell mobilization/differentiation andextracellular matrix formation.The purpose
of this study wasto analyze the influence of the treatment with HBOon thrombus
formation time and resolution of C57bl/6 male mice. Animals were divided into 3
groups: (1) control; (2) injured + 3 days and (3) injured + HBO treatment for 3
days.Arterial injury was made in the right carotid for 2 minwith ferric chloride 15%
and blood flow was measured using an ultrasound probe.Two hours after the surgery,
mice received 1 hour HBO session of 100% O2 at 2.5 ATA, for 3 days in a standard
hyperbaric chamber. The area of thrombus formation was measured on hematoxylineosin stained histologic sections taken from lesioned carotid arteries.We found a
48% reduction of thrombus formation in HBO treated animals (p<0.001)when
compared to injured animals without treatment. In conclusion, HBO treatment
reducedthrombosis in mice and provides new important insights to be investigate in
the pathogenesis of arterial thrombosis.
(Project approved by Ethics Committee of UNICAMP, protocol no 3550-1).
Financial support: Fapesp/CNPq.
Antnio Felix da Silva Filho (1); Ana Cristina Falco Esteves (2); Deniele Bezerra
Ls (2); Jos Lamartine de Andrade Aguiar (3); Maira Galdino da Rocha Pitta (1);
Moacyr Jesus Barreto de Melo Rgo (1); Silvia Regina Arruda de Moraes (2);
Departamento de Bioqumica, Universidade Federal de Pernambuco (UFPE);
(2) Laboratrio de Plasticidade Neuromuscular (LAPLAN) - UFPE
(3) Departamento de Cirurgia Experimental - UFPE
Author presenting: Antnio Felix da Silva Filho
Address: Laboratrio de Imunomodulao e Novas Abordagens Teraputicas,
Universidade Federal de Pernambuco. Av. Prof. Mores Rgo, 1235 - Cidade
Universitria, Recife - PE, 50670-901.
Email: tonifelix4@gmail.com Telephone: (81) 997097087
In cases of severe tendon damage, it is necessary the use of implantable devices
having favorable mechanical strength to serve as a support in tendon healing.
Between different materials, sugar cane biopolymer has demonstrated significant
applicability in tissue recovery procedures. Thus, this study aimed to evaluate the
histomorphological consequences brought about by the use of sugar cane biopolymer
in the treatment of induced lesions in calcaneal tendon. Seventeen male Wistar rats
were used, with 60 days, randomly distributed in tree groups: uninjured control
group (GC, n = 5); experimental group 1 (EG1, n = 6), only submitted to tenotomy;
and the experimental group 2 (EG2, n = 6) whose animals underwent to tenotomy
and treatment with biopolymer thirty days after. Tissues obtained were fixed and
stained for histomorphometric evaluation in light microscope Leica DM 5000.
Statistical analysis was performed using the t test student in SPSS software. For
immunohistochemistry, it was used anti-actin-6 antibody and DAB revelation. The
amount of fibrocytes was lower in EG2 compared to EG1 (p = 0.002) and higher
than the GC (p=0,002) (512,70 72,30; 1000,33 132,08; 766,50 61,33,
respectively). The same pattern was observed in the fibroblasts and blood vessels
counting, although not significant. Immunohistochemistry showed that the blood
vessels present in the injured tendon tissues had actin-6 staining +2 in comparison
with the vessels EG1 (+1) and GC (0). We conclude that sugar cane biopolymer
induces an increase on fibrocytes number and a possible molecular rearrangement
regarding to the vascular actin-6 in tendon lesions treated.
Key-words: Tissue regenerator; angiogenesesis; immunohistochemistry.
Ethical approval: n23076.038181/2012-19
Financial support: PROPESQ - UFPE

Q63
BEHAVIOR OF THE LYSOSOME RELATED ORGANELLE DURING

DIFFERENTIATION OF GIARDIA INTESTINALIS
Victor Midlej (1, *), Wanderley de Souza (1) and Marlene Benchimol (1,2)
1
2
Instituto de Biofsica Carlos Chagas Filho, UFRJ Brazil

UNIGRANRIO, Rio de Janeiro Brazil
vmidlej@hotmail.com, phone contact: 21 3938-6583 (Institutional) / 73 991226493 (mobile)

Giardia intestinalis is a parasite that exhibits two forms in its life cycle: flagellated
trophozoite and cyst. Although typical lysosomes are not found, the peripheral
vesicles (PV) behave as lysosomes. The knowledge of the endomembrane system
during G. intestinalis encystment is limited. To analyze the behavior of these vesicles
during the differentiation process, markers of acidic compartments such as Lucifer
yellow (LY) and Acridine orange (AO) were used to track the PVs; cytochemistry
for acid phosphatase and a tridimensional reconstruction of these vesicles were
performed. Results were observed using fluorescence, transmission electron (TEM)
and Focused Ion Bean (FIB) microscopies. Biochemistry analysis of phosphatase
activities was done, measuring the rate of p-nitrophenol (p-NP) production.
Microvesicular bodies inside PVs were revealed by FIB. LY and AO fluorescence
decreased during encystment process. The same results were observed with acid
phosphatase technique. It was observed a labeling of acid phosphatase on the
plasmatic membrane on 21h-encysted cells. This finding was corroborated with
biochemical analysis. The production of p-NP was increased in non-permeabilized
cells, showing the presence of phosphatase in the plasma membrane in Giardia after
21h of encystment. By TEM we identified PVs with electrondense profile near the
wall of cysts induced to excyst. In conclusion, a change in PVs behavior happens
during parasite differentiation, with a translocation of phosphatases from PVs to the
plasma membrane. Moreover, there is a participation of these organelles during the
exit of the parasite from cyst wall.
This work was supported by CNPq, FAPERJ, CAPES and INBEB.

R NEUROBIOLOGY
R1
R2
PRION
PROTEIN
MODULATES
MONOAMINERGIC
NEUROTRANSMITTER SYSTEMS AND DEPRESSIVE-LIKE BEHAVIOR
IN MICE
MEMORY RETRIEVAL OF INHIBITORY AVOIDANCE REQUIRES

HISTAMINE H1 RECEPTOR ACTIVATION IN THE HIPPOCAMPUS
Danielle Beckman(1), Lus Eduardo Santos(1), Tatiana Alves Americo(1), Jos

Henrique Ledo(2), Fernando Garcia de Mello(1), Rafael Linden(1)
(1) Institute of Medical Biochemistry Leopoldo de Meis, Federal University of Rio
de Janeiro, Rio de Janeiro, Brazil
(2) Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de
Janeiro, Rio de Janeiro, Brazil
Contact details: Danielle Beckman, Rua Alberto de Campos, nmero 107, apto 202,
Ipanema, Rio de Janeiro, RJ. CEP:22411-030, daniellebeckman@gmail.com, tel: 2139386594, 21-994627398
Dysfunction of monoaminergic systems is a common feature of several CNS
pathologies, and may underlie clinical depression. The course of transmissible
spongiform encephalopathies is associated with conformational conversion of the
prion protein (PrPC), the physiological functions of which are also implicated in
Alzheimers Disease. To help understand the functional interactions of PrPC with
monoaminergic systems, we compared both behavioral responses and neurochemical
markers of monoaminergic function in wild-type and PrPC-null mice of a controlled
genetic background. A battery of tests showed depressive-like behavior in knockout
animals, when compared with wild-type, with no detectable motor defects. The
contents of dopamine, tyrosine hydroxylase and the 5-HT5A serotonin receptor were
increased in the cerebral cortex of PrPC-null mice, as compared to wild-type
controls. Accumulation of cAMP in cerebrocortical tissue was distinct between the
two genotypes following stimulation with either serotonin or dopamine, but not with
norepinephrine. Under confocal microscopy, PrPC colocalized with the
dopaminergic D1 and the serotonergic 5HT5A receptors. These data are consistent
with a role for PrPC in the scaffolding of monoaminergic signaling modules with
functional consequences for neurotransmission. Our results may be relevant for the
understanding of psychiatric disorders such as major depression as well as the
pathogenesis of neurodegenerative disorders like Prion and Alzheimers Diseases.
All
procedures
followed
institutional
guidelines
and
were
approved by the Animal Care and Use Committee of the Federal University of Rio
de Janeiro. This work was supported by grants from the Conselho Nacional de
Desenvolvimento Cientfico e Tecnolgico and National Institute of Translational
Neuroscience.
Disclosures: The authors declare that they have no conflicts of interest with the
contents of this work.
Jociane de Carvalho Myskiw1, Roberta Fabbri1,2, Cristiane R.G. Furini1, Maria

Beatrice Passani3, Gustavo Provensi2, Elisabetta Baldi4, Corrado Bucherelli4, Ivan
Izquierdo1, Patrizio Blandina1
1
Memory Center, Brain Institute of Rio Grande do Sul, Pontifical Catholic

University of Rio Grande do Sul (PUCRS), Av. Ipiranga, 6690, 90610-000 Porto
Alegre, RS, Brazil. 2Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e
Salute del Bambino, Sezione di Farmacologia e Tossicologia, Universit di Firenze,
Viale G. Pieraccini 6, 50139 Firenze, Italy. 3Dipartimento di Scienze della Salute,
Sezione di Farmacologia e Chemioterapia, Universit di Firenze, Viale G. Pieraccini
6, 50139 Firenze, Italy. 4Dipartimento di Medicina Sperimentale e Clinica,
Universit di Firenze, Largo Brambilla 3, 50134 Firenze Italy
To whom correspondence may be addressed:
E-mail: jociane_carvalho@hotmail.com
Memory Center, Brain Institute of Rio Grande do Sul, Pontifical Catholic University
of Rio Grande do Sul (PUCRS), Av. Ipiranga, 6690 2nd floor, 90610-000 Porto
Alegre, RS, Brazil. Phone (+55) 51 3320 3336; fax (+55) 51 3320 3312.
Retrieval represents a dynamic process that may require neuromodulatory signaling.
Here, we report that the integrity of the brain histaminergic system is necessary for
retrieval of inhibitory avoidance (IA) memory, since rats depleted of histamine
through lateral ventricle injections of alpha-fluoromethylhistidine (a-FMHis), a
suicide inhibitor of histidine decarboxylase, displayed impaired IA memory when
tested 2 days after training. a-FMHis was administered 24 h after training, when IA
memory trace was already formed. Infusion of histamine in hippocampal CA1 of
brain histamine-depleted rats, hence amnesic, 10 min before the retention test
restored IA memory, but was ineffective when given in the basolateral amygdala
(BLA) or in the ventral medial prefrontal cortex (vmPFC). Intra-CA1 injections of
selective H1 and H2 receptor agonists demonstrated that histamine exerted its effect
by activating the H1 receptor. Noteworthy, the H1 receptor antagonist pyrilamine
disrupted IA memory retrieval in rats, thus strongly supporting an active involvement
of endogenous histamine. Ninety min after the retention test, c-Fos positive neurons
were significantly less in the CA1 of a-FMHis-treated rats, that displayed amnesia,
compared with control group. We also found reduced levels of phosphorylated
cAMP responsive-element-binding protein (pCREB) in the CA1 of a-FMHis-treated
animals compared with controls. Increases in pCREB levels are associated with
retrieval of associated memories. Targeting the histaminergic system may modify the
retrieval of emotional memory, hence histaminergic ligands might reduce
dysfunctional aversive memories and improve the efficacy of exposure
psychotherapies.
Ethical approval by CEUA registration: 13/00330
Work supported by research grants from the National Council of Research of Brazil,
CNPq, Compagnia di San Paolo (Italy), and Ente Cassa di Risparmio di Firenze
(Italy).

R3
R4
EXTINCTION LEARNING, WHICH CONSISTS OF THE INHIBITION OF

RETRIEVAL, CAN BE LEARNED WITHOUT RETRIEVAL
Jociane de Carvalho Myskiw1, Cristiane R.G. Furini1, Bianca Schmidt1, Flvia
Ferreira1, Clarissa Penha Farias1, Eduarda Godfried Nachtigall1, Ivan Izquierdo1
1
National Institute of Translational Neuroscience (INNT), National Research Council

of Brazil, and Memory Center, Brain Institute of Rio Grande do Sul, Pontifical
Catholic University of Rio Grande do Sul (PUCRS), Av. Ipiranga, 6690 2nd floor,
90610-000 Porto Alegre, RS, Brazil.
To whom correspondence may be addressed.
E-mail: jociane_carvalho@hotmail.com and izquier@terra.com.br
Memory Center, Brain Institute, Pontifical Catholic University of Rio Grande do Sul
(PUCRS), Av. Ipiranga, 6690 2nd floor, phone (+55 51) 3320 3336; 90610-000.
Porto Alegre, RS, Brazil.
In the current study we test the hypothesis that extinction is not a consequence of
retrieval in unreinforced conditioned stimulus (CS) presentation but the mere
perception of the CS in the absence of a conditioned response. Animals with
cannulae implanted in the CA1 region of hippocampus were subjected to extinction
of contextual fear conditioning. Muscimol infused intra-CA1 before an extinction
training session of contextual fear conditioning (CFC) blocks retrieval but not
consolidation of extinction measured 24 h later. Additionally, this inhibition of
retrieval does not affect early persistence of extinction when tested 7 days later or its
spontaneous recovery after 2 weeks. Furthermore, both anisomycin, an inhibitor of
ribosomal protein synthesis, and rapamycin, an inhibitor of extra-ribosomal protein
synthesis, given into the CA1, impair extinction of CFC regardless of whether its
retrieval was blocked or not by muscimol. Therefore, retrieval performance in the
first unreinforced session is not necessary for the installation, maintenance or
spontaneous recovery of extinction of contextual fear conditioning.
Ethical approval by CEUA registration: 11/00262
Work supported by research grants from the National Council of Research of Brazil,
CNPq.
MITOCHONDRIAL RESPIRATORY CHAIN ENZYME ACTIVITIES IS

IMPAIRED BY GLYPHOSATE-BASED HERBICIDES IN ZEBRAFISH
BRAIN
Aline Guimares Pereira (1), Aline Pertile Remor (2), Alexandra Latini (2), Yara
Maria Rauh Mller (1) and Evelise Maria Nazari (1).
(1) Laboratrio de Reproduo e Desenvolvimento Animal, Departamento de
Biologia, Embriologia e Gentica, Universidade Federal de Santa Catarina,
Florianpolis, Santa Catarina, Brasil.
(2) Laboratrio de Bioenergtica e Estresse Oxidativo, Departamento de Bioqumica,
Universidade Federal de Santa Catarina, Florianpolis, Santa Catarina, Brasil.
alinegp77@gmail.com
Glyphosate (N-[phosphonomethyl]-glycine) (GLY) is a broad-spectrum herbicide,
and their use has increased extensively with the development of genetically modified
GLY-resistant crop varieties. GLY is classified as an organophosphate, which are
recognized by their toxic effects on the central nervous system (CNS). However, few
studies have investigated whether low environmentally relevant concentrations of
glyphosate-based herbicides (GBH) can lead to neurotoxicity of non-target
organisms and also how the CNS cells respond to this herbicide. Zebrafish Danio
rerio was used as a model to study the neurotoxic effects induced by sublethal
concentrations of GBH on the respiratory chain enzyme activities. The animals were
exposed to concentrations of 0.065, 1.0 and 10.0 mgGLY/L of GBH (Monsanto do
Brasil Ltda, containing 720g/Kg acid equivalent to GLY), for 7 and 15 days
(CEUA/UFSC N 5466040416). Non-exposed animals were used as controls. After
exposure periods, the brains were dissected and respiratory chain enzyme activities
were measured in brain homogenate preparations. The enzyme activities of
complexes I, II and IV were measured using a Microplate Readers M200 Tecan. The
animals exposed to lower concentrations (0.065 and 1.0 mgGLY/L of GBH) showed
impaired respiratory chain enzyme activities, evidenced by reduced activities of
complexes I, II and/or IV (*P<0.05; **P<0.01) in both exposure times. However, the
animals exposed to higher concentration (10.0 mgGLY/L of GBH) showed no
significant difference, when compared to controls. These results suggest a inverted
doseresponse relationship of GBH, wherein the low concentrations of GBH induces
inhibition of mitochondrial respiratory chain enzyme activities in zebrafish brain.

R5
R6
A RECEPTOR-INDEPENDENT AND NON-SPECIFIC MECHANISM FOR

ENDOCANNABINOID ACTION BASED ON THE MODULATION OF THE
HYDROPHOBIC COUPLING MEMBRANE/PROTEIN
EXPRESSION AND DISTRIBUITION OF STEAROYL-COA DESATURASE

(SCD2) IN THE BRAIN OF RODENTS: SIGNS OF COLOCALIZATION IN
PROLIFERATIVE CELLS
Djalma Medeiros (1,2)*, Laz da Costa Silva-Gonalves (1), Mirian Elisa Rodrigues
Guerra (3), Annielle Mendes Brito da Silva (1), Jos Roberto Ruggiero (3), Marcia
Perez dos Santos Cabrera (3,4), Manoel Arcisio-Miranda (1)
(1)
(1) Departamento de Biofsica, Escola Paulista de Medicina, Universidade Federal(2)
de
So Paulo, So Paulo, SP, Brasil
(2) Filosofia, Faculdade de So Bento, So Paulo, SP, Brasil
(3) Departamento de Fsica, IBILCE, Universidade Estadual Paulista, So Jos do
Rio Preto, SP, Brasil
(4) Departamento de Qumica e Cincias Ambientais, IBILCE, Universidade
Estadual Paulista, So Jos do Rio Preto, SP, Brasil
Felipe Corra da Silva (1), Roberta Haddad Tvolli (1), Joseane Morari (1), Lucas
Francisco Ribeiro do Nascimento (2), Licio Augusto Velloso (1)
*e-mail: djalmamedeiros@uol.com.br; telephone: 55 11 55764848 #2337

Endocannabinoids are lipidic messengers that exercise important physiological
functions in the nervous system by binding to receptors CB1 and CB2. Antagonists
and knock-down of these receptors do not completely abolish endocannabinoid
activities, suggesting a direct mechanism (not cannabinoid receptor mediated) by
regulating the function of ion channel proteins. However, an ordinary regulation
model by specific binding of the endocannabinoid to its target does not promptly
explain its modulatory activity on different classes of ion channels. Our research
proposes an alternative endocannabinoid mode of action based on a receptorindependent and non-specific mechanism involving the adjustment of the
membrane/protein hydrophobic coupling, which arises from amphiphilic-induced
local changes in bilayer elastical properties that alter the energetic coupling between
the protein and their host bilayer. We studied the regulatory action of AEA (the best
characterized endocannabinoid) in planar lipid bilayers composed of DOPC and
DPhPC, combining membrane capacitance measurements, gramicidin single channel
conductance and molecular dynamic simulations. AEA does not change the
membrane capacitance or the amplitude of single channel current, but produces
increase in the channels appearance frequency and its open state average lifetime.
The molecular dynamic shows that AEA has no substantial effect on the overall
structure of lipid bilayers, but locally change its lateral pressure. These findings, in
conjunction with theories of elastic deformation of bilayers, supports the nonspecific action mechanism of endocannabinoids via modulation of the hydrophobic
coupling membrane/protein, providing a quantitative and predictive tool that will
broaden our understanding on how endocannabinoids affect neurophysiological
processes.
(1) Laboratory of Cell Signaling, University of Campinas, Campinas, Brazil.

(2) Institute for Diabetes Ressarch, Helmholtz Zentrum, Munich.
Stearoyl-CoA desaturase-2 (SCD2) is the main 9 desaturase expressed in the central
nervous system (CNS). Since de novo lipogenesis, and the subsequent desaturation
of lipids, contributes to events leading to cell proliferation especially in the synthesis
of membrane phospholipids. In this work, we propose to investigate the SCD2 gene
expression and its cellular distribuition in different anatomical regions of CNS.
Swiss male mice were used and SCD2 were analyzed by real-time PCR and
immunohistochemistry. CNS anatomical regions (spinal cord, cerebellum, cortex,
hippocampus, olfactory bulb, thalamus, hypothalamus, midbrain and striatum) were
analyzed. Our results show that SCD2 is present in all of the regions evaluated, being
more abundant in spinal cord, hypothalamus and midbrain. Next, we have elected the
hypothalamus for an in depth analyses of SCD2 expression at diferent ages. There
were no significant diferences between the ages tested (postnatal day 0 P0, P21 and
seven week old mice adult), although there was a trend of increasing in P21 and
seven weeks as compared to P0 (p=0,06 and p=0,07, for P21 and adult respectively).
Regarding the cellular distribution of SCD2, our experiments reveal that this protein
is expressed in all cell types of central nervous system, including those responsive to
proliferation (FGF10+) as well those that are proliferating (Ki67+). Thus, SCD2 is
highly expressed in CNS of rodents, being present in all cell types, including those
involved with proliferation.
All experimental procedures involving mice were approved by the Ethics Committee
at the University of Campinas (4138-1) and supported by FAPESP.
Financial Suport: FAPESP, CNPq

R7
R8
IMMUNOREGULATORY
DRUG
INDUCES
EXPRESSION
OF
NEUROTROPHINS AND PROMOTES NEURONAL DEVELOPMENT IN
RETINA CELLS
Vanessa do Socorro Cabral Miranda (1)*; Giselle Cristina Brasil Carvalho (2);
Aldanete Santos Rosrio (2); Barbarella Mattos Macchi (2); Chubert Bernardo
Castro de Sena (1,2); Jos Luiz Martins do Nascimento (1, 2)
(1) Laboratory of Structural Biology, Institute of Biological Science Federal
University of Par, Belm-Par, Brazil
Science, Federal University of Par, Belm-Par, Brazil.
Address: Rua Augusto Corra, 01 Guam 66075110 - Belm, PA Brasil. E-mail:
vanessacabralmiranda@gmail.com Phone Numbers: +559132017545 (lab)/+55
91993690552 (mobile)
Cyclosporin A (CsA) is an immunosuppressive drug found in the fungus
Tolypocladium inflatum. CsA is used in post-organ transplantation to prevent
rejection. The known action mechanism is related to the inhibition of nuclear factor
of activated T cells (NFAT). Our research group has reported an alternative role of
CsA in sensory and sympathetic neurons of ganglion root dorsal from embryo
chickens after induction of neuronal differentiation related to expression of Nerve
Growth Factor (NGF) and its high affinity receptor (TrkA). In this work, we
evaluated the effects of CsA in the Central Nervous System during and after in vitro
retinal cell differentiation from chicken embryo (E6). The cells were treated with 1 40M CsA at different days of in vitro differentiation for three days. After the
treatments we analyzed the expression level of NGF and TrkA, cell differentiation
and viability by RT-PCR, photomicrographs and MTT assay, respectively. Our
results showed that only 1M of CsA at fourth day of in vitro differentiation induced
significantly increased of cell viability, comparing with untreated cells (39% of
increased, p< 0.05). The photomicrography of this treated culture showed increased
of neuronal population. These results were related with high expression level of NGF
after CsA treatment (84,78%, p< 0.05). No published data has been showed this
specific neuromodulatory action of CsA during the development of retina cells.
These data suggest that CsA works differently at different stages of cell
differentiation, inducing alternative mechanisms to promote neurogenesis.
Ethical approval: 85/15. Financial support: CAPES and CNPq.

THE ROLE OF KOJIC ACID (HMP) IN THE ANTIOXIDANTS AND

REDOX SIGNALING IN MLLER CELLS FROM CHICKEN EMBRYOS
Giselle Cristina Brasil Carvalho (1)*, Aldanete Santos Rosrio (1); Vanessa do
Socorro Cabral Miranda (2); Barbarella Mattos Macchi (1); Chubert Bernardo
Castro de Sena (1,2); Jos Luiz Martins do Nascimento (1, 2)
Science, Federal University of Par, Belm-Par, Brazil.
(2) Laboratory of Structural Biology, Institute of Biological Science Federal
University of Par, Belm-Par, Brazil
Address: Rua Augusto Corra, 01 Guam 66075110 - Belm, PA Brasil. E-mail:
brasil.cristina@gmail.com. Phone Numbers: +559132017545
Kojic acid or 5-Hydroxy-2-hydroxymethyl--pyrone (HMP), is a fungal metabolite,
known as tyrosinase inhibitor. Recently, we have shown that HMP is able to activate
macrophage promoting significant cell activation and cytoplasmic accumulation of
reactive oxygen species (ROS). However, no information is available regarding its
effects on central nervous system (CNS). Neuroglial cells are important to
homeostasis in the brain and defense against pathological insults. In the retina of
vertebrate, Mller cells have a protective role similar to macrophages, in response to
infection and injury. Therefore, the aim of this study was to evaluate the effect of
HMP in glial cell culture from chicken embryos. Mller cell cultures were treated
with HMP during 24 hours and compared with untreated cells (control group). NBT
(nitro blue tetrazolium) assay showed significant increasing of ROS in
concentrations of 10, 25, 50 and 100 M of HMP (89, 74, 80, 79 and 98%
respectively, p 0.05). Enzymatic assay showed that HMP (100 M) induced
significant inhibition of catalase and superoxide dismutase activity (47.41% and
53.93%, respectively), as well as decreased the total glutathione levels (89.61%). The
results suggest that HMP promotes accumulation of ROS and inhibition of
antioxidant profile of Mller cells. In summary, HMP may be therapeutically used as
an alternative drug to treat eye infections due its modulatory role in Mller cells.
This study was approved by Ethics Committee of UFPA (85/15).
Financial support: CAPES, CNPq and PROPESP


R9
R10
ANTIOXIDANT DEFENSE SYSTEM PRESERVES THE INTEGRITY OF

RETINA CELL IN CULTURE AFTER HYDROXYCHLOROQUINE (HCQ)
TREATMENT
P2Y1 RECEPTOR MODULATES THE TRANSITION FROM G1 TO S

PHASE OF CELL CYCLE OF RAT RETINAL PROGENITOR CELLS
Aldanete Santos Rosrio (1), Giselle Cristina Brasil Carvalho (1), Vanessa do
Socorro Cabral Miranda (2), Chubert Bernardo Castro Sena (1,2), Anderson Raiol
Rodrigues (3), Barbarella de Matos Macchi (1), Jos Luiz Martins do Nascimento
(1,2).
Science, Federal University of Par-PA, Brazil.
(2) Laboratory of Structural Biology, Institute of Biological Science, Federal
University of Par-PA, Brazil.
(3) Tropical Medicine Center NMT, Federal University of Par-PA, Brazil.
Address: Rua Augusto Corra, 01 Guam 66075110 - Belm, PA Brasil.
E-mail: alda-santos@live.com Phone Numbers: +559132017545
The treatment of autoimmune diseases with antimalarial drugs as chloroquine (CQ)
and its derivative hydroxychloroquine (HCQ) is very common. However, these drugs
can trigger serious ocular side effects in special the retina. The injury mechanisms of
these drugs are not known on retinal cells. Thus, the aim of this study is to evaluate
the effects of CQ and HCQ drugs in the cell viability and on the antioxidant system
of retinal cells. Retinal cell cultures from chicken embryos (E8) were treated with
different concentrations (0, 25, 50 and 75M) of CQ or HCQ for 24 hours to
evaluate the cell viability, enzymatic activity of catalase and the total glutathione
levels. Our data showed reduction of cell viability that was dose-dependent manner
when treated with CQ, but without significant changes for catalase activity and
glutathione levels in low doses. However HCQ-treated cells showed no reduction in
cell viability, but the activity of catalase and total glutathione levels was increased
after treatment with 50M (46% and 33%, respectively). These results suggest that
HCQ is not cytotoxic for retinal cells due to the role of antioxidant defense system,
showing that this system is essential to preserve the integrity of retina cells.
This study was approved by the Ethics Committee of UFPA (85/15). Financial
support: CAPES, CNPQ and PROPESP.
1.
2.
Luana de Almeida Pereira, Marinna Garcia Repossi, Ana Paula F. Paz dos Santos,
Alfred Sholl-Franco, Ana Lucia Marques Ventura and Lucianne Fragel-Madeira
1
Department of Neurobiology, Institute of Biology, Fluminense Federal University; 2

Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro.
In vertebrates the retina histogenesis is guided by extrinsic and intrinsic mechanisms,
in which ATP has been widely studied as being important to many processes
involved with nervous system development. Previous studies from our group
demonstrated that endogenous adenine nucleotides are able to regulate the
proliferation of retinal progenitor cells in vivo through P2Y1 receptor. Based on this
data, we evaluated which cell cycle phase is regulated by P2Y1 receptor in the retina
of newborn rats. Intravitreal injection of 100 M of MRS 2179, a P2Y1 receptor
antagonist, in rats at two postnatal days (P2) was performed and after 24 hours the
retinal protein extract was analyzed by western blotting. The analysis revealed an
increase in expression of p57KIP2, whereas no change in p27KIP1 was observed.
Furthermore, there was also an increase in the levels of cyclin D1 and a reduction of
cyclin E. In order to analyze cell cycle phases transitions, BrdU (60mg/Kg) was
intraperitoneally injected for 3 hours before the end of treatment with MRS 2179 and
was observed a decrease of 18% in BrdU-positive cell number at S-phase stratum but
there was no change in the cell migration through G2 to M phases of cell cycle. This
data was confirmed by phospho-histone H3 immunolabeling in which was not
observed change in the number of labeled treated cells compared to control. In
conclusion, the P2Y1 receptor controls the transition to G1 from S phase in retinal
progenitor cell by p57KIP2 modulation.
CEUA-UFF: 547/2014. Funding support: Capes, FAPERJ and CNPq.

R11
R12
PARACETAMOL AUGMENTS THE NEUROTOXICITY OF MeHg IN

RETINAL CELL CULTURE
ASCIDIAN BLOOD CELLS MIGRATION TO THE CENTRAL NERVOUS

TISSUE AFTER LESION WITH A NEUROTOXIN
Iago Rodrigues Blanco (1)*; Herley Machado Nahum (1); Barbarella Mattos Macchi
(1); Chubert Sena (1,2); Jos Luiz Martins do Nascimento (1,2)
Isadora Santos de Abreu (1), Ins Julia Wajsenzon (1), Rosalia Mendez-Otero (1) e
Silvana Allodi (1)
(1)
Institute
of
Biological
Sciences,
Laboratory
of
Molecular and Cellular Neurochemistry, University of Par, PA, Brazil
(2) Institute of Biological Sciences, Laboratory of Structural Biology, University
of Par, PA, Brazil
(1) Instituto de Biofsica Carlos Chagas Filho, Universidade Federal do Rio de

Janeiro, Rio de Janeiro, Brazil
3.
Presenter contact details: Address: Rua Augusto Corra, 01 Guam 66075110 Belm, PA Brazil
Methylmercury (MeHg) toxicity is governed by cellular thiol compounds and its
capacity to generate reactive oxygen radicals and oxidative stress. Due to its
analgesic properties, a drug widely used to mitigate pain symptoms is
Acetaminophen (AAP, paracetamol), considered to be safe in therapeutical doses.
However, in high concentrations, AAP proved to be cytotoxic to hepatic and neural
cells, and experimental animals. This study aims to investigate whether AAP can
enhance MeHg cytotoxicity on Retinal Cells of Chick Embryo. Retinal cells were
obtained after dissection and enzymatic dissociation of the tissue in the 7th day of
development of the Chick Embryos. Cells were treated with MeHg (1, 2.5, 5 and
10M) and AAP (1, 10, 100M and 1mM), or AAP (100 M) + MeHg (5M) for
24h at 7th day of culture. After treatment, the cells were subjected to MTT (3-(4, 5dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) viability Assay, or
Measurement of Total Glutathione (GSH) Assay. MeHg proved to be cytotoxic to
the cells in a concentration-dependent manner, while AAP did not induced
significant cell death in tested concentrations. Also, both MeHg and AAP reduced
GSH levels in a concentration dependent manner. These results suggest that AAP
can enhance MeHg toxicity, probably due to its pro-oxidant activity. Animal
handling was approved by the Ethics Comittee of the University of Par (85/15).
Funding Support: CNPq, PROPESP
Corresponding author: Isadora Santos de Abreu; abreunutri@gmail.com; +55

2139386539.
Tunicates are marine animals capable of neuroregeneration after damage. Hemocytes
(blood cells) are involved in neuroregeneration, because they act in the phagocytosis
and repair of damaged tissue and differentiation of neural cells.
Objective To study the migration, involvement and contribution of hemocytes in
the Central Nervous Systems (CNS) neuroregeneration of the tunicate Styela plicata.
Methodology Neurodegeneration was produced by a systemic dose of 3acetylpyridine (3AP), described as toxic to the CNS. The hemocytes were collected
from the heart, labeled with FeraTrackTM (iron nanoparticles coated with dextran)
and were submitted to: prussian blue staining for light microscopy (LM),
immunocytochemistry against dextran, transmission electron microscopy (TEM) and
microanalysis, and scanning electron microscopy (SEM). The cells were then
injected back into the heart. One, 4, 7 and 10 days after 3AP injection, controls and
injured animals were observed by Magnetic Resonance Imaging (MRI). The CNS
was dissected and analyzed by LM and TEM.
Results LM, immunocytochemistry and TEM revealed the internalization of
nanoparticles by hemocytes. Microanalysis revealed the presence of intracellular
iron. SEM (acquired with backscattered electrons) revealed FeraTrackTM as white
dots inside hemocytes. MRI revealed migration of hemocytes to CNS in all the times
studied. LM and TEM showed that there were more hemocytes in injured CNS than
in controls.
Conclusion FeraTrackTM was capable to be internalized by hemocytes and did not
alter their viability and migration. The presence of hemocytes in the CNS suggests
that they were attracted by the damaged CNS tissue participating in its repair.
Key words : MeHg, Paracetamol, Retinal cell culture and Gluthatione

Funding support: CNPq /FAPERJ.

R13
R14
MORPHOLOGICAL CHARACTERIZATION OF THE MOUSE MOTOR

CORTEX
BRAIN AREAS INJURY IN FOCAL ISCHEMIA
Juara Loli de Oliveira1, Rejane Maria Cirra Scaff1, Andrea Gonalves Trentin2,
Marcio Alvarez-Silva2
1.
2.
(1) UFSC Departamento de Cincias Morfolgicas

(2) UFSC Departamento de Biologia Celular, Embriologia e Gentica
Background: Motor cortex recordings have revealed complex relationships between
cortical dynamics and movements (Graziano, 2009). Activity motor cortex neurons
in mammalian correlates with muscle force and joint movement with complex
control hand and arm (Afshar et al., 2011).
The aim of our study was to demonstrated the histological characteristics of the
secondary and primary cortex in adult mice C57/BL6.
Methods: The sections 5m left-brain were deparaffinized, hydrated, submitted to
Nissl and hematoxylin-eosin staining. Slices to immunohistochemistry, were
deparaffinized, hydrated in xylene and alcohol series, rinsed with phosphate buffer
solution (PBS) and incubated with NeuN primary antibody, following secondary
antibodies streptavidin-peroxidase (3,3' Diaminobenzidine/DAB). Experiments were
performed in accordance with the guidelines by Colgio Brasileiro de
Experimentao Animal (COBEA) and were approved in Universidade Federal de
Santa Catarina Animal Care Committee (CEUA PP00943).
Results: Based on histomorphological analysis of the I,II/III IV, V and VI cortical
layers from the motor cortex, identified neurons, astrocytes and microglia-like cells.
We characterized pyramidal neurons by the triangular cell body and non-pyramidal
neurons by the round nuclei with one, two or three nucleoli. Astrocytes were
recognized by the spherical nucleus and the heterogeneous arrangement of chromatin
and microglia-like cells by the elongated and tapered nucleus, quite distinct from the
neurons and astrocytes.
Conclusion: our results revealed that in the secondary cortex superficial layers was
characterized by the predominance of pyramidal neurons, while in the deep layers
neurons showed a pyramidal and rounded aspect. The superficial primary motor and
deep layers showed predominance of rounded neurons.
Keywords: cerebral cortex, neurons, cortical cytoarchitecture
(1) Department of Morphological Science, University Federal of Santa Catarina,

(2) Department of Cell Biology, Embryology and Genetics, University Federal of
(3) Postgraduate Program in Cell Biology and Development, University Federal of
University Federal of Santa Catarina, 88049-900, CP 476, Florianpolis, SC, Brazil,
jucara.oliveira@ufsc.br
Background: The middle cerebral artery occlusion (MCAO) model in rodents has
been widely used to study focal cerebral ischemia (Liu at al., 2009). The evolution of
infarct within the area blood supplied by MCAO not been well elucidated.
The aim of present study was to identified the extent of lesion in the brain under
Ischemic condition.
Methods: Briefly, 810 weeks old C57/BL6 mice were anesthetized with
intraperitoneal (IP) injection of ketamine (50mg/kg) and xylazine (5mg/kg). After 48
hours surgery of the occlusion artery by electrocauterization with an eletronic
scalpel, animals were anesthetized for the intracardiac perfusion with saline solution
and 4% paraformaldehyde solution in 0.1 ml of phosphate buffer, pH 7.2. The brains
were removed and sectioned coronally at 2 mm intervals.
Experiments were performed in accordance with the guidelines by Colgio Brasileiro
de Experimentao Animal (COBEA) and were approved in Universidade Federal de
Santa Catarina Animal Care Committee (CEUA PP00943). For vital staining, the
sections were incubated for 30 min in a solution of 2,3,5- Triphenyltetrazolium
chloride (TTC, Sigma) at room temperature, (Benedek, et al., 2006). The samples of
cerebral cortex were deparaffinized, hydrated, submitted to Nissl staining.
Results: The infarct was demonstrated in the motor cortex (primary and secondary)
and sensory cortex. In ischemic tissue, lack of TTC staining was considered nfarcted
and viable tissue is stained red.
Conclusion: this study showed that following focal ischemia of distal portion of the
middle cerebral artery, affected of cerebral tissue of the motor and sensitive cortex.
Keywords: TTC, middle cortical brain lesion, focal ischemia.

R16
R15
3-ACETYLPYRIDINE-INDUCED
CRUSTACEAN DECAPODS
Juara Loli de Oliveira (1), Rejane Maria Cirra Scaff (1), Elisa Cristiana
Winkelmann Duarte (1), Andrea Gonalves Trentin (2,3), Rodrigo Lucas (3), Marcio
Alvarez-Silva (2,3).
DEGENERATION
IN
ADULT
INTERACTION OF MESENCHYMAL AND NEURAL STEM CELLS WITH

POLYMERIC NANOFIBER PBAT/PPy FOR NEURAL APPLICATIONS
Wajsenzon, IJR (1); Wajsenzon-Lopes, I (1,3); Santos de Abreu, I (1,2); Allodi, S

(1),
Alessandro Eustaquio Campos Granato (1), Anderson de Oliveira Lobo (2),

Marimlia Porcionatto (1)
(1) Laboratrio de Neurobiologia Comparativa e do Desenvolvimento, Instituto (1)

de
Biofsica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, RJ, Brasil
(2)
(2) Ps-Graduao em Cincias Biolgicas-Fisiologia, Instituto de Biofsica Carlos
Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil (3)
(3) Graduao em Medicina, Universidade Federal Fluminense, Niteri, RJ, Brasil
(1) Laboratrio de Neurobiologia, Universidade Federal de So Paulo, So Paulo,

SP, Brazil
(2) Laboratrio de Nanotecnologia Biomdica, Universidade do Vale do Paraba, So
Jos dos Campos, SP, Brazil.
Corresponding author: Ins Julia Wajsenzon; ines_wajsenzon@hotmail.com; +55

2139386539
Introduction: The neurotoxin 3-acetylpyridine (3AP) is a niacinamide antagonist that
produces selective lesions in the central nervous system with a pronounced effect on
neurons both of vertebrates and invertebrates. Here we used 3AP to study
neurodegeneration in the protocerebrum of the adult crab Ucides cordatus, as the
first step to study neural regeneration.
Methods: We produced lesions by administering systemically a single dose of 3AP
diluted in crustacean PBS. We then anesthetized experimental crabs (n=15) and a
control and dissected their protocerebrum. We observed the tissue 1, 7, 14, 21, 28
days after the procedure. Then, we carried out immunohistochemical reactions using
the following antibodies: anti-NeuN (to identify mature neurons), anti-GFAP (to
identify glial cells) anti-synaptophysin (SYP; to evidence synapses).
Results: One day after the lesion, the animals injected with 3AP showed the most
severe motor impairments, which lasted until 3 days. Microscopy revealed a trend
toward the increase of the number of cells labeled with anti-GFAP and toward a
significant reduction of cells labeled with anti-NeuN. Additionally, we did not
observe any labeling with anti-SYP when compared with the other groups or with the
control. Crabs started to recover 7 days after the injection and 28 days after, we
observed a spontaneous motor recovery accompanied by an increase in the number
of neurons labeled with anti-NeuN. SYP labeling was also seen, suggesting
synaptogenesis as a mechanism involved in motor recovery.
Conclusion: Neurodegeneration caused by 3AP provoked cellular and behavioral
changes and recovery involved reactive gliosis, synaptogenesis and possibly,
neurogenesis.
In the last decade, there is increasing interest in the use of nanotechnology solutions
to correct injuries and degeneration, and eventually, regenerate the central nervous
system. Topographical and mechanical stimulation based on the control of
biomaterial features is a promising approach. This work presents results of the study
of biocompatibility of polybutyrate/polypyrrole (PBAT/PPY2%) nanofibers. The
conductive PBAT/PPY2% nanofibers were used as scaffolds to grow mesenchymal
stem cells (MSC) and neural stem cells (NSC). MTT and cell counting were used to
evaluate the cytotoxicity of the nanofibers. Later, we investigated cell adhesion and
differentiation of MSC grown on the nanofibers by scanning electron microscopy
(SEM) and staining with alizarin red and oil red, respectively. We observed that
MSC adhered to the nanofiber surface and maintained their multipotent character.
The NSC adhered and proliferated when cultured on nanofibers coated with laminin,
and remained undifferentiated for up to five days. NSC cultured on PBAT/PPy2%
nanofibers were electrically stimulated for five days to investigate differentiation,
and we found no difference in differentiation when compared with control group (no
stimulation).Therefore, we conclude that PBAT/PPY2% nanofibers are
biocompatible, being suitable for the future use in neural applications.
Financial Support: FAPESP, CNPq
Ethics Committee Approval: 0397/11
Funding support: CNPq/FAPERJ.

R17
R18
EFFECTS OF SIMULTANEOUS CONSUMPTION OF ETHANOL AND

CAFFEINE ON APOPTOSIS IN THE CEREBELLUM OF UCHB RATS
(VOLUNTARY ETHANOL CONSUMERS): PROTEIC ANALYSIS OF
CASPASE 3, XIAP, AND IGFR-1
SENILITY AND ALCOHOLISM: GENE EXPRESSIONS ON APOPTOSIS IN

UChB RAT CEREBELLUM (VOLUNTARY ETHANOL CONSUMERS)
Isabela Maria Urra Rossetto (1), Luis Fernando Tirapelli (2), Valeria H.A. CagnonQuitete (3), Luiz Gustavo de Almeida Chuffa (4), Francisco Eduardo Martinez (4),
Marcelo Martinez (1).
(1) Department of Morphology and Pathology, Federal University of So Carlos, So
Carlos, Brazil - Via Washington Lus, Km 235 -13565-905 - So Carlos, SP - Brazil
- Phone: (16) 3351 8325
(2) Department of Surgery and Anatomy, University of So Paulo, Ribeiro Preto,
Brazil - St. Prof. Dr. Antonio Celso Wagner Zanin - 18618-689 Botucatu, SP
Brazil - Phone: (14) 3880-0012
(3) Department of Anatomy, Cellular Biology, Physiology and Biophysics,
University of Campinas, Campinas, SP, Brazil - Ave. Bandeirantes, 3900, Monte
Alegre, 14049900 - Ribeiro Preto, SP - Brazil, Phone: (16) 3315 3305
(4) Department of Anatomy, Institute of Biosciences, University of State of So
Paulo, Botucatu, Brazil - Av. Bertrand Russel, s/no, Cx. Postal 6109, Campinas, SP,
Brazil, 3521-6184
Contact details:
Presenting author: Ave. So Carlos, 2724, Centro, 13560-002, So Carlos SP,
Brazil. Phone: (19)988216806. Email: isabela.urra.rossetto@gmail.com
Background: The intake of energy drinks with caffeine simultaneously with the
consumption of alcoholic beverages increases the risk of alcohol abuse and
dependence, as it is associated with increased alcohol consumption both in quantity
and frequency. Aims: We aimed to determine the pattern of apoptotic and antiapoptotic protein expressions after ethanol abuse and caffeine intake in the
cerebellum. Methods: The rats were divided into three groups (n=10/group): 1.UChB
rats fed with 1:10 (v/v) ethanol ad libitum (free choice for water or ethanol) drinking
7g of ethanol/Kg body weight/day; 2.UChB rats fed with 1:10 (v/v) ethanol ad
libitum (free choice for water or ethanol) drinking 7g of ethanol/Kg body weight/day
+ caffeine 300ml/l); 3.Wistar rats group with voluntary water consumption, ad
libitum. Cerebellar fragments were analyzed by immunohistochemistry for Caspase3, XIAP and IGFR-1. Results: For Caspase-3, the UChB group had greater
immunoreactivity for Granular and Golgi cells, indicating neurodegeneration
processes. For XIAP, UChB group presented greater expression in the glomerular
zone, indicating compensatory response to increased apoptosis. In Purkinje cells,
XIAP expression was bigger in UChB+caffeine group. To IGFR-1, the group with
the highest expression of this receptor was the UChB+caffeine group. Medular body
UChB+caffeine showed higher expression of IGFR-1 and XIAP. Conclusion: The
simultaneous ingestion of ethanol and caffeine reversed the ethanol damages acting
caffeine with a possible neuroprotective effect.
Isabela Maria Urra Rossetto (1), Fermino Sanches Lizarte Neto (2), Luis Fernando
Tirapelli (2), Daniela Pretti da Cunha Tirapelli (2), Luiz Gustavo de Almeida Chuffa
(3), Francisco Eduardo Martinez (3), Marcelo Martinez (1).
(1) Department of Morphology and Pathology, Federal University of So Carlos, So
Carlos, Brazil - Via Washington Lus, Km 235 -13565-905 - So Carlos, SP - Brazil
- Phone: (16) 3351 8325
(2) Department of Surgery and Anatomy, University of So Paulo, Ribeiro Preto,
Brazil - St. Prof. Dr. Antonio Celso Wagner Zanin - 18618-689 Botucatu, SP
Brazil - Phone: (14) 3880 0012
(3) Department of Anatomy, Institute of Biosciences, University of State of So
Paulo, Botucatu, Brazil - Ave. Bandeirantes, 3900, Monte Alegre, 14049900 Ribeiro Preto, SP - Brazil, Phone: (16) 3315 3305
Contact details:
Presenting author: Ave. So Carlos, 2724, Centro, 13560-002, So Carlos SP,
Brazil. Phone: (19)988216806. Email: isabela.urra.rossetto@gmail.com
Background: Elderly are particularly susceptible to the harmful effects of ethanol
consumption, because of biological changes associated with aging such as decrease
of water in the body, efficiency of liver enzymes and hepatic blood flow, the
responsiveness of the brain, reinforced by interaction with drugs. The adverse effects
of acute or chronic exposure to ethanol on the functions of the cerebellum have been
recognized for decades, affecting movement and balance. In addition to the motor
impairment, cerebellar degeneration contributes to distinct neuropsychological
deficits in chronic alcoholics, as well as in children with prenatal exposure to
ethanol. However, the effects on senile individuals are scarce. Aims: Our objectives
were to determine the pattern of apoptotic and anti-apoptotic gene expressions after
ethanol abuse on the senile cerebellum. Methods: The senile rats were divided into
two groups (n=10/group): 1. UChB: rats fed with 1:10 (v/v) ethanol ad libitum (free
choice for water or ethanol) drinking from > 1.9 g of ethanol/Kg body weight/day. 2.
Control: free choice for water. Cerebellar sections were subjected to gene expression
(RT-PCR) for Caspase-3, XIAP and IGFR-1 and protein analysis. Results: The
results showed Caspase-3, Xiap and IGFR-1 gene expressions similar in both groups.
Conclusion: Ethanol consumption is able to modulate expressions interfering on
apoptotic pathways in senile rat cerebellum.
Acknowledgements: FAPESP process n 2013/13604-1 and 2015/07807-2
Acknowledgements: FAPESP process n 2013/11095-2 and 2014/06240-6

R19
R20
NEUROPROTECTIVE EFFECTS OF BRADYKININ-POTENTIATING

PEPTIDES AGAINST HYDROGEN PEROXIDE-INDUCED CELL INJURY
IN NEUROBLASTOMA HUMAN CELL SH-SY5Y
INDUCTION OF ASTROCYTE DEDIFFERENTIATION IN VITRO
Querobino, S. M. (1), Alberto-Silva, C.(2)
Laboratrio Neurobiologia, Departamento de Bioqumica, Universidade Federal de

So Paulo (UNIFESP)
(1) Graduate Program in Neuroscience and Cognition, Mathematics, Computing and

Cognition Center, Federal University of ABC (UFABC), So Bernardo do Campo,
SP, Brazil. samyr.machado@ufabc.edu.br
(2) Morphophysiology Experimental Laboratory, Natural and Humanities Sciences
Center (CCNH), Federal University of ABC (UFABC), So Bernardo do Campo, SP,
Brazil.
Background. The low molecular weight fraction (LMWF) from Bothrops jararaca
(Bj) snake venom has a great variety of bioactive peptides, including the bradykininpotentiating peptides (BPPs). LMWF can inhibit changes in mitochondrial
membrane permeability. In addition, BPPs can increase the activity of the enzyme
argininosuccinate synthetase (AsS), increasing the availability of L-arginine and
consequently the polyamine synthesis with neuroprotective activity.
Aim. Neuroprotective effects of synthetic BPPs [BPP-5a (<EKWAP), BPP-7a
(<EDGPIPP), BPP-10c (<ENWPHPQIPP), BPP-11e (<EARPPHPPIPP), BPP-12b
(<EWGRPPGPPIPP), BPP-AP (<EARPPHPPIPPAP) was investigated in human
neuroblastoma cells SH-SY5Y against H2O2-induced injury.
Methods. To evaluate the neuroprotective effect of BPPs, neuroblastoma human cell
SH-SY5Y were pre-treated with BPPs 1M and after 4h were induced oxidative
stress with H2O2 450 M. After 20h, MTT analysis was performed to evaluate the
cell viability. The inhibitory effect of BPPs against H2O2-induced ROS generation
was evaluated by DCFH-DA staining assay. For statistical comparisons, analysis of
variance followed by Turkeys post hoc test was used.
Results. Pre-treatment with BPPs reduced significantly death cell induced by H2O2.
Cells treated only with H2O2 (450 M) showed 32,23 0,96% of viability while
cells pre-treated with BPP-5a (81,41,7%), BPP-7a (77,40,9%), BPP-10c
(76,33,5%), BPP-11e (45,76,9%), BPP-12b (44,9963,19%) and BPP-AP (47,5
4,2%). Interestingly, the pre-treatment with BPP-5a, BPP-7a and BPP-10c reduced
significantly ROS generation in relation the cells treated only with H2O2.
Conclusion. The results suggest that all BPPs demonstrated neuroprotective activity
against oxidative stress, but the BPP-5a, BPP-7a and BPP-10c were more
neuroprotective properties than others BPPs tested.
Sardinha, A.A., Mundim, M.V., Porcionatto, M.A.
Astrocytes play an important role in the central nervous system (CNS), taking part in
determination of cellular fate and proliferative control at neurogenic regions.
Although astrocyte reaction is well recognized in several injury types, its role in
regeneration is not completely understood. A recently evidenced astrocyte response
to injury is the transition of a mature subpopulation into an undifferentiated state.
This process is characterized by phenotypic changes and entrance in a proliferative
state, suggesting the occurrence of cellular dedifferentiation, i.e. astrocytes begin to
present neural stem cell phenotype. In order to investigate molecular mechanisms
involved in dedifferentiation, we aimed to standardize primary astrocyte culture and
scratch wound healing protocol to induce astrogliosis in vitro. Briefly, cortical cells
were extracted from neonatal mice and isolated through plating on poly-L-lysine
precoated plates for 10-14 days until confluence. Astrocyte purity was verified by
GFAP-immunostaining. Cells were dissociated and plated in precoated PolyHema
plate, which prevents attachment of adherent cells. As a result, we observed that
astrocytes formed neurospheres similar to those formed by neural stem cells. When
submitted to a migration assay, astrocytes expressed both GFAP and nestin (stem
cell marker), suggesting astrocyte plasticity. In the scratch wound assay, we observed
morphological changes as well as intensified proliferation throughout time in injured
areas. These results indicate successful induction of dedifferentiation process in
vitro. Understanding the molecular mechanisms involved in this process is crucial to
optimize the generation of new multipotent cells from this alternative stem cell
source.
Financial Support: CNPq and FAPESP
Approved by Animal Experimentation Ethics Committee (CEUA/UNIFESP):
1364051015

R21
R22
EFFECTS OF PHYSICAL EXERCISE ON MEMORY PERFORMANCE

AND TRANSCRIPTIONAL VARIANTS OF THE BDNF GENE IN FEMALE
MICE EXPOSED TO A POSTNATAL STRESS PROTOCOL
A TRAUMATIC, EXTINCTION-RESISTANT EXPERIENCE TRIGGERS

SKILLED WALKING IMPAIRMENT IN RATS
Rodrigo Orso (2), Luis Eduardo Wearick-Silva (1,2), Paul Marshall (3), Thiago
Wendt Viola (1,2), Anderson Centeno da Silva (2), Lucas Arajo de Azeredo(1,2),
Xiang Li (3), Timothy W. Bredy (3), Rodrigo Grassi-Oliveira (1,2).
(1) Graduate Program in Pediatrics and Child Health, Pontifical Catholic University
of Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil;
(2) Developmental Cognitive Neuroscience Laboratory (DCNL) Pontifical Catholic
University of Rio Grande do Sul (PUCRS), Porto Alegre, Brazil;
(3) Department of Neurobiology and Behavior, University of California - Irvine,
Irvine, CA
Presenting author:
Rodrigo Orso
Avenida Ipiranga, 6681, prdio 11, sala 936 - Partenon, Porto Alegre, RS - Brazil
+55 51 95640-414 - Tel.: +55 51 3320-3633 ext. 7740
E-mail address: rodrigo.orso@acad.pucrs.br
Exposure to stress during early stages of development has been associated with
memory impairments through altered brain-derived neurotrophic factor (BNDF)
signaling. Parallel to that, it is known that physical exercise could facilitate learning
and memory processes. The aim of this study was to investigate the impact of
physical exercise after exposing mice to early life stress, and analyzing the possible
alterations on the mRNA transcription of exons I, IV and IX of the BDNF. Female
Balb/c mice were subjected to a Maternal Separation (MS) protocol in which pups
were isolated from their dams for 180 min from PND 2 to PND 15, and then
performed a 3-week protocol of treadmill running. To evaluate recognition memory,
it was executed the object recognition task. After the test, was performed the
extraction of the hippocampus for analysis of transcripts of the BDNF (exons, I, IV
and IX) by qPCR. We found that MS caused a memory impairment, which was
reversed by physical exercise. Moreover, we found an upregulation of BDNF exon I
mRNA levels in mice that performed physical exercise. In addition, we demonstrated
a downregulation of BDNF exon IX mRNA levels in the exercise groups. Our
findings indicate that MS alters memory performance of female mice, and this effect
can be neutralized after performing physical exercise. The transcripts of the BDNF
presented a heterogeneous pattern, demonstrating different levels of expression after
exposure to MS and physical exercise. All procedures were in accordance with the
universitys ethical committee.
Lucas Athaydes Martins1, Filipe Medeiros1, Jociane de Carvalho Myskiw2, Pedro

Porto Alegre Baptista1,Laura Tartari Neves1, Cristiane Regina Guerino Furini2, Ivn
Izquierdo2, Lder Leal Xavier1, Rgis Gemerasca Mestriner1.
1. Cell and Tissue Laboratory, Biosciences College. Pontifical Catholic University of
Rio Grande do Sul (PUCRS).
2. Memory Center of Pontifical Catholic University of Rio Grande do Sul (PUCRS).
Corresponding Author: Lucas Athaydes Martins
Nursing, Nutrition and Physical Therapy College - Pontifical Catholic University of
Rio Grande do Sul - Avenida Ipiranga, 6681 - Prdio 12 / 8. andar - Porto Alegre,
RS, Brazil - CEP: 90619-900
e-mail: lucas.athaydes18@gmail.com
Phone: (55) (51) 33203646 or (55) (51) 82978648
Motor disorders co-exist in many neuropsychiatric diseases, such as
functional/psychogenic movement disorders, post-traumatic stress disorder as well as
in other fear and anxiety-related phobias. While the physiology of neuropsychiatric
movement disorders (NMDs) is still unclear, there are some well-established factors
that can increase the risk of developing it, for example, stressful events, childhood
neglect, drugs abuse and, most commonly, experiencing a traumatic event that could
results in a traumatic, extinction-resistant trauma. It is widely known that suffering a
traumatic experience triggers a series of fear-conditioning mechanisms closely
associated with increased sensitivity to fear and freezing behavior. However, the
clinical profile of the NMDs suggests their neurobiology is beyond the freezing
behavior and other defensive behaviors. In the present study we test the hypothesis
that aversive, resistant to extinction experience could impair skilled walking
performance and change neuron and glia densities as well as neuron-glia ratio in
medial amygdala. Thus, we established a major trauma protocol able to induce a
resistant to extinction trauma followed by testing its effects on skilled walking
performance and medial amygdala histophysiology. Footshocks exposure reduces
skilled walking performance 3 days after the hit. Additionally, the adoption of a
safety behavior in the plus maze test was associated with worse ladder walking
performance. Statistical trend of significance were founded for changing glia and
neuron-glia ratio in medial amygdala. Therefore, suffering traumatic experiences
able to generate resistant to extinction memories can result in long-term impairment
of gait control, which affects the walking ability.
(CEUA 15/00442-PUCRS). Support: CNPq,CAPES,FAPERGS.
Financial Support: CNPq

R23

R24
CANNABIDIOL EXPOSURE DURING NEURONAL DIFFERENTIATION

SENSITIZES CELLS AGAINST REDOX-ACTIVE NEUROTOXINS
STUDY OF E-NTPDase ROLE IN THE DEVELOPMENT OF RETINAL

PROGENITORS OF RAT
Patrcia Schnhofen (1,2), Liana Marengo de Medeiros (1,2), Ivi Juliana Bristot
(1,2), Fernanda Martins Lopes (1,2), Marco Antnio De Bastiani (1,2), Flvio
Kapczinski (2,3), Jos Alexandre S. Crippa (2,4), Mauro Antnio A. Castro (5),
Richard B. Parsons (6), Fbio Klamt (1,2).
Marinna Garcia Repossi (1), Ana Paula Fernandes Paz dos Santos (1), Luana de
Almeida Pereira (1) and Lucianne Fragel Madeira (1)
(1) Department of Biochemistry, Laboratory of Cellular Biochemistry,

ICBS/UFRGS, Porto Alegre, RS 90035-003, Brazil
(2) National Institutes of Science and TechnologyTranslational Medicine (INCTTM), Porto Alegre, RS 90035-903, Brazil
(3) Molecular Psychiatry Laboratory, HCPA/UFRGS, Porto Alegre, RS 90035-903,
Brazil
(4) Neuroscience and Behavior Department, Faculty of Medicine of Ribeiro Preto,
USP, Ribeiro Preto, SP 14049-800, Brazil
(5) Laboratory of Bioinformatics, Professional and Technological Education Sector,
Centro Politcnico, UFPR, Curitiba, PR 81531-970, Brazil
(6) Institute of Pharmaceutical Science, Kings College London, 150 Stamford
Street, London SE1 9NH, UK
patrcia_schonhofen@yahoo.com.br ; institution fone: +55 51 33085555 ; cell fone:
+55 51 96298116
Background: Cannabidiol (CBD), one of the most abundant Cannabis sativa-derived
compounds, has been implicated with neuroprotective effect in several human
pathologies. Until now, few undesired side effects have been associated with CBD.
Aims: In this study, we aimed to evaluate CBDs neuroprotective/neurotoxic effect
in mature and in underdevelopment neurons. Methods: CBD effect was assessed by
using the human neuroblastoma SH-SY5Y cell line terminally differentiated (mature
neurons model) and during neuronal differentiation (neuronal developmental toxicity
model). Results: A dose-response curve was performed to establish a sub lethal dose
of CBD with antioxidant activity and 2.5 M was selected for further experiments. In
terminally differentiated SH-SY5Y cells, incubation with CBD was unable to protect
cells against the neurotoxic effect of glycolaldehyde, methylglyoxal, 6hydroxydopamine, and hydrogen peroxide (H2O2). Moreover, no difference in
antioxidant potential and neurite density was observed. When SH-SY5Y cells
undergoing neuronal differentiation were exposed to the same dose of CBD, no
differences in antioxidant potential and neurite density were observed. However,
CBD potentiated the neurotoxicity induced by all redox-active drugs tested.
Conclusion: Our data indicate that 2.5 M of CBD, the higher dose tolerated by
differentiated SH-SY5Y neuronal cells, does not provide neuroprotection for
terminally differentiated cells and shows that exposure of CBD during neuronal
differentiation could sensitize immature cells to future challenges with neurotoxins.
(1) Department of Neurobiology, Fluminense Federal University, Rio de Janeiro,

Brazil.
E-mail: marinna.repossi@gmail.com. Phone: (5521) 3674-7844 / (55) 21 987236877
In vertebrates, histogenesis of the retina is orientated by intrinsic and extrinsic
mechanisms of which extracellular nucleotides play various functions acting through
P2 receptors. The study of these receptors is prejudiced by the presence of
ectonucleotidases that break down nucleotides into nucleosides. Previous results
from our group reported that P2Y1 receptor modulated progenitors cells proliferation
during rat retina development. Based on this, our objective was to analyze if
ectonucleotidases were able to modulate the proliferation during the late
development of retina. Lister hooded rats at four postnatal days (P4) were
anaesthetized by hypothermia followed by intravitreal injection ARL 67156
(ectonucleotidases inhibitor) alone or together with MRS 2179 50M (P2Y1 receptor
antagonist) for different survival times. The proliferation was assessed by
immunohistochemistry for Ki-67 and cell death was evaluated by TUNEL. The
treatment with 100 M, 200 M or 400 M ARL 67156 at P5 rats increased
proliferating cell number by almost 50% and MRS 2179 plus ARL 67156 had no
effect. After 24 hours, 200 M ARL decreased cell death about 50% but after 48
hours, there was an increase about 50% in TUNEL positive cells compared to
control. Our data suggest that ectonucleotidases increase proliferation rate of rat
retina at later stage of development independently of P2Y1 receptor but retinal
signaling somehow compensates that increase probably triggering a cell death
program.
This project obtained approval by Ethics Committee on Animal Use (CEUA) under
protocol number 547/2014. Funding support: Capes, FAPERJ, CNPq and ProppiUFF.
Funding: This work was supported by grants from the Brazilians agencies
MCT/CNPq Universal (470306/2011-4), PRONEM/FAPERGS (11/2032-5),
CNPq/MS/SCTIE/DECIT - Pesquisas Sobre Doenas Neurodegenerativas
(#466989/2014-8) and MCT/CNPq INCT-TM (573671/2008-7). No ethical
committee approval was needed.


R25
R26
INVOLVEMENT OF P2X7 RECEPTORS ON SATELLITES CELLS OF DRG

IN ACUTE PAIN INDUCED BY CAPSAICIN
EFFECTS OF HYPOTHYROIDISM ON THE DEVELOPMENT OF

PERIPHERAL NEUROPATHY BY SCIATIC NERVE CHRONIC
CONSTRICTION IN RATS
Jlia Borges Paes Lemes1, Tas Campos Lima1, Fernando Oliveira1, Amanda Ferreira
Neves2, Carlos Almicar Parada2, Celina Monteiro da Cruz Lotufo1.
1
Department of Physiological Sciences, Uberlandia Federal University, Minas

Gerais, Brazil.
2
Department of Structural Biology, University of Campinas, So Paulo, Brazil.
julia.borges92@yahoo.com.br (34)99976-2119
Recent studies indicate that satellite glial cells present in the dorsal root ganglia,
communicate with the primary sensory neurons through the release of ATP by
neurons and activation of P2X7 receptors present on glial cells. This communication
seems to involve the participation of gap junctions. However, this communication
has still not described in acute pain. In present work, we investigate the role of P2X7
receptors present in satellites glial cells on dorsal root ganglia and gap junctions in
acute nociception induced by capsaicin injection. The animals received A740003 (1
nM), P2X7 receptor antagonist, and Carbenoxolone (1 nM), blocking of gap
junctions, by intraganglionar injections (right L5). After thirty minutes, capsaicin
was injected (subcutaneous) into the right paw and the number of flinches registered
during 5 minutes. Mechanical sensitivity was accessed using Von Frey electronic test
15, 30, 60 and 90 minutes after capsaicin injections. Blockage of P2X7 receptors by
A740003 as well as of gap junctions by carbenoxolone in the dorsal root ganglia
reduced capsaicin induced nociceptive behavior. Mechanical sensitization was also
reduced after 15 minutes of capsaicin injection by the P2X7 antagonist administered
at the dorsal root ganglia. Communication between neurons and satellite cells
involving the release of ATP and activation of P2X7 receptors and also the
intercellular communication via gap junctions seems to be important in processing
capsaicin induced acute pain in the dorsal root ganglia.
Ethical approval: CEUA UFU (Protocol 008/14).
Funding support: CAPES, FAPEMIG.
Kalina Kelma Oliveira de Sousa (1), Kildere Marques Canuto (1) Helson Freitas
Silveira (4), Mario Roberto P Lisboa (4), Francisco Fbio Bezerra de Oliveira (3)
Luiza Clertiani Vieira Alves (4), Anamaria Falco Perreira (3) Luane Macdo de
Sousa (4), Diego Bernarde Souza Dias (3), Bruno Wesley de Freitas Alves (3),
Delane Viana Gondim (4), Mariana Lima Vale (3),Reinaldo Barreto Ori (1)*
Federal University of Ceara, Fortaleza, CE, Brazil
(2) Microscopy and Imaging Core, Department of Morphology and Institute of
Biomedicine, School of Medicine, Federal University of Ceara, Fortaleza, CE, Brazil
(3) Department of Physiology and Pharmaocology, Faculty Medicine, Federal
University of Cear, Fortaleza, CE, Brazil
(4) Department of Morphology, Faculty of Medicine, Federal University of Cear,
Fortaleza, CE, Brazil
*email: kalinakelma@hotmail.com, telephone: (85) 33668239
The deficiency of thyroid hormones leads to pathological conditions in the nervous
system. The effects of hypothyroidism (HYP) on peripheral neuropathy are poorly
studied. We have investigated the effect of HYP in experimental neuropathy induced
by chronic constriction of the sciatic nerve (CCSN) in rats by evaluating nociceptive
behaviors and nerve damage morphology. The work was approved by the Ethics
Committee from UFC. For this, HYP was induced in male Wistar rats by daily intake
of 0.05% propylthiouracil (PTU) in drinking water for 6 weeks, which was
confirmed by body weight reduction and size change of the thyroid gland. CCSN
was performed 3 weeks after PTU. For neuropathy evaluation, we have used Von
Frey plantar test and cold allodynia after HYP and CCSN induction. Subsequently
the nerve, dorsal root ganglia (DRG) and lumbar spinal cord were removed for
morphological analyses by nerve toluidine blue-semithin section staining, TEM, and
immunofluorescence for myelin basic protein (MBP) and ATF-3 and c-fos in the
DRG. Our results showed an increase in the nociceptive threshold in the HYP and
reduction in the CCSN group when compared to the control. Morphological analyses
showed reduced myelin thickness and defects in the myelin sheath in both HYP and
HYP and CCSN groups. ATF-3 immunostaining was significantly increased in the
CCSN group. C-fos immunostaining was increased in HYP, CCSN and HYP and
CCSN groups. MBP was increased in CCSN and HYP and CCSN rats. Our findings
suggest that HYP has important effects on the sciatic nerve experimental neuropathy.

R28
R27
EXPRESSION OF THE CANNABINOID RECEPTORS DURING THE
RETINA DEVELOPMENT IN A MURINE MODEL OF RETINITIS
PIGMENTOSA
Daniella Senos Lopes (1), Camila Feitosa Magalhes (1) and Lucianne FragelMadeira (1)
Brazil.
ROLE OF HIPPOCAMPAL CONNEXIN 43 IN CELL DEATH AND GLIAL

POPULATION AFTER NEONATAL ANOXIA
Natlia Myuki Moralles Dias (1); Juliane Midori Ikebara (1); Dbora Sterzeck
Cardoso (1); Beatriz Crossiol Vicente de Campos (1); Talitha Amanda Sanches
Bretherick (1); Fernanda Caboclo (1); Silvia Honda Takada (1); Alexandre Hiroaki
Kihara (1).
(1) Centro de Matemtica, Computao e Cognio, Universidade Federal do ABC,
So Paulo, Brazil
E-mail: danisenos3@gmail.com. Phone: (5521) 3674-7844 / (55) 21 97017-5788

The cannabinoid receptors, CB1 and CB2, are key parts of the endocannabinoid
system and have been related to neuroprotection, neural differentiation and synaptic
transmission. The expression of cannabinoid receptors was demonstrated in several
vertebrates during retinal development. However, their presence in mice retina
during the development remains unknown. We also used an animal model for
retinitis pigmentosa, PDE6rd10 (rd10), in order to know if the photoreceptor
degeneration alters the cannabinoids receptors expression. C57/Black6 (Bl6) and
rd10 mice at postnatal days (P) 3, 5 and 9 (early stage of development) and 19, 25
and 30 (late stage) had their retinas harvested for immunohistochemistry and western
bloting. The immunofluorescence showed CB1 and CB2 were present in the
plexiform layers of retina of Bl6 and rd10 animals. However, CB1 was found since
P5 for Bl6 and since P19 for rd10 animals and CB2 immunolabeling was not found
at early stages of rd10 development. By western blotting, CB1 expression decreased
on both Bl6 and rd10 animals at P25 compared to P19 but the rd10 expression was
lower compared to Bl6. The expression of CB2 showed an opposite pattern,
increasing on Bl6 and rd10 at P25 when compared to P19 but rd10 expression was
higher than to Bl6. Thus, the expression of cannabinoid receptors seems to be altered
in the retina of rd10 mice maybe due to death of photoreceptors.
protocol number 679/2015. Funding support: Capes, FAPERJ, CNPq and ProppiUFF.
Address: Rua Arcturus, 03. Laboratrio 112, Bloco Delta. 09606-070. Jardim
Antares, So Bernardo do Campo, SP. Brazil.
Email: nataliamyuki@hotmail.com - Tel.: +55 11 23206170
Background: The neonatal anoxia is considered an important public health problem
because it affects approximately 60% of premature infants with low birth weight.
Those who survive, 25% have some permanent neurological sequelae such as
cerebral palsy, cognitive, hearing and visual deficits. Neonatal anoxia causes
neuronal and glial cell death, especially in vulnerable regions such as hippocampus.
One hypothesis for the contribution of neurodegeneration after oxygen deprivation
involves the participation of connexins (Cxs), which are transmembrane proteins
with the ability to form intercellular channels in the gap junctions (GJs). Aims: to
analyze possible changes in glial GJs resulting from neonatal anoxia. Methods: we
analyzed by immunohistochemistry the distribution of Cx43 and GFAP and cell
death using Fluoro-Jade C (FJC) after Cx43 blockade by intrahippocampal injection
of carbonexolone after neonatal anoxia in male Wistar rats. Results: 24 hours after
anoxia, there was an increase of Cx43 immunoreactivity in the hippocampal oriens
layer and a reduction of GFAP in the pyramidal layer of CA3. 72 hours after
neonatal anoxia, GFAP was increased in CA1 and decreased in dentate gyrus.
Following Cx43 blockade, there was a reduction in FJC+ cells. Conclusion: based on
these results, we suggest the participation of glial GJs in the hippocampal cell death
following neonatal anoxia.
Information on ethical approval and funding support: All animal studies were
approved by Ethics Committee for Animal Experimentation of the Universidade
Federal do ABC (protocol 005/2014). Project supported by Fapesp (2014/16711-6).


R29
R30
THE ROLE OF STRESS INDUCIBLE PROTEIN 1 IN BRAIN TUMOR

ANGIOGENESIS
TOPIC DEXAMETHASONE IMPAIRS PROTEIN SYNTHESIS AND

NEURONAL REGENERATION IN THE OLFACTORY EPITHELIUM
Rodrigo T. Cartaxo (1*), Vilma R. Martins (1), Tiago G. Santos (1)
Umberto Crisafulli (1), Andr M. Xavier (2), Tavane D. Cambiaghi (3), Fabiana B.
dos Santos (2), Beatriz A. Castilho (3), Marimlia Porcionatto (2), Bettina Malnic (1)
and Isaias Glezer (2)
A. C. Camargo Cancer Center, So Paulo, Brazil

* rcartaxo@cipe.accamargo.org.br / (35)98413-9956
The cochaperone STI1 forms a protein complex with Hsp70 and 90 that supports
protein folding. STI1 is secreted associated to endosomal vesicles, once in
extracellular space, it interacts with prion protein (PrPC) on plasma membrane and
triggers several trophic effects, such as proliferation, protection against cell death,
differentiation and migration. Hypoxia-driven STI1 secretion allows the interaction
with PrPC triggering angiogenesis stimulation in experimental ischemia, contributing
to post-ischemia brain recovery. Together, these findings suggest the involvement of
STI1-PrPC complex to the mechanisms related to angiogenesis. The ability of tumor
cells in stimulate blood vessel formation is an essential hallmark for tumor growth
and, consequently, a matter of intense research for new therapeutic targets. The aim
of this study is to evaluate the angiogenic potential of STI1-PrPC complex using in
vivo and in vitro approaches. The mouse brain tumor cell line GL261-luciferase was
chosen to study in vivo angiogenesis in transgenic mice expressing different levels of
STI1 and PrPC. Preliminary results indicate that STI1 haploinsufficient (STI1+/-) and
overexpressing STI1 mice inoculated with GL261 cells have increased survival rate
when compared to wild-type ones. In vivo imaging indicated increased
bioluminescence signal in STI1+/- and overexpressing mice. STI1+/- mice present
larger tumors with increased blood vessel formation, as assessed by CD31
immunstaining. Moreover, in vitro analyses in HUVEC (human umbilical vein
endothelial cells) tube formation assay suggest that recombinant STI1 has angiogenic
activity. Additional in vivo and in vitro experiments will be carried out to understand
the functions of STI1 and its receptor PrPC in angiogenesis associated processes.
(1) Biochemistry Department, Instituto de Qumica, Universidade de So Paulo

(2) Biochemistry Department, Escola Paulista de Medicina, Universidade Federal de
So Paulo
(3) Department of Microbiology, Immunology and Parasitology, Escola Paulista de
Medicina, Universidade Federal de So Paulo
Contacts:
e-mail: andremxdm@gmail.com
phone number: 0055 11 55764445 R: 1096
mobile: 0055 15 981379598 Endereo: Rua Trs de Maio, 100 - 5 Andar Lab. De
Neurocincias e Genmica Funcional - Vila Clementino, So Paulo - SP, 04044-020
ABSTRACT WITHHOLD UPON AUTHORS REQUEST
Financial Support: FAPESP, CAPES.
R31
THE
ORDERED
METABOLIC
DISTRIBUTION
OF
FLUORODEOXYGLUCOSE IN THE BRAIN OF WISTAR RATS
18

R32
F-
ANANDAMIDE ENDOGENOUS DELAYED PHOTORECEPTOR CELL

DEATH OF A MURINE MODEL OF RETINITIS PIGMENTOSA
Camila Feitosa Magalhes (1), Daniella Senos Lopes (1) and Lucianne FragelMadeira (1)
Stfanie Ingrid dos Reis Schneider, Pedro Porto Alegre Baptista Samuel Greggio ,
Gianina Teribele Venturin, Isadora Lopes Alves, Cristina Maria Moriguchi Jeckel,
Jaderson Costa Da Costa, Rgis Gemerasca Mestriner, Lder Leal Xavier.
Laboratrio de Biologia Celular e Tecidual, Departamento de Cincias
Morfofisiolgicas, Pontifcia Universidade Catlica do Rio Grande do Sul, Porto
Alegre - RS, Brazil.
Instituto do Crebro do Rio Grande do Sul INSCER-PUCRS, Porto Alegre - RS,
Brazil.
3
Nuclear Medicine and Molecular Imaging Department, University Medical Center
Groningen, Groningen Netherlands.
Corresponding Author: Stfanie Ingrid dos Reis Schneider, MSc.
Departamento de Cincias Morfofisiolgicas - Faculdade de Biocincias
PUCRS - Avenida Ipiranga, 6681 - Prdio 12, Sala 104 - Porto Alegre, RS, Brasil
CEP:90619-900 - e-mail: stefanie.schneider@acad.pucrs.br
phone: +55 (51) 3320-3545
mobile: +55 (51) 82391889
Imaging tests of the small animal positron emission tomography (microPET) using
the 18f-fluorodeoxyglucose (FDG) has advantages over classical technique
histochemical as a marker cytochrome c oxidase (COX) for the in vivo analysis, in
which the tissue remains intact and functional. In this research, we sought out to
provide more detailed information on the distribution of FDG uptake in different
areas of the brain of Wistar rats; additionally, we compare the distribution to
previously published works that mapped out the distribution of COX in the rat brain.
We used four Wistar rats, adults, males. The animals were injected with 1mCi of
FDG through the vein flow, and then they underwent a 30-minute period of
conscious tracer uptake and scanned for a 1-hour period in the microPET equipment
individually anesthetized. We evaluated a total of 56 brain regions. Firstly, we tested
for laterality and found no difference. Second, we compared our results to the five
COX studies, and found only one to be correlated (r = 0.702; P = 0.002). In addition,
we noticed some characteristics worth mentioning since FDG has a predilection for
gray matter, thus (1) the cortex areas should not be clearly visible, (2) white matter
areas and ventricles should not present the fainter intensity of uptake, and (3) the
basal ganglia and midbrain should not have a homogeneous uptake. This study is the
first to quantitatively map the FDG distributions in Wistar rats, and we trust it will
aid future researches on the same topic.

Brazil.
camilafm@id.uff.br (21) 3674-7844 (21) 96844-6731
Retinitis pigmentosa is an inherited disease manifested by progressive degeneration
of photoreceptors cells and does not have cure yet. Among the molecules with
neuroprotective activity, cannabinoids became known for its beneficial effects in the
central nervous system diseases, more specifically in retinal injuries. The two best
characterized endocannabinoids are anandamide and 2-arachidonoylglycerol. Our
work aimed to modulate pharmacologically the anandamide levels in a murine model
of retinitis pigmentosa, the Pde6rd10 (rd10) in order to prolong the survival of
photoreceptors cells. Thus, we performed daily treatment with intraperitoneal
injections of 0.3mg/kg URB597, an inhibitor of FAAH (enzyme that degrades
anandamide). The treatment began when the animals open their eyes and finished at
19, 22 or 25 postnatal days (P19, P22 or P25, respectively). After, the photoreceptor
cells were evaluated by immunofluorescence for recoverin. The number of these
cells and the thickness of the photoreceptor cellular layer increase about 50%
compared to controls animals at P19, but this effect was lost at P22 and P25 animals.
We conclude that anandamide may provide a neuroprotector effect in the
photoreceptors cells only on an early stage of the disease. Therefore, the
endocannabinoid system can be an interesting target of research in the context of
retinitis pigmentosa.
This project agrees with the Ethical Principles for Animal Experimentation and
obtained approval for implementation by Ethics Committee on Animal Use (CEUA)
under protocol number 679/2015. The funding support was made by Capes,
FAPERJ, CNPq and Proppi-UFF.

R33
R34
NEURONS HAVE AN EXTRA miRNA REGULATION LEVEL REGARDING

XRN2 AND PAPD4
ENDOGENOUS ADENINE NUCLEOTIDES AVAILABILITY DECREASED

DURING THE DEVELOPMENT OF RAT RETINA IN VIVO
Victor Allison da Silva (1), Felipe Fernandes Correia (1), Danilo Luna Campos (1),
Natalia Dal Re Nogueira (1), Alexandre Hiroaki Kihara (1), Vera Paschon (1)
Ana Paula Fernandes Paz dos Santos, Luana de Almeida Pereira (1), Marinna Garcia
Repossi and Lucianne Fragel Madeira
(1) Ncleo de Cognio e Sistemas Complexos, Universidade Federal do ABC, So

Paulo, Brasil

Brazil.
Address: Rua Arcturus, 03 Jardim Antares, So Bernardo do Campo, Laboratrio

112, Bloco Delta
E-mail: victor_alisson96@hotmail.com
Telephone: +55 11 2320-6171 (institucional) +55 11 9 9507-2500 (mobile)
E-mail: anapaulapaz.95@hotmail.com. Phone: (5521) 3674-7844 / (55) 21 993800321
The action of miRNAs on target genes depends on intracellular concentration, which

reflects the balance of biosynthesis and degradation. Small non coding RNAs are
highly expressed in the central nervous system, however, the mechanisms regulating
the stability of miRNAs in the spinal cord are not stabilished. The aim of this project
was to describe the expression of XRN2 and PAPD4 (genes involved in degradation
and stability of miRNAs), during spinal cord development, neuronal activation and
after injury. PCR analises revealed high expression for XRN2 (263%, P<0.05) and
PAPD4 (527%, P<0.05) at P0 that in P60. Imunnofluorescence assay showed distinct
sub cellular localization for XRN2 and PAPD4 that were transiently accumulated in
nuclei and cytoplasm. Combined analyzes with anti-Ki67 and -glutamine synthetase
(GS) revealed poorly expression in progenitor and glial cells. Moreover, experiments
with Choline acetyltransferase (ChAT), calretinin (CR) and calbindin (CB), specific
spinal cord neuronal markers, showed that XRN2 and PAPD4 are expressed by
motorneurons labele with ChAT, and inter neurons labeled with CR or CB. Those
results indicate that XRN2 and PAPD4 provide an extra level of regulation for
miRNA activity in neurons when compared with glial cells. The neuronal activation
by physical exercise did not change XRN2 labeling but increased the number of cells
expressing PAPD4. Finally, the spinal cord injury increased the number of TUNELpositive cells after 24-hours whatever fell off then accumulated XRN2 and PAPD4.
This possible mechanism could be neuroprotector in spinal cord damage. Financial
support: FAPESP.
Keywords: spinal cord; miRNA; PAPD4; XRN2; neurodevelopment, neuronal
activation, spinal cord injury, apoptosis.
In vertebrates, the histogenesis of the retina is guided by several mechanisms, in

which adenine nucleotides are responsible for modulating many events involving the
development of the central nervous system. Previous studies showed that ATP
modulated rat retinal progenitors proliferation in vivo, via P2Y1 metabotropic
receptor. Based on this information, the objective of this study was to evaluate the
availability of endogenous ATP and ADP, by assessing its release and degradation
during the development of newborn rat retina in vivo. The ectonucleotidases (ENTPDases) activity was analyzed using malechite green assay and we observed an
increase during the first five postnatal days (P) of animals life (P0= 2.45 0.48
nmol; P3= 3.37 0.75 nmol and P5 =11.9 2.75 nmol to ATP; P0= 1.90 0.60
nmol, P3= 3.33 0.52 nmol and to P5= 6.95 1.25 nmol to ADP), suggesting a
reduction in the availability of both purines as retina develops. The measurement of
the extracellular ATP amount in the rat retina showed no difference between the ages
(P0= 1.40 0.47 pmol/L; P3= 0.59 0.30 nmol/L and P5 =0.69 0.18 nmol/L).
We conclude that the availability of extracellular ATP is higher during early stages
of the retina development and that this availability tends to decrease due to an
increase in E-NTPDases activities. This project obtained approval by Ethics
Committee on Animal Use (CEUA) under protocol number 547/2014.
Funding support: Capes, FAPERJ, CNPq and Proppi-UFF.

R35
R36
HIPPOCAMPAL DAMAGE AND MEMORY DEFICITS CAUSED BY

CHRONIC EXPOSURE TO METHYLMERCURY IN RATS
ULTRASTRUCTURAL CHANGES IN DEVELOPING SPINAL CORD

AFTER IN OVO METHYLMERCURY (MeHg) EXPOSURE
Bittencourt, L. O.1; Santana, L. N. S1; leo, L. K. R.1; Luz, D. A.2; Silva, M. C. F. 1;

Lund, L. A.3; Crespo-Lopez, M. E.4; Maia, C. S. F. 2; Lima, R. R.1*
Fabiana de Ftima Ferreira (1); Evelise Maria Nazari (2); Yara Maria Rauh Mller
(2).
(1) Institute of Natural, Human and Social Sciences, Federal University of Mato
Grosso, Sinop, Brazil.
(2) Department of Cell Biology, Embryology and Genetic, Federal University of
Laboratory of Functional and Structural Biology, Institute od Biological Sciences,

(1)
Federal University of Par, Belm, Par, Brazil;
2
Laboratory of Pharmacology of Inflammation and Behavior, Institute od Biological
(2)
Sciences, Federal University of Par, Belm, Par, Brazil
3
Laboratory of Ecotoxicology, Institute od Biological Sciences ,Federal University
(3)
of Par, Belm, Par, Brazil.
4
Laboratory of Molecular Pharmacology, Institute od Biological Sciences, Federal
University of Par, Belm, Par, Brazil.
*
Corresponding Author. Eletronic adress: rafalima@ufpa.br
Due to the increase of methylmercury (MeHg) in the marine food chain, the humans
are exposed to low doses of MeHg continuously through the consumption of fish and
seafood. However, the effects of chronic exposure to low doses of MeHg in the
hippocampus are little known. This work aimed to investigate the effects of a low
dose chronic methylmercury exposure on oxidative biochemistry in hippocampus
and cognitive parameters. 15 adult male Wistar rats were exposed to 0.04 mg/kg/day
of MeHg by intragastric gavage for 60 days. Another 15 animals received only oil
vehicle. After the exposure, the animals were subjected to social recognition and
morris water maze test for cognitive assessment. Upon completion of testing,
animals were sacrificed and the hippocampus was collected. The samples were
intended for quantification of mercury deposits by atomic absorption spectrometer.
Antioxidant capacity against peroxyl radical group (ACAP), nitrites levels and lipid
peroxidation (LPO) were evaluated. Data was statistically evaluated with Test TStudent (p <0.05). The animal exposed to MeHg showed significant deposits of
Mercury and on the same group we found lower levels of ACAP and higher levels of
LPO and nitrire; On behavioral results, the exposed group indicated decrease on
short and long-term memory, verified by social recognition and morris water maze
test. Our data demonstrates that MeHg chronic exposure, promoted deposits of total
mercury in the hippocampus, which may be associated with mechanisms of oxidative
stress diagnosed and related to the modulation of memory parameters.
fffferreira@hotmail.com; evelise@ccb.ufsc.br; yararm@ccb.ufsc.br

Phone: (48) 37219799
The neurotoxic effects of methylmercury (MeHg) are well known, but more
detailed information on its action during embryonic development are still scarce.
This study aimed to characterize the effects of MeHg on the structural and
ultrastructural organization of the developing spinal cord, using Gallus domesticus
embryos as a model (Ethics Committee 355/CEUA/UFSC). At third embryonic
day (E3), the embryos were treated in ovo with a single dose of
methylmercury chloride (ClMeHg) (0.1 g diluted in 50 L saline) or saline only
(n=5 per group), and evaluated at the tenth embryonic day (E10) by structural and
ultrastructural techniques. The mitochondrial quantification it was performed from
20,000x electronmicrographs. No structural alterations were observed in MeHgtreated embryos, however, increased number of TUNEL-positive cells was identified
in the intermediate (mantle) layer, colocated with greater amount of mercury
deposits. Ultrastructural analysis showed changes in some subcellular compartments,
especially in mitochondria. MeHg-treated embryos presented higher number of
altered mitochondria, with membrane ruptures and crests disorganization.
Furthermore, in this group were observed not common mitochondrial shapes, such as
donut and cup-like, and reduction in the number of fusion and fission mitochondrial
prolifes.
Our date shows that although the MeHg dose has not caused
morphologycal alteration, the ultrastructural changes and the increased cellular death
observed in the embryonic spinal cord, can cause disturbances in the developing
central nervous system with impairments in the postnatal life.
Support: Capes
Federal University of Par Animal Care and Use Commitee BIO 225-14

R37
NEURONAL LESION AND INNATE IMMUNE RESPONSE ACTIVATION
STIMULATE PROGENITOR CELL PROLIFERATION IN THE
HYPOTHALAMIC NEUROGENIC NICHE
Seo Young Chang (1), Juliana Suzuki (1), Marimelia Porcionatto (1) and Isaias
Glezer(1).
(1) Department of Biochemistry, Escola Paulista de Medicina, Universidade Federal
de So Paulo.
Contacts:
e-mail: chsy827@gmail.com phone number: 0055 11 55764445 R: 1096 mobile:
0055 11 972452906 Endereo: Rua Trs de Maio, 100-5andar- Lab. Neurocincias e
Genmica Funcional -Vila Clementino, So Paulo SP, 04044-020
ABSTRACT WITHHOLD UPON AUTHORS REQUEST

S PLANT CELL BIOLOGY
S1
S2
EFFECTS OF EGTA ON CALCIUM SIGNALING DURING EARLY

DEVELOPMENT TETRASPORES OF PALISADA FLAGELLIFERA
(CERAMIALES, RHODOPHYTA)
PRE-CLINICAL STUDY OF THE TOXICITY OF THE ETHYL ACETATE

EXTRACT RICH IN BETULINIC ACID FROM DILLENIA INDICA FRUITS
APPLIED TOPICALLY IN MICE
Luciane Cristina Ouriques1, Joo Pedro Krauspenhar Barros 1, Elisa Poltrionieri1,

der Carlos Schmidt2, Zenilda Laurita Bouzon1, Carmen Simioni2.
Flvia de Souza Fernandes (1), Isabela Machado Barbosa David (1), Vincius Freitas
Marcos (2), Ingrid Carla dos Santos (3), Maicon Roberto Kviecinski (1)
Laboratory of Cell Biology Plant, Department of Cell Biology, Embryology and

Genetics, Federal University of Santa Catarina, 88049-900, CP 476, Florianpolis,
SC, Brazil
2
Post-Graduate Program in Cell Biology and Development, Department of Cell
Biology, Embryology and Genetics, Federal University of Santa Catarina 88049-900,
CP 476, Florianpolis, SC, Brazil
luciane.ouriques@ufsc.br ( 48 3721 5149 e 99630994)
E-mail: flaviafernandes101081@gmail.com; Phone: +55 48 32791167 or +55 48

84094066
Post-Graduate Program in Health Sciences. Universidade do Sul de Santa Catarina

(UNISUL). Palhoa, SC, Brazil
2
Odontology School,Universidade do Sul de Santa Catarina (UNISUL). Palhoa, SC,
Brazil.
3
Medicine School,Universidade do Sul de Santa Catarina (UNISUL). Palhoa, SC,
Brazil.
The dependence of calcium ions as an intracellular regulator polarization and

formation of rhizoids are unknown during germination of red algal spore. This study
informs about the calcium chelantor (EGTA) to characterize the involvement of Ca2+
ions during the adhesion and germination of tetraspores of P. flagellifera.
Tetrasporophitic plants control were placed on slides in Petri dishes with sterilized
sea water and the treated samples were incubated with the different concentrations of
EGTA (0,5mM, 1,0mM, 1,5mM an 2,0mM). After 12 hours of incubation the
tetraspores of the control samples were already released, attached and in the initial
process of germination. The tetraspores released are surrounded by adhesive
mucilage. After adhesion, the cell wall is synthesized and then germination occur
with the tetraspores became elongate. The first division is transversal forming two
different cells. One of these cells becomes more slender giving rise to a rhizoid.
Germination continued with different division planes. In the treatment with 0.5 mM
and 1.0 mM EGTA the tetraspores don't become elongated and the cells divisions are
irregular. With 0.5 mM EGTA, rizoidal cell is long and slender, and 1.0 mM EGTA
rhizoidal cell is short. With 1.5 mM and 2.0 mM EGTA treatment, the tetraspores
elongate are more elongated compared to the control. In both treatments exist
inhibition of cell division and rhizoidal cell is short. These results suggest that the
initial phases of germination in P. flagellifera are calcium dependent.
Background: Previously, promising data were obtained showing anti-inflammatory

effect for the ethyl acetate extract rich in betulinic acid from Dillenia indica fruits,
which was applied topically on skin wounds in rats. Aims: To evaluate the safety of
the treatment done with extract applied on the skin of mice. Methods: Healthy mice
were divided (n=10) into one negative control treated with excipient and one testgroup treated with extract (150 mg/ml). Treatments were applied on the tail every 24
h for 10 days. Parameters assessed: weight of body and organs, water and food
consumption, mortality, piloerection, dullness, diarrhoea, spasms. Blood was
collected for biochemistry and haematological assays. Results: Average body weight
gain was 1.2 1.3 g. Average liver weight was 1.5 0.1 g, heart (0.14 0.01 g),
spleen (0.07 0.01 g), kidneys (0.53 0.05 g), consumption of water (223.2 40.3
ml) and food (241.9 27.2 g). No mortality, piloerection, dullness, diarrhoea or
spasms were observed. Average glycemia was 52.8 5.0 mg/dL, cholesterol (91.2
6.9 mg/dL), triglycerides (46.6 4.0 mg/dL), urea (52.8 4.9 mg/dL), aspartate and
alanine-aminotransferases were 27.1 3.8 and 30.7 8.4 U. Average count of
erythrocytes was 7.0 0.8 million/mm3, leukocytes (4.0 1.1 thousand/mm3),
erythrocyte sedimentation rate > 5 mm/h, haematocrit (58.4 5.3 %) and mean
corpuscular volume was 82.2 6.9 fL. Conclusion: No alterations were verified for
the endpoints of toxicity compared to the control.
Ethical approval CEUA/UNISUL 15.011.4.01.IV. No conflict of interest.

S3
EFFECTS OF UV RADIATION ON THE CELLULAR ORGANIZATION IN
THE MACROALGA ACANTHOPHORA SPICIFERA
Dbora T. Pereira (1), Luciane C. Ouriques (1), Carmen Simioni (1), Zenilda L.
Bouzon (1), der C. Schmidt (1)
(1) Plant Cell Biology Laboratory, Department of Cell Biology, Embryology and
Genetics, Federal University of Santa Catarina, Florianpolis, SC, Brazil.
E-mail address: edcash@ccb.ufsc.br/ eder.schmidt@pq.cnpq.br (E.C. Schmidt) Tel:
+55 48 3721 5149
Sunlight is composed of a continuous spectrum of electromagnetic radiation that is
divided and designated in accordance with the wavelength range, and can be
ultraviolet radiation (UVR) (100-400nm), visible photosynthetic radiation (PAR)
(400-780nm), and infrared (> 780nm). UVR is the least reaches the Earth's surface,
but is the most harmful radiation. This radiation is subdivided into three different
spectral regions: UVA (400 to 315 nm), UVB (315 to 280 nm), and UVC (280 to
100 nm). UVA radiation is closest to the visible spectrum, and it is not attenuated by
ozone (O3). UVB radiation is not completely attenuated by O3, and it is harmful to
living organisms. Acanthophora spicifera specimens were collected from two sites
in Florianpolis, Santa Catarina, Brazil: Conceio Lagoon and Sambaqui Beach.
Samples of A. spicifera were examined under two different conditions of radiation,
PAR and PAR+UVA+UVB (PAR+UVAB). Daily doses of PAR, UVA and UVB
irradiances were 657.40 kJ m-2 (70 10 mol photons m-2s-1), 7.56 kJ m-2 (0.70 W
m-2), and 3.78 kJm-2 (0.35 W m-2), respectively, during a 12-h photocycle for 3 h
each day for 7 days. The aim of this study focused on the effects of UVAB on cell
morphology and cell ultrastructure through transmission electron microscopy.
UVAB treatment caused changes in the ultrastructure of cortical and subcortical
cells, including increased cell wall thickness and accumulation of starch grains, as
well as disrupted thylakoids into the chloroplast. We concluded that The present
study demonstrates that ultraviolet radiation negatively affects many morphological
parameters in A. spicifera.
Funding support: The authors acknowledge the CNPq and the CAPES.


T REPRODUCTIVE BIOLOGY
T1
T2
RESPONSES OF THE SOMATIC AND GERM CELL AGAINST

GLYPHOSATE EXPOSURE ON MALES OF ZEBRAFISH Danio rerio
CYCLOPHOSPHAMIDE INDUCES DBA LECTIN+ UTERINE NATURAL

KILLER CELLS MODIFICATIONS AND PREGNANCY LOSS IN MICE
Luciane Nezzi* (1),Neide Armiliato (1, 2),Carla Eliana Davico (1), DibAmmar
(1,3),Yara Maria RauhMller (1)and Evelise MariaNazari (1)
Renato de Oliveira Horvath1, Letcia Penna Tamburus1, Andrea Mollica do

Amarante Paffaro, Valdemar Antonio Paffaro Jnior1
(1) Laboratrio de Reproduo e Desenvolvimento Animal, Universidade Federal de

Santa Catarina, Departamento de Biologia, Embriologia e Gentica, Florianpolis,
Santa Catarina, Brazil.
(2) Laboratrio de Anlise Ambiental, Universidade do Contestado, Concrdia,
Santa Catarina, Brazil.
(3) Centro Universitrio Catlica de Santa Catarina, Joinville, Santa Catarina, Brazil.
(1) Laboratory of Integrative Animal Biology, Department of Cell and

Developmental Biology, Federal University of Alfenas, Alfenas, Brazil
*lucianenezzi@posgrad.ufsc.br - Laboratrio de Reproduo e Desenvolvimento

Animal - (48) 3721-9799
Background: Glyphosate is a herbicide soluble in water, used to inhibit the growth of
weeds. When applied in terrestrial systems, glyphosate penetrates into soil, reaching
the aquatic ecosystems, and affecting nontarget organisms, as fishes. Aims:
Investigate the reproductive toxicity of glyphosate in males of zebrafish Danio rerio,
evaluating the ultrastructure and the apoptotic responses of somatic and germ cells.
Methods: Adult males were chronically exposed for 15 days to 65 g glyphosate/L
aquarium water, according to resolution 357/2005/CONAMA for Brazilian inland
waters. Non-exposed males were used as controls. Testicles were dissected and
submitted to ultrastructural analysis. In order to investigate the expression of antiand pro-apoptotic proteins, cytotoxic assays were performed (Ethic Committee
n.0746/CEUA/UFSC). Results: A significant increase (p < 0.0001) of micronuclei in
blood cells was recognized, indicating the effective exposure. Ultrastructural analysis
allowed us to identify subcellular impairments induced by glyphosate, as disruption
on structure of the spermatogenic cysts, accompanied by reduction on intercellular
bridges between the germ cells. Additionally, Sertoli cells showed non-electrondense
cytoplasm with large vesicles.Glyphosate also induced a significant increase (p <
0.001) of Fas-ligand in Sertoli cells. However, no changes were observed in antiapoptotic protein Bcl-2 and in pro-apoptotic proteins Bak and active-caspase-3.
Conclusion: In this study we recognized adverse effects caused by glyphosate on
zebrafish testicles, mainly in the morphofunctionality of Sertoli cells.
renato_horvath@yahoo.com.br
Mobile Phone: (35) 99197-5538; Institutional Phone: (32) 3299-1360
Mouse Uterine Natural Killer Cells (uNK) are the largest immune cells in decidua. A
great amount of this cells (up to 90%) express N-acetyl-D-galactosamine in their
granules and membrane which could be stained by dolichos biflorus agglutinin
(DBA) lectin (DBA+uNK cells). Cyclophosphamide is teratogenic, induces
pregnancy loss and affect immune cells. In this study we aim to evaluate the effects
of cyclophosphamide in the gestational viability and in the uNK N-acetyl-DGalactosamine expression, distribution and differentiation.After UNIFAL-MG Ethics
approval (546/2013), supported by CAPES and FAPEMIG, SWISS mice were mated
and the gestation day (gd) 1 was assumed when the vaginal plug was observed.
Pregnant mice were 40mg/kg cyclophosphamide treated by 48h, 2h and 1h before
implantation sites (IS) collection at gd10. Gestational viability was assessed
macroscopic and morphologically. DBA lectin histochemistry and caspase-3
immunohistochemistry was undertaken and statistical analysis was by unpaired t test
(p<0,05). Cyclophosphamide induced drastic gestational viability reduction
(32,91%) 48h after treatment, 100% of fetal loss in pregnancy to term and several
pregnancy loss-related modifications including apoptosis. In 1h treated mice we have
observed an increasing of the DBA+ uNK number followed by a reduction in the 48h
treated mice. In the implantation sites of all treated mice were observed critical
changes in the uNK differentiation and distribution including the observation of a
high number of DBAlow uNK (DBAlow granules and membrane). Therefore
cyclophosphamide can impair uNK DBA reactivity and differentiation while causing
pregnancy loss in pregnant mouse suggesting that this drug could activate NK cell
cytotoxicity.
Support: CAPES

T3
T4
FIRST
REPORT
ON
SPERM
CELLS
MODIFICATIONS
POSTCOPULATION AND POST-OVIPOSITION IN THE BROWN SPIDER
LOXOSCELES INTERMEDIA (ARANEAE: SICARIIDAE)
EVALUATION OF GRAPE JUICE CONCENTRATE ON EPIDIDYMIS

MORPHOLOGY OF WISTAR RATS INTOXICATED WITH CADMIUM
CHLORIDE
Camila A. dos Reis (1), Maria A. M. Soares (2), Jos R. Gomes (2), Cristina L. S.
Costa-Ayub (2)
Gabriel da Silva Cordeiro (1), Maria Aparecida S. Diamante (2), Celina A. Lamas
(2), Heidi Dolder (2), Fabrcia de Souza Predes (1)
(1) Academic 's Degree in Biological Sciences from the State University of Ponta
Grossa ( UEPG ), Ponta Grossa, Paran, Brazil.
(2) Department of Structural Biology, Molecular and Genetics ( DEBIOGEM )
Sector of Biological and Health Sciences ( SEBISA ), UEPG, Ponta Grossa, Paran,
Brazil.
(1) State University of Paran Campus Paranagu, Paranagu, Paran, Brazil. Rua
Comendador Correia Junior, 117, Centro, Paranagu, Paran, Brasil
camilaaudreyreis@gmail.com
gabrielcordeiro.pr@gmail.com, Institutional number 55 41 3423-3644, Mobile

number 55 41 87996293
The brown spider Loxosceles intermedia is a haplogyne species widely distributed in

southern Brazil. In order to understand the conditions of sperm storage inside female
body after copulation and oviposition, we studied adult and young male and female
brown spiders maintained in laboratory conditions, where copulations followed. The
specimens had their reproductive organs processed for light microscopy, with
staining for H.E., P.A.S., and ninhydrin-Schiff. Loxosceles intermedia transfers
spermatozoa to the females spermathecae in the synspermia form surrounded by a
P.A.S. positive secretion and during copulation, female and male secretions are
mixed, but spermathecae secretions showed some alterations in the protein
componentst at this moment. At oviposition, individualized and uncoiled sperm cells
appear at the stalk portion of females spermathecae surrounded by a uterus-like
secretion, even in females that had laid eggs some time ago. Sperm cells are inside
the uterus externus, uterus internus and oviduct of females sacrificed at oviposition.
From these results, we can discuss about the sperm cells activation, and the female
signalizing for oviposition in brown spider. The separation of the sperm cells
(completion of the spermiogenesis), their uncoiling and displacement inside the
female reproductive system is certainly due its interaction with the female secretions,
which activate them. However, those are assumptions, and all evidences point to the
meet of the gametes occurring inside the females body, and for the contribution of
male and female secretions in processes involved with reproduction in this species of
spider.
Studies point to toxicological cadmiums (Cd) influence on human health. The

general population is exposed to Cd through water, food, tobacco, in addition to
occupational exposure. This exposition can cause male infertility and modify the
synthesis of important hormones. Thus the objective of this study was to analyze the
possible morphological, morphometric and biometric changes in the epididymis of
rats intoxicated with cadmium and check the ability of grape juice concentrate
(G8000) to protect this organ. Twenty four male Wistar rats (50 days) were used.
The control group (C) received saline daily by gavage. The cadmium 1.2 group
(Cd1.2) received a single intraperitoneal dose of 1.2 mg/kg body weight of cadmium
chloride. The grape juice concentrate group (SU) received daily gavage of 2g/kg of
G8000. The cadmium plus grape juice group (CdSU) received daily gavage of
2g/kg of G8000 and Cd at a single intraperitoneal dose of 1.2 mg/kg BW. All
statistical analyses were carried out using Statistic 8 (ANOVA and Tukeys Test).
Cd1.2 group showed significantly increased epithelium height and volumetric
proportion, besides reduced lumen volumetric proportion relative to C group.
Otherwise, in CdSU group these parameters were reestablished, showing values
similar to C group. This study confirms cadmium toxicity to the epididymis and
shows the effectiveness of G8000 to protect this organ, also demonstrating the
hazard of this metal in industries and the environment. Continued research should be
directed to the discovery of active compounds such as grape juice concentrate that
promotes health and this protective property.
Keywords: Brown spider, sperm cell activation, females secretions, spider

reproduction, cell and secretions interactions.
Ethical approval: 2900-1

Financial support: Fundao Araucria,CAPES and FAPESP.

T5
T6
BEHAVIORAL AND CITOCHEMICAL STUDIES COMPARING MOUSE

PSEUDOPREGNANCY TO MOUSE PREGNANCY
EVIDENCES FOR FETAL PROGRAMMING OF THE UTERINE NATURAL

KILLER CYTOTOXICITY CAUSED BY HIGH-FAT DIET DURING
MOUSE PREGNANCY
Paulo Fernando Carlstrom, Igor Emiliano Lemos de Souza, vila da Silva Lopes
Salles , Bruno Zavan, Valdemar A. Paffaro Junior
(1)
Instituto de Cincias Biomdicas, Universidade Federal de Alfenas, Alfenas, Brasil.

Pseudopregnancy is a phenomenon where psiconeuroendocrinology modifications
occur similarly to pregnancy. Pseudopregnancy is observed in several mammalian
and has been studied in rodents, especially rats as a pregnancy-like model without
embryo implantation. Because the knowledge about the mouse behavior during
pseudopregnancy is very fragmented and inconclusive and knowing the importance
of this model, we have proposed the comparative study of the anxiety and memory
between pseudopregnant and pregnant mice. After UNIFAL-MG Ethics approval
(336/2011) and supported by FAPEMIG, SWISS mice were mated with
vasectomized or non-vasectomized mice and the pseudogestation day (pgd) 1 or
gestation day (gd) 1 was assumed when the vaginal plug was observed. After the
pgd2, mice were submitted every day to vaginal smear to check the predominace of
leukocytes indicating pseudopregancy. Mice were submitted to the Elevated pluz
maze to anxiety and object recognition (OR) to memory tests at pdg2, 6, 10 and gd2,
6, 10. Mice were killed at pgd10 or gd10 and the uterus were collect, submitted to
paraffin embedding to obtain histological sections where we have developed DBA
lectin cytochemistry to identify uterine natural killer cell (UNK). Statistical analysis
was by ANOVA followed by Scott-Knott (p 0,05). We have found non differences
through anxiety and memory tests between pseudopregnant and pregnant mice
concomitantly to the identification of uNK cells in the pgd 6, 10 and gd6, 10,
indicating the efficiency of this method to generate pseudopregnant mice. Therefore
in our lab we managed to develop an easy way to study mouse pseudopregnancy.
vila da Silva Lopes Salles, Carolina Chaves do Nascimento, Paulo Fernando

Carlstrom , Andrea Mollica do Amarante Paffaro, Alexandre Giusti Paiva, Valdemar
Antonio Paffaro Junior
Institute of Biomedical Sciences, Federal University of Alfenas, Alfenas-MG, Brazil.
About 90% of uterine natural Killer cells (uNK) express N-acetyl-D-galactosamine
in their granules and membrane which could be stained by dolichos biflorus
agglutinin (DBA) lectin (DBA+uNK). DAB+uNK have no cytotoxicity in normal
pregnancy, however pregnancy modifications could changes offspring physiology as
a phenomenon called fetal programming. This study aimed to investigate the effect
of a high fat diet in DBA+uNK from mothers (F0) and their offspring (F1). SWISS
mice were mated, been gestation day (gd) 1 the day of vaginal plug observation
(Ethics approval-656/2015, support: FAPEMIG). Pregnant F0 received control
(MCD; n=6) or High-fat diet (MHF, n=6) and were killed at gd10 to implantation
sites (IS) collection. Two days after birth, F1 was fostered by a control mother. Eight
weeks after birth, females from each F1 (F1CD, n=6 and F1HF, n=6) were mated to
IS collection at gd10. IS were DBA and perforin stained. F0 were weighed at gd1
and gd10 and the perigonadal white adipose tissue (WAT) from pregnant F1 was
subjected to morphometry. Data were analyzed by unpaired t test (p 0.05). We have
observed DBAlowuNK concomitantly to the reduction of uNK perforin reactivity in
MHF and F1HF. A high weight was found in the MHF (MCD=4,6g; MHF=7,3) and
pregnant F1HL had a WAT higher area (F1CD=3935,91 m2; F1HL=6062,72 m2).
HF diet during pregnancy causes changes in uNK N-acetil-D-Galactosamine and
perforin reactivity in F0 and F1 mice, suggesting this could program F1 uNK to the
cytotoxicity during pregnancy.
T7

T8
ANALYSIS
OF
DEVELOPED
MATURATION
OF
FEMALE
SPERMATHECAE OF CRAB GONIOPSIS CRUENTATA (RED ARATU)
MARAJ / SOURE ISLAND
HISTOLOGIC DESCRIPTION OF SPERMATOGENESIS OF ARATU CRAB

(GONIOPSIS
CRUENTATA)
(LATREILLE,
1803)
(CRUSTACEA,
BRACHYURA, GRAPSIDAE)
Letcia da Silva Palheta (1), Adriano Biancalana (1).
Amlia Sousa Goveia (1), Ingrid Santos Palheta (1), Adriano Biancalana (1)
(1) Laboratory of Cellular and Molecular Biology, Federal University of Par
Campus Maraj/ Soure, Archipelago Maraj, Soure, Brazil.

Campus Maraj-Soure, Soure, Brazil.
Address: 13th Street, Umirizal, Soure, PA, Brazil.

E-mail: ameliagoveia@hotmail.com / contact: (91) 989941023

E-mail: leticiasilva14@hotmail.com contact: (91) 980699345
The spermatheca are pairs of spherical structures that store spermatophores until the
female is able to fertilize. The objective of the research was to determine the
maturational stages of development of the spermathecae Goniopsis cruentata, based
on observation of macroscopic and microscopic morphology. Between August 2014
and August 2015, were collected 88 female Goniopsis cruentata coming from
mangrove Extractive Reserve of Soure (RESEX / Soure). The animals were weighed,
measured, photographed and anesthetized by cooling. The gonads were collected,
fixed in 10% formalin, dehydrated, cleared and blocky. Histological cuts were
obtained from 7m thick, processed and stained with hematoxylin-eosin, toluidine
blue and PAS. During the months of study it was recorded 29 ovigerous females,
except in the months of December, January, February and July. Occurring two
reproductive peaks characterized as bimodal reproductive. At the Microscopic
analysis may be noted that the wall spermatheca comprises a prismatic
pseudostratified epithelium which has villi over the entire inner membrane and
projections close to the oviduct membrane. Different the other species has not aratu
spermatophore, it has free spermatozoid and immersed in a dense mass of secretion.
The physiological structures found in Goniopsis cruentata are similar to those found
in other decapod. However presents peculiarities in their histology not yet described
in the literature.
The procedures were performed with the approval of (ICMBio) and with PIBICCNPQ funding.
The spermatogenesis is the process of cell division and differentiation, which

originate spermatozoid. The aim of the study was to describe the spermatogenesis of
the species. The animals were weighed, measured, photographed and anesthetized by
cooling. The gonads were collected, fixed in 10% formalin, dehydrated, cleared and
blocky. Histological cuts were obtained from 7m thick, processed and stained with
hematoxylin-eosin. The results showed that spermatogenesis starts with
spermatogonia, precursor cells that give rise to spermatozoid, rounded with beads
formed by chromatin and are grouped forming germinal centers. The primary
spermatocytes are derived from the first cell type and are distinguished by being
smaller cells without chromatin granules and with more compressed genetic material.
Already the secondary spermatocytes are the third cell type in the differentiation
process and differ from the previous due to intense process of meiotic division. After
the division to form spermatids, reduced cell size and compacted core. The
spermatids after the differentiation, they begin the process of nuclear lateralization,
where occurs the appearance of acrosome, which is initially biased in opposition to
the core. At the end of differentiation arises mature sperm with elliptical shape,
lateralized core, clear cytoplasm and well centered acrosome. Sperm are not
flagellated cells. We have described five stages of differentiation process of
spermatogenesis and the spermatozoid corresponds to typical description for decapod
been described.
This work is the first to describe the complete spermatogenesis species Goniopsis
cruentata.
The procedures adopted had approval (ICMBio). And funded by CNPq / UFPA.

T9
T10
CHANGES IN AUTOPHAGIC CLEARANCE IN SERTOLI CELLS OF

MURINE MODEL OF MUCOPOLYSACCHARIDOSIS TYPE I (MPS I), AN
ULTRASTRUCTURAL INVESTIGATION
TESTICULAR MORPHOLOGICAL ANALYSIS OF A MURINE MODEL OF

MUCOPOLYSACCHARIDOSIS TYPE I
Cinthia C do Nascimento, MS(1), Odair A Junior, PhD(2), Vnia DAlmeida,

PhD(1)
1
Department of Psychobiology. Universidade Federal de So Paulo, So Paulo,

Brazil.
2
Department of Biosciences. Universidade Federal de So Paulo, So Paulo, Brazil.
Presenting author: Cinthia Castro do Nascimento. 925, Napoleo de Barros St.
04024-002 So Paulo, SP, Brazil.
cinthiaccn@gmail.com Telephone: +551121490155. Extention: 234. Mobile:
+5511988139659
Background: Mucopolysaccharidosis I (MPS I) is a genetic disease caused by a
deficiency of alpha-L-iduronidase (Idua), a lysosomal enzyme which hidrolyzes
glycosaminoglycans. It is known that the unsuitable storage of these substrates inside
lysosomes and in extracellular matrix impairs cellular homeostasis in a variety of
tissues and organs, mainly in bones, spleen, liver, heart and brain. Additionally, some
authors have demonstrated an impairment of autophagic clearance in specific tissues,
as a consequence of the lysosomal dysfunction. Due to the susceptibility of
reproductive system to environmental and genetic factors, we also consider
important the investigation of the gonads of the murine model of MPS I. Aims: the
present work aims the examination of the testicular ultrastructure of MPS I mouse
model. Methods: Epon embedded testis from 6-month-old Idua+/+ and Idua-/- mice
were examined by transmission electron microscopy. Images of Sertoli cell
cytoplasm were captured in order to identify and quantify autophagic vacuoles,
lysosomes, autolysosomes and mitochondria. Results: We found a higher number of
autophagosomes (p=0.008) and a lower number of lysosomes (p<0.001) in samples
obtained from Idua-/- mice. The number of autolysosomes and mitochondria were
not statistically different among groups. Besides that, myoid cells were widely
vacuolated and interstitial compartment of seminiferous tubules presented infiltrated
cells through all its extension and Leydig cells appeared to be damaged, with
lamellar bodies and sparse vacuoles. Conclusion: We conclude that autophagic
vacuoles are abundant in Sertoli cells and consequently, lysosomes must be more
demanded.
Ethical approval: 4226130214
Financial support: AFIP, CAPES, CNPq and FAPESP.
Ana Barbosa Mendes(1), Cinthia C do Nascimento, MS(1), Guilherme Baldo,

PhD(2), Odair A Junior, PhD(3), Vnia DAlmeida, PhD(1)
(1) Department of Psychobiology. Universidade Federal de So Paulo, So Paulo,
Brazil.
(2) Department of Physiology. Universidade Federal do Rio Grande do Sul, Rio
Grande do Sul, Brazil.
(3) Department of Biosciences. Universidade Federal de So Paulo, So Paulo,
Brazil.
Presenting Author information: Ana Beatriz Barbosa Mendes. 925, Napoleo de
Barros St. 04024-002 So Paulo, SP, Brazil.
anabeatrizbmendes@gmail.com Telephone: +551121490155 Ext. 234 Mobile:
+5519981100671
Background: Mucopolysaccharidosis I is a lysosomal storage disorder, caused by
alpha-L-iduronidase (Idua) deficiency. It results in the accumulation of
glycosaminoglycans, which has severe consequences in most tissues. In previous
studies, we have observed signs of testicular morphological damage in 3 and 6month-old Idua-/- mice, demonstrating the testis susceptibility to the disease
progression. Aims: The present work is focused in the morphological analysis of
seminiferous tubules in 12-months-old Idua-/- mice. Methods: Paraffin embedded
testes from 12-month-old Idua+/+ and Idua-/- mice were examined through light
microscopy. The percentage of degenerated tubules, tubular diameter, epithelium
height, interstitial area and the classification of stages of tubular cross sections were
determined. Results: We observed a greater number of vacuolated cells in the
interstitial compartment of Idua-/- mice. The interstitial area between seminiferous
tubules was increased in Idua-/-mice, as we have previously encountered in younger
Idua-/- mice. Tubular diameter was decreased, suggesting tubular damage in a more
advanced stage of the disease, which was not detected in younger mice. However, we
did not find any differences in the percentage of degenerated tubules, epithelium
height and the percentages of tubules in each stage of the seminiferous epithelium
cycle between the two groups. Conclusion: We can infer from the present data that
the damage caused by the disorder is not confined to the interstitium, but is also
present in the tubules, morphologically detected only in 12-months-old mice,
evidencing the progression of the disease.
Approved by the UFRGS Ethics committee (06-509 CP-HCPA)
Financial support: AFIP, CAPES, CNPq and FAPESP.

T12
T11
EMBRYO QUALITY AND DEVELOPMENT UNDER HEAT STRESS
Aldcejam Martins da Fonseca Juinior Naianne de Arajo Felix Luis Eduardo
Pereira de Andrade Ferreira
Laboratory of Animal Reproduction Instituto Federal de Educao, Cincia e
Tecnologia da Paraba - Campus Sousa
Contact: aldcejamjunior@hotmail.com/ (+55 83)996667189
Background and aims: Several studies have attested that heat stress is responsible for
harmful effects on cell functions, interfering with fertilization and embryo
development. This study aimed to evaluate the effect of heat stress on quality and
development stages of bovine embryos. Methods: Crossbred dairy cows have been
established for the experiment, being the test group subjected to semi-intensive
handling to sunlight, while the control group had access to artificial shading all day
long in the same breeding system. They received reproductive monitoring and
superovulation based on luteinizing hormone (LH) and human chorionic
gonadotropin (HCG) and subsequent artificial inseminations. The collection of
embryos occurred seven days after induction in superovulation protocol (D7) through
uterine washes with DPBS solution. Results: As regards the developmental stages of
embryos recovered, most of the structures (54.5%) were classified as morula,
referred to as the optimal bipartition phase in cattle embryos. Other significant
figures were 27.2% of early blastocysts and 18.1% of initial morula. Regarding
embryo quality, 70% of the structures were categorized as excellent, 20% regular and
10% as poor, with no degenerate. Conclusion: The animals subjected to heat stress
suffered probable interference on cell bipartition phases generating low morula index
to D7 whilst embryo quality, which maintained the viability according to its rate, it
may have been influenced by adaptive homeostatic processes of animals.
Financial support: PIBICT/IFPB
Reproductive biology
MORPHOMETRY AND ULTRASTRUCTURE OF THE SPERMATOZOON

OF
THE
BAT
ARTIBEUS
LITURATUS
(PHYLLOSTOMIDAE:
CHIROPTERA)
Cornlio Souza Santiago1,*, Alderrosy Fragoso Rodrigues1, Cleber Silva Andrade1,
Eliana Morielle-Versute2, Sebastio Roberto Taboga3, Mateus Rodrigues Beguelini1
1
Center of Biological and Health Sciences, UFOB Universidade Federal do Oeste

da Bahia, Barreiras, Bahia, Brazil.
2
Department of Zoology and Botany, UNESP Univ. Estadual Paulista, So Jos do
3
Department of Biology, UNESP Univ. Estadual Paulista, So Jos do Rio Preto,
So Paulo, Brazil.
*Presenting author: Cornlio Souza Santiago, mail: nelios.santiago@hotmail.com,
tel: (77) 36143215, cel: (77) 991662484.
Background: Despite bats exhibit the second largest number of mammal species, the
second largest territorial dispersion pattern and many unique reproductive
specializations, within the order Chiroptera, the studies related to the spermatozoon
ultrastructure are still scarce. Aims: The aim of this study was to characterize the
ultrastructure and morphometry of the spermatozoon of the bat Artibeus lituratus.
Methods: Ten specimens were collected in the Northwest of So Paulo State, Brazil,
and their testes were analyzed by transmission electron microscopy. Results: The
spermatozoon of A. lituratus has a dorso-ventrally flattened, arrow-like head
(4.72m 0,05). Its acrosome is endowed with a prominent perforatorium (0.49m
0,04), which has a spear form. The midpiece is not so long (8.12m 0,29) and
contain approximately 41 mitochondrial gyres (41 1,12). In cross section, the outer
dense fibers were arranged in a horseshoe fashion, with the fibers 1, 5, 6 and 9 larger
than other. An annulus is found caudal to the last mitochondrial gyre and, caudally to
it, the tail diameter lessens abruptly and gives rise to the principal piece. This is
longer than the midpiece. Discussion: The spermatozoon of A. lituratus is similar to
other species of phyllostomid bats, but presents considerable differences in relation
to species from other families, such as Noctilionidae, Molossidae and
Vespertilionidae. Conclusion: Our data demonstrate that the ultrastructure of the bat
spermatozoon has a pattern more closely related to the human than to other
mammals, with an accentuated interfamilial variation.
Ethics approval: Process: 013/09 CEEA, ethics committee at So Paulo State
University (UNESP).
Funding support: FAPESP (processes: 2012/09194-0 and 2009/16181-9).

T13
ALUMINUM
ORAL
EXPOSURE
ALTERS
DEVELOPMENT OF THE GERBIL PROSTATE
T14
THE
PRENATAL
Liana da Silva Gomes(1), Danilo da Silva Lima (1), Yasmin Inocncio Fernandes e
Silva (1), Manoel Francisco Biancardi (1), Mara Rbia Marques (1), Paulo Csar
Ghedini (2), Fernanda Cristina Alcantara dos Santos (1).
Gois, Goinia-GO, 74001970, Brazil
(2) Department of Pharmacology, Laboratory of Molecular and Biochemistry
Pharmacology, Institute of Biological Sciences, Federal University of Gois, Goinia
- GO, 74001970, Brazil.
OVARIAN SEX STEROID RECEPTORS AND HORMONE LEVELS IN

ANDROGENIZED RATS AFTER TREATMENT AND RECOVERY
PERIODS
Vinicius Augusto Simo (1), Luiz Gustavo de Almeida Chuffa (2), Isabel Cristina
Cherici Camargo (1).
(1) Department of Biological Sciences, Universidade Estadual Paulista - UNESP,
Faculty of Sciences and Letters, Assis, So Paulo, Brazil
(2) Department of Anatomy, Universidade Estadual Paulista - UNESP, Institute of
Biosciences, Botucatu, So Paulo, Brazil.
(1) Department of Biological Sciences, Universidade Estadual Paulista UNESP,
Faculty of Sciences and Letters, Assis, So Paulo, Brazil.
email: liana.anato@yahoo.com.br; Phone +556292782545; +556235211765

E-mail: viniciusmorma@gmail.com; Telephone: +55 (18) 98128-8592.
Prostate development is dependent of an equilibrated hormonal regulation, so that
sensible interferences during this period may predispose the gland to lesions during
ageing. Industrial activities increased the exposure of this gland to active elements
found in environment, such as aluminum (Al). Al presents toxic effect for living
beings, having the potential to disrupting the development and growth of several
organs and systems. Therefore, the aim of this study was to evaluate whether the
prenatal exposure to Al may alter the prostate development. Pregnant females were
orally exposed to aluminum chloride (100 mg/kg/day) from 17th to 21th gestational
day. Following the birth, the male and female pups were euthanized with one-dayold. The prostates were collected for biometrical, three-dimensional reconstruction,
morphometrical, and immunohistochemical analysis. The results demonstrated that
the Al decreases the body weight of males and females, and also reduces the
anogenital distance of females. Three-dimensional reconstruction and morphological
data showed that Al exposure changes the prostate development pattern in gerbils.
Prostates of females and males showed an increase of the epithelial buds frequency
and PCNA-positive cells. Moreover, we observed a decrease of the AR
immunostaining in both sexes, and an increase of the ER-positive cells in
females.Therefore, the data suggest that Al acts either as an antiandrogenic
compound, blocking androgenic action at AR level, or as an estrogenic agonist in
prostate. These findings are important for public health, since Al exposure increased
during last years, especially in newborns and pregnant women.
Ethical approval: CEUA/UFG: n024/13. Support: FAPEG n01/2016.
Background: despite the increasing use and reported side effects of the anabolic
androgenic steroid Nandrolone Decanoate (ND) on reproduction, little attention is
given to the regulation of sex hormones and ovarian steroid receptors.
Aim: this study aimed to evaluate the effects of different doses of ND on ovarian
function in rats, after treatment and recovery periods.
Methodology: Adult Wistar rats (n=6/group) received ND at doses of 1.87, 3.75, 7.5
and 15 mg/kg body weight or mineral oil (Control group) for 15 days,
subcutaneously and the groups were divided into three periods of evaluation: ND
treatment for 15 days; ND treatment and recovery for 30 days; and ND treatment and
recovery for 60 days. Estrous cycle was monitored daily. At the end of each period,
the females were euthanized for collection of blood serum and ovarian tissue for
immunohistochemical and western blot analysis of steroid receptors.
Results: persistent diestrus occurred during ND treatment and 30-day recovery. The
highest dose of ND was able to maintain diestrus phase until 60-day recovery. The
expression of ovarian steroid receptors varied in a dose and period-dependent
manner, having a more pronounced response with 15 mg ND/kg. Although ND
treatment increased serum levels of luteinizing hormone, testosterone, 17-estradiol
and dihydrotestosterone, mainly in the highest doses of 7.5 and 15 mg ND/kg, no
change was observed in the levels of follicle-stimulating hormone.
Conclusion: we concluded that ovarian sex steroid receptors and sex hormones are
able to restore only in the lower doses of ND and after a longer period of recovery.
Ethical Approval: CEUA (Permit number: 005/2011; 31 August).
Financial Support: FAPESP (Proc. 2012/01747-0 and 2013/14510-0) and
FUNDUNESP (Proc. 2178/002/14).

T15
T16
EXPOSURE OF HUMAN SERTOLI CELLS TO TCDD (2,3,7,8TETRACHLORODIBENZO-P-DIOXIN): EVALUATION OF THE ARYL

HYDROCARBON RECEPTOR PATHWAY
Carolina Fazio Campos* (1), Mariana Antunes Ribeiro (1), Wellerson Rodrigo
Scarano (1)
(1) Department of Morphology, Institute of Biosciences, Sao Paulo State University,
UNESP, Botucatu, SP, Brazil.
* Correspondence: Carolina Fazio Campos, Rua Professor Doutor Antonio Celso
Wagner Zanin, s/n. Institute of Biosciences of Botucatu - UNESP, Department of
Morphology,
CEP:18618-689.Botucatu,
SP,
Brazil.
Email:
carolinafaziocampos@hotmail.com; Phone: +55 14 38800491.
Background: Studies in animals have shown that TCDD could disrupt
spermatogenesis. TCDD interacts with aryl hydrocarbon receptor (AhR). AhR is
controlled by its repressor AHRR. When AHRR is not able to effectively suppress
the TCDD action, it may occur an increase in oxidative stress by the production of
reactive oxygen species and consequently interfere with spermatogenesis and
therefore male fertility. Aim: Evaluate the effect of TCDD exposure in Sertoli cells
regarding the expression of genes and proteins AhR and AHRR. Methods: Primary
human Sertoli cells (HSeC) were exposed to 10 nM of TCDD during 72h in
triplicates. AhR and AHRR expression analysis was based on RT-qPCR (ReverseTranscriptase quantitative Polymerase Chain Reaction) approach. Protein expression
of those corresponded genes was performed by Western Blot. Results: After 72h of
exposure to TCDD, the expression of AhR had a significant decrease compared to
control while AHRR showed no expression differences. Regarding protein
expression, AHR showed increased expression although not statistically significant
compared to control cells while AHRR was down regulated after 72 hours of
exposure to TCDD. Conclusion: Our findings show that a negative feedback is
returning AHR to levels similar to the control group. Regarding AHRR, a post
transcriptional regulation could be involved in protein expression downregulation.
The engagement mechanism of TCDD in gene expression in Sertoli cells needs to be
further investigated considering the importance of this cell type to the whole process
of spermatogenesis. Financial support: (FAPESP: 2015/06149-1); Ethical Protocol:
Not applicable, because no animals were used.
The authors report no conflict of interest.
EFFECTS OF CAFFEINE CONSUMPTION DURING PREGNANCY ON

PLACENTAL AND FETAL DEVELOPMENT IN MICE
Thas Paula (1), Fabola Nihi (1), Fernando Felicioni (1), Marcos Gomes (2), Hlio
Chiarini-Garcia (1), Fernanda Almeida (1).
(1) Departmento de Morfologia, Universidade Federal de Minas Gerais, Minas
Gerais, Brasil.
(2) Departamento de Biologia Estrutural, Universidade Federal do Tringulo
Mineiro, Minas Gerais, Brasil.
Authors contact:
Thas de Mrici Domingues e Paula ; thaismerici@hotmail.com
55.31.9.9732 5458
55.31. 3409 2994
Laboratrio de Biologia Estrutural e Reproduo, Instituto de Cincias Biolgicas
ICB/UFMG - Av. Pres. Antnio Carlos, 6627 - Pampulha, Belo Horizonte/MG,
31270-901.
Background: Caffeine is consumed by pregnant women to reduce fatigue. Due to its
ability to cross the placental barrier, it should be restricted during pregnancy, despite
women usually consume large amounts daily. Aim: To investigate the effects of
different doses of caffeine during pregnancy on placental and fetal morphology.
Methods: Female Swiss mice were divided into four groups (n=5), which received
different caffeine doses (0-CC; 60-T60; 120T120; 240T240 mg/kg/day) during
one week. Daily vaginal smears were collected to identify the estrous cycle phase,
and the females continued receiving the same amounts of caffeine after mating until
day 18 of pregnancy, when they were euthanized. Data on estrus frequency,
pregnancy rate, body, brain, liver and placental weight of each fetus were recorded.
Results: The T240 group showed a high frequency of females in arrested diestrus,
which lead to a low conception rate. In this group only two, out of five females,
showed vaginal plugs, however, no fetuses were present at the time of euthanasia.
Placental weight was lower in the T120 group (P<0,0001). Interestingly, the T60
group showed heavier placentae (P<0,0001). Fetal weight was lower in all treated
groups compared to the control (P<0,0001).The brain/liver weight ratio, a predictor
of intra-uterine growth restriction, was similar among the experimental groups
(P=0,08). Conclusion: Daily consumption of caffeine during pregnancy, even in
lower doses, may have negative effects on placental and fetal development. Further
investigations are necessary to study caffeine intake effects on conceptus
development.
This project was approved by CEUA/UFMG (395/2015).
(1)
(2)
T17
T18
EFFECTS OF HIGH PROTEIN DIET IN PREGANT MICE AND THEIR

OFFSPRING
EFFECTS OF MONOBUTYL PHTALATE (MBP) AND PANAX GINSENG

EXPOSURE ON HUMAN SERTOLI CELLS IN VITRO
Glcia Marlia Zambroti Greco* (1), Ana Patrcia Barbosa Silvrio (1), Adriana Dias
(2)
; Valdemar Antonio Paffaro Junior (1); Andra Mollica do Amarante-Paffaro (1)
Andr Teves Aquino Gonalves de Freitas (1); Cristiane Figueiredo Pinho(1);; Yuri
Alves Silva(1); Wellerson Rodrigo Scarano(1)*
(1) Laboratory of Integrative Animal Biology, Department of Cell and

(1)
Developmental Biology, Federal University of Alfenas, Alfenas, Brazil.
(2)
(2) Departament of Exact Sciences Federal University of Alfenas
Department of Morphology, Institute of Biosciences, UNESP, Botucatu, SP, Brazil
Adress: Gabriel Monteiro da Silva, n 920A, Alfenas, Minas Gerais, Brazil

Institutional phone: (35) 3299.1360
Mobile Phone (35) 991395775
e-mail: gliciagreco@gmail.com
Gestation is a singular moment in which several woman body modifications occurs
and pregnant women can choose different diets in order to maintain body shape.
High protein (HP) diets have been used by humans as benefical. We aimed to
analyze the influence of HP diet in pregnant mice and their offspring development.
SWISS mice (Ethics approval-68/2015, support:PIB-PS/UNIFAL; FAPEMIG)
were mated and Gestation day (gd) 1 was the vaginal plug observation day. Mice
received control or HP diet (42%HP) from gd1 until pregnancy to term. Food intake
and body weight were analyzed every day until parturition, when puppies were
counted, weighed and standardized (three female and three male from each
offspring). Puppies were weighed and measured every two days and submitted to
physical and neurological developmental tests. At day 32 of life, puppies were killed
and Brown adipose tissue (BAT), white adipose tissue (WAT) and kidney from
mothers and puppies were collected for morphological and morphometric analyses.
Data were analyzed by unpaired t test (p 0.05). HP mothers food intake, weigh
gain and glomerular number were smaller than control. Puppies from HP mothers
have opened the eyes later and exhibited smaller BAT compared with control. The
vaginal opening of HP female offspring occurred later and the kidney size was lower
than control. Our data suggested that HP diet during mice pregnancy has effect on
mother organism and delayed the development of the puppies. Further studies are
necessary to investigate the effect of this unbalanced diet on mice adulthood.
Contact: andretagf@gmail.com
Phone: 55 11 999804900 / 55 11 38800491
Sertoli cells are connected by gap and tight junctions, forming the blood-testis barrier
(BTB), responsible for the spermatogenic process. The dynamics of BTB formation
is directly related to the balance between synthesis and degradation of specialized
molecules. Overlooking the importance of endocrine signals in the modulation of
Sertoli cells, the aim of this study is to determine the action of the endocrine
disruptor Monobutyl phthalate (MBP) and possible protective effect of Panax
Ginseng (GIM-1), in different exposure periods, from a low toxicity dose on the
HSec line of human Sertoli cells in vitro. Sertoli cells were propagated in 3D model,
above artificial matrix, in DMEM Medium supplemented with 10% fetal bovine
serum. For GIM-1 cytotoxicity evaluation and dose definition MTT assay was
performed. For MBP, recent studies of our group showed 10M is not cytotoxic for
HSec cells. MTT results for GIM-1 showed an increase in cell viability at 5M.
Then, cells were exposed to 10M MBP for 12 and 48 hours and submitted to
morphology evaluations. Hematoxylin and Eosin assay was performed for cell
behavior, adhesion complexes and morphology observations. For F-actin evaluation,
cells were stained with a 500ug/ml fluorescent phalloidin conjugate solution.
Phalloidin and Hematoxylin and Eosin assay revealed phenotically disruption in
Sertoli cells adhesion, without changes in F-actin expression or localization. Our data
suggested that MBP presents potential for disrupting the structure of BTB adhesion
complex in HSec cells. Exposure to GIM-1 and a combined exposure of both
substances is being now performed for further evaluation.
Keywords: endocrine disruptors, monobutyl phthalate, male reproductive function,
blood-testis barrier, (HSec) Human Sertoli cells.

T19
T20
EFFECTS OF PREPUBERTAL EXPOSURE TO LOW DOSES OF

ASSOCIATED
METHYLMERCURY
AND
POLYCHLORINATED
BISPHENOLS ON THE MORPHOLOGY AND NUMBER OF
CHROMATOID BODIES IN ROUND SPERMATIDS
TROPHOBLAST CELLS UNDER ENDOPLASMIC RETICULUM STRESS

ARE INDUCED TO APOPTOSIS BY SDF2 (STROMAL CELL DERIVED
FACTOR-2)
Aline R. Lorenzon-Ojea (1),Clarissa Ribeiro Rocha (2), EstelaBevilacqua (1)
Mariana S Garcia1, Rita L Peruquetti1, Wilson A Orcini1, Juliana E Perobelli2.

1
Laboratrio de Biologia Molecular e Citogentica, Universidade do Sagrado

Corao, Bauru, So Paulo, Brasil;
2
Departamento de Cincias do Mar, Universidade Federal de So Paulo, Santos, So
Paulo, Brasil.
Correspondent author information: aupairmariana@hotmail.com Fone number: +5514-997748858
Background: Chromatoid body is a cytoplasmic structure of male germ cells that has
a role in the regulation of mRNA transcription during spermatogenesis. This event
can be affected by exposure to methylmercury and polychlorinated bisphenols
(PCBs), which are important environmental toxicants, since they are persistent and
suffer biomagnification along the food chain. Aims: To investigate whether
prepubertal exposure to solutions containing PCBs and MeHg at low doses causes
immediate and/or delayed effects on morphological and molecular organization of
CBs in the germinal epithelium of rats. Methods: 87 male Wistar rats, 21 days old,
divided into 5 groups: G1 (negative control), G2 (MeHg pure), G3 (Aroclor pure),
G4 (Mixture 1), G5 (Mixture 2). The animals were treated daily from postnatal day
(PND) 23 to 53 by gavage. At PND 53 half of the animals were euthanized. There
was an interval of 62 days without exposure to chemicals and then the other half of
the animals were euthanized at PND 115. Results: Immunofluorescence analysis
allowed us to observe that the organization of the CBs in all groups treated with
Aroclor or Aroclor+MeHg has become compromised in the seminiferous epithelium
of 53 days old animals. Quantitative analyzes concerning the morphological and
numerical organization of CBs are being finalized to support qualitative observations
as well as analyzes in the seminiferous epithelium of animals at PDN115.
Conclusion: These preliminary results show that Aroclor or Aroclor+MeHg may
affect short term CB assembly during rat spermatogenesis.
Financial Support: FAPESP 2013/26797-2; 2013/14477-3. Ethics committee
approved: CEUA-USC (30/13).

Sciences, University of So Paulo, So Paulo, Brazil
(2) Department of Microbiology, Institute of Biomedical Sciences, University of So
Paulo, So Paulo, Brazil
Background: Endoplasmic reticulum stress results from changes in ER homeostasis
and folding of secretory proteins and initiates adaptive cellular mechanisms to rescue
cell homeostasis or to elicit apoptosis. SDF2 is expressed in placenta and keeps
similar structure with proteins known to be involved in ER stress.
Aims: We investigated a possible role of SDF2 in trophoblast cells.
Methods: HTR8-Svneo trophoblast cells were induced to ER stress, and its markers
were evaluated by qRT-PCR (sXBP1, CHOP and SDF2) and Western Blot
(GRP78/BiP and SDF2). Cells were also silenced, and superexpressed for SDF2; the
same factors were evaluated. Apoptosis and cell survival were evaluated by caspase3 and ATP production luminescence assay. Ethical approval CEP-ICB No 702/14.
Results: SDF2 is downregulated during ER stress treatment. In an early time point,
SDF2-silenced cells show upregulation of the survival marker, GRP78/BiP; in a later
time, sXBP1 is upregulated while CHOP is downregulated. Caspase 3/ATP
production was lower in the group silenced and induced for ER stress while this ratio
was increased in SDF2 superexpression.
Conclusions: Cells treated with tunicamycin and silenced for SDF2 showed a prosurvival fate (changes in GRP78/BiP, sXBP1, CHOP and caspase 3/ATP production)
while superexpression of SDF2 correlated to a pro-apoptosis fate (an increase of
caspase 3/ATP production). Our data support a role of SDF2 in trophoblast cell
survival/apoptosis in ER stress response. The onset of ER stress during pregnancy is
related with several pathologies, including preeclampsia. Identifying new factors of
this response may contribute to the understanding of gestational diseases.
*Supported by FAPESP and CNPq
(1)
(2)
T21
T22
ANALYSIS OF MMPS REGULATORS TIMPS, CANONICAL AND NONCANONICAL SPLICING RECK ISOFORMS AND SPARC IN DEEP
ENDOMETRIOSIS
MORPHOMETRIC ANALYSIS OF THE PROSTATE EPITHELIUM OF

ADULT AND SENILE GERBILS EXPOSED TO ETHINYLESTRADIOL IN
THE NEONATAL PERIOD
Ana Claudia Oliveira Carreira (1,2); Lucas Bassi (1); Marina Trombetta-Lima (1,2);
Maurcio Simes Abro (3); Mari Cleide Sogayar (1,2)
(1)
(1) NUCEL (Cell and Molecular Therapy Center) and NETCEM (Center for Studies
in Cell and Molecular Therapy), Internal Medicine Department Department, Medical
(2)
School, University of So Paulo, So Paulo, Brazil
(2) Departament of Surgery, School of Veterinary Medicine and Animal Science,
Paulo, Brazil
Mariele Ilario Zuco(1), Luiz Roberto Falleiros Junior(1), Luiz Henrique Alves
Guerra(1), Nayara Fernanda da Costa Castro (1), Patricia Simone Leite Vilamaior(1),
Department of Biology, So Paulo State University, So Jos do Rio Preto, So
Paulo, Brasil.
To whom correspondence should be addressed: Ana Claudia O. Carreira

(anaclaudiaoli@gmail.com), NUCEL-NETCEM, Rua Pangar, 100, Cidade
Universitria, So Paulo, SP, Brazil. 05360-130, Phone: +55 11 2648-0230
(3)
Mobile Phone: +55 11 983229615
Background: Endometriosis is a benign gynecological disorder characterized by
ectopic growth of endometrial cells. The ectopic endometrium degrades the
extracellular matrix, through the activity of metalloproteinases (MMPs) and other
molecules, and invades the surrounding tissue with corresponding cell proliferation
and neoangiogenesis. These degradation by MMPs is closely regulated by TIMPs
under normal physiological conditions. Other MMPs regulators, RECK, which
negatively regulates MMPs in tumors, and SPARC, which upregulates MMPs, have
never been analyzed in deep infiltrating endometriosis (DIE).
Aims: No studies are available on expression of canonical and non-canonical
splicing RECK isoforms, SPARC or their correlation with other regulators in DIE.
The objective is to analyze the expression profiles of MMPs and their regulators, to
describe their involvement in endometriosis.
Methods: The expression profiles of MMPs-2,-3,-7,-9,-10,-14, TIMP-1,-2,-3,
SPARC, RECK and their isoforms in eutopic and ectopic endometrium (N=40) were
analyzed by qRT-PCR. The gene expression of ectopic endometrium was compared
with a pool of endometrium from healthy fertile women (control N=25) and bowel.
Results: MMP7, MMP14, SPARC, MMP2 and their regulator TIMP1 were
(4)
upregulated in ectopic endometrium when compared to bowel. These results indicate
(5)
the possible involvement of these genes in the progression of the DIE. RECKB was
modulated in controls endometrium. Immunohistochemistry was performed for
MMPs-2,-9, RECK and SPARC, and the results are being analyzed to correlate gene
and protein expression.
Conclusion: Our results indicate that new genes, SPARC and RECK and its isoforms,
are involved in DIE.

T23
Luiz Henrique Alves Guerra (1), Sebastio Roberto Taboga (1), Patrcia Simone
Leite Vilamaior (1)
(1) Department of Biology, So Paulo State University, So Jos do Rio Preto, So
Paulo, Brasil.
Luiz Henrique Alves Guerra (lhaguerra@gmail.com)
Department of Biology- Rua Cristvo Colombo, 2265. Bairro: Jardim Nazareth.
CEP:15054-000 - So Jos do Rio Preto, So Paulo, Brasil.
Telephone contact: (+5517) 3221-2200 branch 2733
Studies have shown mineral oil presents estrogenic activity in vitro, however, the
influence of purified mineral oil, a vehicle used in endocrine-disrupting researches, is
unknown on the prostate. This gland is dependent on steroid hormones for it
maintenance, being sensitive to substances that interfere with endocrine pathways.
So, this study evaluated the pure mineral oil action on the gerbils prostate. For this,
adult male gerbils (90 days old, n=6) were divided into 4 groups: Intact (I), mineral
oil (MO), mineral oil II (MOII), and corn oil (CO). Mineral and corn oils were
administrated from 90 to 115 days old, via gavage. I, MO, and CO animals were
killed at 116 days old, and MOII animals at 123 days old. Prostates were processed
for histological analyses. Morphometric, biometric, stereological, and
immunohistochemistry for AR detection analyses were performed. Serum
testosterone and estradiol levels were determined by ELISA assays. There was no
variation in the biometric analyses, as well as in the hormonal analyses, among all
groups. MOII group showed higher epithelial and lower luminal compartment
volume. This group also presented higher epithelium height, and the muscular layer
thickness increased in all groups in comparison with I animals. Exposure to mineral
oil increased the frequency of AR-positive cells. This data show that both mineral
and corn oil caused morphological alterations in gerbils prostate. Nonetheless, the
morphological alterations derived from mineral oil exposure were more expressive
than the corn oil exposure.
Ethical approval: protocol 061/ 2012-CEUA. Founding support: PROPe /UNESP,

FAPESP (Proc. 2012/12607-4).
T24
MINERAL OIL AS POTENTIAL ENDOCRINE DISRUPTER: EFFECTS ON

VENTRAL PROSTATE GERBILS
CEUA protocol: 124/2015. Financial support: CAPES/FAPESP
The prostate depends on androgens principally, but it is also dependent of estrogens.

The 17--ethinylestradiol (EE) is a synthetic estrogen that is present in
contraceptives. Neonatal exposure to EE, in gerbils (Meriones unguiculatus) results
in benign and premalignant disorders in adult individuals, but the effects of exposure
in senile stage is unknown. Therefore, the objective of this study was to evaluate the
morphologic effects in the prostate epithelial cells of adult and senile gerbils exposed
to EE in the neonatal period. In order to do this, 20 adult male gerbils were used, 10
were exposed to EE and 10, the control group, were treated with the vehicle mineral
oil via breastfeeding. Both were analyzed in adulthood and senility. Adult and senile
gerbils were euthanized at the age of 120 and 365 days old, respectively. Then they
were weighed, the prostate was removed and processed for histological analysis. The
morphometric and kariometric analysis were made with the help of the program
Image Pro Plus in sections stained with hematoxylin-eosin and Feulgen reaction.
Data were statistically analyzed. The groups treated with EE showed significant
differences in the height of the epithelium and in the nuclear area comparing to the
control groups. These differences can indicate a reduction in the synthetic and
secretory activity of prostate epithelial cells caused by exposure to EE. Therefore,
neonatal exposure to the EE resulted in alterations in the glandular homeostasis and
in the glandular activity.
Support: FAPESP, CNPq, CAPES, FINEP, BNDES, MCT, MS-DECIT.

Ethical Approval: CAPPESq-0386-11
Keywords: MMPs system, Extracellular Matrix, Deep Endometriosis.
(1)
Mariele Ilario Zuco

Address: Frei Valrio Kirch, 05 So Jos do Rio Preto- SP
Email address: mariele.ilario@gmail.com
Phone: +55 18 982065260
Institutional: Address: Laboratrio de Microscopia e Microanlise - Cristvo
Colombo, 2265 So Jos do Rio Preto-SP
Phone: +55 (17) 3221-2200 ramal 2733
ANALYSIS OF THE TRANSCRIPTIONAL ACTIVITY DURING THE

PHENOMENON OF NUCLEOLAR PERSISTENCE IN THE GERM CELLS
OF TRIATOMINES
Fernanda Fernandez Madeira (1), Kaio Cesar Chaboli Alevi (1), Ana Letcia Guerra
(1), Joo Aristeu da Rosa (2) and Maria Terclia Vilela de Azeredo Oliveira (1)
(IBILCE), Universidade Estadual Paulista UNESP, So Jos do Rio Preto, SP,
Brasil. (2) Departamento de Cincias Biolgicas, Faculdade de Cincias
Farmacuticas (FCFAR), Universidade Estadual Paulista UNESP, Araraquara, SP,
Brasil.
Fernanda Fernandez Madeira (Presenting author) - Mailing address: Rua Cristovo
Colombo, 2265 Jardim Nazareth - CEP: 15070-040 - So Jos do Rio Preto/SP - Email address: fernanda.bio56@hotmail.com - Telephone contact: (17) 99625-0011
(mobile) (17) 3221-2200 (institucional) Ramal: 2787
Kaio Cesar Chaboli Alevi E-mail address: kaiochaboli@hotmail.com
Ana Letcia Guerra ; E-mail address: analebio@yahoo.com.br
Joo Aristeu da Rosa ; E-mail address: joaoaristeu@gmail.com
Maria Terclia Vilela de Azeredo Oliveira ; E-mail address: tercilia@ibilce.unesp.br
Triatomines are hematophagous insects of great importance for public health,
considered the main form of transmission of Trypanosoma cruzi, etiologic agent of
Chagas disease. In addition to the epidemiological importance, these insects are
important biological models for cell studies because they display holocentric
chromosomes, sex chromosomes with inverted meiosis, and nucleolar persistence
during meiosis. The nucleolar persistence phenomenon is characterized by the
presence of nucleoli or nucleolar corpuscles during all phases of meiosis. This
behavior is quite unique in comparison to other eukaryotes, in which the nucleolus is
fragmented at the end of prophase and rearranged at the end of anaphase. Thus, this
study aimed to analyze if the persistent nucleolus is transcriptionally active. Five
adult males of the species Triatoma infestans, Rhodnius montenegrensis, and
Panstrongylus megistus were dissected, the testicles were removed and, through cell
crushing, slides were mounted and stained with critical electrolyte concentrations
(CEC), which allows the distinction between DNA (green) and RNA (violet). The
analysis of the metaphases of the three species stained with CEC indicated that the
persistent nucleolus has no transcriptional activity, since it was not possible to
observe the presence of RNA in the slides, as already noted, for example, in initial
phase of prophase. Thus, through the initial cytochemical analysis, we found that
although the nucleolus persists during meiosis in triatomines, this nuclear structure is
not transcriptionally active, which shows that new studies are required in order to
assess factors related to this phenomenon present in the cells of these vectors.
Financial Support: FAPESP (Process number 2015/14762-5), CAPES and CNPq.

T25
T26
NUCLEOLAR PERSISTENCE: PECULIAR CHARACTERISTIC OF

SPERMATOGENESIS OF THE VECTORS OF CHAGAS DISEASE
(HEMIPTERA, TRIATOMINE)
DIFFERENT TYPES OF MACROPHAGES IN TISSUE REMODELING IN

THE VENTRAL PROSTATE OF CASTRATED GERBILS (MERIONES
UNGUICULATUS)
Fernanda Fernandez Madeira (1), Kaio Cesar Chaboli Alevi (1), Ana Letcia Guerra
(1), Joo Aristeu da Rosa (2) and Maria Terclia Vilela de Azeredo Oliveira (1)
Nayara Fernanda Costa Castro (1), Luiz Roberto Falleiros Jr.(1), Cssia Regina
Suzuki Caires (1), Sebastio Roberto Taboga (1), Patrcia Simone Leite Vilamaior
(1).

(IBILCE), Universidade Estadual Paulista UNESP, So Jos do Rio Preto, SP,
Brasil. (2) Departamento de Cincias Biolgicas, Faculdade de Cincias
Farmacuticas (FCFAR), Universidade Estadual Paulista UNESP, Araraquara, SP,
Brasil.
Name: Fernanda Fernandez Madeira (Presenting author) - Mailing address: Rua
Cristovo Colombo, 2265 Jardim Nazareth - CEP: 15070-040 - So Jos do Rio
Preto/SP - E-mail address: fernanda.bio56@hotmail.com - Telephone contact: (17)
99625-0011 (mobile) (17) 3221-2200 (institucional) Ramal: 2787
Name: Kaio Cesar Chaboli Alevi ; Mailing address: Rua Cristovo Colombo, 2265
Jardim Nazareth - CEP: 15070-040 - So Jos do Rio Preto/SP - E-mail address:
kaiochaboli@hotmail.com
Name: Ana Letcia Guerra ; Mailing address: Rua Cristovo Colombo, 2265
Jardim Nazareth CEP: 15070-040 - So Jos do Rio Preto/SP - E-mail address:
analebio@yahoo.com.br
Name: Joo Aristeu da Rosa ; Mailing address: Rod. Araraquara - Ja Km 1 Machados - CEP: 14800-901 - Araraquara, SP - E-mail address:
joaoaristeu@gmail.com
Name: Maria Terclia Vilela de Azeredo Oliveira ; Mailing address: Rua Cristovo
Colombo, 2265 Jardim Nazareth CEP: 15070-040 - So Jos do Rio Preto/SP - Email address: tercilia@ibilce.unesp.br
All the species of triatomines are considered potential vectors of the Chagas disease
and the reproductive biology of these bugs has been studied by different approaches,
such as cytogenetic, structural, and ultrastructural analysis. In 1999, nucleolar
persistence during meiosis was observed in the Triatominae subfamily for the first
time. It was noted that these triatomines exhibited a different nucleolar behavior than
the one described for other eukaryotes: the nucleolus persisted during all stages of
meiosis. Recently, it has been observed that all species within the genus Rhodnius
exhibit the same phenomenon, suggesting that it may be a synapomorphy of the
triatomines. Therefore, this study aimed to describe the nucleolar behavior in new
species of at least seven new genera (Cavernicola, Psammolestes, Dipetalogaster,
Eratyrus, Meccus, Mepraia, Nesotriatoma). We analyzed at least two adult male
specimens of 59 species Cavernicola (1), Psammolestes (1), Dipetalogaster (1),
Eratyrus (1), Meccus (2), Mepraia (1), Nesotriatoma (1), Rhodnius (14),
Panstrongylus (2) and Triatoma (35). The analysis of the nucleolar behavior during
the meiosis of these bugs has highlighted that all species feature the nucleolar
persistence phenomenon, and the results were the same for all triatomines. Recently,
it has been suggested that nucleolar persistence may be fundamental for the
spermatogenesis of these vectors, since it is related to the formation of the
chromatoid body. Therefore, we emphasize that this phenomenon is a peculiarity of
the Triatominae subfamily and that further studies are required in order to analyze if
the nucleolar material that persists is active or not.
(1) Department of Biology, Institute of Biosciences, Letters and Exact Sciences, So

Paulo State University IBILCE/UNESP, So Jos do Rio Preto, So Paulo, Brazil;
nayaracastro2011@gmail.com, (55) (17) 3221-2511. Rua Cristovo Colombo, 2265,
CEP 15054-000, So Jos do Rio Preto, So Paulo, Brazil.
The deprivation of androgens may occur through orchiectomy (surgical castration),
a process that can suppress the levels of testosterone. Thus, this study evaluated the
effects of castration on prostate histophysiology, focusing on the observation of the
role of different cell types, in specially macrophages, in the process of tissue
remodeling. Were used 40 gerbils (Meriones unguiculatus) adult males divided into
two groups: control and castrated (euthanized on the 3rd (Ca3d), 7th (Ca7d) and 14th
(Ca14d) day after orchiectomy). The ventral lobe of the prostate of these animals was
removed, weighed and processed for light microscopy. Stereological, morphometric,
immunocytochemical and immunofluorescence analyzes were performed. The data
indicate that castration influenced the changes on the relative ratio of the tissue
compartments of the ventral prostate gland in different experimental groups. The
stereological analysis indicate a decrease in the epithelial compartment in Ca3d and
Ca7d groups compared to other groups. We observed a decrease in epithelial height
in castrated compared to controls. Immunofluorescence for -actin allowed
observing the increase in the thickness of CML in Ca14d group. Was observed also
an increase in the number of macrophages (stromal and intraepithelial) of different
populations (M1 and M2), cell types that alter the stromal microenvironment and
modify the interaction between epithelium and stroma. Thus, the results contribute to
a greater understanding of the action of different cell types in the process of tissue
remodeling forward the realization of orchiectomy.
CEUA protocol: No. 123/2015. Financial support: CNPq, CAPES e FAPESP.
Financial Support: FAPESP (Process number 2015/14762-5), CAPES and CNPq.
T27
T28
NEONATAL EXPOSURE TO ETHYNYLESTRADIOL INCREASES

VENTRAL PROSTATE GROWTH AND PROMOTES EPITHELIAL
HYPERPLASIA AND INFLAMMATION IN ADULT MALE GERBILS
CHRYSIN AS AN ENDOCRINE DISRUPTER IN THE DEVELOPMENT OF

PROSTATE MALE MONGOLIAN GERBILS
Luiz Roberto Falleiros-Jnior (1), Ana Paula da Silva Perez (2), Neusa Alexandra
Zavanelli Martins Falleiros (1), Nayara Fernanda da Costa Castro (1), Fernanda
Cristina Alcntara dos Santos (3), Sebastio Roberto Taboga (1), Patrcia Simone
(1)
Leite Vilamaior (1).
(1) Department of Biology, Institute of Biosciences, Letters and Exact Sciences, (2)
So
Paulo State University IBILCE/UNESP. Rua Cristovo Colombo, 2265, CEP
15054-000, So Jos do Rio Preto, So Paulo, Brazil
(3)
(2) Department of Morphology, Federal University of Gois, Jata, Brazil.
(3)Department of Histology, Embriology and Cell Biology, Federal University(4)of
Gois, Goinia, Brazil.
(5)
Email: faleiros@ibilce.unesp.br, Phone number + 55 17 3221-2511, + 55 17 988078895 (mobile).
Prostate development occurs under the influence of an androgen and estrogen
regulated and precise control, so sensible interferences may predispose this gland to
developing diseases such as benign prostatic hyperplasia and cancer during adult and
senile life. The aim of this study was to analyze morphologically the ventral prostate
of adult gerbils exposed to ethynylestradiol (EE) during the first week of prenatal
development. To this, we employed morphological, stereological-morphometrical,
immunohistochemical and ultrastructural methods. The results showed that the
postnatal exposure to EE duplicated the prostatic complex weight, increasing the
relative frequency of epithelial and stromal compartments, besides the secretory
activity of the ventral lobe of the prostate. All glands exposed to EE showed strong
stromal reshuffling and some foci of epithelial hyperplasia and inflammatory
infiltrated in both luminal and epithelial or stromal compartments. Cells positive for
AR and PCNA increased into the epithelial and stromal tissues. ER-positive cells,
which are normally found into stromal compartment of intact prostates, were
frequently observed in the prostatic epithelial of treated animals. This study
demonstrated that the exposure to EE during the postnatal development causes
histophysiological alterations of this gland, predisposing to the development of
prostatic lesions during life. These results are important taking account public health,
considering the EE has been largely used by women worldwide. Moreover, the
bioaccumulation of this chemical has been increased in different ecosystems. CEUA
protocol: No.061/2012.
Financial support: FAPESP.
Cssia R S Caires (1), Ana P S Perez (2), Nayara F C Castro (1), Sebastio R Taboga
(1,3), Patrcia S L Vilamaior (1)
(1) Department of Biology Institute of Biosciences, Letters and Exact Sciences,
So Paulo State University - IBILCE/UNESP. Rua Cristovo Colombo, 2265, CEP
15054-000. So Jos do Rio Preto-SP, Brazil.
(2) Federal University of Gois (UFG) Special Institute of Health Sciences,
Medical School, Jata-GO, Brazil;
(3) Institute of Biology, PPG in Cell and Strutural Biology Unicamp, CampinasSP, Brazil.
ca.scaires@gmail.com/ Phone number: (17) 32212511 (17) 991191030 (mobile)
The chrysin, a flavonoid present in high levels in honey and propolis, has capacity to
bind the estrogen receptors and plays anti-estrogenic effects, interfering in activity of
aromatase, considered a potential endocrine disruptor. Our purpose was to evaluate
the effects of neonatal exposure to chrysin on ventral prostate in adult male gerbils
determining the frequency (%) of cells positive for ERS1, ERS2, PCNA e p63. In
treated group (CR), females received 50 mg/ kg/ day of chrysin diluted in vehicle,
from 1 to 14 day of lactation period. Vehicle group (CV), females received only
corn oil. In control group (CO), females no received treatment. The male offspring
were euthanized at 120 days old and ventral prostate was removed for histological
processing and subjected to immunohistochemical reactions. The PCNA frequency
positive cells significant increase in CR and CV when compared to CO. The basal
cell was not changed by the treatment, because we verified the p63 frequency
unchanged. The immunoreactivity for ERS1 no altered in stromal and epithelial
compartments of experimental groups, while for ERS2 had increased in both
compartments of the CR group. The exposure to chrysin during the neonatal period,
promoted the increase proliferative cells, but not observed prostatic disorders in
adult, showing the action of this compound as an endocrine disruptor. Further studies
should be made to add to this disclosed herein and thereby conclude the effect of
chrysin in the prostate of Mongolian gerbils.
Ethical Approval: CEUA 107/2015
Financial support: Capes/Cnpq/FAPESP


T29
T30
SPERMATOGONIAL KINETIC OF MARMOSETS CAN BE DIVIDED INTO

VI STAGES AS IN HUMANS
SATURATED LIPIDS INTAKE FROM WEANING FAVORS THE

DEVELOPMENT OF LESIONS IN THE PROSTATE OF MONGOLIAN
GERBILS AT ADULTHOOD
Andr Lucas Caldeira-Brant, Fernanda R.C.L. Almeida, Hlio Chiarini-Garcia
Mariana Marcielo de Jesus1,2, Ana Carolina Negrin1,2, Rejane Maira Ges1,2
Department of Morphology, Federal University of Minas Gerais, Belo Horizonte,

MG, Brazil
E-mail: andrelucao@gmail.com. Tel: 55 31 3409-2994; 55 31 99617-1301
Laboratrio de Biologia Estrutural e Reproduo, Departamento de Morfologia, ICB
UFMG.
Av. Antnio Carlos, 6627, Pampulha. CEP 31.270-901 Belo Horizonte, MG, Brasil
The marmoset Callithrix penicillata has been widely used in biomedical research due
to its closely phylogenetic relation to humans, which makes it an interesting
experimental model. In the reproductive field, their testes present seminiferous
epithelium organized in multi-stage tubules, despite having different number of
stages of the seminiferous epithelium cycle - SEC (marmoset IX and human VI). The
present work intend to re-evaluate, in a comparative way, the SEC under a plastic
embedding method called High Resolution Light Microscopy (HRLM), focusing on
spermatogonial lineage. Five adult male marmosets were fixed by heart perfusion
with glutaraldehyde and testes were routinely processed following HRLM protocols,
as previously standardized by our group. First, the IX stages of marmoset SEC was
redefined into VI stages. After that, spermatogonial subtypes were characterized
based on their nuclear morphology and kinetics along both SEC. Spermatogonial
kinetic at both SEC presented the same pattern. Adark spermatogonia maintained their
population constant along the SEC while Apale spermatogonia varied their number
considering that they split into B1 spermatogonia (B1). Subsequent germ cell
divisions were B1 to B2 spermatogonia and B2 to preleptotene primary spermatocyte,
when the peak of each one was almost the double of the predecessor cell, confirming
that one type was splitting into another one. Thus, we observed after high resolution
light microscopic evaluation that original spermatogonial kinetics of marmoset in IX
stages can be adapted into VI, like in human, without changing their pattern along
the seminiferous epithelium cycle.
Financial Support: FAPEMIG, CNPq
Key-words: spermatogonial kinetics, testicular morphometry, marmoset, primate.
Ethical Approval: CETEA/UFMG n077/03
Department of Functional and Structural Biology, Institute of Biology, University of

Campinas (UNICAMP), Campinas, SP, Brazil.
2
Department of Biology, Institute of Biosciences, Humanities and Exact Sciences,
Univ Estadual Paulista (UNESP), Rua Cristvo Colombo, 2265, Jardim Nazareth,
15054-000, So Jos do Rio Preto, SP, Brazil.
E-mail: marimarcielo@hotmail.com, phone numbers: +55 17 32212200 (branch line:
2733), +55 17 981141129 (mobile).
Clinical and experimental studies have correlated obesity with higher incidence of
prostatic diseases; however, the mechanisms responsible for these alterations are not
totally clear. This study investigated the effects of a high-fat diet intake on the tissue
response and lesions incidence in the gerbil prostate at adulthood. Male gerbils (4w
old) were randomly divided into groups Control (C) and Diet (D). Animals (n=10)
were fed a balanced or high-fat diet (31.2% saturated lipids), respectively, for 12
weeks. Gerbils (16w old) were killed, and the ventral prostatic lobe was processed
for paraffin embedding. Glucose levels and lipid profile in blood were evaluated.
Serial prostate sections were stained with HE and Gmris reticulin, submitted to
immunohistochemistry for PCNA, and evaluated by incidence of lesions (n=25
microscopic sections/group). Although the body weight and adiposity index have not
altered between the groups, the high-fat diet intake increased the body weight gain,
retroperitoneal fat deposit, and glucose, total cholesterol, and non-HDL cholesterol
levels at adulthood. There was no alteration in the prostatic weight and cell
proliferation index between the groups. The high-fat diet intake promoted the
development of reactive hyperplasia, PIN, and microinvasive carcinoma in 24%
(6/25), 12% (3/25), and 4% (1/25) of all microscopic sections, respectively. No
lesion was found in the ventral prostate in the C group. These data show that the
saturated lipids intake induces dyslipidemia and damage to prostate health without
interfere in the proliferative activity of gland. CEUA protocol 93/2014. Financial
support:
FAPESP (grant #2014/03300-8), CNPq (grant #147426/2014-6, #308367/2014-6),
CAPES.
T31

T32
IN UTERO EXPOSURE TO DI-N-BUTYL PHTHALATE CAUSES

DYSLIPIDEMIA AND ALTERS SPERM PARAMETERS OF MONGOLIAN
GERBILS
B. JARARACA SNAKE VENOM COMPOUNDS CHANGE THE SERTOLI

CELL FUNCTION, AN IMPORTANT CELL TYPE OF BLOOD-TESTIS
BARRIER
Ana Carolina Negrin1,2, Mariana Marcielo de Jesus1,2, Caroline Maria Christante1,

Maria Etelvina Pinto Fochi2, Rejane Maira Ges1,2.
Arajo, A. C (1,2), Franzin, C.S (1), Simo-Santos, R. B (1), Querobino, S.M (1,3),
Alberto-Silva, C (1).
Departament of Structural and Functional Biology, University of Campinas

(1)
(UNICAMP), Campinas, So Paulo, Brazil.
2
Departament of Biology, Institute of Biociences, Humanities and Exact Sciences,
Univ Estadual Paulista (UNESP). Rua Cristovo Colombo, no. 2265, Jardim
(2)
Nazareth, 15.054-000, So Jos do Rio Preto, So Paulo, Brazil.
(3)
E-mail: acnegrin@gmail.com. Phone numbers: +55 17 32212200 (branch line:
2733), +55 17 991756727 (mobile).
(1) Morphophysiology Experimental Laboratory, Natural and Humanities Sciences

Center (CCNH), Federal University of ABC (UFABC), So Bernardo do Campo, SP,
Brazil.
(2) Metodista University of So Paulo, Biomedicine undergraduate student, So
Bernardo do Campo, SP, Brazil
(3) Graduate Program in Neuroscience and Cognition, Mathematics, Computing and
Cognition Center, Federal University of ABC (UFABC), So Bernardo do Campo,
SP, Brazil.
Toxicological studies indicate di-n-butyl phthalate (DBP) induces reproductive

anomalies when administered during sexual differentiation and may interfere in
adipogenesis and lipid metabolism. This study evaluated the consequences of in
utero exposure to DBP for testicular structure and function in gerbil. Male gerbils
(120d old) were assigned into (C) control (no treatment), (DBP) DBP and (O)
vehicle-treated groups (n=10 per group). 0.1ml of corn oil containing or not DBP
(100mg/kg/day) was administrated to pregnant gerbils from 8th to 23rd gestation day,
by gavage. The main endpoints examined were blood lipid profile, adiposity index,
testes histology, and sperm parameters. There were no alterations in body weight and
adiposity index in O and DBP groups, neither in the testes and epididymis weights.
Triglycerides levels increased in DBP group (C:101.639.8; O:127.963.5;
DBP:179.579.0mg/dL) and non-HDL levels (C:24.45.6; O:44.217.0;
DBP:42.516.7mg/dL) increased in both O and DBP groups, but total cholesterol
and HDL levels were unaffected. O and DBP animals exhibited normal testis
histology, but some seminiferous tubules showed a premature detachment of germ
cells. The expression of connexin 43 protein did not alter among the groups. There
was no change in daily sperm production, but the sperm reserve in the epididymis
decreased from 360.257.1 x106 in C to 285.442.8 x106 in O and to 280.947.3
x106 in DBP groups. Moreover, the sperm motility was negatively affected by both
exposures. In conclusion, DBP-exposure did not impair drastically the testicular
structure, but caused dyslipidemia and reduced epididymal sperm concentration and
sperm motility.
Background. Snake venom comprises a series of bioactive peptides, as BradykininPotentiating Peptides (BPPs). We have shown that some BPPs from B. jararaca (Bj)
venom induce intense disruption of the seminiferous epithelium, presence of atypical
multinucleated cells in the lumen and high degree of seminiferous tubule
degeneration. However, there are no reports in the literature of the effects of venom
on the male reproductive system of accidental Bothrops envenomation victims. Here,
we studied the toxicological effects of crude venom (CV), low molecular weight
fraction (LMWF) and three BPPs (10c, <ENWPHPQIPP; 11e, <EARPPHPPIPP;
AP, <EWGRPPGPPIPP) in the Sertoli cell function.
Methods. Cell cultures (15P-1, ATCC CRL-2618) were treated or not with
0.001, 0.01, 0.1, 1 and 10g/ml of CV or LMWF for 3, 6, 12, 24 and 48h. In
addition, BPPs were tested with 0.1, 1 and 10 M for 3, 6, 12, 24 and 48h. Cell
viability was determined using MTT assay. Quantitative values corresponding to
relative viability were analyzed by two-way ANOVA for statistically significant
differences between groups (Tukey's post-test; p<0.05)
Results. Viability assay indicated that CV was cytotoxic in the concentrations greater
than 0.1g/ml after 12h of treatment in compared to the control. Interestingly,
LMWF showed no cytotoxicity effects in all conditions tested, but increased cell
viability at 0.01 and 10g/mL after 12h of treatment. Preliminary data indicated that
BPP-11e 10 M reduced cell viability, while treatment with BPP-AP 1 M increase
cell viability.
Conclusion. Overall, the results suggest that CV and LMWF alter the Sertoli cell
function.
CEUA
Protocol:
093/2014.
2)/CNPq(308367/2014-6)/CAPES.
Financial
support:
FAPESP(2014/04146-
(1)
(2)
T33
T34
MORPHOLOGICAL AND MORPHOMETRICAL VARIATIONS OF THE

SEMINIFEROUS TUBULES AND EPIDIDYMIS IN THE LIZARD Tropidurus
torquatus (TROPIDURIDAE) DURING SPERMATOGENESIS
ANALYSIS
AND
MORPHOLOGICAL
DESCRIPTION
OF
SPERMATHECAE SESARMA RECTUM RANDALL (BRACHYURA ,
GRAPSIDAE , SESARMINAE ) IN SOURE / MARAJ ISLAND
Dbora Silva (1); Adelina Ferreira (2)
Lase Ramos Dos Santos (1), Letcia da Silva Palheta (1), Adriano Biancalana (1).
PhD student in Cell Biology and Structural in the State University of Campinas
UNICAMP, Campinas, So Paulo, Brazil. Corresponding author: Dbora Silva, email: debora.biocel@gmail.com
Adjunct Professor, Department of Biology and Zoology of the Federal University of
Mato Grosso UFMT, Cuiab, Mato Grosso, Brazil. E-mail: adelina@cpd.ufmt.br

Campus Maraj/ Soure, Archipelago Maraj, Soure, Brazil.
Tropidurus torquatus is a neotropical lizard belonging to Torquatus Group, which

has great physiological plasticity, with variations in spermatogenesis in different
locations in Brazil. The objective of the study was to investigate the variations in the
height of the germinative epithelium of the seminiferous tubules, the epididymal
epithelium and gonadosomatic reason during spermatogenesis. They analyzed 52
individuals in one year (June/2012 to May/2013). Samples of the right testis and
epididymis were dehydrated and embedded in plastic resin. Semi-serial sections in
the thickness of 3 m were stained with 1% Toluidine Blue, and examined by light
microscopy. Height average of epithelium was obtained with four measurements per
seminiferous tubules and epididymis, totaling 120 measurements per individual each
month. The gonadosomatic reason was obtained by monthly average for each
individual. Tropidurus torquatus presented discontinuous spermatogenesis with
sperm production during the months of June to February and regression from March
to April. The epithelium of the seminiferous tubules and the epididymis duct showed
variations in their average monthly with maximum value in October and January,
during the phase of spermiation and regression, respectively. The gonadosomatix
reason showed the highest value in October, coinciding with the rise of the
germinative epithelium height, subsequently decreasing during testicular regression
and atrophy of the epididymis epithelium. These changes correspond to the different
stages of spermatogenesis, with prolonged hypertrophy of epididymis epithelium for
spermatozoa storage during testicular regression.
Spermathecae is the pair of structures responsible for storage of sperm secretion. Due
to the lack of published information, this work aims to contribute data about the
reproductive system of Sesarma rectum. The study aimed to analyze the morphology
of the species spermathecae from macro and microscopic observations. The animals
were collected in mangrove Soure / PA, taken to the laboratory, weighed, measured,
photographed, anesthetized by cooling and dissected for macroscopic observation of
the reproductive system and dissection of spermathecae. The material was fixed in
10% formalin, dehydrated in ethanol, cleared in xylene 100%, embedded in paraffin
to prepare slides with histological sections of 7m. The slides were processed and
stained with HE, XP and AT followed by microscopic observation. Macroscopic
analysis has observed predominantly white coloring spermathecae and some with
brown punctiform pigment. Microscopic analysis of the membrane was observed the
presence of four well developed layers. The epithelium that covers the organ ranges
from stratified to simple cuboidal. The underlying layer of the epithelium has
secreting cells. In the organ lumen are observed cellular projections. The lumen has
secretion and free spermatozoa. It was observed that the morphology of
spermathecae of Sesarma rectum is significantly different from that found in other
crabs. The results could contribute to the understanding and elucidation of the
phenomena involved in the reproductive process of Sesarma rectum. In addition to
assisting in the development of consistent management plans.

E-mail: laiseramos_@hotmail.com / Contact (91) 984435266
The procedures adopted were allowed (SISBIO) and financing CNPq / UFPA.
KEYWORDS: Reproduction, Morphometry, Tropidurus, Lizard
Financial Support: CNPq Process number # 140818/2015-4

T35
INFLUENCE OF DICHLORVOS ON THE DEVELOPMENT
PROSTATIC LESIONS INDUCED BY MNU IN RAT PROSTATE
T36
OF
Fernanda Oliveira dos Santos (1), Camila Tiemi Hirakawa Quadrado (1), Giovanna
Galo Quintino (1), Nara Ruiz Lenharo (1), Maria Eduarda Verderio Espindola (1),
Jos Henrique Forte Stornioli (1), Jlia Chiti Pinheiro (1), Brayan Lucas Lelis (1),
Sergio Pereira (1)
(1)
(1) Department of Biological Sciences, School of Sciences, UNESP, Bauru, Brazil
PROLIFERATIVE ACTIVITY OF DORSAL AND LATERAL PROSTATE

OF ADULT WISTAR RATS AFTER EXCESSIVE DIETARY FAT
THROUGHOUT DIFFERENT PERIODS OF LIFE
Tatiane Pereira Scarpelli (1); Elosa Zanin Pytlowanciv (1); Marina Guimares
Gobbo (1); Rejane Maira Ges (1).
(1) Department of Biology - Ibilce, Univ Estadual Paulista (Unesp), So Jos do Rio
Preto, SP, Brazil.
Contact: Department of Biological Sciences, School of Sciences, UNESP, Bauru,

Brazil, spereirafc@gmail.com, (14) 3103-6078, (14) 997761667.
Contact details: Tatiane Pereira Scarpelli. e-mail: tati.scarpelli@gmail.com, Tel.:

+55 17 32212391. Cel.: + 55 17 991161972.
Background: Organophosphate pesticides are very effective, widely used, and pose
great risk to the environment and health of many organisms, including humans.
Dichlorvos (DDVP) is an organophosphate pesticide and it has mutagenic and
carcinogenic potential for humans. Many pesticides, such as DDVP, act as endocrine
disruptors, and may alter endocrine homeostasis. These compounds promote
endocrine and reproductive disorders such as prostate cancer. Aims: The present
study aims to histopathological evaluate of the rat prostate subjected to chemically
induced by N-methyl-N-Nitrosourea (MNU) associated with pesticide DDVP.
Methods: 20 Fisher rats at 90 days old were randomly divided into 4 experimental
groups: sham (S), Sham + DDVP (SD), MNU induction (M) and MNU induction +
DDVP (MD). In groups S and SD, animals were inoculated into the ventral prostate
capsule sodium citrate. Groups M and MD were inoculated (MNU) at 15 mg/kg to
chemical induction. To the groups M and MD, were given subcutaneous injections of
2.5 mg/kg testosterone cypionate daily for 20 days. Groups SD and MD, at 120 to
240 days age received the basal diet supplemented with 10 mg/kg DDVP.
Histopathological analysis were performed.
Results: They were found several types of lesions in the rat ventral prostate, being
more prevalent in the group M is PIA and PIN and in the group MD PIN and
Adenocarcinoma.
Conclusion: The pesticide dichlorvos contributes to promote prostatic lesions in this
chemical induction model.
Excessive dietary fat (EF) favors benign prostate hyperplasia and prostate cancer.
However, the impacts of EF during different periods of life are unknown. This study
examined dorsal and lateral prostate response to EF for cell proliferation and
frequency of androgen receptor (AR), peroxisome proliferator-activated receptor
gamma (PPAR) and liver X receptor alpha (LXR). Male Wistar rats (19wks-old;
CEUA/protocol n.022/09) were subjected to EF in the following periods: gestation
(G), gestation/lactation (GL), from post-weaning to adulthood (WA), from lactation
to adulthood (LA) and from gestation to adulthood (GA). WA, LA and GA groups
were fed with a high-fat diet (HFD; 20% fat) for 15wks and G, GL, LA and GA
groups included HFD-feeding of their mothers 15wks before gestation (maternal
obesity). Prostate lobes were processed for histological and immunohistochemistry
analyses. In the dorsal lobe, epithelial cell proliferation (PCNA-positive cells)
decreased only in GA group, whereas for the lateral lobe, cell proliferation
augmented in G and WA groups. Despite the testosterone plasma levels reduction in
WA, LA and GA, EF did not alter the AR-positive cells frequency in any group. No
immunolabeling for PPAR and LXR was detected for both lobes. Considering that
PPAR and LXR can mediate the lipids action on cells, it is possible that the lower
responsiveness of these lobes to the EF can be explained by their absence or low
expression. The results also suggested that proliferative alterations on lateral lobe of
G and WA are related to other mechanisms not associated to androgen signaling.
Financial support: FAPESP(2011/01612-4)/ CNPq(308367/2014-6).
Information on ethical approval: CEUA/FC/UNESP Processo 996/46/01/2013.

Funding support: FAPESP Processo 2014/07329-0.

T37
T38
COLLAGEN DISTRIBUTION IN PROSTATE OF GLUTAMATE-INDUCED

OBESE RAT
VERSICAN EXPRESSION IS A TISSUE MARKER FOR ABNORMALLY

INVASIVE PLACENTATION
Srgio Marcelino de Oliveira (1), Kallyne Kioko Oliveira Mimura (2).
Jaqueline Correia Santos (1), Camilla Mendes Gonalves (1), Keyla Silva Nobre
Pires (1), Lais Carvalho Silva (1), Karen Priscilla Tezotto Pendelosky Ribeiro (2),
Silvia Daher (2), Sue Yazaki Sun (2),Estela Bevilacqua (3), Alexandre Urban
Borbely (1)
(1) Health and Biological Science Institute ICBS, Federal University of Mato
Grosso UFMT/CUA, Barra do Garas-MT, Brazil.
(2) United Colleges of the Araguaia Valley Univar, Barra do Garas-MT, Brazil.
(4)
email: sergio-marcelino@ufmt.br
Telephone: +55 66 3402 1108
(5)
Studies indicate increased risk of prostate cancer associated with obesity. Some
(6)
elements of extracellular matrix such collagen, glycosaminoglycans and
metalloproteinases (MMPs) are important modulators of tissue homeostasis.
(7)
Imbalance in these components may stimulate cell proliferation, angiogenesis and
malignant development in various tissues including the prostate. Male rats were
treated neonatally with monosodium-glutamate (MSG) at doses of 4 mg/kg, on
postnatal days 2, 4, 6, 8 and 10. On day 120, obesity was confirmed by Lee index.
Prostate was removed and fragments were fixed in Methacarn and prepared for
paraffin embedding. Sections were stained by: haematoxylin-eosin (HE); picrosiriushaematoxylin for collagen evaluation. The stereological analyses were performed
using Weibel's multipurpose graticulate with 130 points (Image-Pro Plus 6.0.0.260)
to compare relative proportion (%) of each stromal prostate compounds (nonmuscular stroma and collagen content). Stroma of obese animals showed apparent
increased volume. Picrosirius-haematoxylin stained tissue showed higher collagen
content in obese rats prostate, when compared to control animals. Stereology
performed in polarized images showed notable high volume of stromal compartment
in prostate from obese animals (24%) when compared to control (20%).
Additionally, amount of collagen in stroma of prostate obese rats (44%) was higher
than in control tissue (39%). Further studies need to be performed but these
preliminary data lead us to conclude that glutamate-induced obesity seems to
promote increase in stromal volume and collagen content in old rat prostate.
(Ethics committee 23108.097747/2015-64 CEUA-UFMT)
(2) Department of Obstetrics, Paulista Medical School, Federal University of Sao
Sciences, University of Sao Paulo, Sao Paulo, Brazil
To whom correspondence should be addressed:Jaqueline Correia Santos, Cell
Biology Laboratory, Institute of Biological and Health Sciences, Federal University
of Alagoas, Campus A.C. Simes. Av. Lourival Melo Mota, s/n, 57072-970. Maceio,
Alagoas, Brazil. Laboratory Phone: ++558232141704. Mobile: +558296473987. Email: jaque-cs@hotmail.com
Background:Versican is a proteoglycan that integrates the extracellular matrix
(ECM) in a variety of tissues. Versican regulates numerous functions, including
proliferation, migration and invasion, which are key events for correct placentation
in a controlled microenvironment. Increased or impaired trophoblast invasion are
associated to different placental pathologies, maternal morbidity and mortality.Aims:
t was aimed to determine versican localization and expression in healthy and
pathological placentas. Methods:Samples included first trimester,premature and term
placentas, as well as early-onset preeclampsias, placenta accreta or abnormally
invasive
placentation
(AIP),invasive
moles
and
choriocarcinomas.
Immunohistochemistry with two different anti-versican antibodies, staining
quantification and RT-PCR assays for versican and its four isoforms V0, V1, V2 and
V3 were performed. Results:V0 and V1 isoforms were expressed in decidual cells
and their ECM in first trimester placenta. Premature, term placenta and
preeclampsias also expressed both isoforms, but only in decidual cells. All isoforms
were expressed exclusively in AIP samples, especially in EVT cells. Invasive moles
were negative for versican. Choriocarcinomas presented few stained cells only with
one of the antibodies. Quantification of stained cells confirmed AIP staining is
statistically significant in relation to other groups.Conclusion:The exclusively
versican expression pattern in AIP suggest this proteoglycan may have an important
role on AIP physiopathology. Its localization only in EVT cells from AIP, the
mRNA expression of all isoforms and immunoperoxidase quantification support
versican expression as very specific tissue marker for AIP that could assist
physicians in current and predictive AIP diagnosis.
Ethical Approval:CAEE 43605515.9.0000.5013
Financial Support: Cell Biology Laboratory from ICBS/UFAL

T40
T39
STRUCTURAL
TESTICLE
CHARACTERIZATION
OF
ASTYANAX
XAVANTE
SELENIUM IN MAINTENANCE OF MORPHOLOGY OF GERBILS

PROSTATE COMPARTMENTS
Andressa Maria Pereira Sargiani (1); Fernanda Cristina Alcntara dos Santos (2),
Sebastio Roberto Taboga (3) Srgio Marcelino de Oliveira (1)
Glaucia Fernanda Martins Fernandes (1), Fernanda Cristina Alcntara dos Santos (2),
Sebastio Roberto Taboga (3) Srgio Marcelino de Oliveira (1).
Goias-UFG, Goinia-GO, Brazil.
(3) Department of Biology, State University of Sao Paulo-IBILCE/UNESP, So Jos
do Rio Preto-SP, Brazil.
Gois-UFG, Goinia-GO, Brazil.
(3) Department of Biology, State University of Sao Paulo-IBILCE/UNESP, So Jos
do Rio Preto-SP, Brazil.
Contact: sergio-marcelino@ufmt.br
email: sergio-marcelino@ufmt.br
Telephone: +55 66 3402 1108
Spermatogenesis of some fish occurs in spermatogenic cysts located in germinative

epithelium of seminiferous tubules. Astyanax xavante, which is endemic of
Avoadeira creek in Barra do Garas-MT, does not show sexual dimorphism. Due to
limited information about the reproductive biology of this species, this work aims to
describe morphological structure of testicular compartments of A. xavante using
optical microscopy techniques. From collections made in April (rainy season) and
September (dry season), animals were dissected and testicles were removed and
fixed in metacarn. Fragments were dehydrated in ascending ethanol drum 70% to
100% for routine histological processing. As expected, only specimens of A. xavante
were found in collections. The fishes analyzed showed an average weight of 3.23g (
2,18 SD), total length of 58,4mm ( 13,6 SD) and standard length of 48,3 mm (
11,96 SD). Testicles were elongates and showed whitish color. Histological sections,
stained with hematoxilin-eosin (HE), showed a large amount of seminiferous tubules,
bounded by germ epithelium that had numerous spermatogenic cysts in which were
found germ cells. Numerous mature spermatozoids were found inside seminiferous
tubules. These preliminary data lead as to conclude that A. xavante has parceled
spawning because spermatogenic cysts and spermatozoids were found in testicles of
animals from both collections.
Ethical approve: 23108.076602/2015-20.
(Financial support: FAPEMAT - Proc. 224710/2015; PRONEX/FAPEMAT/2009)
Prostate can be focus of innumerous proliferative disorders and epidemiological

studies in humans have associated selenium with reduction of cancer incidence. In
order to analyze prostatic compartments under influence of testosterone (T) and
selenium (Se), 15 adult male Gerbil (90 days-old) were divided as followed (05
animals/group): TG, received testosterone administration (1 mg/kg); TSG, received
testosterone administration (1 mg/kg) and doses of selenium by gavage (10 mg/kg);
CG, intact animals. Prostate was removed and fixed in metacarn. Fragments were
processed for routine histological methods. The morphometric-stereological analyses
were performed using Weibel's multipurpose graticulate with 130 points (Image-Pro
Plus 6.0.0.260) to compare relative proportion(%) of prostatic compartments
(epithelium, lumen, muscular stroma and non-muscular stroma). Morphologicalmorphometric analysis showed epithelium stratification and presence of higher
columnar cells in TG (45,2m/1,2SE) when compared to CG (30,1m/1,0SE) and
TSG (35m/0,9SE). Stereological analysis showed that volume occupied by
epithelium in TG (24.18%/2,3SE) and TSG (25.22%/0,9SE) was higher than in GC
(19.98%/0,9SE). Furthermore, relative non-muscle stroma volume in TG
(30.99%/3,5SE) was higher than in TSG (21.57%/1,2SE) and CG (20.64%/1,5SE).
Although the results presented show a greater height and epithelial volume in TG
and TSG groups it is important to emphasize the lower volume of non-muscle stroma
in TSG group because studies show an interrelationship between increase in volume
of this prostatic compartment with the development of proliferative lesions. Thus,
these data suggest that selenium can be an important factor in maintenance of
extracellular compounds amounts, which can improve prostatic lesions prevention.
Ethics committee: 23108.076606/2015-16. (PRONEX/FAPEMAT/2009)

T41
T42
AGING: THE RELATIONSHIP BETWEEN THIS PHYSIOLOGICAL

CONDITION AND STRUCTURAL ALTERATION OF MAMMAL'S
CHROMATOID BODIES
A BIOINFORMATIC AND BIOCHEMISTRY APPROACH TO IMPROVE

OOCYTE SELECTION
Elisa Gomes Santos1,2, Maraisa Alves2,3, Wilson Aparecido Orcini2, Rita Luiza
Peruquetti1,2,3.
1
Programa de Ps-graduao em Odontologia Sade Coletiva, University Sagrado

Corao, Bauru, Sao Paulo, Brasil.
2
Laboratrio de Biologia Molecular e Citogentica, University Sagrado Corao,
Bauru, Sao Paulo, Brasil.
3
Centro de Cincias da Sade, University Sagrado Corao, Bauru, Sao Paulo,
Brasil.
Correspondent author
+5516981401987
information:
rita.peruquetti@usc.br
Fone
number:
Introduction: Chromatoid body (CB) is a cytoplasmic structure of male germ cells

playing a role in the mRNA regulation during the spermatogenesis. Recent proteomic
analysis revealed 100 CB-associated proteins and it showed that the most abundant
proteins in the CB molecular composition are MVH and MIWI. CB proteome also
included nuclear proteins being transiently translocate to the cytoplasm. Among all
those transient proteins, the circadian proteins CLOCK and BMAL1 called our
attention. Aims: The aim of the study is to find out if the morphological alteration
previously observed in spermatids of BMAL1 KO mice are due to the ablation of
BMAL1 in those models, or if they are related to the aging process that this ablation
produces. Methods: 30 male mice were placed into 3 groups: I: Young (45 days old);
II: Adult (120 days old); III: Old (180 days old). Seminiferous tubules at stages IVVI were taken from all the groups, and they were used to prepare squash slides.
Detection of MVH and BMAL1 was performed by immunofluorescence. Results and
discussion: Preliminary data showed a different pattern in the distribution of MVH in
the cytoplasm of group III spermatids, such as decrease of MVH concentration in the
CB as well as CB morphological and numerical alteration. BMAL1 detection
decreased with time in the cytoplasm of first spermatocytes as well as in the
cytoplasm and CBs of round spermatids. Conclusion: These results show that aging
may have influence on the distribution pattern of MVH and BMAL1 in the male
germ epithelium as well as in the CB assembling.
Ethics committee approval: CEUA-USC (7445240415 and 1192290515). Keywords:
Chromatoid body; Cronobiology; CLOCK; BMAL1; Aging; Fertility.

T43
Flvia B. Constantino(1), Sergio A.A. Santos(1), Ana.C.L. Camargo(1), Ketlin T.
Colombelli(1), Carolina N. Barquilha(1), Suelen Franco (1), Jaqueline C. Rinaldi(1),
Sergio L. Felisbino(1), Luis A. Justulin(1).
1Department of Morphology, Institute of Biosciences, So Paulo State UniversityUNESP- Botucatu - Brazil. flaviabessi@ibb.unesp.br (14) 981269312
Introduction: The prostate development and homeostasis are under androgen control.
However, non-steroidal hormones have been characterized by acting in the prostate,
including prolactin (PRL). AIM: To investigate if the PRL administration alters
ventral prostate (VP) structure in adult castrated rats subjected to the testosterone
replacement (TR). Methods: Male Sprague Dawley rats (n=6/group) was castrated
and after 21 days, these animals received daily injection of prolactin (0,3mg/kg) for
03 or 10 days, associated or not with TR (4mg/kg). After that, the VP lobes were
removed and processed for histology or western blot analyses. Results: The body
weight did not change between experimental groups. Castration reduced VP weight
compared control group. Three days of TR elicited VP regrowth, but only after 10
days of TR, the VP weight was restored to the values of control. PRL treatment did
not recovery VP weight or morphology in castrated rats. Castration reduced the
Stat5a/b expression compared to control group, whereas PRL or TR restored the
Stat5a/b expression. However, PRL associated with TR did not cause an increment in
the expression of Stat5/a. Conclusion: Although PRL acts as growth factor for VP,
the PRL administration at 0,3mg/kg was not sufficient to induce prostate regrowth in
castrated rats. When PRL was administered associated with TR, we did not observe
an increment in prostate growth. These data can be attributed to supraphysiological
dose of TR administered or that PRL was not sufficient to induce an incremental VP
growth in castrated rats submitted to androgen replacement.
Laboratory of Cellular Biochemistry, ICBS/UFRGS, 90035-003 Porto Alegre (RS),

Brazil;
ProSer Fertility Clinic, Porto Alegre (RS), Brazil.
Background Cumulus oophorus cells surround and have an intimate relationship
with the oocyte, whose activity influences directly gene expression and cellular
processes. Since these cells are discarded after in vitro fertilization techniques, they
can be collected noninvasively and used to evaluate oocytes quality.
Aims: Select and evaluate, in cumulus cells, biological processes that indicates
oocytes quality.
Methods: Using public NCBI microarray data (GEO 37277), we analyzed the
processes involving genes differentially expressed in cumulus samples obtained from
follicles classified according to the embryo development: samples from follicles
containing oocytes that generated blastocysts were classified as good quality and
those that did not were classified as poor quality. Following the processes
highlighted and literature, our group examined the redox profile of cumulus samples
from 48 patients submitted to fertilization treatments (ethical committee approval
#29577). We analyzed enzymatic (catalase, glutathione peroxidase and glutathione-stransferase activities), and non-enzymatic (reduced glutathione, sulphydryl levels and
TRAP) antioxidant levels.
Results: Cellular processes upregulated in good quality samples were associated with
plasticity and development. However, genes upregulated in poor quality group
pointed to an unfavorable environment, highlighting metabolism of reactive oxygen
species. The redox imbalances were validated in vitro using the same endpoint.
Conclusion: Cumulus cells gene expression is a reflection of their follicular
microenvironment. There is no agreement between studies suggesting single genes as
biomarkers. Therefore, we propose analyzing processes to identify oocyte quality,
such as the redox profile, since bioinformatics approach successfully recognized
indications of potentially good quality oocytes in cumulus cells.
Support MCTI/CNPq Universal (476114/2008-0)
T44
IMPACT OF PROLACTIN ADMINSTRATION ON THE STRUCTURE AND

STAT5A/B SIGNALING IN CASTRATED RAT VENTRAL PROSTATE
Financial support: FAPESP, CAPEs

CEUA N 767
Lcia Meirelles, Marco A. De Bastiani, Eloisa T. Massignam, Letcia S. Arruda,

Carlos Link, Fabio Klamt
ANTI-INFLAMMATORY AND ANTIANGIOGENIC THERAPIES IN THE

TRANSGENIC ADENOCARCINOMA OF THE MOUSE PROSTATE
(TRAMP) MODEL: EFFECTS ON CANCER PROGRESSION, AR
DISTRIBUTION AND MICROVESSEL DENSITY
Pedro Augusto Marischka Mateus; Larissa Akemi Kido; Rafael Sauce; Valria
Helena Alves Cagnon; Fabio Montico
Presenting author. E-mail: larissa.kido@gmail.com. Telephone: +55 19 3521-6103
(Institutional) / +55 19 98294-7894 (Mobile). Address: Department of Structural and
Functional Biology, Institute of Biology, University of Campinas (UNICAMP),
Corresponding author. E-mail: fabio260687@gmail.com. Telephone: +55 19 3521
6103.
Background: Chronic inflammation has been implicated in cancer etiology and antiinflammatory drugs such as celecoxib have been analyzed as chemotherapic agents.
Angiogenesis is also stimulated in cancer, forming new blood vessels to supply
tumor metabolic demands. Antiangiogenic agents such as nintedanib have emerged
as therapeutic options for delaying tumor progression, including in the prostate.
Aims: To evaluate cancer progression, AR distribution and CD31-positive
microvessel density in the ventral prostate of TRAMP mice following antiinflammatory and/or antiangiogenic therapies. Methods: Male TRAMP mice (12
week-old) were divided into the groups: Control (TRCON): received the vehicles
used for drug dilution; Celecoxib (TRCEL): received celecoxib (15 mg/kg);
Nintedanib (TRNTB): received nintedanib (10 mg/kg); Nintedanib + Celecoxib
(TRNTCEL): received the combination of treatments. After 6 weeks, samples were
collected for morphological and immunohistochemical analyses. Results: Nintedanib
significantly reduced the incidence of well-differentiated adenocarcinoma (PC) foci
in relation to controls, both when administered per se or in association to celecoxib.
Combined therapy also resulted in significant decrease of high-grade pre-malignant
lesions (HGPIN). Such effects observed in TRNTB and TRNTCEL groups were
associated with lower AR distribution in the prostatic epithelium. Moreover, the
latter group also presented significant decrease of microvessel density. Conclusion:
Nintedanib is an effective antitumor agent against prostate cancer progression in
TRAMP mice and may act through its negative influence on AR expression.
Additionally, its association to celecoxib showed the beneficial effects of combining
anti-inflammatory and antiangiogenic strategies to prevent or delay prostatic
tumorigenesis.
(Ethical approval: Unicamp n3807-1. Support: Fapesp and SAE/Unicamp).


T45
T46
EFFECT OF HETEROPTERYS TOMENTOSA (A. JUSS.) INFUSION ON THE

EPIDIDYMIS OF 15 MONTHS OLD WISTAR RATS: SPERM COUNT,
MORPHOMETRY AND ULTRASTRUCTURE
COULD SIMVASTATIN CHANGE THE MORPHOLOGY OF PROSTATE

OF WISTAR RATS?
Camila Merino (1), Maria Aparecida S. Diamante (2), Celina A. Lamas (2), Juliana
Castro Monteiro (3), Heidi Dolder (2), Fabricia de Souza Predes (1).
(1) State University of Paran Campus Paranagu, Paranagu, Paran, Brazil. Rua
Comendador Correia Junior, 117, Centro, Paranagu, Paran, Brasil
(3) Department of Biological and Agricultural Sciences, Federal University of
Esprito Santo, So Mateus, Esprito Santo, Brazil.
merrino.c@gmail.com, Institutional number 55 41 3423-3644, Mobile number 55 41
92010117
Nayara Moraes Rodrigues1, Letcia Prado Oliveira2, Edson Rosa Pimentel2, Heidi
Dolder2, Marcos de Luca Moreira Gomes3, Juliana Castro Monteiro Pirovani*
1
Department of Agrarian and Biological Sciences, UFES, So Mateus, Esprito

Santo, Brazil.
2
Department of Structural and Functional Biology, UNICAMP, Campinas, So
Paulo, Brazil.
3
Department of Structural Biology, UFTM, Uberaba, Minas Gerais, Brazil
Nayara Moraes Rodrigues CEUNES-UFES, Rodovia BR 101 Norte, Km 60, Bairro
Litorneo So Mateus ES, Brasil - Telefone: (27) 3312-1501 (e-mail do autor:
nayara_moraes-R@hotmail.com).
With the increased life expectancy of man it becomes important to maintain fertility
during aging. However, little is known about the epididymis changes and the effect
on sperm maturation. The objective of this study was to evaluate the effect of aging
in the epididymis and the contribution of Heteropterys tomentosa infusion. This
evaluation was performed using sperm count, morphometric and ultrastructural
analysis. Two groups of 12 animals with 15 months of age were treated by gavage.
The control group (C) received 0.5 ml/day of water and the group H. tomentosa (Ht)
received a dose of 104 mg/kg/day of infusion. After 70 days, the animals were
euthanized under anesthesia. From six animals of each group fresh testis and
epididymis were collected for the sperm count. The other animals were perfused and
fixed; the right epididymis was collected and processed for light and transmission
electronic microscopy. All statistical analyses were carried out using Statistic 8
(ANOVA and Tukeys Test).There was a significant increase in the number of sperm
only in the epididymis caput of the Ht group compared to the control group. The
sperm transit time and the epididymal epithelium height did not show significant
changes. The preliminary ultrastructural analyses showed the presence of a great
number of multivesicular bodies, lipid droplets, electron dense bodies and myelin
figures in both groups. Further evidence must be obtained to conclude that the use of
the root infusion of H. tomentosa is effective to improve the performance of aged
epididymis.
Background: Statins are the class of drugs which reduces plasma cholesterol
concentrations by inhibiting the enzyme HMG-CoA reductase in the liver thereby
reducing the LDL levels. The simvastatin is a drug of the statins family, frequently
used the treatment the hypercholesterolaemia, coronary disease and hyperlipidemia.
The prostate is an accessory gland of male reproductive system, it responsible for
store and secrete the prostatic fluid, providing to sperms ideal conditions of survival
and viability during and after ejaculation. This organ is regulated by testosterone,
which is synthesized from cholesterol. Aim: This study was undertaken to evaluate
the effects of simvastatin administration on the prostate of Wistar rats. Methods:
Fifteen males were divided into three experimental groups: S-20, S-80 (treated with
20mg and 80mg respectively); and control group (carboximetilcelulose 0.05%). The
simvastatin dose was calculated by allometric extrapolation according the metabolic
rate of the animals. Treatments were administered daily by gavage, for two months.
Animals were weighed and euthanized; the prostate was removed, dissected and
processed for paraplast embedding. Morphometric and stereological analyzes were
performed using Image ProPlus software. This study was approved by
CEUA/UNICAMP (2473-1). Results: The prostate weight, volumetric proportions
(%) and volume of prostate components were not altered. Therefore, we assume that
the long term administration of simvastatin at 20 and 80mg/kg was not capable of
altering significantly the prostate parameters analyzed so far. Conclusion: Further
histopathological analyzes must be performed to confirm these results.
Ethical approval: 2177-1.
Financial Support: PIIC/UFES
Financial support: Fundao Araucria, CAPES and FAPESP.

T47

T48
MORPHOLOGICAL ANALYSIS OF WISTAR RATS TESTIS TREATED

FOR 15 DAYS WITH YERBA MATE AND CADMIUM CHLORIDE
VERSICAN
ROLE
IN
TROPHOBLAST
DIFFERENTIATION:
IMPLICATION IN GESTATIONAL TROPHOBLASTIC DISEASES
Ianny Brum Reis (1), Luciana Shultais Alto (2), Niumaique Gonalves (2), Juliana
Castro Monteiro Pirovani (2), Mary Anne Heidi Dolder (1)
Keyla Silva Nobre Pires (1), Lais Carvalho Silva (1), Jaqueline Correia Santos (1),
Camilla Mendes Gonalves (1), Silvia Daher (2), Rosiane Mattar (2), Sue Yazaki
Sun (2), Estela Bevilacqua (3), Alexandre Urban Borbely (1)
(1) Departamento de Biologia Celular e Funcional, Unicamp, Campinas, SP, Brazil

(2) Departamento de Cincias Agrrias e Biolgicas, UFES, So Mateus, ES, Brazil
(8)
Correspondent Author Information: Ianny Brum Reis, Universidade Estadual(9)de
Campinas, Departamento de Biologia Celular e Funcional, Instituto de Biologia,
Campinas, SP, (19) 35216116 iannybrumreis@yahoo.com.br.
(10)
Ilex paraguariensis (yerba mate) has proved to be useful as an effective anti-oxidant
for many different systems. These effects have been attributed to the phenolic
compounds present in this plant. Cadmium is a toxic metal and an environmental and
industrial pollutant. Studies showed that it affects the male reproductive system of
humans and animals. This study was undertaken to evaluate the effect of I.
paraguariensis infusion on the testis of Wistar rats treated with cadmium using
morphological analysis (protocol 3898-1). Twenty-five animals were divided into
five groups: I. control (water), II. yerba mate, III. cadmium, IV. cadmium and yerba
mate (15 days), V. yerba mate (for 15 days) and cadmium chloride (single dose after
15 days). A single dose of cadmium chloride (1.2 mg/kg BW) was injected i.p. in
adult rats (groups III, IV and V). The yerba mate used in this research was
commercial tea (110mg yerba mate/1000mL water). Each animal received 0.5mL of
either water (groups I and III) or infusion (groups II and IV and V), during 15 days.
After the treatment, animals were anesthetized with Xylazine and Ketamine
(5:80mg/kg). The testis were removed, weighed, fixed and routinely processed for
light microscopy. The group treated with cadmium lost some weight after treatment
compared to the other groups. Stereological parameters (volume and volumetric
density of testicular parenchyma compounds) were not altered. Histopatological
analysis clearly showed damages induced by cadmium, such as, germ cell damage.
Cell differentiation and spermatozoa development were not observed in affected
testicles.
(2) Department of Obstetrics, Paulista Medical School, Federal University of Sao
Sciences, University of Sao Paulo, Sao Paulo, Brazil
To whom correspondence should be addressed: Keyla Silva Nobre Pires, Cell
Biology Laboratory, Institute of Biological and Health Sciences, Federal University
of Alagoas, Campus A.C. Simes. Av. Lourival Melo Mota, s/n, 57072-970. Maceio,
Alagoas, Brazil. Laboratory Phone: ++558232141704. Mobile: +558299323415. Email: keylanobrep@gmail.com
Background: Gestational trophoblastic diseases (GTD) comprise partial, complete
and invasive moles, choriocarcinoma, placental-site trophoblastic tumor and
epithelioid trophoblastic tumor. GTDs are characterized by abnormal trophoblast
proliferation and differentiation in benign tumours, but also invasion in the
malignant. Versican is a proteoglycan that integrates the extracellular matrix in a
variety of tissues and it is known to regulate several cell functions, amid them
differentiation. Aims: It was aimed to determine versican location in molar
pregnancies and if its expression influentiates trophoblast differentiation. Methods:
Samples included first trimester placenta, partial moles, complete moles, invasive
moles and choriocarcinomas. Immunohistochemistry for versican and staining
quantification in chorionic villi (CV) compartment were performed. The
choriocarcinoma-derived BeWo cell line was employed for flow cytometry,
immunofluorescence and differentiation assay with forskolin. Results: Versican
stained faintly in syncytiotrophoblast from first trimester CV. In partial and complete
moles, versican was moderately stained both in cytotrophoblast in
syncytiotrophoblast. Invasive moles had absent versican staining and
choriocarcinoma stained scarcely in some neoplastic cells that resembled
syncytiotrophoblast. BeWo only expressed versican when differentiated into
syncytiotrophoblast as showed by flow cytometry and immunofluorescence.
Versican silencing regulated BeWo differentiation into syncytiotrophoblast.
Conclusion: Versican seems to have a regulatory role in trophoblast differentiation,
particularly in GTDs with abnormal differentiation. The reason why invasive moles
have absent versican expression remains unclear. Further studies need to be
conducted in order to access its function in a molecular level.
Ethical Approval: CAEE 43605515.9.0000.5013.
Financial Support: Cell Biology Laboratory from ICBS/UFAL

T49
T50
CARACTERIZATION OF THE INTERHEMAL BARRIER FORMATION

AND THE POTENTIAL OF CASPASE-8 EXPRESSION IN SYNCYTIAL
LAYER DEVELOPMENT OF MICE PLACENTA
EVALUATION OF THE CELL POPULATION OF THE SEMINIFEROUS

EPITHELIUM OF ADULT WISTAR RATS AFTER EXPOSURE TO
SODIUM ARSENITE AND SUPPLEMENTATION WITH VITAMIN C AND
ZINC
Gabriela Aparecida Jorge Daolio (1), Aline Rodrigues Lorenzon-Ojea (1),

Estela Maris Andrade Forell Bevilacqua (1).
(1)
(1) Department of Cell Biology and Development, University of So Paulo, So

Paulo, Brazil.
(2)
Departmento de Cincias Farmacuticas, UFES, Vitria, Brasil,

Departmento de Biologia Estrutural e Funcional, UNICAMP, Campinas, SP, Brasil,
Departmento de Cincias Agrrias e Biolgicas, UFES, So Mateus, ES, Brasil,
4
Departamento de Biologia Estrutural, UFTM, Uberaba, MG, Brasil
2
gabriela.daolio@usp.br ; (11) 30917307; (19) 997109614
Background: Placental differentiation is a highly regulated process, which includes

the maturation of an interhemal membrane for exchange between maternal and fetal
organisms. It is formed by two syncytiotrophoblast layers and one layer of
trophoblast giant cells, lining a vascularized mesenchyme. Details of this
differentiation mechanism is not entirely elucidated in mice. In humans, the fusion of
trophoblast cells to form a syncytial layer involves the activation of caspase 8.
Aims: Characterization of the labyrinthine zone differentiation steps and analysis of
caspase 8 activity during syncytial layer development.
Methods: Mice pre-placental samples from gestation day (gd) 8.5 to 11.5 was
morphologically analyzed and correlated to the caspase-8 expression by Western
Blot.
Results: Morphologically, the initial labyrinthine differentiation on gd 8.5 was
sequentially characterized by: arrival of the allantois at the base of the ectoplacental
cone; emergence of chorionic fetal vessels and, entry of this vascularized
mesenchyme into the chorionic plate layer, pushing clusters of undifferentiated cells.
On gd 9.5, cells are organized interposed between the invaginated mesenchymal
tissue. On gd 10.5, maternal blood flows through spaces between the cell columns,
delimited by larger trophoblast cells. On gd 11.5, three layers of the interhemal
barrier are already constituted. Meanwhile, caspase 8 expression shows a progressive
increase at gd 8.5 and 9.5 gd, followed by a decrease on gd 10.5 and 11.5.
Conclusion: Our preliminary data show the expression of caspase 8 expression
during mouse syncytial layers differentiation, suggesting an involvement in the
trophoblast fusion process as seen in humans. This possibility is upon current
investigation in our lab.
Funding support: CNPq, Ethical approval: CEUA-ICB/USP N 104/2015
Correspondent Author information: Luciana Schulthais Alto. PPGCFar/CCS/UFES,

Av Marechal Campos, 1468, 29043-900 - Vitria - ES, Brasil (e-mail:
luciana.altoe@gmail.com).
Arsenic is commonly associated to natural and anthropic processes such as volcanic
emissions, mining and herbicides production, being an important pollutant. Several
studies have associated arsenic intake to male fertility reduction, thus the aim of the
present study was to evaluate whether vitamin C and/or zinc would counteract
arsenic side effects within the population of germ cells in testicles. Adult male
Wistar rats were divided into six experimental groups (n=6/group): control, sodium
arsenite (5mg/kg/day), vitamin C (100mg/kg/day), zinc chloride (20mg/kg/day),
sodium arsenite + vitamin C and sodium arsenite + zinc chloride. Oral treatments
(gavage) were performed for 60 days, when the animals were euthanized by
anesthetic overdose. Testis and epididymis were harvested and either frozen or
routinely processed to be embedded in glycolmethacrylate resin. The population of
each germ cell type was estimated by counting the germ cell nuclei and Sertoli cell
nucleoli present in stage 1 of the seminiferous epithelium cycle using 10 tubules
cross-sections per animal. Arsenic reduced the seminiferous epithelium and tubules
diameter due to germ cell loss. The reduction in the total number of germ cells was
due to the smaller number of spermatocytes in pre-leptotene/ leptotene and/or round
spermatids. It can be concluded that chronic exposure to sodium arsenite alter the
spermatogenic process, it affects even with administration of antioxidants zinc and
vitamin C. However zinc is more efficient, since the number of germ cells was not
reduced in the group As + Zn, despite the reduction of the tubular diameter.
The experimental design was approved by the Ethics Committee on Animal Use of
Universidade Federal do Esprito Santo (protocol number 041/2014).
Financial support: Scholarship Fundao de Amparo Pesquisa do Esprito Santo.

T52
T51
EFFECTS OF IMMUNOSUPPRESSIVE
PLACENTA
Luciana Schulthais Alto1*, Ianny Brum Reis2, Niumaique Gonalves da Silva3,

Maxsirlan Dantas Santos2, Marcos de Lucca Moreira Gomes4, Juliana Castro
Monteiro Pirovani3
DRUGS ON
THE
HUMAN
Sara Maria Zago Gomes (1) Rossana P.V. Francisco (2) Simone Corra da Silva (3)
Estela Maris Forell de Andrade Bevilacqua (1)
(1)Department of Cell and Tissue Biology, University of So Paulo, So Paulo,
Brazil.
(2) Department of Obstetrics and Gynecology of Faculty of Medicine, University of
So Paulo, So Paulo, Brazil
(3) Children's Institute of the Clinics Hospital, University of So Paulo, So Paulo
So Paulo, Brazil.
Background: Placental villi maintain an intimate relationship with the maternal blood
in the intervillous space and play roles in molecular exchanges, control of blood
flow, immune regulation, and endocrine secretion. Immunosuppressive drugs
(administrated in patients with autoimmune diseases/after organ transplant) are
considered compatible with pregnancy. However, such drugs has also been
associated with hypertension, fetal growth restriction, and preeclampsia, as a
consequence of placental dysfunction.
Aims and Methods: To investigate the effect of immunosuppressant drugs in
placental function and, the expression of vascular factors and cytokines associated
with hypertension in immunosuppressed pregnant women.
Term placentas (n=5) from healthy patients were dissected, and the villi were
cultured for 24 and 48 h. The immunosuppressive drugs Azathioprine (AZA, 10
ng/mL, 100ng/mL) and Cyclosporine (CSA, 125ng/mL, 1250ng/mL) were added to
culture media. Cell viability were assessed by MTT assays; expression of IL-10, IL8, IL-6 and TNF-a cytokines by CBA.
Results: AZA and CSA caused a decrease in cell viability and an increase of the proinflammatory cytokines IL-8, IL-6, TNF-a and IL-10 in 24 h and 48 h-cultured villi.
Conclusion: The villous culture shows to be a useful tool to study the placental
response to the immunosuppressive drugs and can contribute to the pathogenesis of
pregnancy diseases. There was a close similarity between the cytokine response of
the immunosuppressed placental villi when compared to previous studies with
immunosuppressed pregnant women. Also, the high levels of placental cell death
may also be a secondary and important factor in placental damage during
immunosuppressive treatments contributing to the pathogenesis of several other
associated-gestational diseases.
Supported: CAPES, FAPESP, Ethical protocol 0820/11
PRENATAL
DEXAMETHASONE
INDUCES
ALTERATIONS
OF
ANDROGEN AND GLUCOCORTICOID RECEPTORS ALONG THE
WOLFFIAN DUCT
Bruno F A Calegare, Camilla M Ribeiro, Maria Christina W Avellar. Section of
Experimental Endocrinology, Department of Pharmacology, Universidade Federal de
So Paulo, Escola Paulista de Medicina, So Paulo, Brazil.
Correspondent author information: Bruno F A Calegare. Rua 03 de Maio, 100.
Depto. de Farmacologia, Unifesp-EPM, CEP: 04044-020. So Paulo, SP, Brasil. Tel:
+55 1155764448. E-mail: brunopo@gmail.com
The respiratory distress syndrome is a severe complication related to prematurity and
treatment with corticoids decreases its incidence, being prescribed to all patients with
prematurity risk. Administration of hydrocortisone acetate during rat late pregnancy
decreased fertility and sexual behavior of adult males. Thus, we aimed to investigate
whether the use of corticoids during late rat pregnancy affect the androgen-induced
Wollfian duct (WD) morphogenesis, and the distribution of androgen (AR) and
glucocorticoid (GR) along the WD. Pregnant Wistar rats were treated subcutaneously
with dexamethasone at 1mg/kg from e17.5 to19.5 (DexS), at 0.1mg/kg from e13.5 to
e20.5 (DexL) or saline (control). Dams were euthanized 24 h later. Blood and tissues
were harvested; WD cryosections were used for histology and immunolocalization of
AR and GR. Compared to control groups, DexS and DexL dams presented lower
plasma corticosterone levels and did not gain the expected pregnancy body weight.
Dam adrenal glands were smaller in the DexL group. DexS and DexL decreased
body weight in the offspring, while the testis and WD relative weight are increased
in DexL offspring. We observed that the AR and GR distribution patterns varied
along control WD, which also was influenced by DexS and Dex. These observations
suggest the organization of WD in functional units, controlled by steroid hormone
signaling. Moreover, the hormonal status disruption in the WD by prenatal
dexamethasone may influence long-term reproductive aspects.
Ethics
Approval:
CEUA-Unifesp-EPM#8759031215.
Financial
PNPD/CAPES, CNPq/CSF (401932/2013-3, 150066/2016-3).
support:

T53
T54
CHANGES IN ESTROGEN RECEPTOR ER (ESR2) EXPRESSION

WITHOUT CHANGES IN THE ESTRADIOL LEVELS IN THE PROSTATE
OF AGING RATS
THE ENDOMETRIOTIC MILIEU CHANGES THE LEVELS OF

CYTOKINES IN ENDOMETRIAL STROMAL CELLS 3D-CO-CULTURED
WITH ENDOTHELIAL CELLS
Mnica Morais-Santos (1)*, Aryane E. B. Nunes (1), Andr G. Oliveira (1), Jnia
Dayrell Moura-Cordeiro (1), Germn A. B. Mahecha (1), Maria Christina W. Avellar
(2), Cleida A. Oliveira (1)
Caroline Borgato (1); Aline Lorenzon-Ojea (1); Elaine Cardoso (1); Tatiana Bonetti
(2); Eduardo Leme Alves da Motta(3); Paulo Cesar Serafini (3); Vanessa Freitas (1);
Alexandre Borbely (1,4); Estela Bevilacqua (1)
(1) Department of Morphology, Universidade Federal de Minas Gerais, Belo

Horizonte, Brazil.
(2) Department of Pharmacology, Universidade Federal de So Paulo, So Paulo,
Brazil.

Sciences, University of So Paulo, SP, Brazil
(2) Department of Obstetrics, Federal University of So Paulo, SP, Brazil
(3) Department of Gynecology, University Federal of So Paulo; Huntington Center
for Reproductive Medicine, So Paulo, SP, Brazil. Huntington Center for
Reproductive Medicine, So Paulo, SP, Brazil
(4) Institute of Biology and Health Sciences, Federal University of Alagoas, Macei,
AL, Brazil.
* Presenting author: Mnica Morais Santos

Universidade Federal de Minas Gerais, Instituto de Cincias Biolgicas, Av. Antnio
Carlos 6627, CEP 31270-901, Belo Horizonte, MG, Brazil
Phone: +55.31.3409-2808 Cel phone: +55.31.98859-0852
E.mail: monicamoraissantos@gmail.com
Although the prostate is androgen-dependent, it is also influenced by estrogens,
which act via estrogen receptors ER and ER. ER is highly expressed in the
prostate epithelium participating in the regulation of cell proliferation, apoptosis and
differentiation. Evidence shows that ER is decreased in malignant prostate,
suggesting that it plays an antineoplasic role. Despite the relationship between
reductions in ER and abnormal growth of the gland, little is known about the agedependent variation of this receptor. Therefore, we aimed to investigate ER
expression in the prostatic lobes of aging Wistar rats (3 to 24 months).
Histopathological alterations, including hyperplasia, nuclear atypia, prostate
intraepithelial neoplasias (PIN), as well as epithelial atrophy were observed in aging
prostates. ER Immunoreaction was reduced in specific areas related to PIN,
atrophic abnormalities and cellular atypia in senile rats. The punctual reduction in
ER paralleled the increase in cell proliferation especially in areas of PIN and
nuclear atypies. Basal cells intensity positive for aromatase forming a continuous or
stratified layer similar to those induced by estrogenization were also detected. These
alterations occurred in a hormonal milieu characterized by a constant concentration
of estradiol and decreased DHT. This is a pioneering study revealing focal ER
reduction in the aging prostate, thus indicating a potential disorder in the ER
pathway and a possible local hormonal imbalance, favoring estrogen action and the
development of pathologies. These results corroborate previous data from humans
and dogs that silencing of ER may be associated with premalignant or malignant
conditions in the prostate.
Ethical approval: The experimental procedures were approved by the Ethical
Committee for Animal Experimentation of the Universidade Federal de Minas Gerais
(CETEA/UFMG - process 286/2008).
Funding support: Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico
and Fundao de Amparo Pesquisa do Estado de Minas Gerais.
Endometriosis is a common gynecologic benign disease in reproductive age women,

characterized by the presence of endometrial-like tissue outside the uterine cavity
and associated with infertility.
Aim: We aimed to establish a 3D culture model using essential components of the
endometrial stroma (fibroblast/decidual cells, endothelial cells, and extracellular
matrices) to study the inflammatory profile of health stroma in response to serum and
leukocytes of endometriotic women.
Methods: The 3D model comprised of primary fibroblasts extracted from normal
human endometrium embedded in an ECM (collagen I, III, V, and fibronectin) and
covered by a layer of primary human endometrial endothelial cells. After assembled,
the 3D culture, morphologically similar to a health endometrium, received serum and
leukocytes from patients with endometriosis and from healthy individuals. The
profile of the cytokines and chemokines IL-2, IL-11, IL-12, IFN-, and RANTES
were determined by ELISA.
Results: Serum from patients with endometriosis presented higher levels of IL-2 and
reduced levels of IL-11 when compared to controls (p<0.005), there were no
differences regarding IL-12, IFN- and RANTES (p>0.05). Treatment of 3D cocultures with endometriotic serum induced IL-11 production and reduced the
expression of IL-12 and IFN- when compared to controls (p=0.05). IL-12, IL-11,
and RANTES were higher in the presence of leukocytes of patients with
endometriosis (p<0.05), whereas IL-2 and IFN- levels, did not change (p>0.05).
Conclusion: In summary, the 3D model used in this study showed to be a useful tool
to understand the endometrial response to serum/leukocytes of women with
endometriotic lesions. Also, the dysregulation in endometrial IL-11 and IL-12
expression by serum and leukocytes associated with endometriosis can be related to
infertility in some endometriotic women.
Keywords: Co-culture, 3D culture, endometrium, endometriosis, leukocytes,
cytokines.
T55

T56
SERUM FROM PREGNANT WOMEN WITH PE STIMULATES THE

EXPRESSION OF GRP78 PROTEIN IN CHORIONIC VILLI FROM
HEALTHY PREGNANT PLACENTA
IMMUNOEXPRESSION OF ANDROGEN AND ESTROGEN RECEPTORS

IN ADULTS GERBILS AFTER PROLONGED EXPOSURE TO LOW DOSE
OF BISPHENOL A AND HIGH-FAT DIET
Castro KR1, Prado KM1, Lorenzon-Ojea AR1, Gomes SZ1, Francisco RP2, Zugaib
M2, Hoshida MS2, Alves EA3, Veras MM4, Bevilacqua E1.
Camila Helena Facina (1), Bianca Facchim Gonalves (2), Silvana Gisele Pegorin de
Campos (1), Sebastio Roberto Taboga (1)
1.Department of Cell and Developmental Biology, Institute of Biomedical Sciences,

2. Department of Obstetrics and Gynecology, School of Medicine, University of So
3. Department of Pathology, School of Medicine, University of So Paulo, So
Paulo, Brazil.
(1) Departamento de Biologia, Instituto de Biocincias, Letras e Cincias Exatas,

IBILCE/UNESP, So Jos do Rio Preto, SP, Brasil.
(2) Instituto de Biocincias, IBB/UNESP, Botucatu, SP, Brasil.
Background: Preeclampsia (PE) is a gestational disease characterized by maternal

high blood pressure and proteinuria and fetal impaired trophoblast invasion into the
uterine spiral arteries. PE may lead to intrauterine growth restriction. In stress
conditions, cells are able to activate the Unfolded Protein Response (UPR), a cascade
of cellular signaling induced by the Endoplasmic Reticulum (ER) stress, in order to
restore cell homeostasis or activate apoptosis. The chaperone GRP78 (glucoseregulated protein 78) is the main chaperone sensor of ER stress and its upregulation
is associated with several pathologies.
Aims: Evaluate the expression of GRP78 protein in placental chorionic villi from
non-PE and PE pregnant women, stimulated with non-PE and PE pregnant women
serum.
Methods: Chorionic villi were dissected and cultured from term placentas (PE and
non-PE, n=3 each) and stimulated with serum from pregnant patients with PE, nonPE, and fetal bovine serum. GRP78 expression was assessed by Western blotting 24
h after stimulation. B-actin was used as loading control. Bands densitometry were
quantified by Image J software.
Results: Expression of GRP78 increased in non-PE placenta stimulated with PEserum compared to control groups (p 0.05).
Conclusion: Our findings suggest that serum from pregnant women with PE may
contain factors that activate the Unfolded Protein Response. Secreted and membrane
proteins are folded and released by ER. During ER stress the expression of these
proteins are affected by the UPR and may induce or aggravate the pathological
condition of preeclampsia.
Financial Support: CAPES, FAPESP. Ethical Approval CIAPP-394/14
Correspondent author information: Camila Helena Facina, Rua Cristvo Colombo,

2265, Jardim Nazareth. Departamento de Biologia, Instituto de Biocincias, Letras e
Cincias Exatas, IBILCE-UNESP, CEP: 15054-000. So Jos do Rio Preto, SP,
Brasil. Tel: +55 17 32212380. E-mail: camilafacina@gmail.com
Background: The exposure to endocrine disrupters, such as bisphenol A, can cause
morphophysiological changes in the prostate. Another environmental factor
associated with carcinogenesis is the high-fat diet. Aims: Evaluate the ventral and
dorsolateral prostate lobes after prolonged exposure to bisphenol A and high-fat diet
and the behavior of steroid receptors. Methods: Twenty-four gerbils were divided in
four groups: Control(C); Treated groups: Diet(D):animals that received high-fat diet
and filtered water; Bisphenol A(BPA):animals that received control diet and
bisphenol A-50g/kg/day in drinking water; Bisphenol A+Diet(BPA+D):animals
that received high-fat diet+bisphenol A-50g/kg/day in drinking water. The methods
involved morphological analysis of prostatic lesions and immunohistochemistry for
Androgen Receptor(AR) and Beta Estrogen Receptor(ER). Results: We observed
an increase in the number and severity of prostatic lesions in both lobes of the treated
groups. Both lobes of these groups also had a very similar frequency of positive AR
cells and it was greater than the control, mainly in areas of histopathological lesions.
The frequency of ER-positive cells was less than or similar to the control. In some
neoplastic foci stimulated by diet, bisphenol A or the combination of both, the
presence of positive ER cells was low or undetectable. Conclusion: The results
indicated that the methodology used was effective in generating metabolic changes,
which directly compromised the prostatic homeostasis. Diet and BPA appear to
modulate the activation of AR pathway and thereby optimize the tumor
establishment on gerbils prostate, while, simultaneously, promoting the inhibition of
the antitumoral activity of ER.
Financial Support:Capes/FAPESP.
CEUA/UNESP.
Ethics
Committee
Protocol:079/2013

T57
T58
MORPHOLOGICAL EFFECTS OF ACUTE FOOD RESTRICTION IN MICE

PREGNANCY
DOCOSAHEXAENOIC ACID AND REPRODUCTIVE BIOLOGY: NEW

INSIGHTS ABOUT POLYUNSATURATED FATTY ACIDS IN HUMAN
PROSTATE CELLS
Fernanda Cristina Silva de Oliveira, Ariana Musa de Aquino, Jefferson Merencio dos
Reis, Valdemar Antonio Paffaro Junior, Andrea Mollica do Amarante Paffaro
Department of Cell Biology and Development, Federal University of Alfenas, Minas
Gerais, Brazil.
Address: Gabriel Monteiro da Silva 700, Centro Alfenas MG
Institutional telephone: (35) 32991360 - Mobile telehone: (35) 992306430
email: nandah.cris@hotmail.com
Food restriction (FR) during pregnancy can cause serious problems in mice
embryo/fetal development and postnatal growth. In this context we evaluated the
morphological alterations in the embryonic and fetal development sites of pregnant
femeals submitted to food restriction. Females were caged overnight with males (1:3)
following morning the presence of vaginal plug was consider the first day of
pregnancy (dop). The food were removed for 48h in three periods (6dop-GI; 9dopGII; 13dop-GIII). After FR the females were euthanized, the uterine horns were
removed, the sites were isolated, weighted, fixed in paraformaldehyde 4% and
routinely processed to paraffin embedding. The histological sections were stained
with Hematoxylin and Eosin, submitted of Periodic Acid (PAS) reaction, or DBA
lectin cytochemistry. Procedures were accepted for Ethical Board of Federal
University of Alfenas (449/2012). The morphological analysis demonstrated a
decreased pregnancy viability in GI and GII. The embryos showed a important mass
reduction in GI and the placentas had regions with delay cell differentiation in GII
and GIII. The placental labyrinth zone in GIII was reduced and degeneration areas
with pyknotic nuclei were found near trophoblastic giant cells. The PAS reaction
showed a reduction in the number of glycogen cells in all the groups and de DBAlectin cytochemistry demonstrated a increasing of the total number of unK cells only
in GII. In conclusion, the FR in mice can make changes in embryo and placenta
development and interfere in maternal/fetal interactions.
Supported by CAPES
Guilherme H. Tamarindo4, Marina G. Gobbo, Daniele L. Ribeiro, Marlia de

Freitas Calmon Saiki, Paula Rahal, Eduardo A. Almeida, Sebastio R. Taboga,4
and Rejane M. Ges,4.
Department of Biology, Institute of Biosciences, Humanities and Exact Sciences,
So Paulo State University (UNESP), So Jos do Rio Preto, So Paulo, Brazil.
Department of Chemistry and Enviromental Sciences, Institute of Biosciences,
Humanities and Exact Sciences, So Paulo State University (UNESP), So Jos do
Histology Sector, Institute of Biomedical Sciences, University of Uberlndia (UFU),
Uberlndia, Minas Gerais, Brazil.
4
Institute of Biology, University of Campinas (UNICAMP), Campinas, Brazil.
Address of Author: Cristvo Colombo Street, 2265, Jardim Nazareth, CEP 15054000 - So Jos do Rio Preto, So Paulo, Brazil. / E-mail:
gui_tamarindo@yahoo.com.br / Telephone contact details: +55 17 981456204
Dietary fat exert negative role in prostate health and facilitate cancer development.
However, the action of specific lipids such as polyunsaturated fatty acids are far from
be elucidated. Docosahexaenoic acid (DHA) is an -3-polyunsaturated fatty acid and
its function in prostate cancer have been strongly discussed. Nevertheless, its effects
in normal conditions are unknown. We determined DHA effects in cell proliferation
and lipid peroxidation of human prostatic normal cells as well as potential related
mechanisms. PNTA1 cells were treated with 10, 20, 50 and 100M of DHA for 24,
48 and 72h. Cell proliferation was analyzed by MTS assay. Lipid peroxidation was
determined by malondialdehyde levels in cell lysate by HPLC-FL and total lipid
hydroperoxides levels in medium by FOX method. Oil Red O assay was applied to
evaluate lipid intake. Morphology was analyzed after hematoxylin-eosin staining and
phase contrast images. Peroxisome Proliferator-activated Receptor gamma (PPAR)
and Liver X Receptor alpha (LXR) content were assessed by western blotting as
well as confocal microscopy. DHA induced cell proliferation in almost all
concentrations, especially at 20M-24h and 50M-48h(~24%). Lipid peroxidation
was higher at 48h after DHA incubation in both, lysate and medium. Lipid
accumulation in cytoplasm increased in dose and time dependent manner. At 50M48h cells appeared more fasciculate and the content of PPAR and LXR apparently
increased. DHA at supra-physiological dose increased proliferation of PNTA1 cells
and these findings may be related to nuclear receptors activation. This proliferative
effect of DHA on PNTA1 cells differs from observed in tumor cell lineages.
Funding Support: FAPESP and CAPES.

T60
T59
MORPHOFUNCTIONAL AND THREE-DIMENSIONAL ANALYSIS OF
THE MALE AND FEMALE GERBIL PROSTATE (MERIONES
UNGUICULATUS) EXPOSED TO DIFFERENT LEVELS OF BISPHENOL A
DURING THE NEONATAL DEVELOPMENT
Rodrigo Fernandes de Lima (1), Mnica Sousa Campos (2), Mara Rbia Marques
(1), Manoel Francisco Biancardi (1), Fernanda Cristina Alcantara dos Santos (1)
Gois, Goinia-GO, Brazil;
(2) Department of Biology, So Paulo State University, So Jos do Rio Preto SP,
Brazil.
email: rodrigolima_biologia@hotmail.com; phone +556235211765, +556281535797
Prostate development is regulated by hormonal interactions. Bisphenol A (BPA) is
an estrogenic disruptor (DE) that alters the prostate development. The objective of
this study was to determine whether the exposure to BPA affects the prostate
development of gerbils. Neonates were exposed to environmental (LBPA
40/kg/day) and high levels (HBPA 4mg/kg/day) of BPA, from the 1st to the 7th
day of life. The prostatic complexes were collected for three-dimensional
reconstruction and immunohistochemical analysis.Three-dimensional reconstructions
showed a reduction in paraurethral bud (PaB ) in all females and HBPA and LBPA
groups a reduction in the mesenchyme paraurethral (PaM). In males the ventral
prostate buds have elongated towards the ventral condensed mesenchyme (VMP). In
females the paraurethral prostatic lobes (PaL) emerged from the urothelium forming
the PABs. AR-positive cells were present in all prostatic compartments in male and
female with higher frequency in the periurethral mesenchyme (PeM), ventral buds
(VB) and smooth muscle (SM) in males. The same was observed in the PeM but
with a immunonostaining decrease in the periurethral bud (PeB) and PaM in the
LBPA females. In the HBPA there was an increase of AR-positive cells in the PeM,
PaB, PeB and PaM. PCNA-positive cells were reduced in the PeB and SM of LBPA
males. We conclude that postnatal female prostate development is earlier and
morphologically distinct from males. Our data show that BPA exerts an antiproliferative effect on the prostate gland. Moreover, females are more susceptible to
this DE.
Ethical committee approval: CEUA/UFG: N 024/13). Financial Support:
CAPES/FAPEG.
MATERNAL PROTEIN RESTRICTION IMPAIRS PROSTATEIN AND pAKT EXPRESSION IN PROSTATE

Srgio Alexandre Alcantara dos Santos 1*; Jaqueline Carvalho Rinaldi1; Suelen
Franco1; Luiz Marcos Frediani Portella; Ana Carolina Lima Camargo1; Flavia Bessi
Constantino1; Ketlin Thassiani Colombelli1; Caroline Nascimento Barquilha1; Srgio
Luis Felisbino1; Luis Antonio Justulin Jr1;
1
Departament of Morphology Institute of Bioscience, UNESP, Botucatu, So

Paulo, Brazil.
Srgio Alexandre Alcantara dos Santos, Extracellular Matrix Laboratory,
Department of Morphology, Institute of Biosciences of Botucatu, UNESP.
(14)38800502/ (14) 981612599. Email: sergioale@ibb.unesp.br (presenting and
corresponding author). Address: Rua Loureno Castanho, 1533 Jardim Flamboyant.
Botucatu/SP-Brazil
Background: Adverse perinatal conditions may lead to irreversible systemic changes
in the offspring, a condition known as Fetal Programming (FP). Intrauterine protein
restriction has been associated with low birth weight and metabolic syndromes
during aging. It has been demonstrated that FP also alters steroid hormones and
growth factors such as insulin/IGFs in the offspring from restricted dams. Aims: To
investigate if perinatal protein restriction (gestational and lactational periods) affects
the ventral prostate (VP) structure and the insulin/IGF signaling in male offspring.
Methods: Pregnant Sprague Dawley rat were distributed into three groups: Control
(CTR: fed normal 17% protein diet); Gestational Protein Restriction (RPG, fed 6%
protein diet during gestation); Gestational and Lactational Protein Restriction
(RPGL, fed 6% protein during both periods). After delivery, biometrical parameters
and VP were harvested at post-natal day (PND) 540 and processed for morphological
and biochemical analyzes. Results: At PND 0, the offspring from RP groups
demonstrated significant lower body weight and smaller ano-genital distance (AGD)
compared to CTR group. Morphological difference in VP on PND 540 was not
observed among the experimental groups. The western blot analysis demonstrated
reduction in AKT phosphorilation (p-AKT) and prostatein expression in RPG and
RPGL compared to CTR group. No difference was observed in the expression
insulin receptor (IR), Pi3k and total AKT. Conclusion: The decrease in p-AKT in
restricted groups may represents, at least in part, a disruption of IGF signaling, which
also affects secretory VP function in these animals.
Ethical approval CEUA UNESP-Botucatu (Protocol CEUA-573).
Funding support: FAPESP (2013/24230-5; 2014/08531-8).

T61
T62
H2 RECEPTOR ANTAGONIST (CIMETIDINE) AFFECTS STRUCTURAL

INTEGRITY AND IMMUNNOLOCALIZATION OF AR AND SHBG IN THE
RAT EPIDIDYMIS
CHORIONIC VILLOUS sFLT-1 EXPRESSION IS CHANGED BY SERUM

OF PREECLAMPTIC WOMEN
Fabiane de Santi (1), Flvia L Beltrame (1), Paulo S Cerri (2), Barry T Hinton (3),
Estela Sasso-Cerri (2)
(1) Federal University of So Paulo, Department of Morphology and Genetics, So
Paulo - Brazil
(2) Dental School of So Paulo State University, Department of Morphology,
Araraquara Brazil
(3) University of Virginia School of Medicine, Department of Cell Biology,
Charlottesville, USA
fabianedesanti@yahoo.com.br, Dental School of So Paulo State University. Rua
Humait, 1680. CEP 14801-903. Araraquara, SP, Brazil. Phone number: 55 16
33016491 / 55 16 996364190.
In male patients treated with cimetidine - an H2 receptor antagonist, impotence,
gynecomastia and changes in testosterone levels have been detected and related to
antiandrogenic effect. In rat testes, cimetidine caused seminiferous tubule atrophy
due to cell loss, as well as Leydig cell impairment and reduction of testosterone
levels. As the epididymis is an androgen-dependent organ essential for male fertility,
epididymal structure and immunolocalization of androgen receptors (AR) and steroid
hormone binding globulin (SHBG) were evaluated following cimetidine treatment.
Male rats were distributed into control (CG; n=10) and cimetidine (CMTG; n=10)
groups. CMTG received cimetidine (100mg/kg b.w.) for 50 days and CG received
saline. In HE-stained epididymal cauda sections, the diameter of epididymal duct,
number of principal cells (NPC) and the birefringent collagen content (BCol) were
evaluated. TUNEL method, AR and SHBG detection by Western blot and
immunofluorescence for quantitative analyses were performed. In CMTG, the
epididymal duct diameter, NPC and BCol were significantly reduced. TUNELpositive epithelial cells were found. An abnormal and diffuse AR
immunofluorescence was detected in the PC cytoplasm while nuclei were weakly
immunolabeled. These findings were confirmed by quantitative data. The SHBGimmunolabeled area in connective tissue was also reduced. Cimetidine treatment
induces atrophy of the epididymal epithelium and connective tissue. The epithelial
damage is related to impairment of the androgenization process due to disruption of
intracellular AR shuttling between the nucleus and cytoplasm. Since SHBG is bound
to extracellular proteins, the reduction of this globulin may be consequence of
collagen loss.
Financial support: FAPESP (2012/23845-3; 2013/25322-0; 2015/09341-0), CAPES
and CNPq.
This work was approved by Ethical Committee for Animal Research of Federal
University of So Paulo (CEUA/ UNIFESP n 1794060415).
Prado KM1, Castro, KR1, Lorenzon-Ojea AR1, Gomes SZ, Hoshida MS2, Alves E2,
Zugaib M2, Francisco RP2 and Bevilacqua, E1
1

University of So Paulo, SP, Brazil
2
Department of Obstetrics and Gynecology of Faculdade de Medicina da
Universidade de So Paulo, Sao Paulo, SP, Brazil
Correspondence to: Karen Prado (email: karenprado@usp.br)
Institute of Biomedical Sciences - University of So Paulo - Av. Prof Lineu Prestes,
1524 Butantan - 05508-000 SP-SP Brazil - Tel +55-11-3091-7307
Introduction: Preeclampsia (PE) is a gestational disease associated with high blood
pressure and proteinuria. In addition, also occurs reduced trophoblast invasion and
incomplete remodeling of uterine spiral arteries in the placental bed, changing
placental functions and fetal development. PE has been associated with increased
production of anti-angiogenic soluble VEGF receptor 1 (sFlt-1), a VEGF antagonist,
involved in endothelial dysfunction and decreased angiogenesis.
Aims: This study aims to analyze whether maternal conditions in PE are a relevant
factor in the control of sFLT-1 expression by placental villous compartment. This
study was approved by Ethic Committees from Biomedical Sciences Institute and
University Hospital of Sao Paulo University.
Method of Study: Chorionic villi were dissected and cultured from term normal
(control, nonPE) and PE placentas during 24 hours. Analysis of sFLT-1 placental
protein expression was performed in control villous compartment cultured with PE
serum and in preeclamptic villi cultured with normal serum (n=4).
Results: Our preliminary results showed that sFlt-1 expression increased
significantly in normal placentas (p = 0.0052) when nonPE villi were cultured in the
presence of pregnant serum with preeclampsia. Meanwhile, in placentas from PE
women, sFlt-1 expression decreased (p = 0.0002) in the presence of control serum.
Conclusion: Our findings suggest that excess sFlt-1 produced by the placenta in PE
may be induced by factors present in the maternal blood, likely due to the several
maternal dysfunctions. The modulation of sFlt1 placental expression warns for a
possible mechanism of self-maintenance/aggravation of PE directly orchestrated by
maternal conditions.
Financial support: CAPES, CNPq and FAPESP
Ethical Approval: CIAPP 394/14
T63
T64
SPERM DAMAGE CAUSED BY CIMETIDINE IN ADULT RATS
GESTATIONAL AND LACTATIONAL PROTEIN RESTRICTION ALTERS

BIOCHEMICAL PARAMETERS AND PROSTATE GROWTH IN THE
MALE RAT OFFSPRING
Flvia Luciana Beltrame (1)*, Fabiane de Santi (1), Vanessa Vendramini (1); Regina
Elizabeth Loureno Cabral (1); Paulo Srgio Cerri (2), Sandra MariaMiraglia (1),
Estela Sasso-Cerri (2)
(1) Department of Morphology and Genetics, Federal University of So Paulo, So
Paulo/SP, Brazil.
(2)Department of Morphology, Dental School of So Paulo State University,
Araraquara/SP, Brazil.
*fla_biomed@yahoo.com.br -Dental School of So Paulo State University RuaHumait, 1680. CEP 14801-903. Araraquara (SP), Brazil. Phone number: +55 16
3301-6491.
The H2 receptor antagonist cimetidine has been clinically used as antiulcer,
competing with H2 receptors in the parietal cells. Nowadays, this drug has also been
used as adjuvant in therapy for cancertreatment due to its antiangiogenic effect.
Cimetidine antagonizes androgen receptors, exerting antiandrogenic effect, which
has been related to damage in the male reproductive system. In this context, we
purposed to evaluate if cimetidine causes morphological and quantitative changes in
spermatozoa as well as alteration of the sperm DNA integrity.Adult male rats
received intraperitoneal injections of 100mg/kg bw of cimetidine (CMTG; n=6) and
saline (CG; n=6).The sperm was collected from epididymis cauda for the following
analysis: concentration, morphology, motility, mitochondrial activity and sperm
DNA integrity (comet assay). The statistical analysis was performed by Students ttest (p<0.05). The animals from CMTG showed decreased sperm concentration,
motility and mitochondrial activity, besides the significant increase in the number of
abnormal formsof spermatozoa, including damage in head and in tail. An increase in
the percentage of sperm DNA fragmentation, assessed by comet assay, was also
observed in CMTG. The results show that cimetidine treatment causes decrease in
sperm quality, suggestingthat this drug may also interfere in the events related to the
maintenance of spermatogenic process and sperm maturation. These findings provide
new insights into the importance of the cimetidine study for the male fertility.
Funding support: FAPESP (2012/23845-2; 2013/25322-0) and CNPq.
This work was approved by Ethical Committee for Animal Research of Federal
University of So Paulo (CEUA n 7950060514) and by Ethical Committee of
Dental School of So Paulo State University (CEUA n 28/2014).
Suelen Franco1, Srgio Alexandre Alcantara dos Santos1 Luiz Marcos Frediani
Portella, Ana Carolina Lima Camargo1, Flvia Constantino Bessi1, Ketlin Thassiani
Colombelli1, Caroline Nascimento Barquilha1, Fernanda Mani2, Luis Antonio
Justulin Jr1
1
Institute of Biociences of Botucatu, Morfology department UNESP de Botucatu,

So Paulo, Brazil.
2
Institute of Biociences of Botucatu, Department of Chemistry and Biochemistry UNESP de Botucatu, So Paulo, Brazil.
Suelen Franco, Extracellular Matrix Laboratory, Department of Morphology,
Institute of Biosciences of Botucatu, UNESP. (14)38800502/ (14) 98181-4757.
Email: suelen.franco4@gmail.com (presenting and corresponding author). Address:
Rua Loureno Carmlo,510, Jd. Paraso. Botucatu/SP-Brazil
Background: Exposure to intrauterine protein restriction (PR) causes irreversible
morphological changes in the fetus. The PR changes the nutritional and hormonal
levels in the offspring, leading to alterations in specific organs in adulthood. Aims:
To evaluate the biochemical parameters of pups from dams subjected to perinantal
(gestation and lactation) protein restriction and the effects on the ventral prostate
(VP). Methods: Male Sprague Dawley pups born to mothers fed a normal diet (17%
protein, CTR group) or hypoproteic (6% protein) during pregnancy (RPG group),
and during pregnancy and lactation (RPGL group) were used. The animals were
weighed, euthanized and blood was collected on postnatal day (PND) 21 and 540 for
biochemical analysis and the PV was harvested to histology. Results: The body
weight of both restricted rats at PND21 was reduced compared with CTR, whereas at
PND540 this difference was seen only in RPGL than CTR. At DPN21, the VP
weight in restricted groups decreased to the CTR. Histological analysis showed
reduction in glandular compartment and increased in the stroma of RPG and RPGL
groups at PND21 than CTR. Serum analysis showed reduction in total protein in
both restricted groups compared to CTR, whereas triglycerides and glycemic index
were lower only in RPGL group. There is no difference all parameters analyzed at
PND 540. Conclusion: Perinatal protein protein restriction impacts nutritional
parameters of offspring at PND21 and these results can be associated with delay in
prostate growth. However, there is an evident recover of VP structure and
biochemical parameters at PND540.
Ethical approval CEUA UNESP-Botucatu (Protocol CEUA-573).
Funding support: FAPESP (Proc. N. 2013/24230-5; 2014/08531-8; 2015/18999-0).


T65
T66
HISTOPHYSIOLOGICAL CHANGES OF THE GERBIL (MERIONES

UNGUICULATUS) VENTRAL PROSTATE AFTER THE STRESS
INDUCTION BY SLEEP RESTRICTION
FOOD RESTRICTION (FR) IN MICE PREGNANCY INDUCES uNKS

MATURATION
Ricardo Alexandre Fochi (1), Julia Quilles Antoniassi (2), Mariana Marcielo de
Jesus (2), Luiz Roberto Falleiros Junior (1), Sebastio Roberto Taboga (1)
Jefferson Merencio dos Reis, Glcia Marlia Zambroti Greco, Fernanda Cristina Silva
de Oliveira, Ariana Musa de Aquino, Fernando Fellicionni, Valdemar Antnio
Paffaro Jnior; Andra Mollica do Amarante Paffaro
(1) Department of Biology, So Paulo State University, IBILCE/UNESP, So Jos

do Rio Preto, SP, Brazil, So Jos do Rio Preto, Brazil.
(2) Department of Structural and Funcional Biology, Institute of Biology, State
University of Campinas (UNICAMP), Campinas, Brazil.
Address: Gabriel Monteiro da Silva 700, Centro Alfenas MG

Institutional telephone: (35) 32991360
Mobile telehone: (35) 988392982
email: jef.merencio@hotmail.com
ra.fochi@yahoo.com.br, +55 17 99158 6127
The Food Restriction (FR) of pregnant female requires alternatives to maintain the
conceptus metabolism. This proper metabolism is usually a result of interactions with
the mother's body carried out by trophoblast cells. However, these cells often
undergo influences of uNKs cells that are responsible for a host of cytokines and
angiogenic factors. Within this context, this study evaluated the effect of the FR in
uNKs cells at the beginning and the end of the embryonic period of mice pregnancy.
Females were mated with males, and when the vaginal plug was founded it was
considered the first day of pregnancy (1 dop). The food was removed for 24 or 48h
before euthanasia in 8 dop or 15 dop. The pregnant uterus was collect and process for
paraffin embedding and DBA lectin cytochemistry. The uNK1, uNK2, uNK3 and
uNK4 subtypes were count in three endometrium regions (near the embryo, near the
myometrium and in the intermediate area). Mann-Wthitney Wilcoxon test and
software Graph Pad Prism 5 was used for statistical analysis. The FR mother during
24h caused uNK2 and uNK3 increased in all uterus regions in 8 dop and this fact,
can demonstrated a possible acceleration in maturation of this cell subtypes for assist
the maintenance of pregnancy. After female were submitted to RF for 48h was
observed a decrease of uNK2 and a large increase of uNK4 in both groups observed.
Probably this changes can be associated with death and tissue degeneration.
The sleep deprivation promotes important changes in the synthesis of steroid

hormones and also induces the secretion of pro-inflammatory cytokines. Thus, we
investigated the influence of sleep restriction on the ventral prostate histophysiology
of Mongolian gerbils. The gerbils were kept under paradoxical sleep deprivation
during 18 hours straight on platforms surrounded by water, and then under rest for 6
hours in regular cages. The serum testosterone and corticosterone were quantified, as
well as the intratissue levels of IL-1 and TNF-. Prostatic fragments were analyzed
by morphometry techniques and immunohistochemical for androgen receptor (AR),
glucocorticoid receptor (GR), IL-1RI and TNF-R1 receptors. The cell proliferation
was evaluated by PCNA. After the treatments, there was a body weight reduction,
ventral prostate weight decrease and an increase of the glycaemia. Atrophic regions
were observed in glandular epithelium of the treated gerbils, in addition to reducing
the amount of AR-positive cells and proliferative index with an increase of GRpositive cells. The reduction of the AR amount is related to lower concentrations of
testosterone found in treated animals and also with the elevation of corticosterone
levels. In the treated gerbils was observed an increase in the IL-1 and TNF- levels,
besides the presence of its receptors both in the epithelium and in the prostatic
stroma. This hormonal scenario resembles that of senile or castrated animals, which
added to the increase of inflammatory cytokines may cause important
histophysiologic imbalances, fact that may trigger the development of more severe
lesion.
CEUA protocol: 095/14. Financial support: FAPESP and CAPES.

T67
UNIFAL-MG Ethical Board: (690/2015).

Support by: FAPEMIG and CAPES

T68
ETHYNILESTRADIOL
PROMOTES
DIFFERENT
EFFECTS
PROSTATE OF MALE AND FEMALE GERBIL DURING AGING
ON
EFFECTS OF METHYLPARABEN EXPOSURE ON THE ADULT GERBIL

PROSTATE (MERIONES UNGUICULATUS)
Ana Paula S. Perez (1), Cssia R. S. Caires (2), Elisa B. Rezende (1), Fernanda G.
Fleury (1), Lusa R. F. Guimares (1), Tracy M. M. Martins (1), Manoel F. Biancardi
(3), Fernanda C. A. Santos (3), Sebastio R. Taboga (4)
Janana Ribeiro Costa (1), Eliana Gomes Pinto (1), Manoel Francisco Biancardi (1),
Pedro Vale de Azevedo Brito (1), Sebastio Roberto Taboga (2), Fernanda Cristina
Alcantara dos Santos (1)
(1) Federal University of Gois (UFG) Special Institute of Health Sciences Medical
School, Jata-GO, Brazil.
(2) IBILCE/UNESP - Department of Biology, So Jos do Rio Preto-SP, Brazil.
(3) Federal University of Gois (UFG) - Department of Histology, Embryology and
Cell Biology, Goinia-GO.
(4) IBILCE/UNESP - Department of Biology, So Jos do Rio Preto-SP, Brazil and
Department of Structural and Functional Biology (UNICAMP), Campinas-SP,
Brazil.

Gois, Goinia-GO, Brazil;
(2) Department of Biology, Laboratory of Microscopy and Microanalysis, University
Estadual Paulista UNESP, Rua Cristvo Colombo, 2265, So Jos do Rio Preto,
So Paulo, 15054000, Brazil.
paulabio_perez@yahoo.com.br/ Tel: (64) 36068130.

17-ethinylestradiol (EE) is a component of the oral contraceptive that plays actions
as endocrine disruptor. The aim of this study was to analyze the response of male and
female prostate of senile gerbils to oral doses of EE (15g/kg/day) in prenatal and
pubertal periods (EE/PRE-PUB). For this, the prostates, after being subjected to
histological processing, were submitted to morphometric and immunohistochemical
(AR, ERS1, ERS2) analysis. Blood samples were collected and destined to
serological analysis for estradiol and testosterone hormones. Our results showed that
the EE exposure during prostate development decreased testosterone levels in senile
male and increased the estradiol levels in senile female. Exposure to synthetic
estrogens during development interfered in the pathways of sex hormone production
and contributed to changes in hormone levels during aging. We observed a decrease
in the epithelial and muscle thickness of senile male prostate in EE/PRE-PUB group.
In the senile female prostate we observed an increase in epithelial thickness both in
EE/PRE-PUB group. The immunoreactivity of AR in male prostate of EE/PRE-PUB
group decreased and the immunostaining for ERS1 increased in prostatic epithelial
cells of senile male and female. These alterations contributed to the development of
prostatic disorders, observed in the prostates of both sexes. The EE exposure during
prenatal and pubertal period interferes in the morphophysiology of prostate,
promoting different effects in male and female during aging. This study shows that
the gerbil is a good experimental model for studies that evaluate the effects of
ethinylestradiol as endocrine disruptor.
Funding Support: FAPESP 2009/16790-5 and 2011/06335-9
Ethical Approval: N. 020/2009 CEUA
email: janainaribeiro18@hotmail.com; Phone +556235211765, +556299594590

The parabens are a class of preservatives with antimicrobial and antifungal action
widely used in cosmetic, pharmaceutical and food industry. Parabens are known for
disturbing the reproductive system and act as endocrine disruptors, mimicking the
physiological effects of estrogens. However, it is unclear whether parabens can alter
the prostate gland morphophysiology. Thus, the aim of this study was to evaluate the
prostate of adult gerbils exposed to the methylparaben. For this, 90-days-old males
and females received, through gavage, 500 mg/kg of methylparaben diluted in 1% of
Hydroxyethyl cellulose. These animals were divided into three subgroups and
euthanized after 3, 7 and 21 days of treatment (3P, 7P and 21P). The prostates were
collected for structural, cytochemical and immunohistochemical analysis. Results
showed a body weight loss in 3P and 7P males, and testis weight loss in 7P and 21P
groups. Females had an increase in the prostatic complex weight in 21P group.
Moreover, males and females presented morphological alterations such as
hyperplasic growth foci of the epithelium. In females it was also observed the
presence of prostatic intraepithelial neoplasia and stromal inflammatory foci.
Additionally, there was an increase in the frequency of AR-positive cells and cellular
proliferation index in prostates of both sexes. These results suggest that the
methylparaben induces hormonal alterations in the prostate, interfering with the
regulation of androgenic receptors and predisposing the gland to the development of
hyperplasia and intraepithelial neoplasia.
Ethical committee approval: CEUA/UFG: N 024/13.
Financial Support: CAPES, CNPq and FAPEG.

U STEM CELL BIOLOGY
U1
U2
ESTROGEN UTILIZATION OF IGF-1R TO SIGNAL IN HUMAN

PROSTATE STEM/PROGENITOR CELLS
PHOTODYNAMIC ACTIVATION AS A MOLECULAR SWITCH TO

PROMOTE STEM CELL REGULATION OF GENE EXPRESSION OF
SIGNALING PATHWAYS
Jaqueline C Rinaldi*(1,2), Wen-Yang Hu(1), Shyama Majumdar(1), Luis A Justulin(2),

Gail S Prins(1)
(1)
(2)
(3)
(1) Department of Urology, UIC/Chicago, IL, USA

(2) Department of Morphology, UNESP/Botucatu, SP, Brasil
(1)
(2)
*email: jak.rinaldi@gmail.com, phone: +55(14) 3880 0502
(1)
(2)
Leonardo Barcelos de Paula (1), Maryanne Trafani de Melo (1), Fernando Lucas
Primo (2), Antonio Claudio Tedesco (1)
(1) Department of Chemistry, University of Sao Paulo, Ribeirao Preto, Brazil.
(2) Department of Bioprocess and Biotechnology, University of Paulista State Jlio
de Mesquita Filho, Araraquara, Brazil.
The biological actions of estradiol (E2) are mediated by genomic and non-genomic
effects. We previously demonstrated that E2 through estrogen receptors (ER) play
pivotal roles in E2-induced prostate stem/progenitor cell (PSC/PPC) amplification.
Since ER actions can be modified by growth factor receptors through ligandindependent ER phosphorylation, we herein sought to characterize the E2 potential
utilization of IGF-1R to signal in PSC/PPC. To accomplish that, human prostate
PSC/PPC were isolated from primary prostate epithelial cells using 3-D prostasphere
culture. Two human cell lines benign WPE-Stem cells over-expressing ER and
HuSLC cancer stem cells expressing ER but not ER were used to explore the
specific role of ERs. In addition to ERs, we found that human PSC/PPC also express
robust level of IGF-1R. Similar to estradiol-17 (E2), 5nM IGF-1 increased the
number of prostaspheres as well as BrdU-retaining PSC. Conversely, IGF-1R
knockdown decreased both parameters and consistently increased ER expression.
Suggesting IGF-1R activation may drive PSC/PPC amplification by suppressing
ER. Further studies revealed that E2 (10nM) induced pIGF-1R through ERmediated rapid signaling. IGF-1R Knockdown inhibited E2-induced pER, indicating
a positive interaction between IGF-1R and ER. Moreover, IGF-1 treatment induced
pIGF-1R which subsequently activated pAkt/pERK, IGF-1R knockdown inhibited
E2-induced pAkt/pERK, implying a downstream cross-talk. Finally, IGF-1R
knockdown decreased PHLDA1 mRNA, which indicates E2/ER and IGF-1/IGF-1R
may modulate prostate stem cell self-renewal through common target genes
including PHLDA1. In conclusion, our data shows a robust cross-talk between
estrogen and IGF-1 signaling at multiple levels that modulate PSC/PPC numbers to
effectively maintain glandular homeostasis.
barcelos@usp.br
Supported by NIH/NCI award R01 CA172220; scholarship from FAPESP

2014/10965-6. All authors have nothing to disclosure.
References:
Prins GS, Hu WY, Shi GB, Hu DP, Majumdar S, Li G, Huang K, Nelles J, Ho SM,
Walker CL, Kajdacsy-Balla A, van Breemen RB. Bisphenol A Promotes Human
Prostate Stem-Progenitor Cell Self-Renewal and Increases In Vivo Carcinogenesis in
Human Prostate Epithelium. Endocrinology. 2014;155(3):805-17.
Hu WY, Shi GB, Lam HM, Hu DP, Ho SM, Madueke IC, Kajdacsy-Balla A, Prins
GS. Estrogen-initiated transformation of prostate epithelium derived from normal
human prostate stem-progenitor cells. Endocrinology. 2011;152(6):2150-63.
Background: Adult stem cells exist in differentiated organs or tissues such as brain,
bone marrow, blood vessels, skin and liver maintain tissue integrity through
remodeling and injury repair. Among the several types of adult stem cells,
mesenchymal stem cells (MSCs), has attracted great interest. Photodynamic Therapy
(PDT) is a promising strategy to treat a variety of oncological diseases including
dermatological diseases such as skin cancer as well as other non-malignant diseases.
Aims: PDT share knowledge in MSCs at the molecular level allowed to discovery
of genes that participate in the regulation of gene expression of other RTK/Ras/PI3K and AKT/MAPK signaling pathways. Methods: Human bone marrow stromal
MSC (BM-MSC) grown in culture were treated with chloroaluminumphthalocyanine (ClAlPc) incorporated into a polymeric NE (NE/ClAIPc) and further
irradiated with monochromatic visible laser light. Gene expression was analyzed
before and after irradiation, using the Taqman Array Human EGF Pathway 96well plate kit. Results: PDT irradiation decreased the Epidermal Growth Factor
(EGF) pathway gene expression in NE/CIAIPc-treated BM-MSC cells, as compared
to non-irradiated cells. This effect can impact the tumor growth and progression,
because the EGF signaling pathway is directly involved in these processes.
Conclusion: PDT modulates the EGF pathway gene expression in NE/CIAIPctreated BM-MSC. This effect can impact the tumor growth and progression, because
the EGF signaling pathway is directly involved in these processes. This finding
contributes to develop new and advanced clinical protocols that employ nanocarriers
and PDT to treat highly invasive and resistant tumors.
(3)
U3
U4
MESENCHYMAL STEM CELLS PROTECT HIPPOCAMPAL NEURONS

FROM OXIDATIVE STRESS AND SYNAPSE DAMAGE INDUCED BY
AMYLOID- OLIGOMERS
MICROENVIRONMENT STRESS INFLUENCES STEM CELL GENES

EXPRESSION
Carlla A. de Araujo e Silva1, Mariana A. de Godoy1, Leonardo M. Saraiva2, Luiza

R.P. de Carvalho1, Andreia Vasconcelos-dos-Santos1, Hellen J.V. Beiral1, Livian R.
1
(1)
de Paula Silva1, Renata B. Leal1, Victor H. S. Monteiro1, Carolina V. Braga
,
Leandro C. Sinis1, Ana Paula C.A. Lima1, Narcisa L. da Cunha e Silva1, Antonio
2
1,3
2
1,2
Galina-Filho , Adalberto Vieyra , Fernanda G. De Felice , Sergio T. Ferreira(2),
Rosalia Mendez-Otero1
(3)
From the Institute of Biophysics Carlos Chagas Filho, Institute of Medical
3
Biochemistry Leopoldo de Meis, and National Center of Structural Biology and
Bioimaging/CENABIO, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Carlla Assis de Arajo e Silva, Institute of Biophysics Carlos Chagas Filho, Federal
University of Rio de Janeiro, Rio de Janeiro, RJ 21944-590, Brazil. Tel. institutional:
+55-21-3938-6554; mobile: +55-21-98211-9747 E-mail: cahh.araujo@gmail.com
Alzheimers disease (AD) is a disabling and highly prevalent neurodegenerative
condition, for which there are no effective therapies. Soluble oligomers of the
amyloid-beta (A) peptide (AOs) are increasingly thought to be the main
neurotoxins involved in early neuronal oxidative stress and synapse damage in AD.
The aim of the current study was to evaluate the neuroprotective actions of MSCs
against the deleterious impact of AOs on hippocampal neurons. To this end, we
established transwell co-cultures of rat hippocampal neurons and MSCs. We show
that MSCs protect neurons against AO-induced oxidative stress and synapse
damage, revealed by loss of pre- and post-synaptic markers, synaptophysin and PSD95, respectively. The mechanism of protection appears to involve internalization and
degradation of AOs by MSCs, and release of extracellular vesicles containing
catalase. Results suggest that MSCs may represent a promising alternative for cellbased therapies in AD.
Information on ethical approval and funding support - All procedures were approved
by and followed the guidelines of the Institutional Animal Care and Utilization
Committee of the Federal University of Rio de Janeiro (Protocol IBCCF 076).This
work was supported by grants from the Conselho Nacional de Desenvolvimento
Cientco e Tecnolgico (CNPq), Coordenao de Aperfeioamento de Pessoal de
Nvel Superior (CAPES), Fundao Carlos Chagas Filho de Amparo Pesquisa do
Estado do Rio de Janeiro (FAPERJ) and Fundao Universitria Jos Bonifcio
(FUJB).
Keywords: Alzheimers disease, hippocampus, amyloid- oligomers, endocytosis,
mesenchymal stem cells, extracellular vesicles, catalase, oxidative stress, synapse
Ana Carolina Lima Ralph (1), Iuri Cordeiro Valado (1), Murilo Vieira Geraldo(2),
Vanessa Morais Freitas (1)
Sciences, University of So Paulo, Brazil.
(2) Department of Structural and Functional Biology, Institute of Biology, State
University of Campinas, Brazil
Correspondent author:
E-mail: anaralph@usp.br
Phone: 55 11 3091 7778
Background - Cancer stem cells (CSC) have self-renewal and differentiation capacity
along with high chemoresistance, resulting in both poor survival and high recurrence
rates. Development of CSC depends on the tumor microenvironment that consists of
nutrients deprivation, acidosis and hypoxia. Aims To evaluate stem cell gene
expression of breast cancer cells exposure to low glucose, acidosis and hypoxia.
Methods Breast cancer cells (BCCs) MDA MB 231 and MCF7 were maintained
with physiological DMEM - pH (7.4), FBS 10%, penicillin-streptomycin 1%, 37C,
glucose (1.0 g/L), CO2 5% and normoxic environment. Cell treatment consisted of
low glucose (0.5 g/L), acidic (pH 6.2) or hypoxic (200 M CoCl2) environment.
After 72 hours, we proceeded to cell lysis, total RNA extraction, cDNA synthesis,
and then analyzed the expression of a panel of 84 genes related to cancer stem cells
with a PCR array. Results From the 84 analyzed genes, we found 41 (45,55%)
differentially expressed. For further investigation, we choose eight genes with higher
fold change, associated to breast cancer and stem cell biology. These genes are
related to several cellular functions including growth and migration (AXL, MERTK,
CXCL8); epithelial-mesenchymal transition, invasion and metastasis (ZEB1);
endothelial cell differentiation (CD44, GATA3); cellular and tissue development
(NOTCH1, SOX2). All of these genes has some importance in cell-cell and cellmatrix interactions. Conclusion These results highlights the microenvironment
importance to the cancer stem cell development and may point some clues about how
low glucose, acidosis or hypoxia may influence tumor resistance.
Funding support: CAPES, CNPQ and FAPESP. The authors have no conflict of
interest to declare.

U5
U6
STUDY OF THE EFFECT OF CELULAR THERAPY USING

ENDOTHELIAL PROGENITOR CELLS (EPCs) IN THROMBOSIS TIME
AND PLATELET AGREGATION
CONTROL OF
METABOLISM
Micheli Severo Sielski1, Giane Daniela Carneiro1, Camila Wielewski Leme2, Claudio
Werneck2, Cristina Pontes Vicente2.
12-
(1) Department of Cell Biology and Structural - Institute of Biology State

University of Campinas (Unicamp), So Paulo, Brazil
(2) Department of Biochemistry and Tissue Biology - Institute of Biology - State
University of Campinas (Unicamp), So Paulo, Brazil
3Contact: e-mail: micheli_sielski@hotmail.com, Adress: Rua Charles Darwin s\n,

bloco N, 3 andar, Phone: 55(19)3521-6106
Endothelial progenitor cells are immature progenitor cells present in bone marrow
that able to proliferate and diferentiate into mature endothelial cells and participate in
endothelial regeneration. The objective of this work is to analyse the effect of
cellular therapy using EPC in arterial thrombus formation and platelet aggregation in
aterosclerotic mice. We used LDL receptor knockout mice feed with high fat diet.
We isolated mononuclear cells from mice bone marrow and differentiate it using
EGM2 medium from Lonza for 30 days. Arterial thrombosis was induced using
ferric chloride and the thrombosis time determined using an ultrasound probe. We
injected the cultivated EPC intravenuously 15 min before the lesion. To determine
platelet aggregation we used platelet rich plasma (PRP) and the Chrono-log
agregometer using thrombin as agonist. We observed that the injection of EPCs
increased thrombosis time when compared to control and that EPC can be
localized tunica adventitia 7 days after lesion. Also we observed that platelet
aggregation in vitro was decreased 54%, when PRP was incubated with EPC and that
aggregation was not altered when PRP was pr-incubated with HUVECs (human
umbilical Cord endothelial cells) or fibroblasts, indicating that the anti-aggregation
effect is specific of EPCs. We conclude that the injection of EPCs prolonge
occlusion time probably by interfering in thrombus formation and that this effect
may be related to the interaction of EPCs and platelets altering platelet activation.
0
This work was approved by the ethics comitee of UNICAMP (CEUA n 3504-1).
Financial support: Capes, FAPESP

U7
GENE EXPRESSION ANALYSIS DURING HUMAN STEM
CARDIOMYOGENESIS THROUGH POLYSOME PROFILING
Instituto Carlos Chagas, Fiocruz-Paran, Curitiba-PR, Brazil

Unidad de Bioinformtica, Institut Pasteur Montevideo, Montevideo, Uruguay
CELLS
BY
GABA
Luiz Gustavo Dubois (1)(2), Elias H. El-Habr (2), Joanna Lipecka (2), Laurent
Turchi (2), Alexandra Bogeas (2), Franois-Xavier Lejeune (2), Tomohiro Yamaki
(2), Mohammed Fareh (2), Maxime Janin (2), Joan Pallud (2), Bertrand Devaux (2),
Vivaldo Moura Neto (1), Herv Chneiweiss (2) and Marie-Pierre Junier (2).
(1) Instututo Estadual do Crebro Paulo Niemeyer, Secretaria Estadual de Sade do
Estado do Rio de Janeiro.
(2) Neuroscience Paris Seine, Inserm U1130 CNRS UMR 8246 University Pierre &
Marie Curie, Paris, France.
Background: Most neoplasms contain heterogeneous populations of cancer cells with
varying degrees of aggressiveness notably in terms of proliferation and tumorigenic
abilities. Like normal tissues, tumors are the result of a complex developmental
process. This process mixes clonal selection and cell genesis from cancer stem cells,
resulting in tumors composed of heterogeneous cancer cell populations.
Aims: Considering the importance of metabolism reprogramming in supporting
tumor growth, we envisaged the converse possibility that defined metabolic states
restrict the tumorigenic potential of cancer cells.
Methods and Results: We addressed this question by exploring the changes in
metabolism that accompany the differentiation of glioma stem cells (GSC) into
poorly aggressive cells. Gliomas, primary tumors of the brain, are paradigmatic
examples of heterogeneous tumors. First, we characterized the metabolic changes
that accompany miR-302-367-induced differentiation of GSC into cells deprived of
stem and tumor initiating properties. We then verified the relevance of our findings
for cancer cell behavior using a panel of adult and pediatric GSC isolated from
distinct malignant glioma.
Conclusion: We finally deciphered the links between the metabolic changes we
identified and the epigenetic regulations crucial for maintenance of GSC in an
undifferentiated, tumorigenic state.
Financial Support: Faperj, CNPq, Inserm, CNRS.
HUMAN ADIPOSE DERIVED MESENCHYMAL STEM CELLS (ADSCs)

EXPOSED TO BISPHENOL A (BPA): CHARACTERIZATION AND
TOXICOLOGICAL APROACHES
Helga Caputo Nunes1, Samara Tavares1, Bruno Martinucci1, Elenice Deffune2,
Wellerson Rodrigo Scarano1, Flvia Karina Delella1.
(1) Cell Engineering Laboratory, Blood Transfusion Center, Clinical Hospital,
Botucatu Medical School, Univ Estadual Paulista (UNESP);
(2) Extracellular Matrix Laboratory, Department of Morphology, Institute of
Biosciences, Univ Estadual Paulista (UNESP).
E-mail address: isaabelaa@gmail.com (I. T. Pereira)

+55 41 33163231
Heart diseases are the leading cause of mortality. The low rate of cardiomyocytes
proliferation contributes to the limited regenerative capacity of the heart. Advances
in knowledge about stem cell differentiation process support its potential use in
regenerative therapy. Differentiation of embryonic pluripotent stem cells (ESCs) is a
highly coordinated process involving trigger, maintenance and coordination of gene
expression patterns. Regulation of gene expression acts at both transcriptional and
posttranscriptional levels. Furthermore, previous analysis in eukaryotes comparing
mRNA and protein levels have indicated that there is no direct correlation between
transcript levels and protein synthesis, suggesting an uncoupling between the
transcriptome and the proteome. Therefore, we aimed to analyze the mRNA fraction
associated with translating ribosomes (polysomes) as a strategy to investigate
posttranscriptional mechanisms involved in human ESCs (hESCs) under
cardiomyocyte differentiation. NKX2-5eGFP/w hESCs were differentiated to
cardiomyocytes and submitted to a time course analysis (D0, D1, D4, D9 and D15
days of differentiation). Pluripotency, mesoderm and cardiac markers were used to
follow the cardiomyocyte differentiation by cytometry, immunofluorescence and
RT-PCR. In addition, both the free and polysomes mRNA subpopulation fractions
were isolated using a sucrose density gradient and the polysome profile was
recorded. cDNA libraries were prepared for high-throughput sequencing using the
isolated mRNAs. RNA-seq analysis will allow the identification of
posttranscriptional regulated genes and the characterization of the gene regulatory
networks involved in hESCs cardiomyocyte differentiation. Our results will
contribute to a better understanding of the key regulators, pathways and
posttranscriptional mechanisms involved in human cardiac differentiation and
development.
Financial support: FIOCRUZ, Fundao Araucria, CAPES.
STEM-LIKE
U8
CELL
Isabela Tiemy Pereira1, Anny Waloski Robert1, Lucia Spangenberg2, Marco Augusto
Stimamiglio1, Hugo Naya2, Bruno Dallagiovanna1
1
GLIOBLASTOMA
BPA belongs to a group of compounds capable of disrupting functions by mimicking

or blocking endogenous hormones. By the fact that it has lipophilic properties and
considering the importance of ADSCs both in basic to clinical research, this study
aimed to analyze BPA effects on ADSCs. For this, samples from 4 patients, obtained
after abdominoplasty,were characterized by differentiation tri-lineage and flow
cytometry and exposed to 1M, 10M and 100M BPA within 7, 14, 21 days. Cell
number and viability were evaluated by tripan blue, MTT assay and flow cytometry.
The cells were positive for the Oil Red O, Alizarin Red S and Alcian Blue staining;
and showed high expression of stem cells CD markers (CD13: 98.26; CD73:
94.41%; CD90: 98.26%; CD105: 92%), and negative for non stem cell markers
(CD31: 0.05%; CD45: 2.67%; CD34: 7.21%; and Stro-1: 5.07%).After, ADSCs were
exposed to BPA and100M BPA had a cytotoxic action decreasing at least 1/3 of
initial cell density reaching LD50 for this compound. For 1M and 10M doses,
cells showed very different results, but during 7 days, we observed that cell number
and viability were 5.104cells/ml for 1M and 4.5.104 cells/ml for 10M .For MTT
analysis, 1M showed 6.8%lower absorbance average, and10M33.8% higher
absorbance from control. For apoptosis analysis, ADSCs showed higher apoptosis
rates (1M: 15.92%; 10M: 13.12%) from control. Thus, its ppossible to conclude
that BPA in this conditions is capable of altering important functional patterns in
ADSCs.
Plataforma Brasil CAAE: 32712514.4.0000.5411. CAPES.

U9
U10
GLYPHOSATE FORMULATION INDUCES CELL DEATH AND CHANGES

HUMAN ADIPOSE DERIVED STEM CELLS DIFFERENTIATION
DOSE-DEPENDENT EFFECTS OF TRIIODOTHYRONINE ON THE

CHONDROGENIC DIFFERENTIATION OF RAT BONE MARROW
MESENCHYMAL STEM CELLS
Mariane Izabella Abreu de Melo (1), Thas Maria da Mata Martins (2), Pricila da
Silva Cunha (1),Dawidson Assis Gomes (1,2), Alfredo Miranda de Ges(1, 2),
Eliane Novato-Silva (1).
1
Department of Biochemistry and Immunology , Institute of Biological Sciences,

Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais,
Brazil;
(1)
2
Department of Morphology, Institute of Biological Sciences, Universidade Federal
(2)
de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil.
(3)
The authors declare any financial or personal relationships with individuals or
organizations that could be perceived to bias their work.
Presenting author: Mariane Izabella Abreu de Melo
Address: Av. Presidente Carlos Luz, 6627 - Pampulha, Belo Horizonte - MG, CEP:
31270-901
e-mail:
abreu.mariane@gmail.com
Phone:
+553134092631
Mobile:
+5531991406984
The commercial formulation of glyphosate (Roundup) is a herbicide widely used in
Brazilian crops, however, its potencial human health damage capacity remains
controversial. Human adipose derived stem cells (ASCs) play an important role in
maintaining body homeostasis. Therefore, these cells were used for understanding
the toxic effects of this herbicide. ASCs were exposed to DMEM/
osteogenic/adipogenic differentiation media with Roundup at a concentration of 36
g mL-1 (IC50 42, 98 0, 91 g mL-1) during short (24, 48, and 72 hours) and long
(until 21 days) time of exposure. Glyphosate showed citotoxicity capacity throughout
the days of culture according to MTT assay. Moreover, Annexin V-Alexa Fluor 488
and Propidium Iodide staining suggested that ASCs exposure to Roundup induced
the cells mainly to apoptosis after 24h exposure. Morphological changes were
observed by light microscopy during the ASCs differentiations period with this
herbicide. Otherwise, the expressions of genes related to differentiation, such as
leptin, alkaline phosphatase, and osteopontin, accessed by standart PCR, were similar
compared to the control during toxic challenge. However, despite alkaline
phosphatase (AK) expression, the NBT- BCIP assay have shown that the AK
enzymatic activity were reduced in treated group during osteogenic differentiation.
In conclusion, evidences have shown that Roundup causes mild toxic effects on adult
stem cells derived from adipose tissue. ASCs were derived from volunteers subjected
to liposuction surgery approved by ethics committee n 22912213.3.0000.5149.
Higor Azevedo Assis (1), Nathalia Chicon Elert (1), Andr Luiz Buzato Pereira
Azevedo (1), Rogria Serakides (2), Natlia de Melo Ocarino (2), Alfredo Miranda
de Goes (2), Francisco de Paula Careta (1), Greiciane Gaburro Paneto (1), Adriana
Madeira Alvares da Silva Conforti (1), Jankerle Neves Boeloni (1)
(1) Universidade Federal do Esprito Santo, Alegre, ES
(2) Universidade Federal de Minas Gerais, Belo Horizonte, MG
Email: nathalia.elert@hotmail.com
Telefone: (28) 998816500
Bone marrow mesenchymal stem cells (BMMSC) are potential cells for the
generation of cartilage and have receptors for thyroid hormones. The aim of this
study was to investigate the dose-dependent effect of T3 on chondrogenic
differentiation of BMMSC. BMMSCs of Wistar rats that express CD73, CD54 and
CD90 were cultured in chondrogenic medium and separated into five groups: 1)
BMMSC without T3; 2,3,4,5) BMMSC with T3 (0.01; 1; 100 e 1000nM). At seven,
14 and 21 days were evaluated: histomorphometric analysis and quantification of
gene transcripts for Sox-9 and collagen II by qPCR. The means were compared by
the Student-Newman-Keuls test. At Seven days, there was no formation of
cartilaginous matrix. At 14 and 21 days, all the groups treated with T3 showed
matrix formation greater than that control group, however the group T3 0.01nM
showed higher matrix formation at 14n days than that of other groups. At 7 and 14
days, there was no difference in expression of Sox-9, however at 21 days the group
T3 1000nM showed expression significantly greater than that of other groups. The
group T3 0.01nM showed higher expression of collagen II at seven days than that of
other groups. Furthermore, the group T3 1nM showed higher expression of collagen
II at 14 days than that of other groups, however at 21 days there was no difference.
In conclusion, the effect of T3 on BMMSC differentiation is dose-dependent, with
the 0.01nM e 1nM doses promoting better BMMSC chondrogenic differentiation.
Source of funding: CNPQ, FAP/UFES
This work was supported by CAPES, CNPq and Ministrio da Sade (Brazil).

U11
U12
EFFECTS OF DERMATAN SULFATE AND HEPARIN ON THE

PROLIFERATION OF ENDOTHELIAL PROGENITOR CELLS AND
MATURE ENDOTHELIAL CELLS
Luiz Henrique de Freitas Filho1, Jlia Carnaz Benincasa1, Cludio Chrysostomo
Werneck2, Cristina Pontes Vicente1
11 - Department of Structural and Functional Biology, State University of Campinas
(UNICAMP), So Paulo, Brazil
22 - Department of Biochemistry and Tissue Biology, Institute of Biology, State
University of Campinas (UNICAMP), So Paulo, Brazil
3Email: luizbioufu@gmail.com. Address: Rua Charles Darwin s/n, bloco N, 3 andar,
4Phone: 55 (19) 3521-6106, Mobile: (55) 19 984560270
Dermatan sulfate (DS) and heparin (HEP) are glycosaminoglycans with known
antithrombotic and anticoagulant activity. DS is also able to increase the
mobilization and "homing" of endothelial progenitor cells (EPCs) during vascular
remodeling. The aim of this study is to analyze the effect of these
glycosaminoglycans on endothelial cells (EC) and EPC proliferation in vitro. We
used cultured HUVEC (Human umbilical Cord endothelial cells) or EPCs
differentiated from mice bone marrow mononuclear cells using EGM2 medium
(Lonza) for 30 days with different concentrations of DS or HEP during 72 hours and
determined cell proliferation using sulforodhamine staining. We observed no change
in proliferation after 24 hours with all treatments. HUVEC proliferation increased
25% in 48 hours and 29% after 72 hours with 50g/ml DS. Treatments with 25g/ml
or 50g/ml of HEP, increased HUVEC proliferation 55.5% and 45%, after 48 hours
and after 72 hours 45.7% and 27.4% respectively. EPCs proliferation increased 31%
with DS 50g/ml after 48 hours and 38% after 72h. Treatment of EPCs with 25g/ml
or 50g/ml of HEP increased 23% and 47% after 48 hours and 32% and 34% after
72 hours respectively when compared to control. In conclusion, HEP increases cell
proliferation more efficiently than DS, but high concentrations of HEP (50g/ml) can
inhibit cell growth in cultured HUVECs and EPCs after 72 hours.
This project was approved by the Ethics Committee on Animal Use (CEUA),
protocol number 3916-1.
Support: CNPq, FAPESP
THE ROLE OF EPC GROWN IN DIFFERENT CULTURE MEDIA ON THE

ARTERIAL THROMBUS RESOLUTION
Giane Daniela Carneiro1 Fabio Trindade Maranho Costa2, Claudio Chrysostomo
Werneck3 and Cristina Pontes Vicente1
Campinas, Brazil.
(2) Department of Genetics, Evolution and Bioagents, State University of Campinas,
Campinas, Brazil.
(3) Department of Biochemistry e Tecidual Biology, State University of Campinas,
Campinas, Brazil.
Email: cvicente@unicamp.br. Address: Rua Charles Darwin s/n, bloco N, 3 andar,
Phone: 55 (19) 3521-6106, Mobile: (55) 19 997726655
Endothelial progenitor cells (EPC) can be cultured from bone marrow mononuclear
cells and used for cell therapy use to promote vascular regeneration. In this work, we
analyze the role of EPCs cultured 30 days in two different culture media (EGM2, a
commercial medium or DMEM-M1 - DMEM plus VEGF, IGF and bFGF) on arterial
thrombus formation and resolution induced by FeCl3. Immunofluorescence
demonstrated that EPCs grown in the two media present the main EPCs markers
CD34, VEGFR2 and CD31. Flow citometry showed that 49% of EGM2-EPCs and
26.65% of DMEM-M1-EPCs are CD31 positive. Intravital microscopy of thrombus
formation in the animals that received EPCs injections 15 min before lesion showed
increased thrombus embolization in M1 cells treated animals. Thrombosis time was
determined using an ultrasound probe and we observed an increase in M1 (26.23
min) and EGM2 (16.29 min) time when compared to control (4.27 min.). Vessel
histological analysis of mice injected 15 min, 1 and 24 hours after injury showed a
reduction of the 90% of thrombus area 7 days after injury with M1 cells. Injected
EPCs were localized in the vessel wall and adventitia. eNOS expression in vessels
treated with M1 and EMG2 increased 4,6 x and 5,6 x, respectively. We conclude
DMEM-M1 generates a less differentiated population of EPC that are able to
increased thrombosis time and thrombus embolization increasing eNOS expression
in the injured vessel.
This project was approved by the Ethics Committee on Animal Use (CEUA),
protocol number 2988-1. Support: CNPq, FAPESP

U13
U14
COLLAGEN V/MESENCHYMAL STEM CELLS: A NEW PERSPECTIVE

FOR CARTILAGE TREATMENTS
VALIDATION OF A PROTOCOL FOR THE GENERATION OF GOOD

MANUFACTURING PRACTICE (GMP) -GRADE HUMAN BONE
MARROW STROMAL CELL PRODUCTS FOR AUTOLOGOUS USE IN
BONE REPAIR
Isabele Camargo Brindo da Cruz (1), Ana Paula Pereira Velosa (1), Antonio dos
Santos Filho (1), Solange Carrasco (1), Eduardo Pompeu (2), Denise Frediane
Barbeiro (3), Vera Luiza Capelozzi (4), Walcy Rosolia Teodoro (1)
(1)
(2)
(3)
(4)
(1) Discipline of Rheumatology, Medical School of the University of So Paulo, So

Paulo, Brazil
(2) Bioterism Center, Medical School of the University of So Paulo, So Paulo,
Brazil
(3) Discipline of clinical emergencies, Medical School of the University of So
Paulo, So Paulo, Brazil
(4) Department of Pathology, Medical School of the University of So Paulo, So
Paulo, Brazil
isabelecamargo@yahoo.com.br
Institutional Phone: (11)30617211
Mobile Phone: (11)976282999
Background: In cartilage defects there are no treatments with regenerative aspects. In
this way the use of adipose derived stem cells (ADSCs) is emerging on this area due
to their potential for differentiation in many cell types according to a specific
stimulus. Collagen V (COLV) has some particular characteristics and is has an
important role in the development of functional tissue by regulating diameters
collagen fibers in addition to be present on embryonic cartilage.
Objective: Evaluate the expression of extracellular matrix (ECM) components by
ADSCs of rabbits previously cultivated with COLV.
Methods: New Zealand male rabbits were used to obtain ADSCs. Cells were grown
in DMEM (control) and with COLV (50g/mL/72h) and evaluated by histology to
Alcian Blue and Picrosirius, to immunostaining to collagen II (COLII) and
additionally ADSCs were cultivated with TGF (10ng/mL per 72h) to compare
COL2A1 and ACAN genes expression front to control and COLV groups.
Results: ADSCs stimulated with COLV showed more proteoglycans and collagen
expression in relation to control group. In addition, immunostaining to COLII
showed that after COLV stimulation, ADSCs improves is expression. In comparison
to TGF stimulus, COLV was higher as well in gene expression of COL2A1 and
ACAN compared to control group.
Conclusion: The data suggest that COLV, as well as TGF, can act as a
chondrogenic factor to stem cells mediating the increase expression of ECM
components. Therefore, ADSCs/COLV emerges as promise therapeutic in patients
with cartilage degeneration.
Rhayra B. Dias1, Ana C. Leal2, Maria Eugenia L. Duarte2, Rafaela C.

Sartore2,Danielle C. Bonfim2,3
1
Master Program in Musculoskeletal Sciences, National Institute of Orthopedics and

Traumatology, Rio de Janeiro, RJ, Brazil
2
Research Division, National Institute of Orthopedics and Traumatology, Rio de
Janeiro, RJ, Brazil
3
Craniofacial and Skeletal Diseases Branch, National Institute of Dental and
Craniofacial Research, NIH, Bethesda, Maryland, USA
Bone Marrow Stromal cells (BMSCs) contain inherent osteoprogenitors and
constitute a potent tool in the treatment of bone-related conditions, such as criticalsized defects. However, clinical applications require a significant number of cells,
implying the necessity of in vitro expansion. Here we evaluated a protocol for the
generation of GMP-grade BMSCs products, in compliance to the Brazilians
guidelines
(RDC
n
9/2011,
ANVISA)
for
cellular
therapy.
This project was approved by ethics committee of National Institute of Orthopedics
and Traumatology (971.348). Six bone marrow samples were obtained from surgical
waste of primary hip arthroplasties. Donors were aged between 47-61 years,
provided informed consent, and tested negative for blood-borne pathogens. The
frequency of clonogenic cells in each sample ranged from 7,152,5 to 39,65,13 x
per 100,000 nucleated cells, averaging 19,5 1,73. To determine optimal conditions
for cell isolation, two initial seeding densities were tested: 8,000 and 200,000
nucleated cells per cm2. We observed that the higher density yielded the highest
number of BMSCs at passage 1 (10,2 x 106 4,73 vs 2,245x106 1,90). At the end
of expansion in multilayer cell factories, the average yield was 6,2 x 107 cells. Cells
in all products expressed CD73, CD90, CD105 and CD146, were able to differentiate
to the osteogenic and adipogenic lineages, and were negative for aerobic and
anaerobic pathogens. We concluded that BMSCs products generated under our
protocol were in accordance with the requirements of the Brazilians laws and can be
exploited in future autologous applications for bone repair.
Approval of the Ethics Committee: CEUA 123/14

Financial support: Fapesp (2014/11419-5)
U15
METHYLMERCURY
EXACERBATES
CYTOTOXICITY IN MESENCHYMAL STEM CELLS
U16
DIOXIN-INDUCED
Mariana L. Sanches1; Cintia K. Tokuhara2; Camila Peres-Buzalaf1

1
Programa de Mestrado Acadmico em Cincia e Tecnologia Ambiental,

Universidade do Sagrado Corao, Bauru,SP;
2
Faculdade de Odontologia de Bauru, Universidade de So Paulo, Bauru, SP.
Correspondent author information: marianaliessa@hotmail.com Fone number: +55
(14) 2107-7069/ 99687-6813
Background: The prevalence of various human diseases is associated with the
growing incidence of environmental pollutants. Methylmercury (MeHg) and dioxins
are widespread and considered persistent pollutants due to high bioaccumulation in
body tissues. Besides, they coexist in the environment, allowing integration of toxic
effects such as antagonism, addition and synergism. Several studies have focused on
high doses and their particular effect in hematopoietic stem cells. But the impact of
these toxicants in mesenchymal stem cells (MSCs) at low doses, as well as their
differentiation potential is still understood. Aims: To evaluate the in vitro MSCs
viability after combination or not of MeHg and TCDD (2,3,7,8-tetrachlorodibenzo-pdioxin) exposure, evaluated by MTT assay. Methods: For this purpose, bone marrow
cells of 129/Sv strain mice were obtained and cultured in 37C/5% CO2 for several
passages until obtaining a homogenous population of mesenchymal stem cells
phenotypically characterized. Then, adherent MSCs were treated with MeHg (0.0540M), TCDD (0.001-0.1M) or combination of both (at non cytotoxic doses) for 1
and 5 days. Results: Analysis allowed us to observe that the both MeHg and TCDD
showed cytotoxic effect in a dose and time-dependent manner. At similar doses, they
do not present differences in cytotoxicity. When in combination, MeHg decreased
cell viability compared to the TCDD-treated cells in both periods (p <0.05).
Conclusion: These preliminary results show that MSCs are target of cytotoxic effect
followed by combination of MeHg and TCDD even at very low doses, suggesting
toxic synergistic effect between them.
Financial Support: CAPES. Ethics committee: 4267011215.
(4)
RAT MESENCHYMAL STEM CELL BIOSTIMULATION UNDER THE

CONTINUOUS AND PULSED LIGHT ACTION OF 630 nm USING LED
Helosa Vicente Garcia (1), Ana Lvia de Carvalho Bovolato (1), Ana Claudia
Simes (1), Woner Mion (1,2), Brunno Felipe Ramos Caetano (3), Joo Paulo de
Castro Marcondes (4), Natlia Mayumi Inada (2), Marjorie de Assis Golim (1),
Rosana Rossi Ferreira (5), Andrei Moroz (6), Elenice Deffune (1,7).
1. Blood Center, Botucatu Medical School - UNESP.
2. So Carlos Phisics School USP.
3. Institute of Biosciences of Botucatu UNESP.
4. Pathogy Department, Botucatu Medical School UNESP.
5. Science School, Biological Science Departament, UNESP of Bauru.
6. School of Pharmaceutical Sciences of Araraquara UNESP.
7. Urology Department, Botucatu Medical School - UNESP
Ana Lvia de Carvalho Bovolato:
Address: Botucatu Medical School, Blood Center, UNESP.
e-mail: liladcb@gmail.com ; Telephone contact: (14) 3811-6041
Helosa Vicente Garcia: helo.v.garcia@hotmail.com; Ana Claudia Simes:
anaa.csimoes@gmail.com; Woner Mion: wonermion@yahoo.com.br; Brunno Felipe
Ramos
Caetano:
brnncaetano@gamil.com;
Joo
Paulo
de
Castro
Marcondes:jpcastromarcondes@uol.com.br;
Natlia
Mayumi
Inada:
nataliainada@gmail.com; Marjorie de Assis Golim: marjorie@fmb.unesp.br; Rosana
Rossi Ferreira: rossi@fc.unesp.br; Andrei Moroz: moroz@fcfar.unesp.br; Elenice
Deffune: ed12@fmb.unesp.br
Background: Regenerative medicine (RM) has emerged of different sciences boost,
including biology of mesenchymal stem cells (MSCs). They are characterized by
differentiation, self-renewal and the ability to generate different cell lines, involving
damaged tissue repair. However they are dose-dependent, requiring expansion by
cell culture method. The applicability of LED (Light Emitting Diode) as
biostimulator agent has been proposed. Aims: Evaluate the effect of continuous and
pulsed light, 630 nm, comparing this action in cells obtained by the methods of
mechanical (MD) and enzymatic dissociation (ED). Methods: AT was removed from
rats of Wistar lineage with an average age of 3 months. Results: Concentration cells
obtained/gram of tissue was 1 x 105 from MD and 3.34 x 105 from ED. The
phenotypic profile using six markers (BD) showed that ED removes surface
antigens of MSCs. MSCs obtained by MD was beneficiated by the action LED and
proliferating faster (confluence curve at 80%). Most of the samples showed cytokine
(TNF, IFN-, IL-4, IL-10) secretion was obtained by MD. Control samples, notirradiated, also showed cytokines secretion, showing action of the culture medium
additive as biostimulator of paracrine effects. The micronucleus and comet assays,
respectively, showed that pulsed light of 1 J/cm2 determine appearance of
micronuclei, while DNA damage determined decreases by LED action. Conclusions:
MSC photostimulation in culture active molecules, cell sign ways and modulate
MSC functions. It is a not invasive aplication, cheaper and efficient, with
applicability potencial in future researchs.
Ethical Approval: CEP 1009/2013. Funding Suport: FAPESP (2013/19998-1).

U17
U18
PLATELET ACTIVATING FACTOR RESCUES THE PLURIPOTENT

STATE OF MURINE EMBRYONIC STEM CELLS AFTER SPONTANEOUS
DIFFERENTIATION IN VITRO
EXPRESSION OF PLURIPOTENCY AND DIFFERENTIATION-RELATED

GENES DURING IN VITRO MOUSE TROPHOBLAST DIFFERENTIATION
Thais Braga Gomes (1), Ludmilla Oliveira da Silva (1) and Lucianne Fragel-Madeira
(1)
Aline Gomes Melo1; Emanuel Ricardo Monteiro Martinez1; Gabriela Aparecida

Jorge Daoli1; Rafael Henrique Nbrega1
Department of Morphology, UNESP Botucatu, So Paulo, SP.

Brazil.
E-mail: thaisbragagomes@id.uff.br
4229
Phone: (55-21) 3674-7844 / (55) 21 96976-
Embryonic stem cells have two main features: self-renewal and differentiation
potential. Their pluripotent state is maintained in vitro by addition of Leukemia
Inhibitory Factor (LIF). Sphingosine-1-phosphate and lysophosphatidic acid,
bioactive phospholipids, are being described as important maintainers of stem cells
pluripotency. However, little is known about the role of another bioactive
phospholipid, Platelet Activating Factor (PAF), in this process. Preliminary results
from our group suggest that PAF rescued the murine embryonic stem cells (mES)
pluripotent state after spontaneous differentiation in vitro increasing the expression
of OCT-4 and decreasing the expression of Nestin. However, these proteins have
been shown not to be good markers of pluripotency and neural commitment,
respectively. Therefore, our aim was to confirm the effect of PAF upon cell fate of
mES. USP1 and E14TG2a mES lineages underwent into spontaneous differentiation
without LIF for 7 or 4 days, respectively. Subsequently, mES were treated with
10nM PAF alone or with WEB2086 (PAF receptor antagonist) at various
concentrations for 24 hours. USP1 and E14TG2a lineages spontaneously
differentiated expressed PAFR. When these cells were treated with 10nM PAF we
observed an increase on SSEA-1 immunofluorescence and AP activity and a
decrease on PAX-6 (neural marker) immunolabeling and these effects were reverted
by WEB2086 at 100 nM and 10 nM. Taken together we concluded that PAF, by its
receptor, could rescue the pluripotent state of mES in vitro.
protocol number 193/2013.Funding Support: Capes, FAPERJ, CNPq and ProppiUFF.
lih.melo@hotmail.com
Trophoblast stem cells are defined by their capacity of self-renewal and
differentiation into one or more specific cell types. According to literature data, stem
cells have been characterized in amniotic tissue, yolk sac and chorion. These cells
are derived from mesoderm associated with these structures at different stages of
gestation. Here, we aimed to evaluate the expression of pluripotency and
differentiation-related genes during trophoblast differentation in vitro. For that, mice
ectoplacental cones explants were removed from pregnant female mice at 7.5 days,
cultivated for 24h, 48h, 64h and 72h, and collected for total RNA extraction and
cDNA synthesis. Primers for Nanog, Eomesodermin, Fgf4, Fgfr2, Cdx2, and -actin
were designed based on Mus musculus sequences and used in RT-PCR. Results
showed expression for all genes during all time points with slight variations for Fgf4
(increased expression after 24h of culture) and Eomes (slight increase after 48h of
culture). From these data, we conclude that all studied genes were expressed for 72h
in vitro, and during trophoblast differention Nanog expression were maintained
suggesting the existence of pluripotent cells in the ectoplacental cone after 72h of
culture. Eomes increased its expression from 48h of culture confirming trophoblast
differentiation. The presence of Fgf4 and its receptor, Fgfr2, confirms the presence
of cells from the inner cell mass in vitro. These results provide a better understanding
of the dynamics of trophoblast stem cell differentiation of and help us to propose an
in vitro model to study the role of growth factors in the paracrine/autocrine
regulation of these cells posteriorly.
Keywords: stem cells, trophoblast cells, mouse, pluripotency
Financial Support: FAPESP (2015/12104-0)

U19
U20
CICATRICIAL POTENTIAL OF ASCS ISOLATED FROMHUMAN FACIAL

AND ABDOMINAL ADIPOSE TISSUES
STUDY OF THE NUCLEAR RECEPTOR COUP-TFII IN MOUSE

EMBRYONIC
STEM
CELL
USING
CRISPR/Cas9
SYSTEM
Priscilla Barros Delben1, Helena Debiazi Zomer1, Camila Acordi1, Aruana Hansel1,
Fernanda Rosene Melo1, Rogrio SchutzlerGomes2, Deborah A. Cornlio3, Gabriel
da Silva Pescador1, Talita Jeremias1,Silvia Regina Batistuzzo de Medeiros3, Patrcia
Dillenburg-Pilla1, Andrea Gonalves Trentin1.
(1)
1
DepartmentofCellbiology, EmbriologyandGenetic, Federal Universityof Santa
(2)
2
Rogrio Gomes Cirurgia Plstica, Florianpolis, Brazil.
3
Cell BiologyandGeneticsDepartment, Federal Universityof Rio Grande do Norte,
(3)
Natal, Rio Grande do Norte.
(4)
Henrique Marques Barbosa de Souza (1), Amanda Araujo Gomes Ferreira (1),
Viviane de Souza Rosa (1), Aline Mika Matsuguma (1), Caique Camargo
Malosprito (2).
Gabriel da Silva Pescador1: Email: gabrielspescador@gmail.com Phone: 48 91911068

Priscilla Barros Delben1: Email: priscillabarrosdelben@gmail.com Phone: 48 99695260
The nuclear receptor COUP-TFII (factor Chicken Ovalbumin upstream promoter

transcription factor) is responsible for many functions in embryonic development
being involved in the differentiation of neural precursors, mesenchymal and cardiac
cells, besides being involved in others distincts structures and organs, such as: blood
vessels, pancreas and stomach. Coup-TFII has been shown to be intrinsically
involved in the early steps of human Embryonic Stem Cell (ESC) differentiation, by
forming a regulatory circuit with Oct4 and miRNA302. While Oct4 and miRNA302
repress transcriptionally and post-transcriptionally, respectively, Coup-TFII in
undifferentiated hESC, Coup-TFII represses both genes in order to activate cell
differentiation programs. The role of COUP-TFII between the pathways of
undifferentiated to differentiated cells is still partially unknown. To study the
possible roles of Coup-TFII in mouse ESC differentiation, we adopted as a strategy
CRISPR/Cas9 system to knockout the gene and establish a reporter cell line, using
the homology directed repair mechanism to insert a reporter gene within CoupTFIIs casset. The single guide RNA (sgRNA) for the report cell line was built based
in targeting the stop codon of the last exon, which does not affect the gene
functionality. Two Donor Vectors were built using different report genes: GFP and
mCherry fluorescent proteins. We constructed three distinct vectors containing the 5
homology arm, the reporter gene and 3 homology arm, then we used restriction
enzymes to assembling the final vector, so we lipofected the donor vector and the
vector containing the sgRNA into cells mESC E14tg2a. It was possible to genotype
the knockout cell lines by sequencing and the reporter cell lines are still in progress.
Background: Adipose stromal cells (ASCs) are a promisingty pe of stem cells for
regenerative medicine dueto its healingability. The most studied ASCs sourceis
adipose tissue (AT) obtainedby abdominal liposuction. However, AT differ in their
composition, structure and function in accordance with its housing in thebody. Aims:
In this context, the aim of this work was to compare the healing ability of ASCs is
olated from facial and abdominal TA in vitro. Methods: ASCs isolated from face and
abdomen were characterized in our previous work. These cells were subjected to a in
vitro wound healing test, consisting in a scratch assay in the cell monolayer.
Furthermore, cellular integrity was analyzed by cellular senescence curve and
cytokinesis-blockmicronucleusassay (CBMN). Results: Results show that both cells
are equally effective in closingthein vitro scar, presenting more than 90% of closure
after 21 hours. Also, both cells have a similar curve of senescence. However, the
facial ASCs seemed more genetically stable, presenting a lower number of nuclear
cytoplasmic bridges comparedwith abdominal ASC. Conclusion: We can conclude
that facial and abdominal ASCs are promising for regenerative medicine.Thus, ASCs
from the human face can be an alternative fount of cells for these purposes with the
advantage to maintain genetic stability. These results may indicate that face ASC are
safer for therapeutic use, reducingthe chance of tumor appearances after
transplantation.
Supportedby CAPES and CNPQ. EthicscommitteeCEP-UFSC: 131.512
(1)Department of Biochemistry and Tissue Biology, University of Campinas,

Campinas, Brazil.
(2) Department of Biochemistry and Tissue Biology, University of Campinas,
Campinas, Brazil. Email: Caique Camargo Malosprito.
Institutional telephone: (19)35216244, Mobile Telephone: (11) 950202643
Financial: 2015/22067-5, (FAPESP).
(1)
(2)
(3)
(4)
U21
U22
FROM MESENCHYMAL STEM CELLS TO EPIDERMIS DESIGN IN

VITRO: STUDY OF SKIN BIOLOGY AND PERSPECTIVES FOR THE
UNDERSTANDING OF DISEASES DEVELOPMENT
EXTRACELLULAR VESICLES OF MESENCHYMAL STEM CELLS FROM

HUMAN
ADIPOSE
TISSUE
IMPROVE
MIGRATION
IN
KERATINOCYTES AND FIBROBLASTS AND ACTIVATE AKT AND H3
Jeniffer Farias dos Santos (1, 2), Mariana da Silva Arajo (1) and Viviane Abreu
Nunes (2).
Ferreira, A.F.(1); Cunha, P.S.(1); Mendes, V.C.(2); Frezard, F. (2); Resende, V.

(3);Goes, A.M.(2);Gomes, D.A. (2).
(1) Department of Biochemistry of Federal University of So Paulo, So Paulo,

(1)
Brazil;
(2) School of Arts, Sciences and Humanities of University of So Paulo, So Paulo,
(2)
Brazil.
(3)
Contact: jeni.fs@hotmail.com / (+5511) 94106-2565
(1) Department of Biochemistry and Imunology, Federal University of Minas Gerais,

MG, Brazil
(2) Department of Physiology and Pharmacology, Federal University of Minas
Gerais, MG, Brazil
(3) Department of General Surgery, Federal University of Minas Gerais, MG, Brazil
Andrea Da Fonseca Ferreira; Rua Esprito Santo, nmero 1627, apto 11, Belo
Horizonte, MG, Brazil andreaferreira3@gmail.com; (31)3409-2632; (31)971142168
Background: Epidermal differentiation is a complex process in which keratinocytes

go through morphological and biochemical changes. Several approaches have been
proposed to study skin biology and keratinocytes differentiation; however the use of
epidermal cells and the establishment of stable cultures have been restricted. In this
context, stem cells emerge as a promising alternative and have been used in both
research investigation and regenerative medicine. Aims: The aim of this work is to
evaluate the potential of differentiation of mesenchymal stem cells (MSC) from
umbilical cord into keratinocytes and to study molecular events involved in this
process based on the activity of kallikreins, and expression of cytokeratins (CK).
Material and Methods: MSC were cultured in Dulbecco's modified Eagle's medium
(DMEM), 10% fetal bovine serum until reached the spindle-shaped appearance.
After characterization as MSC, differentiation was induced by culturing cells in a
medium for keratinocyte development (KSFM) supplemented with Epidermal
Growth Factor and 0.09 or 1.8 mM CaCl2. Cultures were evaluated on days 1, 4, 7,
11, 14, 17 and 23, based activity of tissue kallikreins 5, 6 and 7 upon specific
synthetic substrates, as well as expression CK10 and CK14. Cell morphology was
monitored in these time points. Results: Data showed high activity of these proteases
in the final cultivation periods (14, 17 and 23 days), and expression analysis of CK10
and CK14 in the early cultivation. Conclusion: these results open the possibility for
the use MSC as a model for studying the biology of the epidermis in vitro.
Subcutaneous adipose tissue is a good source of mesenchymal stem cells used in

several experimental models to achieve tissues regeneration. Cell therapy strategies
usually employ stem cell injection into damaged areas and those treatments bring
good results, but may also cause adverse effects such as the generation of tumors.
Thus, it is imperative to develop new ways to induce tissue regeneration with less
adverse effects. Stem cells secrete extracellular vesicles (EVs) that carry proteins and
RNAs. These molecules, once internalized by epithelial cells, may assist wound
healing, which is dependent on keratinocytes and fibroblasts physiology. The goal of
this study is to investigate the action of EVs in cellular migration and in the
activation of the H3 and Akt pathways. Human stem cells extracted from adipose
tissue were characterized via their differentiating potential and phenotype. EVs
isolation was done by ultracentrifugation and characterization was performed by
optical microscopy, with Nanosight-LM-10. To evaluate the potential effect of EVs
in human keratinocytes and fibroblasts migration and proliferation, scratch wound
healing assay and growth curves were performed. Western blot was used to evaluate
Akt and H3 activation. Our results suggest that keratinocytes and fibroblasts exposed
to stem cells EVs have improved migration, cell proliferation and Akt and H3
activation. We suggest that stem cells EVs could change the microenvironment and
that could lead to improved cell migration in skin cells.
Supported by FAPESP, CAPES and CNPq.
This work was approved by the UFMG ethics committee (35492714.3.0000.5149)

and financially supported by CNPq, FAPEMIG and Capes.

U23
CHARACTERIZATION OF MESENCHYMAL
DERIVED FROM HUMAN ABDOMINAL DERMIS
STROMAL
CELLS
Helena Debiazi Zomer1, Gisele Cristina Varela1, Priscilla Barros Delben1, Maiara
Marques1, Gabriel da Silva Pescador1, Talita Jeremias1, Patrcia Dillenburg-Pilla1,
Andrea Gonalves Trentin1.
1
Departmentof Cell biology, Embriology and Genetic, Federal University of Santa

Gabriel da Silva Pescador1: Email: gabrielspescador@gmail.com Phone: 48 91911068
Mesenchymal stromal cells (MSC) are multipotent stem cells found in various adult
tissues. They are easy to obtain and have high proliferation capacity in vitro. When
transplanted, MSC have the potential to migrate to injury sites, and they are not
immunogenic, or tumorigenic. The abdominoplasty is one of the most frequent
surgery in Brazil, where large amount of dermal tissue is discarded. Thus, it consists
in a promising source of dermal derived MSC. The objective of this study was to
characterize mesenchymal stromal cells derived from human abdominal dermis
(DSC) as a potential source of human MSC to future cell therapies. The DSC were
isolated and characterized in vitro by morphological analysis, colony forming ability,
cell proliferation curve, immunophenotipe markers and cell differentiation potential
to three mesodermal phenotypes. Results shown that the DSC are fibroblastic and
plastic-adherent, have the ability to form colonies and have a high proliferative
potential in vitro. DSC express the mesenchymal markers CD90 and CD73, and do
not express the hematopoietic markers CD45 and CD34. Besides, this cells were able
to differentiate into adipocytes, osteocytes and chondrocytes. In conclusion, the
dermal derived mesenchymal stromal cells share the same characteristics to other
sources of human MSC, consisting in a promising alternative to future therapeutical
aproacches.
Supported by CAPES and CNPQ. Ethics committee CEP-UFSC:1076626

V TISSUE ENGINEERING
V1
V2
EFFECT OF THE AQUEOUS EXTRACT OF COFFEE BEAN RESIDUAL

PRESS CAKE AND CHLOROGENIC ACID ON THE SKIN WOUND
HEALING
EVALUATION OF BOVINE BONE FRAGMENTS AS SCAFFOLDS FOR

OSTEOBLASTIC CELL CULTURE
R. Affonso1, A. P. L. Voytena1, S. Fanan1, H. Pitz1, A.L. Horstmann1, A. Pereira1, D.

S. Coelho1, C. M. Bauer1, V. G. Uarrota1, M.C.R. Hillmann1, L.A.C.Varela1, R. M.
Ribeiro-do-Valle1, G. Calloni1, M. Maraschin1.
1
Plant Morphogenesis and Biochemistry Laboratory, Plant Science Center, Federal

University of Santa Catarina, 1346, Admar Gonzaga Road, Phone number.+55-483721-4813, Fax +55-48-3721-5442, 88048-000, Florianpolis, Santa Catarina, Brazil
Bruno Machado Bertassoli (*1), Millor Fernandes do Rosario (2), Gerluza Aparecida
Borges Silva (1), Erika Cristina Jorge(1)
1 Department of Morphology, Biological Science Institute, Federal University of
Minas Gerais, Belo Horizonte, Brazil.
2 Academic Department, Nature Science Center, Federal University of So Carlos,
Buri, Brazil.
(*) brunobertassoli@gmail.com
This study determined in vivo the effect of the aqueous extracts of green and roasted
coffee bean (Coffea arabica L.) residual press cake (AE) and chlorogenic acid (CA)
on the skin wound healing (SWH). For that, mice with dorsal excisional lesion were
treated with hydrogels containing AE or CA by measuring the reduction of the
wound area of. AE of residual press cake of both green and roasted coffee beans
ameliorate the wound process by reducing the lesion area, with superior performance
for the green coffee AE (78.20% of reduction of the lesion area). Interestingly,
phytochemical analysis detected highest contents of chlorogenic acid (55.60 3.95
mg/g), the major compound, in the AE of the green coffee residue, prompting us to
investigate the effect of that polyphenolic acid in the SWH process. Mice treated
with hydrogel containing CA (3%) were assessed at 3, 7, and 15 days, showing
significantly (p<0.05) reduction of the wound area in the inflammatory phase, which
might be associated with the well known antioxidant and anti-inflammatory action of
CA. The use of the AE of green coffee residual press cake improved the skin wound
healing process and contributes to the reuse of this residual biomass.
Keywords: Coffea arabica L.; chlorogenic acid; post-pressing coffee beans; skin
wound healing.
+55 31 34093033
+55 31 991223045
New strategies for bone tissue engineering are focusing on the development of
biomaterials to be used as scaffolds to improve osteoblastic cell proliferantion,
migration, adhesion, and differentiation. Bovine bone fragments have been showing
positive results when used as grafts in several clinical procedures. This work aimed
to analyze the potential of a bovine bone produced by a Brazilian company called
Critria, as scaffold for osteoblast culture. The pores of these fragments were
mesuared using the Leica LAS-EZ software. Histological sections were evaluated to
detect the presence of bone markers, by immunofluorescence. In vitro analysis was
performed using primary osteoblast cells (from rat calvaria) and MC3T3 cell line.
Cells were seeded onto the fragments and cultured in basal medium for 7,14 and 21
days. The effects of the presence of the bone in culture were evaluated on cell
viability (MTT), and on cell adhesion (eletronic microscopy). Cell differentiation
was assessed by RT-qPCR for bone markers after 7, 14 and 21 days. Pores sizes
measurement revealed that 77% of them were <500m, suggesting favorable
porosity for cell growth and adhesion. BMP4, Osteopontin and type II Collagen were
detected in the bone matrix. An increase in cell viability was observed over time.
Eletronic microscopy showed that cells could adhere to the biomaterial. The
expression of Runx2, Collagen type I, Osterix and ALP was confirmed during 21
days in culture. Our data allowed us to envision the use of this Brazilian bovine bone
as a scaffold for bone tissue engineering methods.
Key words: Bone bioengineering, pre-osteoblasts, scaffold, bovine bone.
V3

V4
ASSESSMENT OF TROPHIC FACTORS ASSOCIATED TO NATURAL

AND SYNTHETIC HYDROGELS FOR DERMAL REGENERATION.
EFFECTS
OF
DIFFERENT
TEMPERATURES
ON
THE
DECELLULARIZATION OF SKELETAL MUSCLE IN A RAT MODEL
Felipe Azevedo Gomes (1), Michele Patrcia Rode (2), Maiara Marques da Silva (3)
Maria Beatriz da Rocha Veleirinho (4), Marcelo Maraschin (4), Leila Hayashi (5),
Rui Daniel Schrder Prediger (6), Marco Augusto Stimamiglio (7), Giordano
Wosgrau Calloni (1).
Carla Maria Figueiredo de Carvalho Miranda1*, Luciano Csar Pereira Campos

Leonel1, Rafael Rossi2, Talya de Moraes Coelho3, Maria Anglica Miglino1, Sonja
Ellen Lobo1,4
(1) Laboratrio de Plasticidade e Diferenciao das Clulas da Crista Neural

BEG/CCB UFSC, Santa Catarina, Brasil. e-mail: feeazevedo@gmail.com
Telefone: 55 (48) 3721 4581; 55 (48) 9991 1826
(2) Grupo de Estudos de Interaes entre Micro e Macromolculas CIF/CCS
UFSC, Santa Catarina, Brasil
(3) Laboratrio de Clulas Tronco e Regenerao Tecidual - BEG/CCB - UFSC,
Santa Catarina, Brasil
(4) Laboratrio de Morfognese e Bioqumica Vegetal - FIT/CCA - UFSC, Santa
Catarina, Brasil
(5) Departamento de Cincias Agrrias UFSC, Santa Catarina, Brasil.
(6) Departamento de Farmacologia, Centro de Cincias Biolgicas, Universidade
Federal de Santa Catarina (UFSC), Florianpolis, Brasil
(7) Fundao Oswaldo Cruz/Instituto Carlos Chagas Paran, Brasil.
The conditioned medium (CM) secreted by multipotent stromal cells of human
dermal origin (MSHD) contains growth factors and extracellular matrix proteins.
Nowadays, CM is receiving attention due to possible roles on tissue
regeneration. Synthetic and natural materials have been used as scaffolds for in vivo
application of CM. Here, we used carrageenan (a natural material) and polyvinyl
alcohol (PVA - a synthetic material) as vehicles to deliver CM of MSHD to injured
mice skin. Therefore, the main objective was to analyze the effects of both: CM and
biomaterials on skin mice regeneration. Briefly, MSHD were cultured in vitro until
the gather of the conditioned medium (approved by the UFSC Human Ethics
Committee 46674215.7.0000.0121). Three steps carried out the in vivo study
(approved by the UFSC Animal Ethics Committee PP810): excisional wound in the
dorsal region of skin mice, application of CM within scaffolds of carrageenan and
PVA and histological analyzes after three and fourteen days. Macroscopic analysis
showed a bigger scar in the animals treated with PVA compared to control and
carrageenan-treated animals. Microscopic analysis showed that skin of mice that
received PVA or PVA+MC scaffolds exhibited a massive inflammatory infiltrate,
while the carrageenan scaffold or carrageenan + MC had a moderate amount of
leukocytes. Furthermore, the thickness of the epidermis was greater in animals
treated with PVA. These preliminary data indicates that the natural material is
desirable upon synthetic material and that CM exhibit different effects according to
the vehicle employed.
Supported by CAPES, CNPQ and FAPESC.
Department of Surgery, Sector of Anatomy, Faculty of Veterinary Medicine and

Animal Science, University of So Paulo, Brazil
2
So Judas Tadeu University, So Paulo, Brazil
3
Metodista University of So Paulo, Brazil
4
School of Medicine, University of So Paulo, Brazil
* carlacarvalhovet@gmail.com
Skeletal muscle has a limited ability to regenerate after volumetric muscle loss,
resulting in scar tissue formation. Tissue engineering has used various therapeutic
strategies to promote muscle regeneration, such as decellularized extracellular matrix
(ECM). Acellular matrix scaffold provides a supportive microenvironmental niche
and has been used to promote the constructive remodeling of injured skeletal muscle.
Here we evaluated the effect of various temperatures on decellularization of skeletal
muscle in a rat model. Tibialis anterior muscle was harvested from adult male Wistar
rats (CEUA N 2989051015). Samples having 4-5 mm thickness were frozen at -20
and -80C for 4 days previously to decellularization and compared to samples that
were kept at room temperature (RT) and 4C. Decellularization was followed by
static immersion in 1% SDS for 4 days, incubation in 5mM EDTA+ 50 mM TRIS
for 2 days and, finally, incubated in 1% Triton X-100 for 2 days. After the
decellularization procedure, the muscles were washed for at least 1 hour in Hepes
buffer, alcohol 70% and PBS 1x. Processed muscles were evaluated by scanning
electron microscopic (SEM), histological and immunofluorescence analysis and
DNA quantification. We observed the cellular content removal of skeletal muscle
decellularized at RT, 4, -20 and -80C and preservation of structure and matrix
proteins (laminin, fibronectin, collagen I and III) at all temperatures, however
samples submitted to -20C exhibited lower concentration of DNA. Decellularization
of skeletal muscle preserved the extracellular matrix proteins and eliminated the
cellular content. Freezing skeletal muscles prior to decellularization procedures
optimized the effects of detergents.

V5
DEVELOPMENT OF AN ORAL CANCER
CULTURE FOR DRUG SCREENING
V6
ORGANOTYPIC
CELL
POLYURETHANE-HYDROXYAPATITE FROM EUTERPE OLERACEA

MART.: A NATURAL ORGANIC POLYMER SYNTHESIZED TO TISSUE
ENGINEERING APPLICATIONS
Bibiana Franzen Matte (1), Lisiane Bernardi (1), Marcelo Lazzaron Lamers (1,2)
(5)
(6)
(7)
(2) Department of Morphological Sciences, Universidade Federal do Rio Grande do
(1)
(2)
bfmatte@gmail.com; +5551 8402-1090; +5551 3308-5011
Development of new therapies is important to all fields of health sciences. However,
drugs tests are usually done in monolayer cell culture that does not represent the
same cytotoxic and effective effects seen in in vivo conditions. The main problem of
in vivo models is that animal and human trials have a high biological, ethical and
financial cost. Therefore, there is a need to develop in vitro models that better
represent human tissues in order to increase the number of drugs with real potential
to be translated to the clinical scenario. To reach this approach, the aim of this study
is to develop an organotypic cell culture of oral cancer. It was used collagen I
extracted from rat tails to produce the extracellular matrix. Then, fibroblasts were
added to the extracellular matrix and incubated for 3 days. On top of the matrix, oral
cancer cells (Cal27) were added and, after 3 more days, the 3D matrix was lifted to
an air-liquid interface to allow epithelium stratification. Then, this structure was
cultured for 7, 14 and 21 days and fixed on paraformaldehyde 4%. The obtained
tissue was processed, paraffin-embedded, sectioned and stained with haematoxylin
and eosin. Preliminary results show that best concentration of collagen I for the
extracellular matrix is 1.8mg/ml. It was observed that the organization of connective
and epithelial tissue in the slides resembled those observed in human tissues. Our
perspective is to use this model for drug screening and evaluate its effectiveness.
Funding Support: CAPES, CNPQ, FAPERGS, UFRGS
Maria Elizabeth Maus dos Santos (1), Dayane dos Reis Costa Dias (1), Maria
Auxiliadora Pantoja Ferreira (2) Carmen Gilda Barroso Tavares Dias (3) Gilmara de
Nazareth Tavares Bastos (1)
(1) Laboratory of Neuroinflamation, Federal University of Para, Belm, Brazil.
(2) Laboratory of Immunohistochemistry and Development Biology, Federal
University of Para, Belm, Brazil.
(3) Laboratory Ecocomposites, Federal University of Para, Belm, Brazil.
Contacts:
Email: maria.elizaabeth@hotmail.com
Phone number: (91) 982488720
Introduction: New polyurethanes synthesized from natural materials have been
developed in various fields to be applied in tissue engineering. Aim: Evaluate the
Polyurethane from Euterpe oleracea Mart. (acai), to synthesize the material
Polyurethane with Hydroxyapatite (HA-PU) and its application as possible
biomaterial. Methods: Biological assays in vitro and in vivo were made in order to
assess the biocompatibility. Fibroblast cells on 3D culture was performed at 24 or 72
hours and the viability assay was evaluated using the MTT assay. To
biocompatibility tests in vivo the materials were implanted in Swiss mices dorsal
area (n=4) at 7 or 15 days and the samples were collected to Hematoxylin-Eosin
histological assay. Results: Our data has shown, that HA-PU material could not
promote cytotoxicity in vitro. The PU group promoted a cell viability of 98% 12,
and HA-PU promoted a viability of 97% 13 at 24 hours. PU and HA-PU had a
viability of 94% 3 and 90% 9 at 72 hours, respectively. To biocompatibility assay
HA-PU did not show inflammation process, at 7 and 15 days in vivo, promoted a
good interaction of scaffolds and subcutaneous tissue. Conclusion: Those results
demonstrated that HA-PU does not presents
cytotoxicity and also show
biocompatibility in vitro and in vivo showing that the material could be promised as a
biomaterial.
CEPAE: 86-2015. Sources of research support: CNPq/UFPA.

V8
V7
APPLICATIONS OF TISSUE ENGINEERING AND ANNEXIN A1 PROTEIN
IN THE ACELLULAR SKIN HETEROGRAFTS
K.K.O. Mimura1, A.R. Moraes2, K.V. Greco3 and S.M. Oliani1,2
Levin G1; Giardulli LF1; Sogayar MC1, 2 ; Carreira ACO1
From the Post-graduation in Structural and Functional Biology, Federal University

of So Paulo (UNIFESP), Paulista School of Medicine (EPM), So Paulo, Brazil
2
Department of Biology, Laboratory of Immunomorphology, University of So
Paulo State (UNESP), So Jos do Rio Preto, SP, Brazil
3
Department of Surgical Research, Northwick Park Institute for Medical Research
(NPIMR), University College of London (UCL), London, UK
The development of skin substitutes is essential to overcome the shortage at organs
transplantation. Therefore, we develop acellular skins matrix (scaffolds), evaluate its
biocompatibility and the role of AnxA12-26 peptide of the annexin A1 protein in
heterologous transplants. Scaffolds (1 and 2) were evaluated histological,
biomechanical and molecularly. Both scaffolds showed DNA absence, extracellular
matrix preservation (collagen and GAGs) and elasticity similar to control. Scaffolds
biocompatibility was assessed, in rats, on the 3rd, 14th, 21st and 90th days after
implantation. Permacol (commercially available dermal scaffold) was used as
control. On 3rd day post-implantation, polymorphonuclear cells were observed inside
scaffolds and were gradually replaced by IL-6 positive cells and M1 macrophages, at
the 14th day. After 21 days, M2 macrophages, myofibroblasts and blood vessels were
observed inside the scaffold. The cellular events observed in heterograft were
consistent with the gene expression of investigated trophic factors, which were
involved in tissue remodeling (IL-1, TGF-1, VEGF-A, COL1A1, COL1A2 and
COL3A1). In the following experiments, the scaffold 1 was used for transplantation
in Balb/c mice, treated or not i.p. with 4 mg/kg AnxA12-26, for 3, 10, 15 and 60 days.
The AnxA12-26 administration promoting the pro-inflammatory mediators reduction
(IL-1, IL-6, TNF-, IL-17 and IFN-) and increasing of cellular infiltration, proangiogenic factors expression (VEGF-A, b-FGF and TGF-1) and myofibroblasts
recruitment. Altogether, our findings show the biologically compatible of both
scaffolds and reveals the AnxA1 protein role in angiogenic and inflammatory
processes on the experimental model of acellular skin heterologous transplants.
Ethical approval: CEP n 0182/12.
PRODUCTION OF RECOMBINANT HUMAN R-SPONDIN1 PROTEIN IN

MAMMALIAN CELLS AIMING AT INTESTINAL REGENERATION IN AN
ANIMAL MODEL
NUCEL/NETCEM (Cell and Molecular Therapy Center) , Internal Medicine

2
Biochemistry Department, Chemistry Institute, University of So Paulo, So Paulo,
Brazil
To
whom
correspondence
should
be
addressed:
Gabriel
Levin
(gabriel.levin2@gmail.com), NUCEL Universidade de So Paulo, Rua Pangar,
100, Cidade Universitria, So Paulo, 05360-130 SP, Brazil., Phone: +55 11 26480230 - Mobile Phone: +55 11 99906-6477
Background: R-Spondins (RSPO) are proteins that induce the WNT pathway,
playing a pivotal and diversified role in cell proliferation, differentiation, migration
and death during embryogenesis and in the adult. The critical role of RSPO1 for
maintenance of small intestine crypt stem cells niche and its mitogenic activity in
intestinal stem cells offer several therapeutic opportunities.
Aims: To generate overproducing cell clones of the recombinant human RSPO1
(rhRSPO1) in CHO-dhfr-/- and HEK293 cells to obtain a purified and biologically
active protein to be used in intestinal regeneration, individually or in combination
with other recombinant peptide growth factors (VEGFs and/or PDGF-BB), to test in
culture of murine intestinal organoid units using a Tissue Engineered Small Intestine
(TESI) model in NOD/SCID receptor mice.
Methods: The coding sequence of hRSPO1 was synthesized and subcloned into the
pNU1 mammalian expression vector. CHO-dhfr-/- and HEK293 cells were stably
transfected with this construct and are undergoing a gene amplification process by
MTX and clone selection, respectively. Dot blot and ELISA assays were used to
evaluate the hrRSPO1 expression levels.
Results: rhRSPO1 is being produced in both expression systems in detectable levels
and shows in vitro biological activity. A new protein purification protocol was
established and RSPO1 will be tested in the TESI model.
Conclusion: Expression of a biologically active rhRSPO1 in mammalian expression
systems allows applications of this protein in Tissue Engineering.
Ethical approval: Ethics Committee for Animal Use of the Medical School,
University of So Paulo.
Support: BNDES, CAPES, CNPq, FAPESP, FINEP, MCTI, MS-DECIT.

V9
V10
QUALITY ASSESSMENT OF SCAFFOLDS OBTAINED FROM BLOOD

VESSELS DECELLULARIZED OF RABBITS
PRODUCTION OF RECOMBINANT TRANSFORMING GROWTH

FACTORS BETA 1 AND 3 USING A CHO CELL EXPRESSION SYSTEM
Ana Lvia de Carvalho Bovolato (1), Matheus Bertanha (1,2), Jaqueline C Rinaldi
(3), Andrei Moroz (4), Marcone L Sobreira (2), Patricia P Reis (2), Elenice Deffune
(5).
1.
1. Blood Center, Botucatu Medical School - UNESP.
2. Surgery and Orthopedics Departament, Botucatu Medical School - UNESP
2.
3. Morphology Departament, Institute of Biosciences of Botucatu UNESP.
4. School of Pharmaceutical Sciences of Araraquara UNESP.
5. Urology Department, Botucatu Medical School - UNESP
Gabriella Christina G. M. de Paula1,2; Ana Claudia Oliveira Carreira2; Renato

Astorino Filho1,2; Mari Cleide Sogayar1,2
Ana Lvia de Carvalho Bovolato:

Address: Botucatu Medical School, Blood Center, UNESP.
e-mail: liladcb@gmail.com
Telephone contact: (14) 3811-6041
Jaqueline
C
Rinaldi:
jak.rinaldi@gmail.com;
Marcone
L
Sobreira:
mlsobreira@gmail.com; Patricia P Reis: preis@fmb.unesp.br; Matheus Bertanha:
matheus.fameca@ig.com.br; Andrei Moroz: moroz@fcfar.unesp.br; Elenice
Deffune: ed12@fmb.unesp.br
Background: Recent research involving stem cells and cell engineering point to a
promising future based on regenerative and replacement therapy of vascular tissues.
Have proposed the use of biological media derived from decellularized organs and
tissue engineering to regenerative pre-clinical and clinical studies. The use of these
natural materials have advantages such as biocompatibility, maintaining conducive to
development and cell differentiation microenvironment. This requires that all cellular
and nuclear material be removed, preserving the composition, biological activity and
mechanical integrity of the extracellular matrix, without stimulating the rejection
reaction. Objective: Produce vascular scaffolds decellularized safe for use as
recelularizado graft. Methods: The samples consist of inferior cava vein of 20 female
rabbits, adult, non-pregnant, weighing around 3kg. The decellularization protocols:
1) sodium deoxycholate (DS) 2% in continuous stirring 1 hour; 2) sodium dodecyl
sulfate (SDS) to 1% in continuous stirring 2 hours. Three samples were subjected to
each treatment and analyzed by histology, H & E, picrosirius, Masson's Trichrome,
Calleja, Masson & Calleja; Immunohistochemistry for Collagen Type III and IV.
Furthermore, quantitative analysis of residual DNA was performed in three samples.
Results: Histological analysis revealed elimination of cellular components and
preservation of the extracellular matrix. Immunohistochemistry showed a significant
reduction in collagen IV fibers and a more mild decrease of collagen III fibers in
both treatments. Quantitative DNA analysis is underway. Conclusion: The methods
of decellularization were effective in removing the cells and preserve the structure of
the extracellular matrix in all methods of analysis performed.

Paulo 05508-000 SP, Brazil
(2) NUCEL/ NETCEM (Cell and Molecular Therapy Center) , Internal Medicine
Department, School of Medicine, So Paulo 05360-130 SP, Brazil
To whom correspondence should be addressed: Gabriella C. G. Manini de Paula
(gabriellamanini@usp.br), NUCEL, Rua Pangar, 100, Cidade Universitria, So
Paulo, SP, Brazil. 05360-130, Phone: +55 11 2648-0230
Mobile Phone: +55 11 95863-9418
Transforming Growth Factors 1 and 3 plays important roles in tissue repair, and
may be used as therapeutic molecules in Regenerative Medicine. The high cost of
these recombinants is the major obstacle to their clinical use, therefore, we aim to
develop a high performance/low cost expression platform for these factors using
CHO-DG44 dhfr-/- cells. TGF-1 and 3 cDNAs were amplified from our Human
Full-Length cDNA Bank and cloned into our pNU1 mammalian expression vector.
The pNU1/TGF-1 construct was used to stably transfect CHO-DG44 cells and
select for transfectant cells with a higher number of cDNA copies using the dhfr coamplification strategy and selection with increasing levels of Methotrexate. Western
Blot and ELISA assays were performed using conditioned media from both the
selected cell populations and overproducing cell clones. Among the 41 cell clones
obtained, five had the highest TGF-1 production levels (1,0 to 2,0(pg/mL)/cell).
These clones were selected for in vitro biological activity using the A549 human
lung carcinoma cell line to evaluate mesenchymal-epithelial transition (underway).
Wound healing assays were stardardized in rat dorsal skin using recombinant PDGFBB to evaluate the in vivo TGF- activity. In parallel, this process is being repeated
for the pNU1/TGF-3 construct. Upon technology transfer to the industrial
pharmaceutical sector, our initiative is likely to provide wounded patients a powerful
alternative for efficient and rapid tissue regeneration and wound healing.
Information on ethical approval: Ethics Committee for Animal Use of the University
of So Paulo Medical School.
Support: FAPESP, CNPq, CAPES, FINEP, BNDES, MCT, MS-DECIT.
Ethical Approval: CEUA 1121/2015

V11
TOPICAL ADMINISTRATION OF HONEY IMPROVES CUTANEOUS
WOUND HEALING IN DIABETIC MICE
Carol V. Serna Gonzlez (1), Alvaro J. Mendivelso Junco (2), Kelly Nascimento
Souza (3), Gabriella M. Moraes (1), Luciene Lauer (3), Lgia B.A. Muradiam (3) ,
Marinilce F. Santos (1*)
(1)
(2)
(3)
(1) Department of Cell and Tissue Biology, Biomedical Science Institute, University
of So Paulo, So Paulo- Brazil.
(2) Faculty of Nursing, National University of Colombia, Bogot D.C.-Colombia
(3) Department of Food Science. Faculty of Pharmaceutical Science, University of
So Paulo, So Paulo- Brazil.
(4)
Presenting Author: email: cvsernag@usp.br Cel:(11)951771064
* Mailing address: Av. Prof. Lineu Prestes, 1524 - Cidade Universitria, Butant So Paulo - SP - CEP 05508-900 Brazil. E-mail: mfsantos@usp.br
Background: Difficult-to-heal chronic wounds in diabetics have been associated with
the oxidative stress caused by chronic hyperglycemia. Honey has been used
therapeutically in wound healing due to different properties, i.e. antioxidant capacity.
Aims: to evaluate the effects of topical administration of Eucalyptus honey (EH)
from Brazil, in diabetic mice wound healing and to compare it with Manuka Honey
(MH) from New Zealand as an international standard. Methods: diabetes was induced
with alloxan in swiss male adult mice. After 30 days, dorsal cutaneous wounds were
performed and treated topically with EH or MH, or artificial honey (AH) as a
control. Wound closure and histology were analyzed, as well as some
physicochemical properties of the honeys. The protocol was approved by the
Institutional Ethics committee. Results: EH and MH treatments accelerated wound
closure in diabetics from 18 to 14 days, similarly to the control animals (treated or
untreated). AH treatment did not accelerated wound healing. The histological
analysis of wounds on day 3 showed a better aspect of injury in diabetic animals
treated with honey, especially regarding inflammation, compared to the untreated
diabetics. MH had a higher phenolic acids concentration (171,6 5,9 vs 109,6 2,3;
control 22,7 0,9 mg/100g) and antioxidant capacity (22,61,6 vs 36,52,1 mg/L;
undetectable in AH) than EH. Conclusion: EH and MH promote cutaneous wound
healing in diabetic mice, likely by their antioxidant activity.
Funding support: CAPES and CNPq.

AUTHOR INDEX (first authors only)

Abdallah, E. A.
Abreu, I. S. de
Acunha, R. M.
Affonso, R.

Alasseri, A.

Alavarce, R. A. S.
Alexandra, T.
Almeida, A. S.
Almeida, J. Z. de
Almeida, M. P. O.
Almeida, S. A. de
Alonso, H. R.
Altei, W. F.

Alto, L. S.

Alvarez, M. M. P.
Alves, A. A.

Alves, C. A. B.
Alves, G. H. V. S.
Alves, L. F.

Alves, M.

Alves, N. de O.
Alves, V. B.

Alves, V. R. G.
Alves-Fernandes, D. K.
Alvim, J. M.

Amaral, L. V. do
Andrade, F. E. C.
Andrade, T. P. de S.
Anjos, L. M. J. dos
Anjos, P. M. F.
Aquino, C. da C.
Aranda-Souza, M. A.
Arajo Jnior, R. F. de
Araujo Jnior, R. T. de
Arajo, A. C.
Araujo, E. C. B.
Araujo, L.

Araujo, T. L. S.
Arizono, C. K.
Assis, H. A.

Assis, L. H. P.
Assis, L. V. M. de
Assuno, L. S.
Azevedo, L. R. de
Azzi, C. M. G.
Bandeira, L. G.
Banionis, M. A.
Banzato, T. P.
Baptista, J.

A8
R12
L7
V1
F14
O3
A32
B15
B21
I3, O12
B5
L8, Q8
A155
T50
B17
N54
I2
J7
A25
T41
B20, D6
A11
A153
A115
N11
N15, Q16
B10
O17
B3
B28
Q42
B37, O18
Q28
A159
T32
O12
L35
G2
Q14, Q18
U10
G1
A5
A42
A21
A125
L10
A169
A70
I6
Barbezan, A. B.
Barquilha, C. N.
Bassani, N.C.
Bastos, A. C.
Becceneri, A. B.
Becker, M.

Beckman, D.
Begalli, I.

Bellato, H. M.
Belotti, L.

Beltrame, F. L.
Benincasa, J. C.
Bernusso, V. A.
Bertassoli, B. M.
Bittencourt, L. O.
Blanco, I. R.

Boia-Ferreira, M.
Borgato, C.

Borges, B. do N.
Borghesi, J.

Bortoli, N. A.
Bourckhardt, G. F.
Bovolato, A. L. de C.
Braga, L. F. P.
Brando-Costa, R. M.
Braz, J.

Briceo, M. P.
Bristot, I. J.

Brito, N. M. de
Brito, P.

Brito, P. L. de
Bueno, P. S. A.
Buranello, P. A. de A.
Buratti, P.

Caetano, B. F. R.
Caires, C. R. S.
Caldeira-Brant, A. L.
Calegare, B. F. A.
Camargo, A. C. L.
Camargo, M. F.
Campeiro, J. D.
Campos, C. F.
Campos, M. S.
Campos, P. S. de
Campos, V. S. de
Candido, N. M.
Canuto, K. M.
Capra, A. A.

Carballo, G. B.
A66
A141
A57
B2
A54
A48
R1
L36
A2
Q30
T63
B14
E5
V2
R35
R11
A170
T54
A162
A68
J6
Q2
U16, V9
L26
A83
Q31
O16
A147
A120
Q10
P5
N24
Q59
Q20, Q21
A1, A4
T28
T29
T52
I12
C7
A99
T15
Q56
A149
L20
A64
B27, R26
Q27
A112

Cardoso, E. C.

Cardoso, M. F. de O.
Carlstrom, P. F.

Carmo, M. M. L. do

Carneiro, G. D.

Carreira, A. C. O.

Cartaxo, R. T.

Carvalho, G. C. B.

Casali, B. C.

Castelucci, B. G.

Castro, K. R.

Castro, M. M. de

Castro, N. F. C.

Castro, P. A. T. de S.
Catae, A. F.

Cato, F. B.

Catroxo, M. H. B.

Cattani-Cavalieri, I.

Cavalcante, D. G. S. M.
Cavalcante, I.P.

Cecchini, M. S.

Chagas, R. S.

Chammas, S. M.

Chang, S. Y.

Chiarantin, G. M. D.
Chudzinski-Tavassi, A. M.
Cisilotto, J.

Coelho, G. D. P.

Coelho, N. L.

Coelho, T. de M.

Cofre, J.

Colombelli, K. T.

Comelis, M. T.

Consonni, S. R.

Constantino, F. B.

Cordeiro, G. da S.

Correa, F. A. C.

Corra, M. P.

Cortez, B. A.

Costa, G. C. da

Costa, J. R.

Costa, L. de A. L.

Costa, N. de S. X.

Costa, V. C. M.

Costanzo, G. M. Di

Couto, N. F. do

Covatti, C.

Crisafulli, U.

Cruz, E. C. S.

Cruz, I. C. B. da

Cruz, K. S.

Cruz, N. C.

O13
N51
T5
C2, L15
U12
T21
R29
R8
L23
L34
T55
A163
T26
Q25, Q26
Q39
A91
O9
L5
N27
A105
C1
Q60
Q11, Q58
R37
N43
H5
A34
L14, N12
A27
L17, L27
A7
Q57
Q38
F9
T43
T4
C5
B4
A156
J3
T68
A121
L29
A150
B24
N22
Q20, Q21
R30
A47
U13
A87, L24
A135
Cucielo, M. S.
Cunha, F. F. M. da
Cury, D. P.

Dal Mas, C.

Dalmaso, B.
Danilucci, T. M.
Dantonio, P. M.
Daolio, G. A. J.
David, I. M. B.
Debone, D.

Deconte, S. R.
Delben, P. B.
Dhyani, A.

Dias, N. M. M.
Dias, R. B.

Diaz, M. T. de M.
Diel, L. F.

Dolci, G. S.

Donato-Trancoso, A.
Duarte, D. da S.
Dubois, L. G.
Dulcey, L. J. L.
Duran, B. O. da S.
Dzik, L. M.

Elert, N. C.

Esquisatto, M. A. M.
Eugnio, A. I. P.
Expedito, A. C.
Fabris, F. C. Z.
Facina, C. H.
Faleiro, A. C.
Falleiros-Jnior, L. R.
Faraco, C. C. F.
Faria, J. A. Q. A.
Faro, T. A. S.
Favero, G. M.
Felisbino, M. B.
Fernandes, C. G.
Fernandes, F. de S.
Fernandes, G. F. M.
Fernandes, J. C.
Ferreira, A. F.
Ferreira, . E.
Ferreira, F. de F.
Ferreira, L. G. A.
Ferreira, M. M. L.
Ferreira, M.T.
Ferrer, V.

Figueira, L. W.
Fiore, A. P. Z. P.
Firmino, T. S. de S.
Fochi, R. A.

A18
D4
Q40
N52
A108
A103
A63
T49
A84
O5
B34, B35
U19
A15
R28
U14
A6
A136, J6, Q55
N53, Q55
L4
A24, A131
U6
A49
P2
A36
U10
L6, Q6
A44
Q22
B18
T56
C7
T27
A92
F3
A162
A71
K1
N48
S2
T40
A30
U22
A79
R36
I15
L37
A117
L28
N33, N45
L11
Q50
T65

Fonseca Junior, A. M. da
Franco, F. O.

Franco, S.

Fratoni, F. M.

Freire, P. P.

Freitas Filho, E. G.

Freitas Filho, L. H. de
Freitas, A. T. A. G. de
Fuzer, A. M.

Gallo, C. C.

Garavelli, G. Y.

Garcia, E. R.

Garcia, H. V.

Garcia, M. S.

Garnique, A. D. M. B.
Genez, L. A. L.

Geraldo, M. V.

Giannotti, K. C.

Giordano, L. de S.

Glinski, A.

Godinho, J. L. P.

Godoi, B. H.

Godoy, J. A. P. de

Goedert, L.

Goes, C. P.

Gomes, C. C.

Gomes, F. A.

Gomes, L. da S.

Gomes, R. C. T.

Gomes, R. N.

Gomes, S. M. Z.

Gomes, T. A.

Gomes, T. B.

Gonalves, B. F.

Gonalves, B. . P.

Gonalves, J.

Gonalves, J. P.

Gonalves, N. do N.

Gonalves, R. V.

Gonalves, S. P.

Gonzlez, C. V. S.

Gonzlez, M. N.

Goveia, A. S.

Granato, A. E. C.

Greco, G. M. Z.

Guerra, H. N.

Guerra, L. H. A.

Herbster, S.

Hollmann, G.

Horvath, R. de O.

M4
Iglesia, R. P.
A134
Introini, G. O.
T64
Jacomassi, M. D.
B25
Jaeger, B. S. R. G.
E10
Janurio, Y. C.
F13
Jensen, L.

U11
Jesus, L. de O. P.
T18
Jesus, M. M. de
A53
Juinior, A. M. da F.
M1
Junho, C. V. C.
A118
Kandalski, P. K.
N18, N41
Kelmer, S. M. G.
U16
Kerche-Silva, L. E.
T19
Kido, L. A.

A75
Kinker, G. S.
A148
Kunde, M. A.
A110
Landim, B. C.
B8
Laureano, N. K.
A122
Leal, C. S.

N19
Leo, G. M. C.
D8
Leito, A.

D1
Lemes, J. B. P.
E8
Leonel, E. C. R.
A165
Leonel, L. C. P. C.
I18
Levin, G.

H4
Liechocki, S.
V3
Lima, A. B. de
T13
Lima, C. F. M. de
L2, L3
Lima, C. R. de
A106
Lima, J. B. de
T51
Lima, J. P. do N.
Q47
Lima, M. A.

U17
Lima, R. F. de
A95
Lino, R. L. B.
A46
Liria, J.G.

L8
Lopes, D. S.

A113
Lopes, F. B.

B19
Lopes, M. A.
L12, L16, N5, N6, Lopes, S.

N14, N15, Q15, Lorenzon-Ojea, A. R.
Q16
Louback, R. de A.
D9
Lucena, M. C. dos S.
V11
Ludwig, R. G.
L33
Luna, A. C. de L.
T8
Lupinacci, F. C. S.
R16
Machado-Neto, J. A.
T17
Maciel, S. P.
L19
Madeira, F. F.
T23
Magalhes, C. F.
A119
Magalhes, M.
F2
Magalhes, Y. T.
T2
Magnusson, A. S.
A31
A57, J4
A129
A9
G5
P7
A124
T30
T11
B11
N26
A12
N1
T44
A93
A73
A89, A132
A149, N53
Q9
J1, J2
A47
R25
Q44
L27
L31, V8
B26
A45
Q24
A157, A158
O10
L6
L18
Q56, T59
A102
A104
R27
L12, Q15
E1
P3
T20
A65
F4
N44
A166
A3
A29, A30
A86
T24, T25
R32
F8
F17
N39

Maia, J. A.

Malosprito, C. C.

Mansano, B. S. D.M.
Manucci, A. C.

Marinovic, M. P.

Marmorato, M. P.

Martens, A. A.

Martin, A. C. B. M.

Martinez, G. R.

Martins, L. A.

Martins-Duarte, . S.
Martinucci, B.

Maschio, D. A.

Mateus, P. A. M.

Matheus, S. M. M.

Matheus, V. A.

Matias, D.

Matos, A. A.

Matos-Rodrigues, G. E.
Matte, B. F.

Mattos, R. M. de

Mazzonetto, P. C.

Medeiros, D.

Meirelles, L.

Mello Jr, L. J. de

Melo, A. G.

Melo, F. C. S. A. de

Melo, M. I. A. de

Mendes, A. B.

Mendona, R.

Meneghetti, D. H.

Menezes, A. C. de

Merino, C.

Mesquita, F. P.

Midlej, V.

Mimura, K. K. O.

Miranda , M. C. de

Miranda, C. M. F. de C.
Miranda, N. C.

Miranda, V. do S. C.
Mols, R. B.

Monteiro, M. M.

Moraes, C. D. F. de

Moraes, G. M.

Moraes, M. N.

Moraes, T. R. de

Morais, K. L. P.

Morais, M. C.C. de

Morais, M. R. P. T.

Morais-Santos, M.

Moreli, J. B.

Morini, B. C.

B22, Q54
U20
M2
I17
F1
B6
A69, P8
A82
A148
Q43, R22
N31
L9
C4
T44
Q19
Q45
A137, A138
A100
I7, I11
A56, V5
N49
C8
R5
T42
M3
U18
M7, N8
U9
T10
B13
N4
A107
T45
A74
Q63
V3, V7
A10
P6, V4
O4
R7
N37
H3
A144
L39
N13
N23
A154
A26, A37
L21
T53
N51
F12
Moritz, M. N. de O.

Moura, F. B. R. de

Moura, G. E. D. D.

Moura, G. M. de M.
Moura, N. A. de

Mller, J.

Myskiw, J. de C.

Nakagawa, I. T.

Nascimento, C. C. do
Naves, M. A.

Negrin, A. C.

Neri, E.

Neves, J. H.

Neves, R. L.

Neyra, J. M.

Nezzi, L.

Nogueira, C. M. B.

Novaes, R. D.

Nunes, F.

Nunes, G. M.

Nunes, H. C.

Ocanha, S. G.

Ojima, K.

Oliveira Jnior, G. P. de
Oliveira, . A. de

Oliveira, F. C. S. de

Oliveira, G. S. N. de

Oliveira, J. L. de

Oliveira, J. R. de

Oliveira, M.

Oliveira, M. B. de

Oliveira, M. de C.

Oliveira, R. B. B. de

Oliveira, R. B. de

Oliveira, S. M. de

Oliveira, T. G. do C.

Ori, R. B.

Ornellas, F. M.

Orso, R.

Ottaiano, T. F.

Ouriques, L. C.

Paiva, I. G. F.

Paiva, L. B. de

Palheta, L. da S.

Pangrazi, E. N.

Pattaro Jnior, J. R.

Paula, G. C. G. M. de
Paula, L. B. de

Paula, T. de M. D. e

Pedrosa, D. G.

Pedroso, D. de L.

Peloggia, J.

L41
L25, L40
F15
D7
A4
F14
R2, R3
L7
T9
A33
T31
P7
I8
A127
A151
T1
M8
N5, N7
A40
N30
U8
C6
H1
O14
M5
T57
N29
R13, R14
N28, N32
A77
A80
Q33
F6
N38
T37
Q23
B21, Q42
M6
R21
F18
S1
A114
A58
T7
A19
N24
V10
A16, U2
T16
Q61
F16
N2

Pentagna, N.
Pereira, A. C. A.
Pereira, A. D.
Pereira, A. de J. S.
Pereira, A. G.
Pereira, D. T.
Pereira, I. T.
Pereira, L .X.
Pereira, L. de A.
Pereira, M. C.
Pereira, R. M.
Pereira, S.

Perez, A. P. S.
Perez, T. S.

Pescador, G. da S.
Pessoa, T. M. R. de P.
Pinto, M. R.

Pires, J. G.

Pires, K. S. N.
Pissarra, M. F.
Pissulin, C. N. A.
Poletti, S.

Popolin, C. P.
Portilho, A. J. de S.
Porto-Carreiro, I.
Prado, K. M.
Prado, M. B.
Prax, M. C. A. de
Przepiura, T. de C. S.
Queiroga, A. S.
Queirz, F. F. de
Queiroz, V.

Querobino, S. M.
Quilles, J. C. J.
Quintana, H. T.
Rablo, L. M. A.
Ralph, A. C. L.
Ramos, G. de O.
Ramos, I. N. de F.
Reina, J.

Reis, C. A. dos
Reis, I. B.

Reis, J. M. dos
Reis, M. D. dos S.
Repossi, M. G.
Rezende, M. M. de
Ribas, T. de A.
Ribeiro, C. M.
Ribeiro, D. L.
Ribeiro, G. O.
Ribeiro, P. R. L.
Ribeiro-Filho, A. C.
I13
K2
I9
F5
R4
S3
U7
B15
R10
D2
A85
T35
T67
A161
I5, U19, U23
B29
Q59
N34, N36
T48
A61
Q19
Q41
A28
A94
A145
T62
A142
K3
N25, N26
A38
O8
P4, Q13
R19
A50, D3
Q3
D7, F16
U4
A56, A101
A59
F7
T3
T47
T66
Q24
R24
E2
E9
I14
A132
A109
L22
A72
Rinaldi, J. C.

Rocha, T. B. L. da

Rocha-Martins, M.

Rockenbach, J. A. Z.
Rodrigues, A. C. C.

Rodrigues, E. C.

Rodrigues, G. J.

Rodrigues, J. A.

Rodrigues, N. M.

Rosa, D. F.

Rosa, M. N.

Rosa-Ribeiro, R.

Rosrio, A. S.

Rosrio, L.

Rosolen, D.

Rossetto, I. M. U.

Russo, L. C.

Russo, T. A.

S, J. F.de

Sabino, A. U.

Saguie, B. de O.

Saito, K. C.

Saleh, N. A.

Salles, . da S. L.

Sanches, M. L.

Sanmukh, S. G.

Santana, J. C. de O.

Santesso, M. R.

Santi, F. de

Santiago, C. S.

Santin, M. da S.

Santos Filho, J. R. de A.
Santos, A. C. D. dos

Santos, A. P. F. P. dos
Santos, B. V. dos

Santos, D. de O.

Santos, E. G.

Santos, F. O. dos

Santos, I. P.

Santos, J. C.

Santos, J. F. dos

Santos, L. R. dos

Santos, M. E. M. dos
Santos, M. H. S.

Santos, M. J. S.

Santos, N. J. dos

Santos, R. V. C.

Santos, S. A. A. dos

Santos, S. C. C.

Santos, V. C. dos

Sarandy, M. M.

U1
B7
I4
H6
A78
A13
P6
A23
T46
N9, N10
A160
A139
R9
A138
A43
R17, R18
C3
L38
A39
A37
Q5
A96
A17
T6
U15
A130
L30
G6
T61
T12
Q29
A167
O6
R34
J8
A168
T41
T35
O2
T38
U21
T34
V6
A41
B27
A152
A123
T60
A76
Q35
C2, L13, L15, M7,
N7, N8, N12, N18

Sardinha, A. A.

Sargiani, A. M. P.

Sartor, L.

Scalabrini, L. C.

Scarano, W. R.

Scarpelli, T. P.

Schfer, B. T.

Schanuel, F. S.

Schmidt, . C.

Schmidt, M. C. B.

Schneider, L. C. L.

Schneider, S. I. dos R.
Schnhofen, P.

Seguin, C. S.

Segundo, F. A. de S.
Sena, W. L. B. de

Sielski, M. S.

Sifontes, Y. J. C.

Silva Filho, A. F. da

Silva Junior, F. C.

Silva, A. H.

Silva, A. R. da C. e

Silva, C. A. de A. e

Silva, C. G.

Silva, D.

Silva, D. C. da

Silva, E. L. da

Silva, F. C. da

Silva, G. . F. da

Silva, G. H. G. da

Silva, H. S. da

Silva, I. A. N. da

Silva, J. A. F.

Silva, J. H. da

Silva, K. M. da

Silva, K. Q. da

Silva, M. de P.

Silva, M. G. K. C.da

Silva, M. T.

Silva, N. A. A. da

Silva, N. G. da

Silva, N. S. de L.

Silva, P. B. G. da

Silva, P. H. de A.

Silva, R. F. da

Silva, R. N.da

Silva, S. I. S. e

Silva, S. V. da

Silva, V. A. da

Silva, V. L. da

Silva, W. B. de S.

Silva-Janurio, M. E. da
R20
T39
L32
A98
A95
T36
Q49
Q4
S3
H5
B1, Q1
R31
R23
B12
M4
B36
U5
O11
A164, Q62
Q46
N16
Q32
U3
O15
T33
A133
A60
R6
A118, A169
N3
I1
Q52
Q34
N50
N21
B22, Q54
A21
A67
E6
L16, N14
Q36
L1, O1
A140
O7
A20
N42
Q12
A116
R33
N56
A81
G3
Silveira, A. A. A.

Simo, V. A.

Sousa, K. K. O. de

Sousa, M. E. P.

Sousa-Squiavinato, A. C. M.
Souza, D. G. N. de

Souza, D. S. P.

Souza, E. R. S. de

Souza, H. M. B. de

Souza, J. M. G. de

Souza, L. D. e

Souza, M. J.

Souza, M. M. de

Souza, R.K.F

Sper, F. L.

Sperandio, L.

Spindola Jr, A.

Spohr, T. C. L. de S. e
Stavare, A. L. A.

Storti, C. B.

Streit, P.

Tamarindo, G. H.

Tamborindeguy, M. T.
Tan, P. B.

Tavares, L. A.

Taveira, G. D. de M.
Teixeira, J.

Terra, M. F.

Tezuka, D. Y.

Theodoro, V.

Tokuhara, C. K.

Toledo, B. C.

Tonelli, F. M. P.

Torquato, H. F. V.

Torres, J. I.

Tortelli-Junior, T. C.

Tsuboy, M.S.F.

Ulsenheimer, B. H.

Valado, I. C.

Vasconcelos-dos-Santos, A.
Ventura, T. M. da S.
Veridiano, J. M.

Verssimo, C. P.

Viana, M.

Vicente, C. P.

Vidal, J. C.

Volpe, C. M. O.

Vuitikaa, L.

Wachesk, C. C.

Wailemann, R. A. M.
Wajsenzon, I. J. R.

Winter, E.

B9
T14
R26
B33
A51
A97
D9
Q48
U20
F10
A128
B16
P8
A22
N46, N47
A55
A143
A111
L2
A146
I10
T58
A101
I11
G4
J5
E3
Q37
D3
Q6
N35
E7
P1
C10
B30, B31, Q53
A52
A90
Q51
A62
A35
N40
L42
I16
B32
U12
H2
B23
N17
B38, N55
D5
R15
A40

Xavier, A. M.
Yamashita, A. M. S.
Zabaglia, L. M.
Zanetti, B. F.
Zangerolamo, L.
Zaniboni, E.

Zazula, M. F.
Zingue, S.

Zomer, H. D.
Zonta, G. M. A.
Zuco, M. I.

Zulian, J. G.

R30
C9
A126
A88
F11
Q17
Q51
A14
U23
N20
T22
E4

Livro de Resumos - Abstracts Book Final 13jul16 PDF

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Poster

Prof. Dr. Hernandes Faustino de Carvalho

Profa. Dra. Patrcia Gama

Marcelo L Lamers (UFRGS)

XVIII Meeting of Brazilian Society for Cell Biology 3

CANCER AND CELL BIOLOGY

CELL BIOLOGY OF INFLAMMATION

CYTOSKELETON AND MIGRATION

EDUCATION IN CELL BIOLOGY

METHODS IN CELL BIOLOGY

CELL CYCLE AND PROLIFERATION

CELL TRAFFICKING AND ORGANELLES

GENE AND CELL THERAPY

GENERAL CELL BIOLOGY

MORPHOLOGY AND CELL BIOLOGY

PLANT CELL BIOLOGY

STEM CELL BIOLOGY

XVIII Meeting of Brazilian Society for Cell Biology 4

Poster presentation guidelines

13h30- 14h15 even numbers

PROTECTIVE EFFECTS OF CAPSAICIN ON DMH-INDUCED

TRANSLATIONAL CONTROL IN INVASIVE BREAST CANCER: A

(1) Department of Pathology, School of Medicine, So Paulo State University,

A.C.Camargo Cancer Center

BACKGROUND: Today, genome-wide projects attempt to define patterns of gene

TUMOR HETEROGENEITY EVALUATION IN GLIOBLASTOMAS USING

DIETARY INDOLE-3 CARBINOL AND SYNBIOTICS PROMOTE TUMOR

(1) Department of Molecular and cell biology, A C Camargo Cancer Center, So

Mobile: (11) 98206-3513

(1) Department of Pathology, School of Medicine, So Paulo State University, Brazil.

XVIII Meeting of Brazilian Society for Cell Biology 6

HEAT SHOCK ANTAGONIZES UVA-INDUCED RESPONSES IN

EVALUATION OF CIRCULATING TUMOR MICROEMBOLI

Leonardo Vinicius Monteiro de Assis, Maria Nathlia Moraes, Ana Maria de

Laboratory of Comparative Physiology of Pigmentation, Department of

International Research Center, A.C. Camargo Cancer Center, So Paulo, Brazil.

METASTATIC COLORECTAL CANCER FOLLOW-UP: EVALUATION BY

Jaime Cofre (1), Eliana Abdelhay (2).

(1) Universidade Federal de Santa Catarina, Laboratrio de Embriologia Molecular

(1) International Center of Research, A. C. Camargo Cancer Center, So Paulo, Brazil.

Conflict of interest statement: None declared.

XVIII Meeting of Brazilian Society for Cell Biology 7

ANALYSIS OF LAMININ-111 CLEAVAGE IN BREAST CANCER

ADIPOSE DERIVED MESENCHYMAL STEM CELL CONDITIONED MEDIA

Basilio Smuczek Ruy G. Jaeger

Department of Cell and Developmental Biology, Institute of Biomedical Sciences,

EXPRESSION AND RELEVANCE OF THE IMPACT PROTEIN IN

ANALYSIS OF KIN17 PROTEIN DETECTION ASSOCIATED WITH

International Research Center, A.C. Camargo Cancer Center, So

New protein synthesis is a highly controlled process whose deregulation may

*Contact: Sabrina Marques Godinho Kelmer, Maria Aparecida Fernandez

XVIII Meeting of Brazilian Society for Cell Biology 8

EVALUATION OF THE RASSF9 ROLE IN MELANOMA BY USING THE

IN VITRO ANTI-TUMOR POTENTIAL OF ABYSSINONE V-4 METHYL

Elizabeth Cristina Rodrigues1, Roger Chammas2, Gustavo Pessini AmaranteMendes1

(1) University of Maroua, Maroua, Cameroon

ANKHD1 SILENCING LEADS TO ACCUMULATION OF DNA DAMAGE

Anamika Dhyani(1,2), Patricia Favaro(3), Sara T Olalla Saad(1,2).

Department of Chemistry, University of Sao Paulo, Ribeirao Preto,

Research was funded by FAPESP and INCT, Sao Paulo Brazil.

XVIII Meeting of Brazilian Society for Cell Biology 9

3D CO-CULTURE: SIMULATING THE TUMOR MICROENVIRONMENT

IMPACT OF FIBROBLASTS IN GENE AND MICRORNA EXPRESSION IN