Libro Resumenes Congreso SEBBM Granada 2014 Post

LIBRO DE RESMENES

Psters
XXXVII Congreso SEBBM
Granada 2014
Psters
Primera edicin: Septiembre 2014
los autores, 2014

XXXVII Congreso de la Sociedad Espaola de Bioqumica y Biologa Molecular
Publica: Sociedad Espaola de Bioqumica y Biologa Molecular (SEBBM)
Produccin editorial: Rubes Editorial, S.L.

Sicilia, 253 6 4. 08025 Barcelona
Psters
ndice
Agradecimientos
.......................................................................
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Comit organizador
Junta Directiva de la SEBBM
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Socios protectores de la SEBBM
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11
Conferencias plenarias
Simposios
S1 Biomedicina molecular
............................................................
S2 Biotecnologa de plantas y productos de valor aadido
11
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15
S3 Estructura y funcin de protenas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19
Psters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23
P00 II Workshop sobre la Innovacin Docente en la Enseanza de la Bioqumica y Biologa Molecular

P01 Apoptosis
......
24
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27
P02 Bases moleculares de la patologa

P03 Biologa del desarrollo
...................................................
...........................................................
P04 Biologa molecular computacional
P07 Bioqumica perinatal
53
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55
........................................................
62
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P08 Bioqumica y biologa molecular de plantas

P09 Biotecnologa molecular
............................................
66
68
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76
P10 Estructura y funcin de protenas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
86
P11 Genmica y protemica
...........................................................
P12 Metabolismo del nitrgeno

P14 Parasitologa molecular
112
..........................................................
117
...........................................................
125
P15 Radicales libres y estrs oxidativo
...................................................
P16 Regulacin de la expresin gnica y dinmica del genoma

P17 Regulacin metablica
P18 Sealizacin celular
103
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P13 Neurobiologa molecular
133
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141
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149
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159
P19 Transgnesis en mamferos
........................................................
P20 Transportadores de membrana
50
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P05 Biomembranas y bioenergtica

P06 Bioqumica de la nutricin
33
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173
175
Granada 2014
Psters
Agradecimientos
Nuestro agradecimiento a las entidades pblicas y privadas que han colaborado econmicamente en la realizacin del XXXVII
Congreso de la SEBBM.
Ministerio de Economa y Competitividad

Consejo Superior de Investigaciones Cientficas (CSIC)
Junta de Andaluca
Universidad de Granada
Parque de las Ciencias
Turismo Ciudad de Granada
Fundacin BBVA
Fundacin Lilly
Fundacin Ramn Areces
LOral For Women in Science
FEBS
PABMB
Bio-Rad
Canvax
BIOTOOLS
Condalab
Cultek
Ecogen
Eppendorf
Gilson
Izasa-Werfen
Neuron
Panreac Applichem
Sarstedt
StabVida
Vitro
Fisher Scientific
Sigma
Psters
Comit organizador
Presidente
Juan Luis Ramos (Estacin Experimental del Zaidn, CSIC,
Granada - Abengoa Research, Sevilla)
Comit ejecutivo
Federico Mayor Menndez (presidente de la SEBBM, Centro
Biologa Molecular Severo Ochoa, Universidad Autnoma
de Madrid)
Miguel Alaminos Mingorance (Facultad de Medicina,
Universidad de Granada)
Mara Dolores Girn Gonzlez (tesorera, Facultad de
Farmacia, Universidad de Granada)
Tino Krell (Estacin Experimental del Zaidn, CSIC,
Granada)
Irene Daz Moreno (responsable de Congresos y Cursos de
la SEBBM, Instituto de Bioqumica Vegetal y Fotosntesis,
cicCartuja, CSIC-Universidad de Sevilla)
Miguel ngel Navarro Carretero (Instituto de Parasitologa y
Biomedicina Lpez-Neyra-CSIC, Granada)
Enrique de la Rosa Cano (responsable de Grupos de la
SEBBM, Centro de Investigaciones Biolgicas, CSIC,
Madrid)
Carme Caelles Franch (responsable de Jvenes de la

SEBBM, IRB-Barcelona, Universidad de Barcelona)
Marta Cascante Serratosa (responsable de Cnsules de la
SEBBM, Facultad de Biologa, Universidad de Barcelona)
Crisanto Gutirrez Armenta (tesorero de la
SEBBM, Centro Biologa Molecular Severo Ochoa, CSICUAB, Madrid)
Almudena Porras Gallo (secretaria Cientfica Electa de la
SEBBM, Facultad Farmacia, Universidad Complutense de
Madrid)
Isabel Varela Nieto (secretaria Cientfica de la SEBBM,
Instituto de Investigaciones Biomdicas, CSIC-Universidad
Autnoma de Madrid)
Otros miembros del Comit organizador - Comit local:
M Jos Alejandre Prez (Facultad de Ciencias, Universidad
de Granada)
Matilde Barn Ayala (Estacin Experimental del Zaidn,
CSIC, Granada)
Andrs Belver Cano (Estacin Experimental del Zaidn,
CSIC, Granada)
Francisco Gamarro Conde (Instituto de Parasitologa y
Biomedicina Lpez-Neyra, CSIC, Granada)
Joaqun Ros i Salvador (coordinador de la Comisin Asesora

de Congresos SEBBM, Facultad de Medicina, Universidad
de Lrida)
Juan Jos Lzaro Paniagua (Estacin Experimental del

Zaidn, CSIC, Granada)
Rafael Salto Gonzlez (secretario, Facultad de Farmacia,

Ana Linares Gil (Facultad de Ciencias, Universidad de

Granada)
Mara Dolores Surez Ortega (vicepresidenta, Facultad de

Farmacia, Universidad de Granada)
Eduardo Lpez-Huertas Len (Estacin Experimental del

Otros miembros del Comit organizador - Junta Directiva de

la SEBBM
Alicia Alonso Izquierdo (vicepresidenta y Responsable de
Relaciones Internacionales de la SEBBM, Departamento
de Bioqumica, Facultad de Ciencias, Universidad del Pas
Vasco)
Esperanza Ortega Snchez (Facultad de Medicina,

Juan P. Bolaos Hernndez (responsable del Portal

Electrnico de la SEBBM, Instituto de Biologa Funcional y
Genmica, IBFG, Universidad de Salamanca)
Mariam Sahrawy Barragn (Estacin Experimental del

Luisa Mara Sandalio Gonzlez (Estacin Experimental del
Alberto M. Vargas Morales (Facultad de Farmacia,
Granada 2014
Psters
Junta directiva de la SEBBM
Presidente
Federico Mayor Menndez (2012-2016)
Enrique de la Rosa Cano (2010-2014)

[Grupos Cientficos]
Vicepresidenta
Alicia Alonso Izquierdo (2010-2014)
[Relaciones exteriores y COSCE]
Juan Luis Ramos Martn (2010-2014)

[Empresa y Patrocinadores]
Secretaria
Isabel Varela Nieto (2010-2014)
[Divulgacin]
Tesorero
Crisanto Gutirrez Armenta (2012-2016)
Vocales
Marta Cascante Serratosa (2010-2014)
[Cnsules]
Juan Pedro Bolaos Hernndez (2012-2016)

[Pgina web]
Carmen Caelles Franch (2012-2016)
[Jvenes Investigadores]
Irene Daz Moreno (2012-2016)
[Congresos y Cursos]
Secretaria Electa
Almudena Porras Gallo (2012-2014)
Socios protectores de la SEBBM
Los Socios protectores de la Sociedad Espaola de Bioqumica y Biologa Molecular (SEBBM) contribuyen al progreso de la ciencia.
Psters
Conferencias Plenarias
Granada 2014
Conferencia inaugural Alberto Sols

Fundacin BBVA
CP01-1 (R01-1)
Selfish genes vs selfish metabolism: how

environmental bacteria conquer the chemical space
Vctor de Lorenzo
Centro Nacional de Biotecnologa, CSIC, Madrid-Cantoblanco, ES
Bacteria that colonize sites polluted by industrial waste are capable of
metabolizing synthetic and recalcitrant chemicals that have been in the
biosphere for only a few years. This capability is orchestrated by the
integration of environmental and physiological signals into regulatory
systems that tightly control the expression of genes that are in charge of
degrading such molecules. The emergence of these capabilities is due not
only to the adaptation of catabolic enzymes acting on new substrates, but
also to the emergence of new regulators that firmly control the expression
of the genes involved in the production of catabolic enzymes.
This scenario provides a good case to examine how transcriptional factors
that respond to small molecules can adjust their specificity to novel signals,
the significance of the effects of individual changes on overall behaviour of
the evolving regulators and the role of other environmental and physiological
circumstances in the process. Biological bottlenecks for biodegradation of
recalcitrant compounds include [i]unfavourable thermodynamics of (bio)
chemical reactions at stake, [ii] lack of specificity of existing pathways and
enzymes for novel substrates, and [iii] physicochemical stress encountered
in polluted sites. Besides these limitations, bacterial cells also experience
increased endogenous oxidative stress during metabolism of aromatic
compounds, which is exacerbated when enzymes meet suboptimal substrates.
Two experimental systems were employed to examine this issue. First, we studied
the regulation of the TOL plasmid-encoded upper and lower operons of the soil
bacterium Pseudomonas putida mt-2, a toluene and m-xylene degrader, on the
background of endogenous production of reactive oxygen species (ROS) during
the process. ROS-responsive dyes and flow cytometry helped to quantify stress
associated to each metabolic block and its relationship with redox metabolism.
We also examined oxidative stress brought about by the still-evolving
2,4-dinitrotoluene biodegradative pathway in Burkholderia sp. DNT. The dnt
pathway of this bacterium apparently evolved from a precursor naphthalene
degradation route and the first enzyme (2,4-dinitrotoluene dioxygenase)
maintains some activity towards its earlier substrate [1]. Examination of both
in vivo reactions and the associated regulatory system suggests that ROS
production is the first bottleneck that evolving pathways have to overcome for
dealing with novel compounds [2]. Evolutionary consequences [3] -and some
hints for engineering new biocatalysts [4] will be discussed.
References
[1] de las Heras, A., Chavarra, M. and de Lorenzo V. Mol Microbiol 2011;
[2] Prez-Pantoja, D., Nikel P.I., Chavarra M. and de Lorenzo, V.
[3] de Lorenzo, V. BioEssays 2014; 36: 226-35.Phil Trans B Roy Soc
2013; 368: 20120377.
Conferencia plenaria LOral-UNESCO

For Women in Science
CP02-1 (R02-1)
Structures and mechanisms of bacterial

transcriptional regulation via oligomeric proteins
Xiadong Zhang
Centre for Structural Biology, Imperial College London, Londres, UK
Bacterial gene transcription depends on the binding of RNA polymerase
to a wide range of sigma factors that recognize specific promoter DNA
sites to achieve specificity. Two classes of sigma factors exist in bacteria.
The 70 class includes most sigma factors and the RNAP 70 -promoter
complex can often spontaneously converts to the open complex, which
is competent for transcription. In contrast, the complex between RNAP
and its major variant sigma factor 54 remains as a closed complex
which is incompetent for transcription until ATP hydrolysis-dependent
remodelling by activator proteins occurs. The activator proteins, which
belong to the AAA+ protein family, bind remotely to the DNA sequences
upstream of the transcriptional starting site and contact RNAP-54
through DNA looping. Most AAA+ activators contain three domains: a
N-terminal regulatory domain, a central AAA+ domain and a C-terminal
DNA binding domain. The regulatory domain senses environmental
changes and controls the activities of the AAA+ domain while the AAA+
domain contacts RNAP-54 and uses ATP binding and hydrolysis to
remodel the closed complex. In addition, bacteria gene transcription can
be inhibited though simple blockage of promoter sites.
Over the last few years we have used a combination of X-ray crystallography,
cryo-electron microscopy single particle analysis and functional analysis to
understand why RNAP-54 remains as a closed complex, how the AAA+
activator interacts with and induces changes in RNAP-54 that ultimately
lead to transcriptional activation and how binding and hydrolyzing ATP are
coupled to the activation process. I will present our results and explain why
RNAP-54 is unable to proceed to transcription, how a hexameric AAA+
protein interacts with and remodels the asymmetric RNAP-54-DNA using
ATP binding and hydrolysis, how these activators are organised at the
enhancer-binding site and how their ATPase activity is controlled by both
the regulatory and DNA-binding domains. In addition, I will discuss how
oligomeric proteins interact with their DNA operators in order to achieve
repressive functions.
Conferencia plenaria Leloir

CP03-1 (R03-1)
The brain speaks back to the ear: molecules,

physiology and pathology of the efferent
olivocochlear-hair cell synapse
Ana Beln Elgoyhen
Instituto de Investigaciones en Ingeniera Gentica y Biologa
Molecular, Dr. Hctor N. Torres, CONICET, Buenos Aires, AR
In bringing information about the world to an individual, sensory
systems perform a series of common functions. Each system responds
with some specificity to a stimulus and each one employs some
specialized receptor cells at the periphery to translate specific stimuli
into electrical signals that all neurons can use. That initial electrical
event begins the process by which the central nervous system constructs
an orderly representation of for example, sounds, odors, tastes and
objects. Thus, basic sound detection begins when sound waves strike
the eardrum, which transmits that physical stimulus to the organ of Corti
within the cochlea, the sensory epithelium of the mammalian inner ear.
Here the primary receptor cells known as inner hair cells transform the
information into electrical signals that are sent to the central nervous
system by the auditory nerve. However, unlike vision, touch and the
chemical senses, sound processing is modulated by efferent signals that
travel in reverse, from the brain back to the inner ear. One fundamental
question in auditory neuroscience is what role(s) this feedback plays in
our ability to hear. The presentation will overview our work over the
years which has contributed to elucidate the molecules which operate at
this efferent olivocochlear-hair cell synapse, how the synapse operates
to fine tune amplification in the inner ear and the role of the efferent
system in protection from acoustic trauma.
Conferencia plenaria Niemeyer - PABMB

CP04-1 (R04-1)
posttranscriptional regulation of gene expression. Examples will be

presented that highlight the role of structural changes and conformational
dynamics in the recognition of the 3 splice site RNA during eukaryotic
pre-mRNA splicing, translational regulation and RNA stability.
Caveolin-1, a pleiotropic molecular switch

in cancer development and progression
Andrew F.Q. Quest
Laboratorio de Comunicaciones Celulares, Centro de Estudios
Moleculares de la Clula (CEMC), Centro de Estudios Avanzados
en Enfermedades Crnicas (ACCDiS), Programa de Biologa Celular
y Molecular, ICBM, Facultad de Medicina, Universidad de Chile,
Santiago de Chile, CL
Cancer is a leading cause of human suffering and death worldwide,
whereby most patient deaths are caused by metastatic dissemination rather
than primary tumor growth. Understanding the mechanisms that favor
such development is currently an area of great interest given the potential it
holds for designing successful therapeutic intervention strategies. A central
dogma in cancer research over the last decades has been that changes at the
molecular level, referred to as gain-of-function in oncogenes or lossof-function in tumor suppressor genes, contribute to the genesis of this
disease. Because it is generally thought that any given protein will belong to
one or the other category, but not to both, treatment strategies will in general
terms seek to enhance tumor suppressor and block oncogene function.
Paradoxically, Caveolin-1 (CAV1), a member of the caveolin family of
scaffolding proteins, is implicated in cancer development and progression
both as a tumor suppressor and promoter of metastasis. In recent years,
research from this laboratory has sought to shed light on this enigma. Tumor
suppression by CAV1 was linked to E-cadherin-dependent suppression of
genes, including survivin and cyclooxygenase-2, and augmented apoptosis.
However, in the absence of E-cadherin, CAV1 promotes metastasis. More
recent studies implicate CAV1 tyrosine phosphorylation and activation
of a novel Rab5-Rac1 signaling axis in enhanced tumor cell migration,
invasion and metastasis. In the presence of E-cadherin, signaling via this
pathway and metastasis are suppressed. Thus, CAV1 switches roles in an
E-cadherin-dependent manner. Understanding such context-dependent
variations in protein function has important ramifications for our basic
understanding of signal transduction processes and cancer, as well as for
the development of successful strategies to treat the disease.
Acknowledgements: FONDECYT-FONDAP
1130250, ACT1111 (AFGQ).
15130011,
FONDECYT
Conferencia plenaria FEBS National

Lecture
CP05-1 (R05-1)
Dynamics of protein-RNA interactions in

posttranscriptional regulation of gene expression
Michael Sattler
Institute of Structural Biology, Helmholtz Zentrum Mnchen,
Neuherberg, and Center for Integrated Protein Science Munich and
Biomolecular NMR, Technische Universitt Mnchen, Garching,
Mnchen, DE
Eukaryotic multi-domain proteins play crucial roles in the regulation of
gene expression and cellular signaling. The structure of these proteins often
depends on dynamic domain arrangements that are modulated by binding
to RNA and/or other proteins. We employ integrated structural biology
combining solution techniques such as NMR-spectroscopy and small
angle scattering experiments with crystallography to study the structure
and dynamics of multi-domain proteins involved in various aspects of
10
Conferencia plenaria de clausura

Fundacin Ramn Areces
CP06-1 (R06-1)
Understanding the role of proteolysis in disease

Charles S. Craik
University of California San Francisco, San Francisco, US
Virtually any biological process can be linked to a proteolytic event. With
approximately 2% of the human genome encoding a protease a plethora
of these enzymes exist, several of which have already been shown to play
roles in diverse physiological processes including development, innate and
adaptive immunity, cell cycle regulation and apoptosis. In situations where
a unidirectional signal is required, the hydrolysis of a peptide bond can
provide an irreversible, one-way event in a complex signaling pathway.
Similarly, the genomes of most other organisms ranging from viruses and
microorganisms to metazoans have 2 to 4% of their genomes dedicated to
proteases. In cases where there is a direct link between the proteolytic event
and disease, significant opportunities exist for therapeutic intervention. For
these reasons, there has been a great deal of interest in protease biology and
in dissecting the complex regulation, localization and activation of protease
function.
Identifying the actual proteases involved in physiological events,
locating the natural substrates of the enzymes and selectively regulating
their activity is of prime importance to the field. This talk will focus on
a highly important subset of the large area of proteolysis to provide a
window into this rich area of research. Methodologies will be presented
that allow the global activity of proteolysis to be determined in complex
biological systems and permit the non invasive imaging of protease activity
in various disease states. These efforts to understand the mechanism of
proteolysis in a biological context and in several cases, the relationship
between proteolysis and disease are providing insights regarding how best
to regulate proteolysis in infectious diseease and cancer.
Granada 2014
Psters
Simposio 1:
Biomedicina molecular
11
Simposios
S1.1 Enfermedades moleculares

S1.1-1 (R1.1-1)
Pancreatic cancer: From molecular

understanding to personalized treatment
Manuel Hidalgo
Centro Nacional de Investigaciones Oncolgicas, Madrid, ES
Pancreatic cancer remains one of the most deadly cancers. Over the last
few years, the genomic landscape of pancreatic cancer as well as precursor
pancreatic cancer lesions have been deciphered in great depth. These studies
show that PDA develops as the consequence of accumulation of mutations in
key oncogenes and tumour suppressor genes. The disease, once established,
is characterized by high complexity, heterogeneity and genomic instability.
Despite this facts, some patients harbour actionable mutations which targeting
has resulted in significant clinical benefit. Indeed, one of the most active
areas of research in PDA is the development of strategies and approaches
to personalize the treatment of patients. This is a complex field that can be
tackle from many complementary angles. Our group has been interested in
using patient derive xenogaft (PDX) models, aka Avatar mouse models, to
guide cancer treatment. A piece of freshly collected tumour is implanted in
immunodeficient mouse models, expanded, treated with different anticancer
agents alone and in combination to select the most effective drug/regimen to
treat the patient cancer. Our data show that the approach is highly predicted
but, because of complexity and cost issues, not widely applicable to clinical
practice at the present stage. To solve some of these limitations we are working
on different aspects. One area is technological development to increase the take
rate of tumours and to speed time to engraftment and expansion time.
Currently, these figures are approximately 60-80 % and 5-7 months. Studies
are in progress to optimize this aspect. Another key question is the selection
of agents, both alone and in combination, to be tested in the model. In this
regard, it is important to integrate biomarker assessment in the tumour to
pre-select a series of treatment candidates that can then be tested in the PDX
models. To this end, we have now integrated next generation sequencing
and assessment of copy number variation in patients tumour. These studies
provide us with an unbiased overview of the tumour genomic landscape.
From this data, using different bioinformatics and biological methods we
extract the most relevant drug targets that are then bench tested against the
patient Avatar mouse model to select the most effective treatment.
Another area of great interest in PDA therapeutics is the cancer
microenvironment. PDA is known for a flourish cancer stroma composed of
extracellular matrix and cells including inflammatory, immune and cancer
associate fibroblast. Strategies aiming to eliminate the cancer stroma with
agents such as hyaluronidase and Hh inhibitors among others have received
substantial attention. nabPTX, an agent proposed to target SPARC, one of
the key elements in the PDA stroma, has shown improvement in overall
survival and has received regulatory approval for this disease. Agents in
this category will also be reviewed.
Finally, strategies to target the cancer stem cell will be presented.
Laboratory studies have shown the presence of a small percentage of cells
in PDA tumours with stem properties. These cells can be identified by
membrane markers and are considered to be resistant to chemotherapy and
radiotherapy. It is possible, that CSC is responsible for cancer failure after
definitive chemotherapy and radiotherapy and there is significant interest
in developing agents to selectively eliminate these cells.
S1.1-2 (R1.1-2)
Assessing the therapeutic role of Vav

oncoproteins in high-incidence diseases
Xos R. Bustelo, Salvatore Fabbiano, Javier Robles, Luis Francisco
Lorenzo, Mara Barreira, Mauricio Menacho-Mrquez
Centro de Investigacin del Cncer, Salamanca, ES
Although there is a wide consensus regarding the potential therapeutic
role of Rho/Rac GDP/GTP exchange factors (GEFs), we have no formal
12

demonstration that such idea is true for most high-incidence diseases.
Tackling this issue has been in fact rather difficult so far, since the human
genome contains more than 70 GEFs that, in many cases, are coexpressed
in the same cell types and tissues. Due to this, it is of paramount importance
to utilize disease-mimetic animal models to characterize unequivocally
the spectrum of GEF-dependent diseases, identify the best therapeutic
targets of the GEF family for specific high-incidence diseases or disease
subtypes and, in addition, unveil the GEF-dependent Achilles heels
present in those diseases that can be used to devise new ways to attack
them pharmacologically. In our lab, we are addressing the above issues
using as working model the Vav GEF subfamily. The three members of
this subfamily present in mammalian species (Vav1, Vav2, Vav3) are
characterized by being directly regulated by phosphorylation on specific
tyrosine residues. Due to this, these GEFs are specialized in connecting
stimulated upstream protein tyrosine kinases with the activation of
downstream Rho/Rac GTPases during both normal and pathophysiological
signaling responses. To this end, we are using both standard and secondgeneration genetically modified mice to assess the pros and cons of the
inactivation of these proteins in a number of high-incidence diseases. Using
this approach, we have discovered that two Vav subfamily members (Vav2
and Vav3) play critical roles in breast and skin primary tumorigenesis,
the metastasis of cancer cells to the lung, obesity, and obesity-linked
comorbodities (i.e., metabolic syndrome, liver esteatosis, type II
diabetes). We have also discovered that the inactivation of any of those
two proteins also leads to negative effects in the cardiovascular system.
However, such collateral defects can be easily bypassed by treatments
with currently available anti-hypertension therapies. Finally, the dissection
of the role of Vav proteins in these diseases has allowed us to discover
biological programs and gene signatures that have favored the molecular
understanding of those diseases, the identification of new targets, and the
diagnosis of some of those diseases. In this talk, we will provide an overall
view of the progress made in these areas.
S1.1-3 (R1.1-3)
Muerte celular, senescencia y autofagia

en la prdida de audicin relacionada
con la edad: regulacin por IGF-1
Isabel Varela-Nieto1, Adelaida Celaya2, Alejandro Gibaja2, Sara
Pulido2, Rocio de Iriarte2, Marta Magarios3
1
Instituto de Investigaciones Biomdicas Alberto Sols (CSIC-UAM),
IdiPAZ y CIBERER, Madrid, ES, 2Instituto de Investigaciones
Biomdicas Alberto Sols (CSIC-UAM) y CIBERER, Madrid, ES,
3
Instituto de Investigaciones Biomdicas Alberto Sols (CSIC-UAM),
Facultad de Ciencias (UAM) y CIBERER, Madrid, ES
La deficiencia congnita en el factor de crecimiento similar a la insulina
tipo 1 (IGF-1) es una enfermedad rara humana que produce alteraciones
severas del crecimiento y sordera neurosensorial. Este factor neurotrfico
es fundamental en la diferenciacin postnatal tarda de la cclea, su
ausencia causa muerte por apoptosis de las neuronas auditivas tipo I, y
otras alteraciones celulares en el receptor perifrico y en las vas auditivas
centrales. Entre los mecanismos implicados se ha descrito la activacin
de quinasas de estrs (MAPK8/9 y 14-FoxP3) y la inhibicin de rutas de
supervivencia (AKT), de proliferacin (MAPK1/3-FoxM1/p27Kip) y de
diferenciacin celular (MEF2).
El IGF-1 en la edad adulta es un protector tico, mantiene la fisiologa
coclear y promueve la supervivencia de las neuronas auditivas. Durante
el envejecimiento, se produce senescencia neuronal, un aumento en la
expresin de genes de la maquinaria autofgica (Becn1, Atg4b yAtg5) y
en los niveles de marcadores proteicos de autofagosomas (LC3-II y p62),
neuroinflamacin y una disminucin en los niveles de IGF-1 que progresa
pari pasu con la prdida auditiva relacionada con la edad (ARHL).
El componente neurodegenerativo de la ARHL empeora en situaciones
patolgicas de dficit de IGF-1, agudizndose el fenotipo molecular,
celular y funcional, e incluso predispone al receptor auditivo a sufrir un
mayor dao cuando se somete a estrs.
Granada 2014
En conclusin, el IGF-1 es un neuroprotector tico que favorece la
supervivencia celular, mientras que su dficit promueve procesos de
inflamacin que producen un aumento del dao en la cclea.
Este trabajo ha sido financiado por los proyectos FP7-innova2 AFHELO
y la MCA TARGEAR.
S1.2 Sealizacin celular cascadas

y dianas
S1.2-1 (R1.2-1)
The loss of memory in Alzheimer disease
Simposios
adenine-dinucleotide (NAD+) to a number of target protein substrates,
leading to the alteration of chromatin associated proteins. PARP-1, the
founder member, is an active player in tumor adaptation to metastasis
and PARP inhibitors, recognized as promising therapeutic agents against
homologous recombination deficient tumors, have novel properties
responsible for the anti-metastatic actions in different tumor settings.
Poly(ADP-ribosyl)ation modulates pro-metastasic activities such as
hypoxic response, angiogenesis and epythelial to mesenchymal transition
(EMT).
Furthermore, key transcription factors are fine-tuned through this action,
helping to the adaptation of tumor to a hostile microenvironment, the
recruitment of new vessels, and stimulate the cellular changes promoting
EMT.
In this presentation we summarized some of the findings that focalize on
PARP-1s action on tumor aggressiveness, suggesting new therapeutic
opportunities against an assembly of tumors not necessarily bearing DNA
repair defects.
Jess vila de Grado

Centro de Biologa Molecular Severo Ochoa (CSIC-UAM), Madrid, ES
Memory impairment in Alzheimer disease (AD) could be related to a defect
in the function of hippocampal region of an AD patient. In this region,
dentate gyrus plays an important role in the formation of new memories.
The study of neurogenesis in dentate gyrus, in mouse models for AD, has
indicated some important features that could also take place in Alzheimer
disease patients.
S1.3 Ingeniera tisular y medicina

regenerativa
S1.3-1 (R1.3-4)
S1.2-2 (R1.2-2)
The role of the JNK pathway in insulin resistance

Carme Caelles
Universidad de Barcelona, Barcelona, ES
During the last decade, the c-Jun N-terminal kinase (JNK) has emerged as
a major regulator of the insulin signalling and, hence, glucose homeostasis.
By directly targeting the insulin receptor substrate (IRS)-1 and -2 for
serine phosphorylation, JNK inhibits insulin signalling at very early steps.
Actually, JNK is activated by insulin and acts as a negative feedback
mechanism to turn off insulin signal under physiological conditions.
However, abnormal JNK activation due to stress, such as oxidative or
endoplasmic reticulum stress, or pro-inflammatory signals induces, by this
IRS-phosphorylation dependent mechanism, insulin resistance. In fact, the
insulin-sensitizing drugs and peroxisome-proliferator activated receptor
(PPAR) ligands thiazolidinediones (TZDs) restore glucose homeostasis
through inhibition of JNK, further supporting the role of this kinase as a
major mediator of obesity-induced insulin resistance. In addition to the
role of JNK in peripheral insulin-target tissues, JNK activity also regulates
insulin action in pancreatic -cells. In this regard, in vivo ectopic activation
of JNK this cell type renders a glucose-intolerance phenotype in mice due
to the impairment of glucose- and insulin-induced insulin secretion and
gene transcription. Moreover, results form these mice demonstrate a direct
action of TZDs on pancreatic -cells as TZD treatment inhibits this ectopic
JNK activation and, thereby, restores insulin signalling and glucose- and
insulin-induced secretion in pancreatic islets, and glucose tolerance in the
mice.
S1.2r-3 (R1.2-3)
Deciphering the insights of poly(ADP-ribosylation)

in metastasis
Francisco Javier Oliver Pozo
Instituto de Parasitologa y Biomedicina Lpez - Neyra, CSIC,
Granada, ES
Poly(ADP-ribose) polymerases (PARPs) are a group of DNA-dependent
nuclear enzymes (also named ARTD) which catalyze the synthesis and
transfer of negatively charged ADP-ribose moieties from nicotinamide-
BIOLAMINATION for customised, rapid tissue

fabrication: printer optional
Robert Brown
University College London. Institute of Orthopaedics &
Musculoskeletal Sciences, RNOH, Stanmore Campus, Londres, UK
A quiet revolution is happening under the umbrella of tissue engineering as
we begin to understand that there are two distinct approaches available for
producing tissues. Not surprisingly, the approach we select has a profound
effect on the technology we use. Firstly, and most familiar, is the idea of
growing tissues by cultivation of cells in/on a substrate (often in bioreactors).
Secondly we can directly fabricate simple living tissues, in the same way
we make cell-phones. In this case there is little or no contribution from
the cells, though they are incorporated as one of the basic building blocks.
Fabricating tissues directly is a concept which has entered largely from public
excitement around the extension of ideas in 3D bio-printing to incorporate
living cells and proteins into the system. In fact 3D cell-printing is only one
of a family of approaches which is better termed BIO-LAMINATION, in
which thin layers (micron scale) of proto-tissue are laid down in series to
build up a stack and produce the gross-scale tissue.
In fact although bio-printing provides the popular image, it is presently
far from the most effective example of fabrication by bio-lamination, as
technical problems of cell survival and natural matrix polymerisation are
complex.
Other bio-lamination approaches include electro-spinning and cell and
matrix layering. Although attractive, the need for bio-printing and spinning
approaches to assemble the X-, Y-, AND Z-planes simultaneously creates
a technical hurdle. Matrix and cell layering techniques avoid this by firstly
mass-fabricating the X-Y plane layers, then stacking them to give the
Z-plane in a separate stage. Cell-rich constructs are fabricated by expansion
of cell sheets on specialised surfaces, matrix-rich X-Y layers by rapid cocompression of cells and matrix proteins (eg collagen). Bio-lamination
fabrication has the capacity to mass produce real tissues (cells embedded
in a native extracellular matrix) in minutes. They can be mass- produced
reproducibly, economically, with simple or complex stacks of tissue
layers. They are already available for 3D tissue drug or disease screening,
customised grafts and advanced drug delivery. But however we produce the
component laminae, they require a number of special processing features.
These include the need (i) to incorporate living cells INTO the fabric of
the matrix at time zero (so no cell-seeding stage but conditions must be
13
Simposios
non-lethal): (ii) to fabricate the matrix primarily from a natural (eg protein)
fibre network; (iii) to provide tight, monitored controls such that the layers
are definable and reproducible when mass produced: (iv) to produce simple
constructs in minutes (rather than days/weeks). Where these criteria can
be met, new opportunities for high impact applications become possible.
S1.3-2 (R1.3-5)
Tejidos artificiales. Del laboratorio a la clnica

Antonio Campos
Facultad de Medicina, Universidad de Granada, Granada, ES
La ingeniera tisular constituye un mbito de investigacin multidisciplinaria
que, sustentada en la histologa, tiene por objeto la construccin de tejidos
artificiales. Para la elaboracin de los mismos, son necesarias clulas madre
con capacidad de proliferacin y diferenciacin selectiva, as como de
biomateriales de distinta naturaleza capaces de reproducir las propiedades
biomecnicas de los tejidos nativos. En la necesidad de articular las
propiedades biolgicas de las clulas y las propiedades derivadas de
la naturaleza fsico-qumica de los biomateriales radica el xito de la
ingeniera tisular y, por tanto, la construccin de un tejido artificial idneo
para la teraputica. Un ejemplo de articulacin especfica es la construccin
de una crnea artificial en la que son necesarios biomateriales de calidad
ptica y clulas diferenciadas para el revestimiento y la regulacin del
transporte inico a partir del humor acuoso. El desarrollo de la ingeniera
tisular est llamado a generar una importante actividad industrial de base
nanotecnolgica, al configurarse, adems de como herramienta teraputica,
como sustituto de los animales de experimentacin en el contexto de la
legislacin europea.
14

S1.3-3 (R1.3-6)
Cardiac reverse remodelling by epigenetic ways

Susana Gonzlez
CNIC, Instituto de la Salud Carlos III, Madrid, ES
The most important determinant of cardiovascular health is a persons
age. Cardiovascular disease (CVD) is a scourge of the developed world,
where it is the leading cause of morbidity and mortality. Despite the
significant progress on many fronts in the cardiovascular field, heart failure
is a particularly debilitating form of cardiovascular disease, with a 50%
death rate in 5 years after diagnosis [Harvey and Leinwand, 2011]. Thus,
numerous gaps in our current knowledge of cardiac aging remain to be
filled. The role of epigenetic circuits has been extensively studied in cardiac
development and inherited cardiac disease [Chang and Bruneau, 2012].
In contrast, critical non-inherited epigenetic modifications controlling
age-associated homeostatic cardiac function in cardiovascular
pathophysiology remain poorly understood. Most studies of
atherosclerosis or cardiomyopathies have been performed in young mice,
while studies of genetic and pharmacological interventions that extend
lifespan rarely assessed whether CVD or heart function was improved
[North and Sinclair, 2012]. Given that the epigenetic Polycomb-member
Bmi1 is central transcriptional instructor in adult stem cell maintenance
[Luis et al., 2012], but unknown cardiac implication, and regulates
lifespan in mammals by targeting components of key longevity pathways
such as p16INK4a, we have explored the possible implication of Bmi1
in the maintenance of adult cardiac function. Using a combination
of conditional knockout models and biochemical analysis, we have
demonstrated that Bmi1 protects against dilated cardiomyopathy (DCM)
by repressing cardiac senescence.
Granada 2014
Psters
Simposio 2:
Biotecnologa de plantas y productos de valor aadido
15
Simposios
S2.1 Ingeniera gentica de plantas

S2.1-1 (R2.1-1)
The creation and utility of synthetic genotypes

in plants: boundaries between metabolic
engineering and synthetic biology
C. Zhu1, T. Capell1, Paul Christou2
1
Department of Plant Production and Forestry Science, ETSEA,
University of Lleida-Agrotecnio Center, Lleida, ES, 2Department of
Plant Production and Forestry Science, ETSEA, University of LleidaAgrotecnio Center, Lleida, ES. Instituci Catalana de Recerca i
Estudis Avanats, Barcelona, ES
An engineered extended ketocarotenoid pathway in rice and maize will be
used as an example to illustrate how synthetic biology principles and multigene transfer can result in the formation of new molecular entities with
useful biological properties. The creation of specialized novel sequestering
structures facilitates the accumulation of metabolic precursors which can
subsequently be decorated be different ketolases and hydroxylases to produce
diverse combinations of ketocarotenoids in an organ- or tissue-specific
manner. The quantitative and qualitative carotenoid and ketocarotenoid
profiles of particular transgenic lines depend on the expression of the
integrated transgene complement. Thus the generation and characterization
of a combinatorial transgenic population is a pre-requisite to select specific
lines with particular chemodiversity. We will discuss the fundamental
mechanisms that underpin these experiments and we will highlight animal
feeding trials to demonstrate the effects of diets fortified with particular
combinations of target metabolites on products derived from such animals.
S2.1-2 (R2.1-2)
Hydrogen peroxide signal transduction in plants.

Gathering the pieces
Frank van Breusegem
Ghent University, Ghent, BE
Different biotic and abiotic stresses adversely affect plant growth and
development leading to worldwide yield losses. A common theme
within these environmental factors is the perturbation of reactive oxygen
species (ROS) homeostasis. Despite the great economic importance,
the signal transduction mechanisms of the oxidative stress response in
plants are poorly understood. We conducted several genetic and chemical
screens, together with proteomic approaches in order to obtain a more
comprehensive overview on the different components of the network that
govern the oxidative stress response. Theses efforts identified several genes
as new members of the oxidative stress gene network in plants, together
with small molecules that are able to interfere with specific events during
the oxidative stress response in plants.
The potential of these genes and molecules for providing abiotic stress
tolerance in commercially relevant plants will be assessed in the future.
S2.1-3 (R2.1-3)
Transcriptional networks and epigenetic

regulation at the core of the plant circadian clock
Paloma Mas
Centro de Regulacin en AgriGenmica (CRAG). Consorcio CSICIRTA-UAB-UB, Bellaterra, Barcelona, ES
The circadian clock is a biological time keeper mechanism able to regulate
biological rhythms with a period of approximately 24-hours. In many
organisms, transcriptional and translational feedback loops underlie the
circadian function at the core of the clock. In Arabidopsis, the evening
oscillator component TIMING OF CAB EXPRESSION 1 (TOC1) was
proposed to activate morning-expressed oscillator genes. In our studies,
16

we have shown that TOC1 does not function as an activator but rather
as a general repressor of oscillator gene expression. Repression occurs
through TOC1 rhythmic association to the promoters of the oscillator
genes, demonstrating that the morning and evening oscillator loops are
connected through the repressing activity of TOC1. Chromatin remodeling
has also emerged as a crucial regulatory mechanism in a number of cellular
processes including the circadian clock. Indeed, the circadian oscillation
of core clock genes correlates with the sequential accumulation of Histone
acetylation and trimethylation. Inhibition of these marks abolishes
oscillator gene expression, indicating that they are critical for gene
activation. Increased clock-repressor binding was observed when Histone
trimethylation was blocked, suggesting that this mark might regulate the
progression from activation to repression. The histone methyltransferase
SET DOMAIN GROUP 2/ARABIDOPSIS TRITHORAX RELATED 3
(SDG2/ATXR3) contributes to this regulation because oscillator gene
expression, Histone trimethylation and repressor binding are altered in
plants mis-expressing SDG2/ATXR3. Our future research goal is to fill
the existing gap in the plant circadian field by identifying other molecular
determinants responsible for particular histone marks at the core of the
clock and their relevance controlling plant responses to the environment.
S2.2 Biologa de sistemas y biologa

sinttica
S2.2-1 (R2.2-1)
Directed enzyme evolution: rational thoughts

for non-rational experiments
Miguel Alcalde
Institute of Catalysis, ICP-CSIC, Cantoblanco, Madrid, ES
Over the past 20 years, directed evolution has been seen to be the most
reliable approach to protein engineering. This tool that has revolutionized
the manner in which proteins are manipulated in the laboratory in order
to improve their application in distinct industrial settings and to engineer
complex metabolic pathways opening a research ground in synthetic biology.
By mimicking the mutation, recombination and selection processes that
occur naturally in evolution, in vitro evolution provides a means of directing
the evolution of genes toward specific goals in a manner that may not occur
in a natural environment. Here, we summarize some successful examples
from our laboratory to tailor ligninolytic genes (laccases, peroxidases,
peroxygenases) in the budding yeast Saccharomyces cerevisiae. This lecture
will pay special attention to the use of the DNA recombination machinery of
S. cerevisiae to accelerate artificial evolution, complementing the traditional
in vitro methods to generate tailor-made enzymes.
References
[1] P. Molina-Espeja, E. Garcia-Ruiz, D. Gonzalez-Perez, R. Ullrich, M.
Hofrichter and M. Alcalde. Appl Environ Microb 2014, 80: 3496-507.
[2] D. Gonzalez-Perez, P. Molina-Espeja, E. Garcia-Ruiz and M. Alcalde.
PloS ONE 2014; 9 (3): e90919.
[3] P. Torres-Salas, D. Mate, I. Ghazi., F.J. Plou, A.O. Ballesteros and M.
Alcalde M. Chem Bio Chem 2013; 14: 903-8.
[4] D. Mate, D. Gonzalez-Perez, M. Falk, R. Kittl, M. Pita, A.L. De Lacey,
R. Ludwig, S. Shleev and M. Alcalde. Chem Biol 2013; 20: 223-31.
[5] D. Gonzalez-Perez, E. Garcia-Ruiz and M. Alcalde M. Bioengineered
2012; 3: 1-6.
[6] E. Garcia-Ruiz, D. Gonzalez-Perez, F.J. Ruiz-Dueas, A.T. Martinez
and M. Alcalde. Biochem J 2012; 441: 487-98.
[7] D. Mate, C. Garcia-Burgos, E. Garcia-Ruiz, A.O. Ballesteros, S.
Camarero, and M. Alcalde. Chem Biol 2010; 17: 1030-41.
[8] M. Zumrraga, T. Bulter, S. Shleev, J. Polaina, A. Martinez-Arias, F.J.
Plou, A. Ballesteros, and M. Alcalde. Chem Biol 2007; 14: 1052-64.
Granada 2014
Simposios
S2.2-2 (R2.2-2)
metabolic machinery. Aside from the carbon and energy stockpiling

function ascribed to the polymers themselves, GAPs play a critical
physiological role in PHA metabolism and granule formation. Phasins
form part of the mediation element network responsible for granule
localization and segregation. PHA synthase and depolymerase are also
attached to the granules, and operate a continuous metabolic cycle that
ensures the turnover of the accumulated polymer and plays a key role in
the maintenance of metabolism homeostasis.
PHAs, traditionally considered bacterial reservoirs of carbon and energy,
are now recognized as having many physiological roles. In P. putida, PHA
production ensures a more robust metabolism and improves bacterial
fitness. Acting as a futile cycle, PHA metabolism balances the storage vs.
spillage equilibrium of both carbon and energy, thus determining cell size
and morphology. PHA metabolism is controlled by the network of local
and global regulators controlling the pathways involved in carbon and
nitrogen assimilation.
PHAs are valuable nutrients for producer strains, but since they can be
extracellularly degraded they are also useful to the wider microbial
community.The predation of PHA- producers by bacterial predators
such as B.dellovibrio bacteriovorus has an important effect on microbial
population dynamics since PHA granules and free HA oligomers are
released into the environment. These can be then incorporated into the
microbial metabolism of not-necessarily accumulating/degrading strains,
enhancing their biological fitness.
The system-wide nature of PHA metabolism makes it a real challenge to
design PHA-producing bacteria using synthetic biology strategies.
Microbial life-styles and adaptive changes

of macromolecular synthesis
Soren Molin
Department of Systems Biology and Novo Nordisk Foundation
Center for Biosustainability, Denmark Technical University of
Denmark, Lyngby, DK
Bacteria belonging to the genus Pseudomonas posses a broad metabolic
repertoire, which allows them to grow in a wide variety of environments.
This versatile potential for growth and persistence is reflected in the large
genome sizes in Pseudomonas bacteria and in a parallel large number of
regulatory genes. Pseudomonas aeruginosa is an opportunistic pathogen
living in the environment, and under some conditions it has the capacity
to colonize humans, where it may develop life long persistent infections.
Moving from the outer environment to a human environment constitutes
a significant change of living conditions challenging the regulatory and
metabolic repertoire of the bacteria.
In patients suffering from the genetic disorder cystic fibrosis (CF) P.
aeruginosa frequently develops persistent infections of the airways, and
over periods of thousands of bacterial generations mutations accumulate in
the bacterial populations, which gradually increase the persistence fitness
resulting in severe chronic lung infections. In a large study of more than 40
young CF patients we have investigated the evolutionary dynamics of more
than 50 different strains of P. aeruginosa, from which we have sequenced
more than 600 genomes covering airway residence times from 1-10 years.
One of the striking observations made from this investigation is that
adaptive mutations in genes encoding global regulatory proteins, including
RNA polymerase associated sigma factors, are very frequent in CF isolates
obtained from patients with early stage P. aeruginosa airway infections. We
are currently studying the relationship between the phenotypes associated
with some of these regulatory mutations and the microbial life-style in CF
airways, and examples of such relationships will be presented. It will be
documented how specific mutations in regulatory genes may switch the
phenotype from one typical life-style to another more compatible with
the novel environment. It is hypothesized that this pattern of mutational
changes observed in infecting bacterial populations may represent bacterial
adaptive evolution in many other scenarios, where bacteria move from one
environment to another.
S2.2-3 (R2.2-3)
The polyhydroxyalkanoate systems metabolism;

much more than a bioplastic production
Mara A. Prieto
Centro de Investigaciones Biolgicas, CSIC, Madrid, ES
Polyhydroxyalkanoate (PHA) are one of the current alternatives to
petroleum-based plastics. From an industrial standpoint, PHAs are
attracting extensive interest as technical-grade polymers due to their
singular set of properties: (i) substitution potential for industrial
thermoplastics such as polypropylene, polyethylene, polyvinylchloride
and polyethylene terephthalate, (ii) biodegradability both in aerobic and
anaerobic conditions, including aquatic environments, (iii) biobased,
renewable origin, (iv) biocompatibility with cells and tissues and (vi)
structural diversity.
The physiology of PHA production has aroused much interest since the
ability to accumulate these compounds is widespread among bacteria
and PHA metabolism influences many cell activities. This is of particular
interest with respect to model bacterial strains such as Pseudomonas putida
KT2440, an outstanding candidate for the development of microbial chassis
with specialized phenotypes. The use of cutting-edge technologies such
as transcriptomics, proteomics and metabolic flux analyses has improved
our understanding of the interplay between PHA metabolism, which is
extremely context-dependent, and other cell functions.
PHA granules represent supramolecular complexes that carry granule
associated proteins (GAPs), essential protein components of the PHA
S2.3 Hispano-Mexicano
S2.3-1 (R2.3-0)
Biotecnologa de anticuerpos: mejora y desarrollo

de nuevos antivenenos
Alejandro Alagn Cano
Instituto de Biotecnologa, Universidad Nacional Autonma de
Mxico, Mxico DF, MX
S2.3-2 (R2.3-1)
Structure and function of bacterial

fructosyltransferases: fructans in traditional
mexican fermentation products
Agustn Lpez-Mungua
Instituto de Biotecnologa, UNAM, Cuernavaca, MX
Fructansucrases (GH68) are enzymes that catalyze the transfer of the
fructosyl unit of sucrose to a growing fructan polymer. Depending on the
chemical nature of the resulting fructosidic bond, these enzymes may be
classified as levansucrases (LS) when the resulting polymer (levan) is
linked through (26) bonds or inulosucrases (IS) when (21) linked
fructosyl units are formed in the main chain (inulin). We first reported the
unusual mosaic structure of inulosucrase (IslA) in Leuconostoc citreum
CW28, a strain isolated from Pozol, a corn traditional lactic fermentation
product traditionally consumed in the south of Mxico. IslA, which is
highly specific for inulin synthesis, has a b-propeller catalytic domain
with high identity to SacB, the levansucrase from Bacillus subtilis.
However, IslA also bears additional domains in both, the amino and
carboxyl terminal regions, domains already described in glucansucrases
from lactic acid bacteria. Glucansucrases are enzymes that carry out the
synthesis of glucans from sucrose, in an analogous mechanism. The same
structural features found in IslA were later identified and characterized in
several bacterial strains, including the industrial dextran producing strain
(Leuconostoc mesenteroides NRRL B-512F) as well as in the sequenced
17
Simposios
genome of Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293.
Both glucans and fructans are present in pulque, a traditional fermentation
product that dates back to the Mesoamerican culture. Pulque is obtained
from aguamiel, a sap extracted from various agave species.
In this conference several aspects related with the structural properties
of these glycosyltransferases, are described, as well as their role in the
traditional Mexican fermented products and their potential applications in
fructan synthesis.
S2.3-3 (R2.3-2)
LINE-1 retrotransposition in Fanconi anemia

patients
Jos Luis Garca Prez
GENYO-University of Granada, Granada, ES
It is becoming increasingly evident that Long Interspersed Element-1
(LINE-1 or L1) retrotransposons have played an important role in shaping
the structure and function of mammalian genomes. L1s are members of
the non-long-terminal-repeat (non-LTR) retrotransposon family, which are
widely spread in the human genome. New L1 mobilization events have
18

resulted in a variety of genetic disorders providing a constant source of
human genetic diversity. L1 might ensure its evolutionary transmission to
new generations through the accumulation of new insertions during early
embryonic development or in germ cells. Thus, L1 insertions can act as
mutagens in the embryonic phase. In fact, endogenous L1s are expressed
in Human Embryonic Stem Cells (hESCs) and human induced pluripotent
stem cells (hiPSCs). Additionally, others and we have shown that engineered
human L1s can retrotranspose in hESCs and iPSCs at a low level. In sum,
these data reveal that both hESCs and hiPSCs are an excellent model to study
LINE-1 accumulation and its genomic impact in humans.
On the other hand, it is well established that L1 retrotransposition occurs
by a mechanism known as target site-primed reverse transcription (TPRT),
which requires both the L1-encoded endonuclease and reverse transcriptase
activities. However, it has been reported that L1 can mobilize through an
endonuclease-independent pathway (ENi), in which the element inserts at
sites of DNA disrepair. Notably, recent data from our lab has demonstrated
that the mobilization of L1 is de-regulated in Fanconi Anemia (FA)
patients, including the ENi pathway. FA is a rare disease characterized
by infant mortality and genomic instability, suggesting that de-regulated
LINE-1 retrotransposition may affect genomic fluidity in FA patients.
Granada 2014
Psters
Simposio 3:
Estructura y funcin de protenas
19
Simposios
S3.1 Receptores y protenas

de membrana
S3.1-1 (R3.1-1)
Transportadores heteromricos de aminocidos:

hacia su estructura atmica
Albert Rosell1, Lukasz Kowalczyk1, Joana Fort1, Ekaitz Errasti1,
Paola Bartoccioni1, Elena Alvarez-Marimon1, Meritxell Costa2,
Marcel Meury3, Guillem Portella1, Laura Prez4, Arturo RodrguezBanqueri1, Ana Obando1, Antonio Zorzano5, Modesto Orozco6,
Xavier Carpena7, Jos Luis Vzquez-Ibar8, Juan Fernndez-Recio4,
Dimitrios Fotiadis4, Ignacio Fita7, Manuel Palacn9
1
IRB Barcelona, Barcelona, ES, 2IRB Barcelona y Swiss National
Centre of Competence in Research TransCure, University of
Bern, Bern, CH, 3Swiss National Centre of Competence in
Research TransCure, University of Bern, Bern, CH, 4Barcelona
Supercomputing Center, Barcelona, ES, 5IRB Barcelona y
Universidad de Barcelona, Barcelona, ES, 6IRB Barcelona,
Barcelona Supercomputing Center y Universidad de Barcelona,
Barcelona, ES, 7Department of Structural Biology, Instituto de
Biologa Molecular de Barcelona (IBMB-CSIC), Barcelona, ES, 8IRB
Barcelona y Institute of Biology and Technology Saclay (iBiTec-S),
Gif-sur-Yvette Cedex, FR, 9IRB Barcelona, Universidad de Barcelona
y CIBERER, Barcelona, ES
Los transportadores heteromricos de aminocidos (HAT) son el nico
ejemplo conocido de transportadores de solutos constituidos por dos
subunidades unidas por un puente disulfuro. En metazoos, la subunidad
pesada es responsable del trfico del heterodmero a la membrana
plasmtica mientras que la subunidad ligera es el transportador. Los
HAT estn involucrados en patologas como aminoacidurias, cncer,
infeccin viral y adiccin a cocana. En los ltimos siete aos se ha
incrementado el conocimiento estructural de estos transportadores de
forma notable. El ectodominio de la subunidad pesada humana 4F2hc
(4F2hc-ED) se ha resuelto a 2.1 revelando homologa estructural con
amilasas bacterianas. 4F2hc-ED adolece de los residuos catalticos clave
pero mantiene la hendidura cataltica para la que no se conocen ligandos.
Homlogos bacterianos con baja identidad de secuencia (<20% con las
subunidades ligeras como el intercambiador arginina/agmatina AdiC
de E. coli) han sido resueltos a 3.0 y revelan un plegamiento de tipo
LeuT, caracterstico de varias familias de transportadores de solutos. La
expresin del heterodmero 4F2hc/LAT2 humano en Pichia ha permitido
obtener el primer modelo a baja resolucin de un HAT (21 ) por tincin
negativa, anlisis por docking y confirmacin por entrecruzamiento de
residuos de cisteina. Este modelo ha revelado por primera vez la posicin
relativa de 4F2hc-ED sobre la subunidad ligera, ofreciendo una explicacin
estructural de la estabilizacin del complejo. Se presentarn las estrategias
que estamos siguiendo para resolver la estructura atmica de homlogos
bacterianos ms robustos y de HAT eucariotas. Estas estructuras facilitarn
la comprensin del papel de 4F2hc en la actividad del transportador
asociado y la conexin funcional con integrinas.
S3.1-2 (R3.1-2)
Implicacin del transportador ABCG2 en la

infeccin por el parsito Leishmania
Francisco Gamarro, David Len-Guerrero, Jenny Campos-Salinas,
Jos Ignacio Manzano, Elena Gonzlez-Rey, Mario Delgado, Jos
Mara Prez-Victoria, Santiago Castanys
Granada, ES
Las protenas ABC (ATP-binding cassette) constituyen una de las mayores
familias a lo largo de toda la escala biolgica, desempeando una gran
diversidad de funciones, entre otras el transporte activo de molculas a travs
20

de las membranas. Los sustratos conocidos son estructuralmente diversos,
encontrando entre ellos lpidos y frmacos. En el parsito Leishmania,
responsable de la segunda enfermedad parasitaria en importancia
conocida como leishmaniasis, existen 42 genes de la familia ABC. Hemos
caracterizado un nuevo transportador ABC de Leishmania, denominado
ABCG2 implicado en el proceso de infeccin. Los anlisis realizados con
parsitos que expresan una versin inactiva del transportador (DN) o con
parsitos mutantes nulos para ABCG2 mostraron que este transportador es
necesario para la exposicin de fosfatidilserina (PS) en la cara externa de
la membrana plasmtica del parsito. Este fenotipo se correlacion con una
deficiente capacidad de infectar macrfagos peritoneales. Se ha descrito
que la exposicin transitoria de PS en la superficie del parsito es necesaria
para el proceso de infeccin celular. Adems, los estudios de infeccin
en un modelo experimental de leishmaniasis cutnea mostraron que los
animales infectados con parsitos DN no desarrollaban ninguna lesin,
mostrando una inflamacin y carga parasitaria ms baja que los animales
infectados con los parsitos control. Todos estos resultados evidencian
la implicacin del transportador ABCG2 en la exposicin de PS en la
membrana del parsito, en la infeccin y virulencia de Leishmania.
S3.1-3 (R3.1-3)
Transferencia de electrones entre protenas:

de la cintica a la molcula nica
Carlos Gmez-Moreno1, Carlos Marcuello2, Rocio de Miguel3, Marta
Martnez Jlvez4, Anabel G Lostao5
1
Universidad de Zaragoza, Zaragoza, ES, 2Instituto de Nanociencia
de Aragn Universidad de Zaragoza, Zaragoza, ES, 3CEA, Paris,
FR, 4BIFI. Universidad de Zaragoza, Zaragoza, ES,5Instituto
de Nanociencia de Aragn/ Fundacin ARAID, Universidad de
Zaragoza, Zaragoza, ES
El proceso de transferencia de electrones entre protenas constituye la
base de muchas reacciones que son fundamentales para los seres vivos. La
fotosntesis, la respiracin, la fijacin de nitrgeno, la desaturacin de los
cidos grasos, etc., son ejemplos representativos. En ellos, las protenas que
intercambian electrones deben interaccionar durante un muy breve espacio
de tiempo para que el electrn/es pase(n) de una protena a la otra. Durante
ms de 25 aos nuestro grupo ha investigado el mecanismo mediante el cual
se produce el reconocimiento entre las protenas que deben interaccionar.
El trabajo se ha centrado en el estudio de la enzima Ferredoxina NADP+
reductasa, que interviene en la produccin de NADPH en la fotosntesis
y sus protenas complementarias, la Ferredoxina (Fd) y la Flavodoxina
(Fld), todas ellas de la cianobacteria Anabaena PCC 7119. Tcnicas
cinticas, tanto en estado estacionario como preestacionario (inducida por
lser y de flujo interrumpido) junto con el empleo de muestras mutadas
en posiciones concretas han permitido concluir que la interaccin seinicia
mediante la orientacin de las superficies de reconocimiento por atraccin
electrosttica seguida por una reorganizacin de ambas molculas mediante
interacciones hidrofbicas.
La cristalizacin y resolucin de la estructura del complejo entre la FNR
y la Fd de Anabaena han ratificado nuestra propuesta al determinar que
los grupos propuestos para la interaccin estn prximos en el complejo y
que los centros redox se encuentran a distancias que son compatibles con
la reaccin.
Recientemente hemos iniciado una lnea de trabajo que se apoya en el uso
de la Microscopa de Fuerzas Atmicas (AFM) para estudiar el proceso
de interaccin desde el punto de vista de la molcula individual. La razn
de esta decisin est en la esperanza de que la observacin directa de las
molculas que interaccionan permita mostrar detalles del proceso que no
son asequibles mediante el estudio del conjunto de los componentes de la
reaccin.
Se ha desarrollado una estrategia que permite inmovilizar las protenas
con la orientacin adecuada para que se produzca la interaccin con
su pareja y medir la eficiencia del proceso de acercamiento as como
la magnitud de la fuerza ejercida. Los resultados de la medida de las
fuerzas intermoleculares de los complejos transitorios entre las protenas
Granada 2014
implicadas en la transferencia de electrones se interpretan de acuerdo
con el modelo propuesto anteriormente que indicaba que la interaccin
FNR:Fd es ms fuerte y especfica que la que se presenta en la FNR:Fld.
Las mayores fuerzas de ruptura medidas entre la FNR y la Fd frente a las
obtenidas entre la FNR y la Fld indican la mayor mecanoestabilidad del
complejo entre la FNR y su pareja natural la Fd.
S3.2 Macromolculas en la enfermedad

y vacunas
Simposios
S3.2-3 (R3.2-3)
NETs from infection to autoimmunity

Arturo Zychlinski
Max Planck Institute for Infection Biology, Berln, DE
Neutrophils are one of the first lines of defence of the immune system agains
tmicrobes. These cells kill microorganisms effectively by phagocytosis and
by the formation of extracellular structures, called Neutrophil Extracellular
Traps (NETs). NETs are made of chromatin and specific neutrophil
proteins and are released after a unique cell death program that requires the
production ofradical oxygen species (ROS) and the relocation of neutrophil
elastase to the nucleus. NETs help limit and control infection and also can
activate the acquired immune system. Thus, formation of NETs appears to
be necessary for an efficient clearing of microbes but can also initiate and
exacerbate autoimmune responses.
S3.2-1 (R3.2-1)
Vacunas contra enfermedades prevalentes

Mariano Esteban
Centro Nacional de Biotecnologa, CSIC, Madrid, ES
La vacunacin es la estrategia ms efectiva y menos cara para controlar
y erradicar las enfermedades humanas. Muchas enfermedades han sido
controladas mediante vacunacin, pero hay otras para las que no hay vacunas
disponibles. Anualmente cerca de 2 millones (M) de personas mueren a
causa del virus de la inmunodeficiencia humana (VIH), cerca de 3M de
tuberculosis, 700.000 de malaria, ms de 7M por cncer, 12M estn infectadas
de leishmania y 170M con hepatitis C (HCV). Adems, enfermedades en
personas de avanzada edad, como Alzheimer y Parkinson, sin tratamientos
efectivos disponibles, estn siendo abordadas mediante vacunacin. Las
dificultades en el desarrollo de vacunas se deben al desconocimiento de
la patologa del microorganismo, diseo del inmungeno, protocolos de
inmunizacin, y a la definicin de la correlacin inmune de proteccin. Con
los nuevos avances tecnolgicos derivados de la biologa de sistemas y las
aproximaciones inmunolgicas, el enfoque de las vacunas ha cambiado.
Nuestro grupo se embarc hace varios aos en el desarrollo de vacunas para
enfermedades para las que no hay vacunas, utilizando vectores poxvirales.
De las lecciones aprendidas de la biologa del vector poxviral virus vaccinia
modificado de Ankara (MVA) y de los estudios en el desarrollo de vacunas,
hemos generado y patentado varios candidatos vacunales contra VIH/SIDA,
hepatitis C (HCV), chikungunya, malaria, leishmania, cncer de prstata,
y algunos han entrado en ensayos clnicos, como con VIH. De hecho,
hemos producido un candidato vacunal para VIH-1 (MVA-B) que ha sido
utilizado en dos ensayos clnicos de fase I en Espaa, con buenos resultados
inmunolgicos. En la conferencia, presentaremos la descripcin de vectores
atenuados de poxvirus, interaccin con el hospedador, preclnica y clnica
frente a modelo de patgenos y estado de vacunas.
S3.2-2 (R3.2-2)
dUTPases, the unexplored family of signalling

molecules
Jos R. Penads1, Alberto Marina2
1
Institute of Infection Immunity and Inflammation, Universidad de
Glasgow, Glasgow, UK, 2Instituto de Biomedicina de Valencia (IBVCSIC), Valencia, ES
Deciphering the molecular mechanisms that control relevant cellular
processes is of utmost importance to understand how viruses, prokaryotic
and eukaryotic cells work. The diversity of living organisms suggests that
there are novel regulators still to be discovered, which may uncover new
regulatory paradigms. dUTPases (Duts) are assumed to be ubiquitous
enzymes regulating cellular dUTP levels to prevent misincorporation of
uracil into DNA. Recently however, Duts have been involved in the control
of several relevant cellular processes, including transfer of mobile genetic
elements, regulation of the immune system, autoimmunity or apoptosis,
suggesting that they perform regulatory functions. This talk aims at
investigating the unexplored impact of Duts as novel signalling molecules.
S3.3 Interaccin protena/protena

y complejos multiproteicos
S3.3-1 (R3.3-1)
Structural characterisation of the chaperone

Hsp70 folding network
Jos Mara Valpuesta
Centro Nacional de Biotecnologa, CSIC, Colmenar Viejo, Madrid, ES
The 70 kDa heat shock proteins (Hsp70s) form a family of highly
conserved molecular chaperones present in all domains of life and in most
subcellular compartments of eukaryotic cells. Hsp70s, acting in concert
with J Hsp40s and nucleotide exchange factors (NEFs) assist a multitude of
different cellular protein-folding processes during protein maturation and
quality surveillance [1,2]. Besides, Hsp70s can interact with other major
chaperones like Hsp90s, Hsp110s and CCT [3-6], in some cases via other
co-chaperones, which act as physical linkers.
We have been working over the last years in characterizing, using mostly
electron microscopy and image processing, the interaction of Hsp70s with
some of these chaperones and co-chaperones in an effort to understand the
interactions that take place between these proteins, that act in coordination,
forming part of an assembly line, to make the protein folding process a
more efficient one.
References
[1] Kampinga, H.H., and Craig, E.A. The HSP70 chaperone machinery: J
proteins as drivers of functional specificity. Nat Rev Mol Cell Biol 2010;
11: 579-92.
[2] Hartl, F.U., Bracher, A., and Hayer-Hartl, M. Molecular chaperones in
protein folding and proteostasis. Nature 2011; 475: 324-32.
[3] Schuermann, J.P., Jiang, J., Cullar, J., Llorca, O. Wang, L., Taylor,
A.B., Demeler, B.,Morano, K., Hart, P.J., Valpuesta, J.M., Lafer, E.M. and
Sousa, R. Structure of the Hsp110:Hsc70 Nucleotide Exchange Complex.
Mol Cell 2008; 31: 232-43.
[4] Cullar, J., Martn-Benito, J, Scheres, S.H.W., Sousa, R., Moro, F.
Lpez-Vias, E., Gmez-Puertas, P., Muga, A, Carrascosa, J.L. and
Valpuesta, J.M. The structure of a CCT:Hsc70NBD complex suggests a
mechanism for Hsp70 delivery of substrates to the chaperonin. Nat Struct
Mol Biol 2008; 15: 858-64.
[5] Cullar, J., Perales-Calvo, J.,Muga, A., Valpuesta, J.M. and Moro, F.
Structural insights into the chaperone activity of the 40 kDa heat shock
protein DnaJ. Binding and remodeling of a native substrate. J Biol Chem
2013; 288: 15065-74.
[6] Lorenz, O.R., Freiburger, LRutz, , D.A., Krause, M., Zierer, B.K.
Alvira, S. Cullar, J., Valpuesta, J.M, Madl, T., Sattler, M, and Buchner, J.
Modulation of the Hsp90 chaperone cycle by a stringent client protein. Mol
Cell 2014; 53: 941-53.
21
Simposios
S3.3-2 (R3.3-2)
Descubriendo interacciones entre enzimas

proteolticas, sus inhibidores y ligandos proteicos
por MS-protemica funcional
Francesc Xavier Avils1, G. Covaleda1, S. Tanco1, O. Tort1, A. Otero1,
J. Vendrell1, J. Lorenzo1, M. Alonso del Ribero2, M.A. Chvez2
1
Instituto de Biotecnologa y de Biomedicina y Dpto. de Bioqumica,
Universitat Autnoma de Barcelona, Bellaterra, Barcelona, ES, 2Centro de
Estudios de Protenas, Facultad de Biologa, Universidad de La Habana,
La Habana, CU
Actualmente se tiende hacia una visin integrada de las enzimas proteolticas
(proteasas) y acompaantes moleculares in vivo, especialmente en el
contexto genmico-protemico-interactmico. Ello tambin se da en unas
de sus variantes ms frecuentes como son las metaloproteasas y, dentro de
ellas, en las metalocarboxipeptidasas (MCP). El fuerte incremento en el
nmero de MCP detectadas en la ltima dcada pone en evidencia de que
an no se comprende su accin simultnea o especfica sobre sustratos,
extensible a sus restricciones por ligandos e inhibidores naturales. El hecho
de que tales sustratos, y sus efectores, sean frecuentemente de naturaleza
proteica, aade grados adicionales de sofisticacin a estas interacciones
(p.e. a niveles qumico-fsicos, de capacidad de discriminacin y cinticos),
as como en la modulacin de actividades y especificidades que promueven
[1-3].
Hoy en da podemos evaluar en cerca de 30, el nmero de variantes
de dichas enzimas MCP, clasificadas entre formas M14A, B y C, y las
recientemente emergentes formas citoslicas (tipo CCP) para la subfamilia
M14D [4,5]. Dado que todas ellas parecen conservar el dominio cannico
de metalocarboxipeptidasa y los sitios de reconocimiento equivalentes,
es un desafo real la comprensin de la relacin compleja entre ellas, su
interaccin discriminativa con sustratos naturales (peptdicos o proteicos,
dado que son proteasas), y con los inhibidores proteicos ambientales.
Tambin lo es para diseadores de frmacos la generacin de ligandos que
especficamente las controlen [2].
Las MCP, como otras proteasas, tienen otra propiedad caracterstica
interactmica: actan de forma transiente sobre sustratos proteicos,
promoviendo cortes sobre ellos que afectan fuertemente su conformacin
y funcionalidad. Cmo detectar dichas interacciones transientes no suele
ser una tarea fcil, aunque factible [6-8]. Tambin se suele desconocer el
papel que juegan en la naturaleza numerosos inhibidores proteicos de ellas,
que se encuentran en sistemas biolgicos muy diversos. Se discutir casos
recientes de descubrimiento de dichos inhibidores y/o ligandos-sustratos, y
de estrategias protemicas e interactmicas, basadas en espectrometra de
masas, as como de biologa estructural seguidas para su identificacin y
caracterizacin [3,6,8-10]. Tambin, si cabe, se presentarn datos de MaldiMS-Imagen (MS-imagen de tejidos) para alguna de dichas biomolculas.
Bibliografa
[1] Arolas, JL, Vendrell, J, Avils, FX, Fricker LD: Curr Pharm Des 2007;
13: 349-66.
[2] Fernandez D, Pallares I, Vendrell J, Aviles FX: Biochimie 2010; 92:
1484-500. // Fernandez D, Pallares I, Covaleda G, Vendrell J, Aviles FX:
Curr Med Chem 2013; 20: 1595-608.
[3] Covaleda G, Alonso-del-Rivero MA, Chvez MA, Avils FX, Reverter
D: J Biol Chem 2012; 287: 9250-8.
[4] Rodriguez de la Vega M, Sevilla RG, Hermoso A, Lorenzo J, Tanco S,
Diez A, Fricker Ll D, Bautista JM, Aviles FX: FASEB J 2007; 20: 851-65.
// Rodriguez de la Vega M, Lorenzo J, Aviles FX, Bautista JM: FASEB J
2013; 27: 424-31.
22

[5] Otero A, Rodriguez de la Vega M, Tanco S, Lorenzo J, Aviles FX,
Reverter D: FASEB J 2012; 26: 3754-64.
[6] Yanes O, Villanueva J, Querol E, Aviles FX: Nature Protoc 2007; 2:
119-130. // Sanglas L et al.: PNAS 2009; 106: 1743-47.
[7] Morell M, Czihal P, Otvos L, Aviles FX, Ventura S: Proteomics 2008;
8: 3433-42.
[8] Van Damme P, et al., Aviles FX, Gevaert K: Nature Meth 2010; 7: 5125.
[9] Tanco S, Lorenzo J, Garcia-Pardo J, Degroeve S, Martens L, Aviles FX,
Gevaert K, Van Damme P: Mol Cell Proteom 2013; 12: 2096-110.
[10] Alonso del Rivero M, Reytor ML, Trejo SA, Chvez MA, Avils FX,
Reverter D: Structure 2013; 21: 1118-26.
S3.3-3 (R3.3-3)
Force generation by FtsZ bacterial cytoskeletal

protein: what we have learned from single
filament analysis
Marisela Vlez
ICP, CSIC, Madrid, ES
FtsZ is a bacterial cytoskeletal protein that polymerizes on the inner surface
of the bacterial membrane and contributes to generate the force needed
for cell division. In the presence of GTP the individual protein monomers
interact longitudinally to form filaments that can then aggregate to form
higher order structures on the membrane surface. These filament aggregates
are dynamic and exchange monomers from the solution. The final outcome
of this dynamic rearrangement on the surface is the generation of force that
bends the cell membrane inward.
The rich self-assembling behavior of the protein is reproduced in vitro
on supported lipid membranes using isolated proteins. Reversible GTPinduced polymerization observed with Atomic Force Microscopy (AFM)
showed that the type of attachment to the surface and the type of lipid
present on the membrane determine the shape of the filament aggregates
observed.
The detailed structural and dynamic information obtained with the AFM
has allowed comparing the experimental results with theoretical models
that describe the polymerization in terms of a simple set of monomermonomer interactions. Experimental results controlling the orientation
of the monomers on the surface, together with molecular dynamics
simulations and theoretical models have revealed that filament curvature,
twist, orientation and the strength of the surface attachment are all
important for determining the amount of force that the filaments can exert
on the surface.
References
Mateos-Gil , P.; Paez ,A.; Hrger, I, Rivas, G.; Vicente, M.; Tarazona, P.
and Vlez, M. Depolymerization dynamics of individual filaments of
bacterial cytoskeletal protein FtsZ. PNAS 2012; 109 (21): 8133 8.
Gonzlez de Prado Salas, P., Encinar, M., Vlez, M. and Tarazona, P.
FtsZ protein on bilayer membranes: effects of specific lateral bonds. Soft
Matter 2013; 9: 6072.
Encinar, M.; Kralicek, A.; Martos, A.; Krupka, M.; Alonso, A.; Cid, Sa.;
Rico, A.; Jimnez, M.; Vlez, M. Polymorphism of FtsZ filaments on lipid
surfaces: role of monomer orientation. Langmuir 2013; 29: 9436-46.
Gonzlez de Prado Salas, P., Hrger, I., Martn-Garca, F., Jess Mendieta,
Alvaro, A., Encinar, M., Gmez-Puertas, P., Vlez, M., and Tarazona, P.
Torsion and curvature of FtsZ. Soft Matter 2014; 10: 1977-86.
Granada 2014
Psters
Psters
23
Psters
P00. II Workshop sobre la Innovacin

Docente en la Enseanza de la
Bioqumica y la Biologa Molecular
P00-1 (R00-2)
Ciencia para Todos. Una experiencia para

aprender divulgando
Pilar Roca, J. Oliver, A. Valle
Grupo Multidisciplinar de Oncologa Traslacional. IUNICS.
Universitat de les Illes Balears, Palma de Mallorca, ES
Entre las competencias genricas que debemos evaluar los profesores de
grados de Ciencias est la trasmisin de informacin, ideas, problemas
y soluciones a pblico especializado y no especializado. Un grupo de
profesores de la UIB nos planteamos trabajar esta competencia en nuestras
asignaturas, por lo que pensamos en desarrollar un encuentro entre jvenes
universitarios y estudiantes de primaria, secundaria y bachillerato, de esta
manera naci CIENCIA PARA TODOS. La experiencia tena un doble
objetivo: fomentar las vocaciones cientficas y la difusin de la Ciencia,
y utilizar el encuentro para que los estudiantes de grado aprendieran
divulgando.
Entre las actividades del encuentro podemos sealar experiencias
cientficas, exposiciones (mujeres y ciencia, alimentacin equilibrada,
animales exticos, minerales, plantas carnivoras), talleres de visualizacin
de molculas, juegos cientficos. Adems se present el blog que recoge los
experimentos propuestos. Los alumnos en el desarrollo de la actividad se
implicaron de manera activa tanto en el planteamiento de las experiencias
de laboratorio (usando materiales caseros o que puedan encontrar en los
colegios e institutos), como en el montaje y presentacin a las personas que
acudieron al encuentro.
En el evento participaron 250 estudiantes de la UIB, y recibimos ms de
3.500 visitantes. La evaluacin del grado de satisfaccin por parte de los
visitantes fue muy alta, al igual que de los alumnos de la UIB. Mientras que
los profesores que participaron con sus asignaturas estamos ya trabajando
en el encuentro de 2015.
Agradecimientos: alumnos de Facultad de Ciencias y Escuela Politcnica
de la UIB, profesores y personal de administracin que nos ayudaron en
el montaje y desarrollo de la misma. FECYT (FCT-13-7535), ICE-UIB.
P00-2 (R00-1)
La bicicleta de Krebs, una iniciativa estudiantil

para el aprendizaje del metabolismo (y ms)
Hugo Pineda1, Lola Nevado1, Sara Bernrdez1, M Jess Pacheco1,
Juan J. Criado1, Florencio Palomas1, Miguel ngel Medina Torres2
1
Alumnos de la asignatura optativa Bioqumica Metablica de la
Licenciatura en Biologa de la Universidad de Mlaga en el curso
2012-13, Mlaga, ES, 2Universidad de Mlaga, Andaluca Tech,
Departamento de Biologa Molecular y Bioqumica, Facultad de
Ciencias, e IBIMA (Instituto de Biomedicina de Mlaga). Unidad
741, CIBER de Enfermedades Raras (CIBERER), Mlaga, ES
Bioqumica Metablica es una asignatura que se ha ofertado como optativa
de segundo ciclo en la Licenciatura de Biologa y como asignatura de libre
configuracin en las Licenciaturas de Qumica e Ingeniera Qumica de la
Universidad de Mlaga durante todos los aos de vigencia del ltimo plan
de estudios de licenciaturas, actualmente en vas de extincin. Configurada
alrededor de tres bloques o unidades temticas (Regulacin integrada
del metabolismo, Regulacin de los ejes centrales del metabolismo y
Metabolismo en accin), la asignatura siempre se ha caracterizado por
su sistema de autntica evaluacin continuada, por su flexibilidad y
libertad en el desarrollo de sus contenidos y por ofrecer a los alumnos un
espacio propio para convertirse en protagonistas corresponsables de la
configuracin de la asignatura. Ello ha brindado ocasin para el desarrollo
24

de numerosas iniciativas estudiantiles para la enseanza-aprendizaje del
metabolismo perfectamente exportables a otros mbitos de la docencia
universitaria. En este marco de referencia se encuadra La bicicleta de
Krebs, una iniciativa alentada por los cinco alumnos que firman esta
comunicacin y que consigui la activa participacin de ms de la mitad de
los alumnos inscritos en el curso 2012-13 (que fue el que cont con mayor
nmero de matriculados en la asignatura). En palabras de los promotores
de esta iniciativa: Quin no ha pensado alguna vez: Qu aburrimiento
de clase!? No habr algn modo de aprender esto de una forma ms
amena y divertida? Aunque a veces nos hagan creer que no, s que la hay.
La bicicleta de Krebs es un buen ejemplo de que hay respuestas positivas
a esta ltima pregunta. Una respuesta, adems, que en este caso ha sido
encontrada, creada, desarrollada y llevada a cabo por los propios alumnos.
P00-3 (R00-5)
Hacia un enfoque evolutivo en la enseanza

de la Bioqumica
Juan Antonio Aguilera Mochn
Departamento de Bioqumica y Biologa Molecular I. Universidad de
Granada, Granada, ES
La clebre sentencia de Dobzhansky, nada tiene sentido en Biologa
si no es a la luz de la Evolucin, apenas se aplica, sorprendente y
lamentablemente, en la enseanza de la Bioqumica.
Para empezar, hay que hacer ver a los alumnos que lo que existe no es
una Bioqumica, sino una enorme diversidad de bioqumicas (que se
desarrollan, es cierto, en torno a una unidad muy notable).
Nuestros alumnos estudian el ciclo del citrato, el cdigo gentico, los
mecanismos de control sin plantearse nunca por qu son como son, esto
es cmo y por qu surgieron en la evolucin? Por ejemplo, por qu el
cdigo gentico tiene precisamente esos aminocidos, y no otros, y no ms,
y slo de la serie L...?
Sin embargo, hay razones para sospechar que el ciclo del citrato pudo
empezar funcionando al revs, que el cdigo gentico coevolucion con
las rutas de biosntesis de aminocidos y se congel, que no es indiferente
desde el punto de vista evolutivo que la induccin o la represin gnicas se
realicen a travs de mecanismos de control positivo o negativo, etc.
Un alumno de Bioqumica que no tenga idea de cmo pudo y puede la
evolucin inventar nuevas actividades enzimticas y nuevas rutas tiene
un entendimiento de corto alcance de la lgica molecular de lo viviente.
Para que esa visin sea suficientemente comprensiva, es necesaria, adems,
una reflexin sobre el problema del origen de la vida.
La visin evolutiva (hasta donde nos es posible en la actualidad) de la
Bioqumica mejora de manera decisiva la comprensin de la vida.
Este enfoque evolutivo se ha aplicado con xito durante aos en la
asignatura Bioqumica evolutiva, impartida por el Dpto. de Bioqumica
y Biologa Molecular I de la Universidad de Granada.
P00m-4
Servicio de Texto Orientado iBiotecSTO:

multiplataforma interactiva para la docencia
de Biotecnologa
Maximino Manzanera Ruiz1, Alberto Vargas2, Rafael Salto2, Antonio
Suarez2, Margarita Aguilera1, Juan Ignacio Vilchez1, Cristina GarcaFontana1
1
Dept. Microbiologa. Universidad de Granada, Granada, ES, 2Depto
Bioqumica y Biologa Molecular II. Universidad de Granada,
Granada, ES
Proponemos el uso de un libro digital interactivo en formato de
multiplataforma para la materia de Biotecnologa Farmacutica que
sea accesible a travs de tabletas, ordenadores y mviles inteligentes
(smartphones). A travs de esta propuesta estamos generando un material
bibliogrfico de actualidad, mediante un sistema que permite una puesta
Granada 2014
al da constante de contenidos, gracias a la incorporacin rpida y
especializada de contenidos de forma constante por parte del profesor.
Igualmente consideramos que es especialmente til para la docencia de esta
materia el suministro de informacin en formato vdeo. Este formato permite
una mejor comprensin y asimilacin de conceptos por parte del alumnado
que en caso contrario pueden ser confusos y llevar a interpretaciones errneas.
Estas interpretaciones errneas son difciles de detectar hasta la evaluacin
de los estudiantes, lo que implica un cierto grado de insatisfaccin por parte
del alumnado, as como por parte del profesorado en cuanto a la adquisicin
de conocimientos y competencias del estudiante de esta materia.
A travs de esta solicitud pretendemos lograr el suministro de herramientas
didcticas con los siguientes objetivos:
(i) Generar un material bibliogrfico en espaol actualizable en sus
contenidos.
(ii) Suministrar contenidos digitales tales como vdeos y esquemas animados.
(iii) Desarrollar un sistema de autoevaluacin continuo por parte del
alumnado con actividades para el desarrollo de las competencias propuestas
en la Gua Docente de la materia.
P00-5
Diseo de clases prcticas de Bioqumica

Metablica: estudio metablico comparativo
de las situaciones de ayuno y alimentacin
Mara del Mar Sola Zapata, Jos Luis Periago Mnguez, Paloma
Hortelano de la Lastra
Departamento de Bioqumica y Biologa Molecular II, Facultad
de Farmacia, Universidad de Granada, Granada, ES
En los Grados de Farmacia y NHyD de la UGR, los contenidos docentes
de metabolismo se imparten en la asignatura Bioqumica Metablica con
4,5 ECTS tericos y 1,5 ECTS prcticos. Hemos diseado unas clases
prcticas que pretendemos sean formativas y amenas al mismo tiempo.
El planteamiento general consiste en desarrollar un estudio comparativo
de algunos parmetros bioqumicos en dos situaciones metablicas
claramente diferenciadas, ayuno y alimentacin. Se han elegido algunos
compuestos y actividades enzimticas que principalmente se afectan por
el ayuno y que ilustran claramente la regulacin adaptativa de las rutas
metablicas. Durante cuatro clases presenciales de laboratorio, explicadas
por un profesor, se realizan: a) Las determinaciones de las concentraciones
(supuestamente sricas) de glucosa, 3-hidroxi-butirato y glicerol mediante
espectrofotometra; b) La determinacin cualitativa de la concentracin
srica de cidos grasos libres mediante cromatografa en capa fina y c)
La determinacin espectrofotomtrica de la actividad enzimtica de la
glucosa 6-fosfatasa heptica. Una quinta clase se dedica a la integracin
metablica de los resultados obtenidos. El material utilizado como muestra
biolgica es diferente en cada caso. Tuvimos en consideracin que la
metodologa estuviera adaptada a los an escasos conocimientos tcnicos
que los alumnos poseen en ese momento, tratando de impedir as que la
complicacin metodolgica desviara la atencin del alumno de los aspectos
metablicos de las prcticas que, creemos, constituyen el principal objetivo
de esta asignatura. Estas clases se han impartido durante los ltimos tres
cursos acadmicos con un considerable grado de satisfaccin de los
alumnos y de los profesores del departamento responsables de ellas.
P00-6 (R00-4)
Utilizacin de cdigos QR en el aprendizaje del

sistema endocrino mediante trabajo en grupo y
presentacin en vdeo
Gracia Morales Kucharski, Ana Mara Snchez Moral
Universidad Europea de Madrid, Villaviciosa de Odn, ES
Objetivos: Como objetivo principal se busca que los alumnos sean capaces
de adquirir conocimiento sobre el tema propuesto mediante la bsqueda
Psters
autnoma de informacin. Pero adems se quieren conseguir otros dos
objetivos relacionados ntimamente con el anterior:
1. Desarrollar la capacidad de comunicacin del conocimiento adquirido
a otros alumnos.
2. Utilizacin de nuevas tecnologas (cdigos QR, vdeos, uso de internet)
para comunicar lo aprendido.
Se pretende con estos objetivos conseguir que el alumno elabore un
pensamiento estructurado y sinttico ya que debe ser capaz de explicar
a sus compaeros el tema propuesto usando un lenguaje cientfico pero
suficientemente claro.
Descripcin: Los alumnos se dividen para formar grupos de trabajo. A
cada grupo de trabajo se le asignar una glndula del sistema endocrino.
Se les facilitan guiones o apuntes que pueden utilizar para guiarse durante
la elaboracin del tema asignado. Asimismo se les facilita bibliografa que
deben consultar para realizar el trabajo.
Una vez elaborado el tema deben grabar un vdeo en el que presentan
el tema elaborado. La grabacin se realiza con un telfono mvil y las
explicaciones pueden realizarse de manera oral ayudndose con diapositivas
del tipo power point o con la aplicacin o medios que consideren oportunos
(dibujos, etc.)
El vdeo se cuelga en una web (youtube, moodle, etc.) a la que el resto
de grupos puede acceder con una clave, de manera que no est abierta de
modo general.
Cada grupo de trabajo se identifica con un cdigo QR. La lectura de este
cdigo muestra cul es el tema que desarrolla este grupo. El tema, a su vez,
se identifica con otro cdigo QR. La lectura de este segundo cdigo lleva
a una direccin URL (normalmente de youtube) en la que se encuentra el
vdeo con la presentacin del tema.
La exposicin de los cdigos QR (mediante una impresin de los mismos)
se realiza en el aula habitual el da que se determine.
La evaluacin de los conocimientos adquiridos por los alumnos se realiza
mediante una prueba objetiva de tipo test. Adems, se pretende que los
alumnos dentro de un grupo evalen el trabajo del resto de componentes
del grupo siguiendo una rbrica.
P00-7
Bioqumica Estructural y Metablica: Un libro

adaptado para la docencia de la Bioqumica en la
Facultad de Farmacia de la Universidad
de Granada
Alberto M. Vargas Morales, Fermn Snchez de Medina Contreras
Departamento de Bioqumica y Biologa Molecular II, Facultad de
Farmacia, Universidad de Granada, Granada, ES
Hemos escrito y editado, en octubre de 2013, un libro de texto de
Bioqumica (Bioqumica Estructural y Metablica, Ed. Tcnica AVICAM,
Granada, 2013) con el principal objetivo de que sea utilizado como
manual de apoyo al estudio por los alumnos de las asignaturas bsicas con
contenidos bioqumicos, Bioqumica Estructural y Bioqumica Metablica,
de los Grados de Farmacia, Nutricin Humana y Diettica (NHyD) y
Ciencia y Tecnologa de los Alimentos (CyTA) de la Universidad de
Granada. El libro est dividido en dos partes diferenciadas para ajustarlo a
la distribucin docente que la Bioqumica tiene en los Planes de Estudios
de estos Grados. Nos propusimos la elaboracin de este libro como
iniciativa de acercamiento docente al alumno que comienza los estudios
universitarios, dado que la mayora de los magnficos textos de Bioqumica
con los que contamos no son consultados por los alumnos porque exceden
sus requerimientos en contenidos y grado de dificultad. Por esta razn,
nuestro planteamiento se basaba en cubrir principalmente los siguientes
objetivos:
- Abarcar de forma resumida todos los objetivos docentes de Bioqumica de
las asignaturas bsicas en los Grados de Farmacia, NHyD y CyTA.
- Concretar la informacin imprescindible.
- Utilizar un lenguaje asequible adaptado al alumno de los primeros cursos
universitarios.
- Amenizar el texto con gran nmero de esquemas e ilustraciones.
25
Psters
- Destacar ejemplos y aplicaciones en Farmacia y Nutricin, para lo que se
han incorporado Recuadros adicionales al texto.
La utilizacin de este libro durante varios cursos acadmicos nos permitir
valorar el grado de consecucin de nuestros propsitos docentes.
P00-8 (R00-3)
Proyecto Netbooks Uno a Uno: entrenamiento

de docentes de escuelas secundarias de
Argentina para el desarrollo y uso efectivo
de recursos informticos en el aula
Ayelen Toro1, Virginia Revora2, Pablo Calzadilla3, Luca Chemes4,
Silvina Ponce Dawson5, Cristina Caputo5, Roberto Gabriel Pozner1
1
Departamento de Qumica Biolgica, Facultad de Ciencias Exactas
y Naturales, Universidad de Buenos Aires, Buenos Aires, AR, 2IIBINTECh (CONICET), San Martn, AR, 3Unidad de Biotecnologa
1, IIB-INTECh (CONICET), Chascoms, AR, 4Fundacin Instituto
Leloir, Buenos Aires, AR, 5Departamento de Fsica, Facultad de
Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos
Aires, AR
El Programa Conectar-Igualdad fue creado en Argentina en 2010 para
reducir las brechas digitales, educativas y sociales en el pas y consiste en
la adjudicacin de netbooks a alumnos y docentes de escuelas secundarias
de gestin estatal. El Programa pretende el uso de las netbooks en la
escuela y los hogares y se propone lograr una sociedad alfabetizada en las
nuevas TIC.
Para impulsar un uso efectivo de estas nuevas herramientas digitales se
dise un curso de capacitacin destinado a docentes de escuela media de
asignaturas relacionadas con ciencias.
El curso consisti en 4 encuentros de 4 horas de duracin cada uno, se
realizaron tutoriales que incluan diferentes secuencias didcticas diseadas
para usar programas especficos. En el caso de los docentes de Qumica
Biolgica, se trabaj con los programas Excel, Image J, Chemsketch y
Avogadro.
El primer encuentro se enfoc en el uso general de la netbook y se
detect una gran disparidad de saberes en el uso de las PC, dificultando la
capacidad de atencin de los docentes que tenan habilidades previas. Sin
embargo, a lo largo del curso la heterogeneidad del grupo se redujo debido
a la complejidad creciente de las aplicaciones.
A modo de cierre, los docentes realizaron un trabajo final que consisti
en una presentacin en modalidad pster de alguna actividad, que cada
docente deba desarrollar con sus estudiantes utilizando la netbook.
Los trabajos presentados muestran que los objetivos del curso se cumplieron
satisfactoriamente, ya que pudieron plasmar sus aprendizajes en el aula y
cules consideraban que eran las ventajas y desventajas de su uso. En las
conclusiones de dichos trabajos la mayora coincidi en el aspecto positivo
de la incorporacin de las netbooks en el aula ya que genera entusiasmo en
los estudiantes.
P00-9
Trabajando competencias docentes mediante el

diseo, realizacin y presentacin de un taller de
criminologa para secundaria y bachillerato
Adamo Valle, Pilar Roca, Jordi Oliver
Grupo Multidisciplinar de Oncologa Traslacional. IUNICS. Universitat
de les Illes Balears. Ciber Fisiopatologa Obesidad y Nutricin
(CB06/03) Instituto Salud Carlos III, Palma de Mallorca, ES
Entre las competencias genricas de los planes de estudio del grado
de Bioqumica estn la de fomentar el trabajo en equipo, la capacidad
organizativa y la expositiva. Dentro del marco del proyecto FECYT
Ciencia para todos nos planteamos que los alumnos de la asignatura
Mtodos y Tcnicas en Biologa Molecular de tercer curso, diseasen
un Taller de criminologa y lo presentasen a alumnos de secundaria y
26

bachiller. El taller deba consistir en un conjunto de experimentos para
determinar la identidad de un asesino a partir de las pruebas halladas
en una escena del crimen. Las actividades deban de ser sencillas, de
bajo coste y que fuesen comprensibles para el pblico en general. Los
alumnos en grupos de 6 bajo supervisin del profesor prepararon el
material y las actividades, que se filmaron e implementaron en un blog
para poder ser consultadas y utilizadas por profesores de secundaria.
Los alumnos plantearon el taller de criminologa como un escenario
distribuido en tres espacios: la escena del crimen donde se recogan
las pruebas (pelo, huellas dactilares, deteccin de sangre con luminol);
el laboratorio de criminologa, donde se analizaban las pruebas (grupo
sanguneo, DNA, pelo, etc.) y la pizarra de los sospechosos donde
por descarte deban de identificar el asesino. Mediante encuesta se
evalu la opinin de los alumnos que visitaron el taller, as como la
de los alumnos de grado que participaron en la actividad. El taller
de criminologa fue una de las actividades mejor valoradas por los
visitantes, y nuestros alumnos reconocieron haber trabajado aspectos
relacionados con competencias del grado como el trabajo en equipo, la
capacidad organizativa y expositiva.
Agradecimientos: alumnos del grado de Bioqumica (BQ3) de la UIB.
FECYT, ICE-UIB.
P00-10
Anlisis del modelo de reparto de tareas

en un trabajo en grupo reglado (TEG-R) para
alumnos de Gentica de primer curso de Grado
de Biologa
Oscar de Luis
rea de Bioqumica y Biologa Molecular, Dpto. de Ciencias Bsicas
de la Salud, Universidad Rey Juan Carlos, Alcorcn (Madrid), ES
En 2010 se present una adaptacin del Aprendizaje Basado en
Problemas para la evaluacin continua de la asignatura de Gentica del
Grado de Biologa de la U. Rey Juan Carlos. La actividad consiste en la
elaboracin, por parte de un grupo de trabajo, de una memoria escrita
consistente en un resumen analtico y un anlisis crtico de un artculo
cientfico publicado en ingls, en revista con ndice JCR. La defensa en
pblico de la memoria por parte de todos los componentes del grupo es
obligatoria. Esta herramienta ha sido perfeccionada para profundizar en la
evaluacin, calificacin y adquisicin por el alumnado de las competencias
generales de la asignatura. Con los datos del perodo 2009-2013 se valor
en un reciente estudio la adecuacin de esta actividad. Sin embargo, no
se contempl determinar si el modelo de organizacin interna libremente
elegido por cada grupo de trabajo puede ser o no determinante a la hora
de alcanzar las competencias propuestas y una calificacin adecuada. Con
los datos del perodo 2011-2014 se han detectado tres tipos principales de
organizacin interna: una dinmica grupal cohesionada donde todos los
componentes del equipo trabajan globalmente, una dinmica de reparto
de tareas por partes temticas reconocibles de corte ms individualista, y
una dinmica de reparto de tareas por fragmentos no temticos de la carga
de trabajo. Cabra esperar que a mayor cohesin interna en el grupo de
trabajo los resultados fueran mejores por que se alcanzan mejor todas las
competencias por parte de cada miembro del equipo, pero si establecemos
como criterio de eficiencia obtener una calificacin superior a la media
del curso se observa que el modelo de reparto temtico de tareas permite
la mxima eficiencia en la calificacin. Estos datos preliminares permiten
proponer una relacin entre el esfuerzo y el rendimiento en cada tipo de
organizacin. Se propone tambin modificar el protocolo de seguimiento
del profesor y el cuestionario de autovaloracin obligatorio de los alumnos
para obtener datos ms precisos con los que contrastar la hiptesis esfuerzorendimiento.
Granada 2014
P00-11
Oportunidades de formacin en biomedicina y

biotecnologa a nivel europeo: el Curso Towards
a Scientific Career: an Introductory Course for
Research in Biomedicine and Biotechnology BIOMED-TECH
Inmacualda Llamas Company1, Mara Dolores Gonzlez Girn2,
Fernando Hernndez-Mateo3, Rafael Salto Gonzlez2
1
Departamento de Microbiologa, Facultad de Farmacia, Universidad
de Granada, Granada, ES, 2Departamento de Bioqumica y Biologa
Molecular II, Facultad de Farmacia, Universidad de Granada,
Granada, ES, 3Departamento de Qumica Orgnica, Facultad de
Ciencias, Universidad de Granada, Granada, ES
Los programas intensivos Erasmus han ofrecido hasta ahora la oportunidad
de desarrollar actividades de formacin en campos concretos. Nosotros
hemos aprovechado este marco para realizar dos ediciones de un curso que
tiene el objetivo principal de dirigir alumnos hacia una carrera profesional
en el mbito de la investigacin en Biomedicina y Biotecnologa. Para ello,
en las diferentes ediciones del programa hemos seleccionado alumnos de
las Universidades de Granada (Espaa), Universidad de Nottingham (Reino
Unido), Universidad de Bayreuth (Alemania) y Universidad de Nova Lisboa
(Portugal), que han convivido durante 2 semanas y que han recibido un
programa intensivo de orientacin y formacin en el rea de Biomedicina
y Biotecnologa. El programa se ha estructurado mediante una serie de
mesas redondas con charlas impartidas por profesores procedentes de las 4
universidades implicadas y figuras relevantes en el rea de conocimiento.
Adems se han realizado visitas a empresas del rea y as, como empresas
agregadas al proyecto, han participado Abbott Laboratories S.A., BioIliberis R&D, Neuron Bio y la Fundacin MEDINA. La formacin de
los alumnos se ha completado con la realizacin de talleres prcticos
sobre quorum sensing, sntesis de neoglicoprotenas, bioinformtica y
mutagnesis sitio especfica de protenas. Los profesores del curso han
tutorado a los alumnos y los han asesorado en cmo orientar su actividad
profesional hacia la investigacin en Biomedicina y/o Biotecnologa,
conocer aspectos prcticos de la carrera cientfica acadmica y en la
industria, analizar los perfiles cientficos profesionales y establecer una red
inicial de contactos en instituciones pblicas as como en la industria. El
hecho de que se hayan seleccionado alumnos de ltimo curso de grado, de
master y alumnos de doctorado ha posibilitado establecer un proceso de
mentorizacin entre ellos. Otros resultados alcanzados, en este caso para
el profesorado ha sido el facilitar la cooperacin entre universidades y con
la industria y establecer procedimientos docentes para la motivacin del
alumnado hacia la investigacin como salida profesional.
Actividad financiada por el Organismo Autnomo de Programas
Educativos Europeos (OAPEE) 2012-1-ES1-ERA10-0083 y 2013-1-ES1ERA10-74542, El Vicerrectorado de Relaciones Internacionales y el
Campus de Excelencia CEI-BIOTIC de la Universidad de Granada.
P00-12
La Bio(Qumica) en la vida cotidiana: mtodo

para estimular el inters de los estudiantes
Rafael Salto Gonzlez1, Luisa Carlota Lpez Cara2, Mara del Mar
Sola Zapata1, Alberto Manuel Vargas Morales1, Mara Dolores Girn
Gonzlez1
1
Farmacia, Universidad de Granada, Granada, ES, 2Departamento
de Qumica Farmacutica y Orgnica, Facultad de Farmacia,
Universidad de Granada, Granada, ES
Divulgar de forma atractiva la ciencia y hacerla asequible a todos los
pblicos no siempre es fcil, especialmente si el rea de conocimiento tiene
una cierta complejidad, lo que hace que sea difcil de comprender para
el estudiante. Tal es el caso de asignaturas como Bioqumica Estructural,
Psters
Bioqumica Metablica y Qumica Orgnica I y II. Todas estn situadas en
el primer y segundo curso de los Grados que se imparten en la Facultad de
Farmacia de la Universidad de Granada. Pensamos que las competencias
adquiridas por los alumnos en su etapa preuniversitaria no siempre son
las ms apropiadas o no son suficientes para obtener el rendimiento
adecuado en estas asignaturas. Nuestra misin como profesores es que el
alumno adquiera las competencias especficas de nuestras asignaturas de la
manera ms asequible y dinmica utilizando todos los recursos que estn a
nuestra disposicin. Las tcnicas y herramientas empleadas para despertar
el inters y motivar a los estudiantes son numerosas. Cuando durante la
docencia de una asignatura, le indicamos al estudiante direcciones de
internet donde pueden consultar modelos tridimensionales o vdeos de
tcnicas de biologa molecular entre otros, comprobamos que despiertan
ms inters que un libro de consulta o divulgativo como La doble hlice de
James D. Watson donde se narran de forma dinmica los experimentos que
llevaron a la dilucidacin de la estructura del DNA. Una forma amena y
divertida para introducir el conocimiento en las aulas es referirnos al cine,
a la televisin o a los peridicos para ilustrar con ejemplos ciertos aspectos
de la asignatura. Las pelculas y series de gran audiencia ofrecen contextos
donde situados los conceptos qumicos y bioqumicos nos permiten mostrar
a los estudiantes la realidad o la ficcin de ejemplos ms o menos cotidianos
que estn acostumbrados a ver pero que no han terminado de asimilar. Los
profesores que presentan esta propuesta saben por experiencia, que ciertos
procesos qumicos y bioqumicos siempre son recordados por los alumnos
cuando recurrimos a casos de la vida cotidiana o del cine y televisin.
Durante el primer ao de implantacin se ha aumentado en un 20% el
rendimiento acadmico de los alumnos.
Proyecto 2013-80. Vicerrectorado de Ordenacin Acadmica y
Profesorado. Secretariado de Innovacin Docente. Universidad de
Granada.
P01. Apoptosis
P01-1
Investigating the membrane topology of BAX/BAK

and the role of mitochondrial membrane lipids in
BAX/BAK activation
Hctor Flores Romero, Juan Garca-Valero, Ane Landajuela, Olatz
Landeta, Itsasne Bustillo, Miguel Garca-Porras, Oihana Terrrones,
Gorka Basaez
Biophysics Unit (CSIC-UPV/EHU), Leioa, ES
Mitochondrial outer membrane (MOM) permeabilization is the point of
no return in many forms of apoptotic cell death. The lethal effect of MOM
permeabilization is twofold, as it allows activation of pro-apoptotic caspase
enzymes and also damages normal mitochondrial function. The BCL-2
family members BAX/BAK are crucial effectors of MOM permeabilization
that adopt inactive conformations in healthy cells. It is well established
that functional BAX/BAK activation is a complicated process, involving
profound conformational changes in these proteins, and culminating with
assembly of a protein-permeable pore in the MOM. However, our current
structural knowledge of BAX/BAK primarily originates from studies with
the protein in solution, although the active locus of these proteins is at
the MOM. In addition, increasing evidence indicates that selected MOMlocalized lipids modulate the BAX/BAK-driven MOM permeabilization
pathway during apoptosis.Given the inherent complexity of the cellular
apoptotic network, we use in vitro reconstituted systems bearing
physiological relevance to try elucidating the membrane topology of BAX/
BAK, and their functional modulation by apoptosis-related lipid effectors.
27
Psters
P01-2 (R01-3)
Cathepsin D promotes the cleavage of PARP-1

mediated by inactivation of BRCA1 in MCF-7
cells
Juan M. Esteve1, Ghita Ghislat2, Erwin Knecht2
1
Facultad de Ciencias de la Salud de la Universidad CEU Cardenal
Herrera, Castelln de la Plana, ES, 2Centro de Investigacin
Prncipe Felipe, Valencia, ES
The breast cancer type 1 susceptibility protein (BRCA1) is a tumor
suppressor involved in the maintenance of genetic stability in response to
DNA damage, due to the control of cell cycle progression and activation
of DNA repair pathways. Cells with BRCA1 mutations manifest DNA
synthetic lethality and apoptosis after pharmacological inhibition of poly
(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that contributes
to the resolution of DNA breaks. Hence, PARP-1 inhibitors represent a
potential novel strategy for the treatment of BRCA1-mutated malignancies.
Our study demonstrates that the silencing of BRCA1 activates the cleavage
of PARP-1, and slows the cell cycle since it increases the percentage of
cells in G1 phase and reduces that in the S + G2/M phase of the cell cycle.
In relation to the mechanism of PARP-1 fragmentation, it has been shown
that caspases-3 and -7 cleave and, consequently, inactivate PARP-1 and
DNA repair, thereby contributing to apoptotic cell death. However, in
MCF-7 cells silenced for BRCA1, we show that the PARP-1 cleavage was
not accomplished by caspases-3 and -7. What type of proteases could then
be responsible for cleavage of PARP-1? The fact that lysosomal proteases,
cathepsins, can be released from lysosome and function in the cytosol
to regulate apoptosis, and that BRCA1 decreases the 52 kDa form of
cathepsin D (CD), suggest a potential role of CD on PARP-1 cleavage. In
this sense, we found that levels of three forms of CD (procathepsin D, the
52 kDa form which leaves the Golgi apparatus and is targeted to lysosomes
(and also to secretion in cancer cells); a lysosomal enzymatically-active
intermediate of 48 kDa; and a lysosomal active mature form of 34 kDa)
were increased in BRCA1-silenced MCF-7 cells, but mainly precursor and
intermediate forms. Furthermore, we also observed that BRCA1 silencing
enhances CD mRNA levels, and our preliminary results suggest no changes
in relation to the rate of the intracellular degradation of CD protein, leading
to the conclusion that BRCA1 inactivation increases CD levels mainly
by the control of its synthesis and not of its degradation. Finally, we
demonstrate that the cleavage of PARP-1 and cell cycle slowing mediated
by silencing of BRCA1 are dependent on CD in MCF-7 cells, and this
PARP-1 fragmentation is accomplished by the catalytic activity of CD. In
summary then, tumor suppressor protein BRCA1 inhibits PARP-1 cleavage
and negatively regulates CD synthesis in MCF-7 cells, and this PARP-1
cleavage is positively regulated by CD. Our results provide new data for
understanding the molecular and cellular mechanisms by which BRCA1
is involved in DNA repair, as well as to explain how PARP-1 inhibitors
trigger apoptosis in BRCA1-mutated cells.
P01r-3
Does sulforaphane protect against MPP+/PQ

oxidative stress effect in mouse embryonic
fibroblasts through the activation of Nrf2/Keap1
pathway?
Elisa Pizarro Estrella1, J.M. Bravo San Pedro2, R. Gmez Snchez3,
S.M.S. Yakhine-Diop1, M. Rodriguez-Arribas1, R.A. Gonzlez-Polo1,
J.M. Fuentes1
1
Centro de Investigacin Biomdica en Red de Enfermedades
Neurodegenerativas (CIBERNED). Departamento de Bioqumica
y Biologa Molecular y Gentica. Facultad de Enfermera y
Terapia Ocupacional. Universidad de Extremadura, Cceres, ES,
2
Neurodegenerativas (CIBERNED). Departamento de Bioqumica y
BIologa Molecular y Gentica. Facultad de Enfermera y Terapia
28

Ocupacional. Universidad de Extremadura. Cceres, ES. IMSERM,
U848, Institut Gustave Roussy, Universit Paris Sud, Villejuif,
Paris, FR, 3Department of Cell Biology, Center for Molecular
Medicine, University Medical Center Utrecht, Utrecht, NL
Several studies have demonstrated the ability of Nrf2 transcription factor
to control the cytoprotective adaptive response in the brain using PD
models. In this sense, brain samples from animals with neurodegeneration
by oxidative stress have shown that increased nuclear Nrf2 may have a
protective effect on dopaminergic neurons in the SNpc, and on the other
hand, in MEFs it is has been observed the induction of antioxidant defenses
from this pathway after exposure to inducing agents. The chemical
characteristics of the agents which have the ability to interact with the Nrf2/
Keap1 pathway show differences from each other in the chemical structure
or how to interact (directly or indirectly) with the pathway. Sulforaphane
(SFN) is a naturally isothiocyanate presents in cruciferous plants. This
substance shows in its structure a highly electrophilic central carbon (-N =
C = S) which can easily react with the sulfhydryl groups of Keap1 to form
dithiocarbamates, so in this fact lies its ability to promote the translocation
of the transcription factor Nrf2 to the nucleus. However, it has been found
that this substance can play a dual role, as a chemotherapeutic agent or as a
promoter of antioxidant defenses.
Based on this powerful cellular defense of Nrf2 factor against oxidative
stress and based on the chemical features offered by SFN, the aim of
this work was to determine whether the activation of this axis exerted a
protective effect or not against MEFs induced PQ and MPP+ cytotoxicity
and on the other way, if that substance could modulate another celular
processes such as autophagy, apoptosis or acetylation, thinking that this
model could be used to elucidate the Nrf2 potential role as a molecular
therapeutic target value.
This work was supported by Gobierno de Extremadura (GR10054);
Instituto de Salud Carlos III (PI11/00040, PI12/02280 and CB06/05/0041).
M.R-A. was supported by a predoctoral fellowship from Universidad de
Extremadura. R.G-S. was supported by a FPU predoctoral fellowship
(Ministerio de Educacin, Spain), E.P-E. was supported by a predoctoral
fellowship (CIBERNED, Instituto de Salud Carlos III, Spain), RA.G-P.
was supported by a Miguel Servet research contract (Instituto de Salud
Carlos III, Spain).
P01-4
IfDotMeter: a new tool for immunofluorescence

analysis
Mario Rodrguez Arribas1, Elisa Pizarro Estrella1, Rubn Gmez
Snchez2, Sokhna Maryama Seidina Yakhine Diop1, Alejandro
Cristo3, Antonio Grajera Hidalgo4, Jos Manuel Bravo San Pedro5,
Jos Manuel Fuentes Rodrguez1, Rosa Ana Gonzlez Polo1
1
2
Neurodegenerativas (CIBERNED). Department of Cell Biology,
Center for Molecular Medicine, University Medical Center, Utrecht,
NL, 3IEEE Aerospace and Electronic Systems. Universidad
de Extremadura, Cceres, ES, 4Departamento de Bioqumica
5
Centro de Investigacin Biomdica en Red sobre Enfermedades
Neurodegenerativas (CIBERNED). INSERM, U848, Institut Gustave
Roussy, Universit Paris Sud, Villejuif, Paris, FR
Most of laboratories interested in autophagy use different imaging
software for managing and analyzing heterogeneous parameters from
immunofluorescence experiments (LC3-puncta quantification and
determination of number and size of lysosomes, among others). In this
sense, one helpful solution would be some software working in users
Granada 2014
laptop or workstation able to access to all image settings and provide a
quick and easy-to-use analysis of these data.
For this reason, we have designed an application called IFDOTMETER
which runs on the major operating systems because it has been
programmed using Java (Sun Microsystems) as the computer platform.
Briefly, IFDOTMETER software has been created to quantify a variety
of biological hallmarks, including mitochondrial morphology, nuclear
condensation, cell area, quantification of number or size of different
puncta staining and so on. Once specific parameters have been defined,
IFDOTMETER allows you to analyze a large number of images in an
objective and automatic way, without the supervision of researcher.
Results obtained are stored in a spreadsheet. Thus, researchers can perform
a comprehensive and precise analysis of a high number of images in an
automated manner, making easier than before this routine chore. Here
we will show results obtained with IFDOTMETER applying different
treatments and staining in different cell lines to demonstrate that this new
tool would be useful to the scientific community because of both being
more objective and saving time.
This work was supported by Gobierno de Extremadura (GR10054);
Instituto de Salud Carlos III (PI11/00040, PI12/02280 and CB06/05/0041).
M.R-A. was supported by a predoctoral fellowship from Universidad
de Extremadura. E.P-E. was supported by a predoctoral fellowship
(CIBERNED, Instituto de Salud Carlos III, Spain), RA.G-P. was supported
by a Miguel Servet research contract (Instituto de Salud Carlos III,
Spain). Authors thank FUNDESALUD for providing helpful assistance.
P01-5
Nuevas funciones gnicas en apoptosis

y autofagia celular identificadas mediante
genmica funcional
Felipe Xos Pimentel Muios
Instituto de Biologa Molecular y Celular del Cncer (IBMCC).
CSIC-Universidad de Salamanca, Salamanca, ES
Previamente en el laboratorio hemos diseado un sistema de alto
rendimiento para identificar funcionalmente molculas capaces de inducir
diferentes modalidades de muerte celular al ser sobreexpresadas. Este
abordaje se basa en el uso de sistemas robticos que facilitan el manejo
sistemtico de genotecas de cDNA clonadas en vectores de expresin, y en
la transfeccin de los clones en clulas susceptibles y en combinacin con
GFP. Tras el escrutinio de 135.000 clones de una genoteca humana, hemos
identificado un total de 90 elementos genticos cuya expresin induce
muerte celular. De forma interesante, aunque la mayora son capaces de
provocar muerte celular apopttica, otros inducen formas de muerte celular
morfolgicamente atpica, diferentes de la apoptosis.
Dentro de los clones apoptticos hemos identificado el transportador
mitocondrial de fosfato, y trabajo adicional demostr que esta molcula
forma parte del poro de transicin de permeabilidad que media la liberacin
de citocromo c y la activacin de las caspasas en algunos paradigmas de
apoptosis. Por otro lado, dentro de los clones atpicos hemos descubierto
una nueva protena transmembrana denominada TMEM59. Estudios
adicionales indicaron que esta molcula induce un fenmeno atpico de
autofagia celular implicado en defensa contra agentes infecciosos, y no
parece tener un papel fisiolgico en muerte celular. Por tanto, a travs de
un sistema no sesgado de identificacin funcional hemos podido adscribir
funciones celulares nuevas, bien a genes conocidos que previamente no se
haban implicado en una determinada funcin (transportador de fosfato),
o a genes nuevos cuya funcin fisiolgica se desconoca por completo
(TMEM59), demostrando el potencial de este tipo de aproximaciones para
anotar funcionalmente el genoma.
Psters
P01-6
Apoptotic efficacy of etomoxir in HL60 human

acute myeloid leukemia cells. Cooperation with
arsenic trioxide and glycolytic inhibitors, and
regulation by oxidative stress and protein kinase
activities
Mara Cristina Esta Omaa1, Eva Calvio Vanegas1, Susana Calvo
Alonso1, Mara del Carmen Boyano-Adnez2, Elena de Blas1,
Eduardo Rial1, Patricio Aller1
1
Centro de Investigaciones Biolgicas, CSIC, Madrid, ES,
2
Universidad de Alcal de Henares, Alcal de Henares, ES
Fatty acid synthesis and metabolism are frequently exacerbated in
leukemia cells, and may therefore represent a target for therapeutic
intervention. Therefore, we analyzed the pro-apoptotic and chemosensitizing action of the fatty acid oxidation inhibitor etomoxir (Eto) in
HL60 human acute myeloid leukemia cells. Etomoxir caused negligible
lethality at concentrations up to 100 M, but greatly potentiated apoptosis
induction by the anti-leukemic agent arsenic trioxide (ATO) and, with
lower efficacy, by other anti-tumour drugs (etoposide, cisplatin). Etomoxir
dose-dependently inhibited mitochondrial respiration while stimulating
glycolysis, but caused minimal alterations in intracellular nucleotide pools
(ATP, ADP, AMP). In addition, etomoxir caused moderate oxidative stress
and stimulated the LKB-1/AMPK pathway, all of which might in part
explain the chemo-sensitizing capacity of the drug with ATO. Etomoxir
also cooperated with the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) to
induce apoptosis, and the combined etomoxir/2-DG treatment stimulated
Akt and ERK phosphorylation/activation. Apoptosis generation by
etomoxir plus 2-deoxy-D-glucose was further increased by co-treatment
with ATO, a response apparently explained by the capacity of ATO to
prevent defensive Akt and ERK activation. In summary, co-treatment
with etomoxir may represent an interesting strategy to increase the cytoreductive efficacy of ATO and 2-deoxy-D-glucose, as well as of etomoxir
itself which, although clinically important anti-tumour agents, exhibit
limited efficacy in monotherapy.
P01-7
Mecanismos involucrados en el efecto

antiapopttico de leptina en placenta humana
a trmino
Ayelen Rayen Toro1, Antonio Prez Prez2, Victor Sanchez
Margalet2, Bernardo Maskin3, Cecilia Varone1
1
IQUIBICEN - Universidad de Buenos Aires, Ciudad Autonoma de
Buenos Aires, AR, 2Departamento de Bioqumica Mdica y Biologa
Molecular, Universidad de Sevilla, Sevilla, ES, 3Hospital Nacional
Profesor A. Posadas, Buenos Aires, AR
Durante la implantacin embrionaria se genera un dilogo materno fetal
que involucra la accin de mltiples reguladores, siendo uno de ellos la
leptina. Esta protena de 16 kDa fue descubierta en tejido adiposo con la
funcin de regular el balance energtico del organismo. Tambin se expresa
en placenta humana donde podra tener efectos sobre el crecimiento,
la supervivencia, la angiognesis y la inmunomodulacin placentaria.
Resultados previos de nuestro grupo demostraron que la leptina aumenta la
proliferacin celular y supervivencia en clulas placentarias.
El objetivo de este trabajo es evaluar el efecto de la leptina sobre la apoptosis
de clulas placentarias.Se utilizaron como modelos de trabajo explantos
placentarios a trmino, los cuales se obtuvieron del tejido trofoblstico de
placentas humanas derivadas de partos vaginales normales. Los explantos
fueron incubados en DMEM-F12 sin SFB durante 24h para inducir la
muerte celular, como control se incubaron los explantos en DMEM-F12
con SFB al 10%. Por Western Blot y qRT-PCR se analiz la expresin de
distintas proteinas involucradas en el proceso de apoptosis, y se evalu la
fragmentacin del DNA.
29
Psters
Encontramos que en presencia de leptina disminuye la proteina p53
y su isoforma fosforilada en Ser46, indicadora de activacin de la va
apopttica. Tambin evaluamos el efecto de leptina sobre mediadores pro y
antiapoptticos. Hallamos que leptina disminuye la expresin de Bax y Bid,
y genera un aumento en la expresin de Bcl-2. Asimismo, el tratamiento
con leptina disminuye la activacin de Caspasa-3. Comprobamos dicho
efecto analizando el fragmento clivado de PARP-1, el cual disminuye en
presencia de leptina. Confirmamos el efecto antiapopttico de leptina,
estudiando la fragmentacin del DNA, la cual disminuye con el tratamiento
con leptina.
Todos estos resultados refuerzan la nocin de la leptina como una importante
citoquina placentaria, con la funcin de promover la supervivencia de las
clulas trofoblsticas.
P01-8 (R01-4)
Lack of oligonucleosomal DNA degradation

during caspase-dependent cell death is a
common trait of human glioblastoma multiformederived cells due to improper activation of
DFF40/CAD
Mara Snchez Osuna1, Fina Martinez-Soler2, Snia Pascual-Guiral1,
Merc Garcia-Belinchn1, Victoria Iglesias-Guimarais1, Elisenda
Casanelles1, Gerard Plans3, Jordi Bruna3, Avelina Tortosa3, Victor J.
Yuste1
1
Cell Death, Senescence and Survival Group, Dept. Bioqumica
i Biologia Molecular and Institut de Neurocincies, Facultat de
Medicina, UAB - Centro de Investigacin Biomdica en Red sobre
Enfermedades Neurodegenerativas (CIBERNED), Barcelona, ES,
2
Department of Basic Nursing, Institut dInvestigaci Biomdica
de Bellvitge-Universitat de Barcelona, Barcelona, AF,3Unit of
Neuro-Oncology, Services of Neurology and Neurosurgery, Hospital
Universitari de Bellvitge, Barcelona, ES
Apoptosis is a tightly regulated process characterized by oligonucleosomal
DNA degradation and nuclear fragmentation. These apoptotic hallmarks
appear when the Caspase-activated DNase (DFF40/CAD) becomes
activated. Here, we show different human glioblastoma multiformederived (hGBM) cells in which caspase-dependent cell death occurs in the
absence of oligonucleosomal DNA degradation. After apoptotic stimuli,
hGBM-derived cells activate the executioner caspases, thus processing
and inhibiting ICAD, the inhibitor of DFF40/CAD. However, injured
cells do not hydrolyze their DNA into oligonucleosome-sized pieces.
Moreover, most injured hGBM cells display heterogeneous nuclear
alterations characterized by highly compacted chromatin, in the absence
of the classical apoptotic nuclear fragmentation. Both in vivo and in vitro
approaches demonstrate that higher levels of DFF40/CAD endonuclease
make hGBM cells competent to undergo oligonucleosomal DNA
degradation after apoptotic stimuli. Intriguingly, the overexpression of
DFF40/CAD does not alter the nuclear morphology observed in apoptotic
wild type hGBM cells. Altogether, these data highlight a new apoptotic
paradigm where nuclear alterations are DFF40/CAD-independent despite
proper caspase activation. This is the first time reporting a common trait
in hGBM cells which may be taken into consideration to design effective
chemotherapy to target these extremely aggressive tumors.
Work supported by Ministerio de Economa y Competitividad, European
Regional Development Fund (ERDF) Grant SAF2012-31485 and
Generalitat de Catalunya Grant 2009SGR-346.
S-O, M. holds an FPU fellowship from Ministerio de Ciencia e Innovacin
(MICINN).
30

P01r-9
Mitochondrial pathway mediators as

pharmacological targets to improve cisplatin
therapeutic utility
Laura Prieto Garca1, Sandra Sancho Martnez1, Jos Miguel Lpez
Novoa1, Francisco Lpez Hernndez2
1
Instituto de Investigacin Biomdica de Salamanca (IBSAL) y
Unidad de Fisiopatologa Renal y Cardiovascular, Instituto Reina
Sofa de Investigacin Nefrolgica, Departamento de Fisiologa
y Farmacologa, Universidad de Salamanca, Salamanca, ES,
2
Instituto de Investigacin Biomdica de Salamanca (IBSAL),
Sofa de Investigacin Nefrolgica, Departamento de Fisiologa y
Farmacologa, Universidad de Salamanca e Instituto de Estudios
de Ciencias de la Salud de Castilla y Len (IECSCYL), Unidad de
Investigacin, Hospital Universitario de Salamanca, Salamanca, ES
Cisplatin is a chemotherapeutic drug widely used against a variety of
cancers. Its clinical utility is limited by its side effects, result of cisplatin
action in normal cells. Cisplatin toxicity is determined by target tissue and
cell accumulation, subcellular handling and trafficking through diverse
subcellular structures. This drug causes cell death both by apoptosis and
necrosis linked to cisplatin concentration. For a better control of cisplatins
side effects, pathways leading to activation of both types of cell death need
to be clarified in order to identify suitable pharmacological targets and
implement appropriate treatments with a theranostic approach. The aim of
this work is to establish a hierarchy in cisplatin cytotoxic mechanisms and
to clarify the role of the mitochondrial pathway in this process.
We used cultured human lymphoma (Jurkat T cells) to investigate the
biochemical and phenotypic characteristics of the death mode induced
by increasing concentrations of cisplatin. For this purpose the cells were
treated for 4, 8, 12 and 18 hours with different concentrations of cisplatin
(0-1000 microM). In order to put in perspective the main pathways of cell
death induced by cisplatin we used different stable trasfectants of Jurkat
cells in which main mitochondrial pathway proteins were modified, such
as Jurkat Bcl-2 (which presents overexpression of an antiapoptotic protein
Bcl-2), Jurkat C9DN (which presents overexpression of a catalytically
inactive form of caspase 9) and their control carrying the empty vector
(pcDNA3.0). Our results indicate that concentrations of cisplatin inducing
a necrotic-like cell death mode are capable of activating the apoptotic
machinery. We demonstrate that caspase 9 activity and antiapoptotic Bcl2 protein play a critical role in the cytotoxicity mechanism induced by
cisplatin, accordingly with the increase in the viability and reduction in the
apoptotic proteins expression. This knowledge may allow us to develop
drugs that block cisplatin-induced apoptosis and minimize cisplatininduced necrosis.
P01-10 (R01-7)
Familial Parkinsonism with G2019S LRRK2

mutation displays a susceptibility to the MPP+
neurotoxin by an mTOR-dependent autophagy
Sokhna M.S. Yakhine Diop1, Jos Manuel Bravo-San Pedro2, Rubn
Gmez-Snchez3, Elisa Pizarro-Estrella1, Mario Rodrguez-Arribas1,
Ana Gmez-Martin4, Ana Aiastui5, Ana Gorostidi5, Adolfo Lpez de
Munain6, Jos Manuel Fuentes-Rodrguez1, Rosa Ana Gonzlez-Polo1
1
y Biologa Molecular y Gentica, Facultad de Enfermera y
Terapia Ocupacional, Universidad de Extremadura, Caceres, ES,
2
y Biologa Molecular y Gentica, Facultad de Enfermera y
Terapia Ocupacional, Universidad de Extremadura, Cceres,
ES. INSERM, U848. Institut Gustave Roussy. Universit Paris
Sud, Villejuif, Paris, FR, 3Centro de Investigacin Biomdica
Granada 2014
en Red sobre Enfermedades Neurodegenerativas (CIBERNED).
Departamento de Bioqumica y Biologa Molecular y Gentica,
F. Enfermera y T.O., Universidad de Extremadura, Cceres,
ES. Department of Cell Biology, Center for Molecular Medicine,
University Medical Center Utrecht, Utrecht, NL, 4Centro
de Investigacin Biomdica en Red sobre Enfermedades
Neurodegenerativas (CIBERNED). Departemento de Enfermera,
Centro Universitario Plasencia, Universidad de Extremadura,
Plasencia, ES, 5Centro de Investigacin Biomdica en Red sobre
Enfermedades Neurodegenerativas (CIBERNED). Cell Culture
Plataform/Neuroscience Area of Health Reseach Biodonostia
Institute, Donostia Universitary Hospital , San Sebastin, ES,
6
Neurodegenerativas (CIBERNED). Cell Culture Plataform/
Neuroscience Area of Health Reseach Biodonostia Institute,
Donostia Universitary Hospital. Department of Neurology, Hospital
Donostia. Ilundain Fundazioa, Department of Neurosciences,
University of the Basque Country UPV-EHU, San Sebastin, ES
Parkinsons disease (PD) is a neurodegenerative disorder characterized
by mitochondrial dysfunction, oxidative stress and later neuronal death.
Several genetics and environmental factors have been implicated in the
pathogenesis of PD.
MPP+ is a neurotoxin widely used to induce parkinsonian cellular model,
it is responsible for cellular damage at different levels, apoptotic death,
depletion of mitochondrial potential membrane and deregulation of cellular
recycling machinery.
In this study, we characterized the MPP+-induced toxicity in fibroblasts
from PD patients with G2019S LRRK2 and control individuals without this
mutation. Obtained results show that MPP+ induces a mTOR-dependent
autophagy in both fibroblasts cells. Further, cell death to MPP+ was higher
in mutant fibroblasts which exhibited a basal level of mTOR-independent
autophagy due to the G2019S LRRK2 mutation.
Inhibition of autophagosome-lysosome fusion by Bafilomycin A1
exacerbated the response to MPP+ exposure in both cell lines, but inhibition
of early state autophagy by 3-methyladenine lessened this difference
between both cell types.
This finding confirms the important implication of the interaction of
genetics and environmental factors in the PD etiology and may help to get
a better understanding in the pathogenic mechanism of this disease.
This work was supported by Gobierno de Extremadura (GR10054); Instituto
de Salud Carlos III (PI11/00040, PI12/02280 and CB06/05/0041). RA.G-P.
was supported by a Miguel Servet research contract (Instituto de Salud
Carlos III, Spain). A.A. was supported by ISCIII (CA00/01506; Ministerio
de Economia y Competitividad) and Instituto Biodonostia and A.G was
supported by the Ilundain Fundazioa. A.L.M. received research support by
the Association Francaise contre les Myopathies (Ref. 12642), the Spanish
Ministry of Health (FIS PS09-00660), the Ilundain Foundation, Isabel Gemio
Foundation, Diputacion Foral de Gipuzkoa (DFG09/001), and SAIOTEK
(SAIO12-PE12BN008). RAG-P received research support from Ministerio
de Ciencia e Innovacin, Spain (PI11/00040). JM. F received research
support from the Ministerio de Ciencia e Innovacin, Spain, CIBERNED
(CB06/05/004), Consejera, Economa, Comercio e Innovacin Junta de
Extremadura (GRU10054), Ministerio de Ciencia e Innovacin, Spain
(PI12/02280). The authors also thank FUNDESALUD for helpful assistance.
P01r-11 (R01-6)
2-phenylethynesulfonamide (PES) uncovers a

non-necroptotic necrotic cell death regulated by
oxidative stress and p53
Paolo Mattiolo1, Ares Barbero-Farran1, Vctor Jos Yuste2, Jacint
Boix1, Judit Ribas Fortuny1
1
Departament de Medicina Experimental, Universitat de Lleida,
Lleida, ES, 2Departamento de Bioqumica y Biologa Molecular,
UAB, Campus de Bellaterra, ES
Psters
2-phenylethynesulfonamide (PES) or pifithrin- is a promising anticancer
agent with preferential toxicity for cancer cells. The type of cell death and
the molecular cascades activated by this compound are controversial. Here,
we demonstrate PES elicits a caspase- and BAX/BAK-independent nonnecroptotic necrotic cell death, since it is not inhibited by Necrostatin-1.
This process is characterized by an early generation of reactive oxygen
species (ROS) resulting in p53 up-regulation. Accordingly, thiolic
antioxidants protect cells from PES-induced death. Furthermore, inhibiting
the natural sources of glutathione with L-buthionine-sulfoximine (BSO)
strongly synergizes with PES in triggering cytotoxicity. Genetically
modified p53-null or p53 knocked-down cells show resistance to PESdriven necrosis. The predominant localization of p53 in chromatinenriched fractions added to the up-regulation of the p53-responsive gene
p21, strongly suggest the involvement of a transcription-dependent p53
program. On the other hand, we report an augmented production of ROS
in p53-positive cells that, added to the increased p53 content in response
to PES-elicited ROS, suggests that p53 and ROS are mutually regulated
in response to PES. In sum, p53 up-regulation by ROS triggers a positive
feedback loop responsible of further increasing ROS production and
reinforcing PES-driven non-necroptotic necrosis.
P01-12 (R01-2)
Influencia de FBWX7 en la adquisicin

de resistencia a paclitaxel en cncer de mama
Jessica Gasca Bellido1, Rainiero vila Polo2, M Luz Flores de
Mera1, Servando Girldez3, Mara Tortolero3, Francisco Romero3,
Miguel A. Japn Rodrguez4, Carmen Sez Torres4
1
1Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario
Virgen del Roco/CSIC/Universidad de Sevilla, Sevilla, Rota, ES,
2
Departamento de Anatoma Patolgica, Hospital Universitario
Virgen del Roco, Sevilla, Sevilla, ES, 3Departamento de
Microbiologa, Facultad de Biologa, Universidad de Sevilla, Sevilla,
Sevilla, ES, 4Instituto de Biomedicina de Sevilla (IBiS), Hospital
Universitario Virgen del Roco/CSIC/Universidad de Sevilla, Sevilla y
Departamento de Anatoma Patolgica, Hospital Universitario Virgen
del Roco, Sevilla, Sevilla, ES
Paclitaxel es un frmaco antimittico que pertenece al grupo de los taxanos
que se emplea en el tratamiento del cncer metastsico de mama, aunque
su utilidad es limitada debido a la aparicin de resistencia a esta sustancia.
La relacin entre la parada de mitosis inducida por paclitaxel y la posterior
supervivencia (o muerte) de la clula no se conoce completamente, aunque
diversos estudios indican que podra estar implicado el sistema ubicuitina/
proteasoma (UPS) que controla el ciclo celular mediante degradacin de
diversos reguladores del ciclo. FBXW7 forma parte del complejo de la
ubicuitn ligasa SCFFBXW7 y es responsable de la unin a los sustratos. Esta
ligasa ubicuitina para su posterior degradacin a oncoprotenas como c-Myc,
Ciclina E, Notch1, c-Jun y Mcl1, entre otras. Se han encontrado mutaciones
o prdida de funcin de FBXW7 en numerosos cnceres, por lo que se
considera generalmente que FBXW7 es un supresor tumoral. En este trabajo
hemos evaluado el posible papel de FBXW7 en la adquisicin de resistencia
a taxanos en el cncer de mama. Estudiamos los niveles de expresin de
FBXW7 y de los sustratos Aurora A, Ciclina E y Mcl1 mediante anlisis
inmunohistoqumico en algunas lneas celulares y en 380 cnceres de mama
observando que la disminucin de expresin de FBXW7 se correlaciona
estadsticamente con un aumento en la expresin de los sustratos estudiados,
as como con el grado tumoral ms agresivo. Tambin estudiamos el
efecto del silenciamiento y la sobreexpresin en algunas lneas celulares
confirmando la influencia de FBXW7 sobre los sustratos mencionados y su
efecto en la muerte por apoptosis tras tratamiento con paclitaxel. Adems,
la sobreexpresin de FBXW7 en una lnea celular resistente a paclitaxel
derivada de clulas MDA-MB-468 provoc un aumento de la muerte
celular y una clara disminucin de la protena anti-apopttica Mcl1 tras
el tratamiento con paclitaxel. Por lo tanto, nuestros datos parecen indicar
que FBXW7 podra influir en la adquisicin de la resistencia a taxanos
principalmente a travs de su papel en la regulacin de Mcl1.
31
Psters
P01-13 (R01-5)
La 2-deoxiglucosa y la privacin de glucosa

inducen diferentes formas de muerte celular
Clara Luca Len-Annicchiarico, Silvia Ramrez-Peinado, Raffaella
Iurlaro, Cristina Muoz-Pinedo
Instituto de Investigacin Biomdica de Bellvitge IDIBELL,
Barcelona, ES
La 2-deoxiglucosa (2DG) es un medicamento ampliamente utilizado en la
investigacin bsica y actualmente en ensayos clnicos como un inhibidor
de la gliclisis, eficaz como agente antitumoral frente a tumores slidos.
Debido al efecto de la 2DG en ruta de la gliclisis, el principal efecto que
se le atribuye es privar a las clulas de ATP. No obstante, hemos visto que el
efecto txico est mediado por el estrs del Retculo Endoplasmtico ms
que por el estrs bioenergtico.
Estudiamos los efectos de la inhibicin de la gliclisis inducida por la 2DG
y por la privacin de glucosa en diferentes lneas celulares de sarcomas
y observamos que ambos tratamientos promueven diferentes formas
de muerte celular. En la lnea celular de rabdomiosarcoma Rh4, ambos
estmulos inducen estrs del retculo endoplasmtico y en ambos casos el
siRNA de ATF4, una protena involucrada en el estrs reticular, protege
de la muerte celular. Adems, ambos estmulos regulan de forma similar
la expresin de Noxa y de las protenas antiapoptticas de la familia
Bcl-2; especialmente con bajada de Mcl-1. Sin embargo, la 2DG y la
privacin de glucosa promueven formas de muerte celular diferentes; la
muerte celular por 2DG pudo ser prevenida por inhibidores de caspasas,
mientras que la privacin de glucosa indujo muerte celular independiente
de caspasas. ste tipo de necrosis no fue consecuencia de una cada en
el ATP, sugiriendo un papel para una necrosis programada o necroptosis.
Sin embargo, la necrostatina-1 o los antioxidantes no redujeron la muerte
celular. Por el contrario, la Ciclosporina A, inhibidor del poro de transicin
de permeabilidad mitocondrial, protegi de la muerte inducida por la
privacin de glucosa.
Por otro lado, encontramos que slo las lneas celulares derivadas de los
rabdomiosarcomas, fueron sensibles a la 2DG y a la privacin de glucosa,
mientras que lneas celulares de liposarcomas o leiomiosarcomas son
sensibles a la falta de glucosa pero no a la 2DG. Nuestros resultados indican
que el tratamiento con la 2DG no es anlogo a la privacin de glucosa y que
la 2DG podra ser eficaz en el tratamiento de rabdomiosarcomas.
P01r-14 (R01-8)
Dissecting the molecular mechanism of cell

death in transformed cells during photothermal
therapy using gold nanoprisms
Marta Prez Hernndez1, Pablo del Pino2, Scott G. Mitchell3,
Beatriz Pelaz4, Eva M. Glvez5, Jess M. de la Fuente6, Julin
Pardo7
1
Instituto Universitario de Nanociencia de Aragn (INA),
Universidad de Zaragoza. Aragon Health Research Institute (IIS
Aragn), Edificio CIBA, Biomedical Research Centre of Aragon
(CIBA), Zaragoza, ES, 2Instituto Universitario de Nanociencia de
Aragn (INA), Universidad de Zaragoza. Fundacin Araid , Zaragoza,
ES, 3Instituto Universitario de Nanociencia de Aragon (INA),
Universidad de Zaragoza, Zaragoza, ES, 4Instituto de Nanociencia
de Aragn, Universidad de Zaragoza, Zaragoza, ES, 5Instituto de
Carboqumica, CSIC, Zaragoza, ES, 6Instituto de Nanociencia de
Aragn, Universidad de Zaragoza Fundacin Araid, Zaragoza, ES,
7
Instituto de Nanociencia de Aragn, Universidad de Zaragoza.
Fundacin Araid. Aragon Health Research Institute (IIS Aragon),
Edificio CIBA, Biomedical Research Centre of Aragon (CIBA),
Zaragoza, ES
Gold nanoparticles (NPs) are promising vehicles to specifically deliver
drugs to cancer cells and in addition to their use in drug targeting, gold NPs
can be used as heaters during photothermal therapy of solid carcinomas
using near-infrared (NIR) laser light.[1,2] We have previously shown that
32

functionalization of gold nanoprisms (NPRs) with glucose selectively
enhances their cellular uptake in transformed cells.[3] Here we reveal for
the first time the molecular mechanism of apoptosis during photothermal
therapy in transformed cells following irradiation with NIR laser light.[4]
To this aim we have established conditions to readily induce apoptosis
on mouse embryonic fibroblast (MEF) cells transformed with the SV40
virus and analyzed the mechanism of apoptosis using MEFs from different
knock out mice, which are deficient in proteins involved in the different
routes of apoptosis (Bak and Bax, Bid, caspase-3 or caspase-9). Our results
show that hot NPRs activate the intrinsic mitochondrial pathway of
apoptosis mediated by Bak and Bax through the activation of the BH3only protein Bid and that apoptosis and cell death is dependent on the
presence of both caspase-9 and caspase-3. Our findings demonstrate how
the functionalization and dose of NPRs, as well as laser power density
and irradiation time exposure, must be regulated to specifically induce
apoptotic cell death. Moreover the molecular mechanism presented here
may help to predict the efficacy of NP-based photothermal therapy to treat
cancer patients.
References
[1] B. Pelaz, V. Grazu, A. Ibarra, C. Magen, P. del Pino, J.M. de la Fuente.
Langmuir 2012; 28: 8965.
[2] C. Bao, N. Beziere, P. del Pino, B. Pelaz, G. Estrada, F. Tian, V.
Ntziachristos, J. M. de la Fuente and D. Cui. Small 2012; 9: 68.
[3] M. Prez-Hernndez, P. del Pino, B. Pelaz, E. M. Galvez, J. M. de la
Fuente, J. Pardo (under review).
[4] M. Prez-Hernndez, P. del Pino, S. G. Mitchell, B. Pelaz, E. M Glvez,
J. M. de la Fuente, Julin Pardo (under review).
P01-15
New oleanolic acid derivatives, with different

polarity, increase apoptotic effects in B16-F10,
murine melanoma cells
Fernando Jess Reyes Zurita1, Marta Medina ODonnell2, Rosa Ferrer
Martn1, Eva Rufino Palomares1, Amalia Prez Jimnez1, Leticia
Garca Salguero1, Pedro P. Medina1, Samuel Martn Fonseca2,
Francisco Rivas2, Andrs Parra2, Juan Peragn3, Jos Antonio
Lupaez1
1
Department of Biochemistry and Molecular Biology, Faculty of
Science, University of Granada, Granada, ES, 2Department of
Organic Chemistry, Faculty of Science, University of Granada,
Granada, ES, 3Depatment of Experimental Biology, Biochemistry
Section, University of Jaen, Jan, ES
Oleanolic acid (3-hydroxyolean-12-e-28-oic acid), a natural hydroxylated
pentacyclic triterpene acid isolated from olive-pressing residues, has been
investigated together with some its derivatives regarding the induction of
apoptosis in B16-F10 melanoma cells. This study examines the response
of B16-F10 murine melanoma cells to oleanolic acid and three derivatives
with an increasing polarity (28-Benzyloleanolic acid, 3-Pthaloyl,
28-Benzylolanolic acid and 3-(3,3-Dimethylglutaryl), 28-Benzyloleanolic
acid). The compounds tested induce citotoxicity with IC50 concentrations
between 9.8 to 48.5 g/mL in a increasing form, depending on the
polarity of the compounds. These results shown that the three derivatives
were more citotoxic that its precursor oleanolic acid. At these drugs
concentrations, all the products tested, induced over 95% of apoptosis,
measure by FACS analysis with annexine V FICT staining. Most of these
compounds, which also exhibited anti-proliferative effects, generated
mitochondrial disturbs, probably involving the activation of an intrinsic
apoptotic route. These mitochondrial disturbs was confirmed by FACS
analysis using rhodamine 123. Morphological changes including cell
shrinkage, chromatin condensation, and loss of nuclear architecture were
observed by Hoechst stained. These finding support a role of oleanolic acid
derivatives as tumor suppressant and as possible new therapeutic tools for
aberrant cell proliferation in skin and may be used as chemopreventives or
chemotherapeutics in melanoma process development.
Granada 2014
This study has been supported by the grants from Ministerio de Ciencia
e Innovacin (Feder-Interconecta Program and CTQ2009-13898) and
the Consejera de Innovacin, Ciencia y Empresa (Junta de Andaluca)
research groups CVI-157 and FQM-7372.
P01-16
The activation of DFF40/CAD and the

cytoplasmic release of nuclear histones during
caspase-dependent apoptotic cell death
Raquel Larramona-Arcas, Laura Martnez-Escard, Mara
Snchez-Osuna, Vctor Jos Yuste Mateos
Departamento de Bioqumica y Biologa Molecular - Instituto de
Neurociencias, Facultad de Medicina, Universidad Autnoma de
Barcelona, Barcelona, ES
The activation of Caspase-Activated DNase (CAD) prompts to the
cytoplasmic release of nucleosomes (DNA and histones). The inhibition
of CAD blocks double-strand oligonucleosomal DNA fragmentation
and histone release. Previous results obtained in our laboratory pointed
to an unexpected apoptotic behavior in glioblastoma-derived cells,
where histones release occurs in the absence of double-strand DNA
fragmentation. Then, we took advantage of different human glioblastomaand neuroblastoma-derived cells, over-expressing or not human CAD.
Only CAD over-expressing cells treated with an apoptotic insult, such
as staurosporine, displayed oligonucleosomal DNA fragmentation during
apoptosis. Then, we compared both, CAD over-expressing and empty
vector-transfected cells, through Western blot. First, we determined the best
condition to perform the subcellular fractionation with different detergents.
The results obtained revealed a correlation between a high expression
of CAD and the release of the histone core variant H2B in several cell
lines, including glioblastoma-derived U87MG and neuroblastoma-derived
IMR-5 cells. Moreover CAD influenced the internucleosomal histone
H1.2 variant release in glioblastoma-derived LN18 and IMR-5 but not in
U87MG cells. Interestingly, CAD-overexpressing U87MG cells were the
only CAD-transfected cells that remained unable to hydrolyze their DNA
into oligonucleosome-sized pieces during apoptosis. Furthermore, a time
course of staurosporine treatment in IMR-5 cells revealed that H1.2 variant
release occurred already at three hours after apoptotic insult.
Supported by MINECO/FEDER Grant SAF2012-31485 and Generalitat de
Catalunya (2009SGR-346). This study corresponds to the work performed
by R.L.-A. under the Master of Neurosciences program from UAB.
P01-17 (R01-1)
Cellular senescence in tissue remodelling: from

physiology to pathology
Daniel Muoz Espn, Manuel Serrano Marugn
Centro Nacional de Investigaciones Oncolgicas (CNIO), Madrid, ES
Recent discoveries are re-defining our view of cellular senescence as a
trigger of tissue remodeling that acts during normal embryonic development
and upon tissue damage. In the case of developmental processes, abrogation
of senescence is partially compensated by apoptosis but still results in
detectable developmental abnormalities. To achieve tissue renewal,
senescent cells arrest their own proliferation, recruit phagocytic immune
cells, and promote the mobilization of nearby progenitor cells that repopulate
the tissue. This sequence of events (senescence, followed by clearance and
then regeneration) may not be efficiently completed in aged or pathological
contexts, thereby resulting in the accumulation of senescent cells. Increasing
evidence indicates that both pro-senescent therapies and anti-senescent
therapies can be beneficial. In cancer and during active tissue repair,
pro-senescent therapies contribute to minimize the damage by limiting
proliferation and fibrosis, respectively. Conversely, anti-senescent therapies
may help to eliminate post-repair senescent areas and recover tissue function.
Psters
P02. Bases moleculares de la patologa

P02r-1
La quinasa humana VRK1 regula la estabilidad

de los Cuerpos de Cajal protegiendo a Coilina
de su degradacin en el proteasoma
Lara Cantarero Abad, Marta Sanz Garca, Pedro A. Lazo
Instituto de Biologa Molecular y Celular del Cncer, Consejo
Superior de Investigaciones Cientficas (CSIC), Universidad de
Salamanca; e Instituto de Investigacin Biomdica de Salamanca
(IBSAL), Hospital Universitario de Salamanca, Salamanca, ES
Los Cuerpos de Cajal (CB) son suborgnulos nucleares implicados
en el procesamiento del RNA, importante en el proceso de splicing. El
ensamblaje de los CB, junto con su protena scaffold Coilina, se regula a
travs del ciclo celular. La quinasa humana VRK1, la actividad de la cual
tambin es regulada dependiendo del ciclo celular, interacciona y fosforila
a Coilina en el residuo serina 184, regulando la estabilidad de los CB. As,
el silenciamiento de esta quinasa, o la falta de suero en el medio, inactivan
a VRK1 causando una prdida en la fosforilacin de Coilina y provocando
el desensamblaje de estos suborgnulos nucleares; efecto rescatado por una
VRK1 activa pero no por la quinasa inactiva. La prdida de fosforilacin
de Coilina y posterior desensamblaje de los CB, son recuperados por los
inhibidores del proteasoma. La falta de VRK1 provoca la ubiquitinacin,
a travs de la lisina 48, de Coilina en el ncleo. Este proceso es mediado
en parte por Mdm2, y el exporte al citosol para su degradacin en el
proteasoma; de tal forma que este proceso se interrumpe al inhibir el
exporte nuclear con LeptomicinaB. De este modo, VRK1 protege a Coilina
de su degradacin y es una quinasa esencial para el mantenimiento de la
estructura de los Cuerpos de Cajal y su regulacin en el ciclo celular.
P02-2
Phosphorylation of mineralocorticoid receptor

at residue S839 impairs aldosterone-dependent
gene transactivation coupling in a dominant
negative manner
Ruben Jimenez-Canino, Miguel X. Fernandes, Teresa Giraldez,
Diego Alvarez de la Rosa
Department of Physiology and Centre for Biomedical Research of the
Canary Islands (CIBICAN), University of La Laguna, La Laguna, ES
The mineralocorticoid receptor (MR) is a member of the nuclear receptor
family of transcription factors that transduces the biological effects of the
steroid hormone aldosterone, including the regulation of transepithelial
sodium reabsorption and blood pressure. Aldosterone interaction with
MR ligand binding domain (LBD) is responsible for nuclear translocation,
dimerization and gene transactivation. It has recently been demonstrated
that phosphorylation of S843, a residue near the aldosterone binding
pocket, inactivates human MR, presumably by reducing affinity for the
hormone. In this work we examined the mechanisms involved in MR
modulation by S843p. To that end we used mouse MR phosphomimetic
mutant S839D (equivalent to S843 in human) cotransfected with wild
type MR in different proportions in COS-7 cells. MR-S839D is inactive
and significantly decreases wild type MR activity when both forms are
coexpressed. Assuming that MR/MR-S839D dimer formation follows a
binomial distribution, our results are consistent with a dominant negative
role of MR-S839D in the dimer. Surprisingly, aldosterone is able to induce
nuclear translocation of MR-S839D, although at a slower rate than wild
type receptors. Therefore aldosterone is still able to bind to MR-S839D but
it is inefficient for gene transactivation. Structure modeling of MR LBD
and docking experiments show that S839D mutation or S839p produce
the same effect, namely a small decrease in agonist docking energy.
Taken together, these results suggest that the effect of S839p cannot be
33
Psters
fully explained by decreased aldosterone binding affinity and may imply a
defect in transactivation coupling.
P02m-3
Initial age-associated metabolic alterations in

different adipose tissue depots: effect of caloric
restriction
Patricia Corrales-Cordn1, Yurena Vivas-Garca1, Cristina MartnezGarca1, Adriana Izquierdo Lahuerta1, Carmen Martnez Martnez1,
Maria Jess Obregn Perea2, Manuel Ros Prez1, Gema MedinaGmez1
1
Universidad Rey Juan Carlos, Alcorcn, ES, 2Instituto de
Investigaciones Biomdicas Alberto Sols, CSIC-UAM, Madrid, ES
Changes in the body composition such as high adiposity and sarcopeny
occur during ageing. These changes are usually associated to metabolic
alterations like insulin resistance, type 2 diabetes and metabolic syndrome.
The fat tissue is not homogeneous and the distribution and function of the
different deposits of white adipose tissue (WAT) and brown adipose tissue
(BAT) change throughout life. In this study, we have studied different
WAT and BAT depots during the first stage of ageing with the objective
to detect the initial signals of alteration. Moreover, we have studied the
effect of the long-term caloric restriction (CR) and its benefits. 3 and 12month-old mice fed ad lib and 12-month-old mice fed ad lib with a 20%
CR have been used. The older animals showed insulin resistance and lower
adiponectin levels in plasma compared with the younger mice. We have
studied expression of different lipid metabolism genes and their regulation
in epididimal WAT (eWAT), subcutaneous WAT (scWAT) and BAT. In
these initial stages of ageing, expression of Glut4, IRSs and lipogenesis and
lipolysis genes were decreased in scWAT of 12-month-old animals, but not
in eWAT. Although visceral WAT has been described as the most dangerous
tissue from the metabolic point of view, our data could suggest an earlier
metabolic alteration in scWAT than in eWAT, which were retrieved by longterm CR. In addition, functional markers of BAT (DIO2, LPL, PGC1)
also showed changes in these first stages of ageing, Furthermore, UCP1 expression was significantly lower in scWAT of aged mice compared
with younger mice, suggesting an age-related loss of sWAT browning.
Our findings suggest that initial age-related metabolic alterations occur in
scWAT, where CR could attenuate them.
Acknowledgements: MINECO (BFU2012-33594), CAM (S2010/BMD2423).
P02-4 (R02-3)
La expresin heptica de la ciclooxigenasa-2

(COX-2) protege de la esteatosis, adiposidad
y resistencia a la insulina inducida por dieta rica
en grasa
Daniel E. Francs1, Omar Motio2, gueda Fernndez-Gonzlez3,
Ana Fernndez-lvarez4, Carme Cucarella5, Rafael Mayoral6,
Mara Fernndez-Velasco7, Luis Castro- Snchez2, Lisardo Bosc8,
Cristina E. Carnovale1, Marta Casado9, ngela M. Valverde3,
Paloma Martn-Sanz8
1
Instituto de Fisiologa Experimental (IFISE-CONICET), Rosario, AR,
2
Instituto de Investigaciones Biomdicas Alberto Sols, CSIC-UAM,
Madrid, ES, 3Instituto de Investigaciones Biomdicas Alberto Sols,
CSIC-UAM; Centro de Investigacin Biomdica en Red de Diabetes
y Enfermedades Metablicas Asociadas (CIBERdem), Madrid,
ES, 4Fundacin Instituto Leloir, IIBBA-CONICET, Buenos Aires,
AR, 5Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia,
ES, 6Centro de Investigacin Biomdica en Red de Enfermedades
Hepticas y Digestivas (CIBERehd); Department of Medicine,
University of California, Madrid, ES, 7Instituto de Investigacin
34

Hospital Universitario La PAZ, IDIPAZ, Madrid, ES, 8Instituto de
Investigaciones Biomdicas Alberto Sols, CSIC-UAM; Centro de
Investigacin Biomdica en Red de Enfermedades Hepticas y
Digestivas (CIBERehd), Madrid, ES, 9Instituto de Biomedicina de
Valencia (IBV-CSIC); Centro de Investigacin Biomdica en Red de
Enfermedades Hepticas y Digestivas (CIBERehd), Valencia, ES
La resistencia a la insulina (RI) participa en la fisiopatologa de enfermedades
relacionadas con la obesidad como la diabetes mellitus tipo 2 y la enfermedad
de hgado graso no alcohlica. Adems, la RI est asociada con un estado de
inflamacin crnica leve que contribuye significativamente a su desarrollo.
Las ciclooxigenasas 1 y 2 (COX-1 y COX-2) catalizan el primer paso en
la biosntesis de los prostanoides. COX-1 se expresa constitutivamente
en diversos tejidos, mientras que la expresin de COX-2 es inducida por
diferentes estmulos. Los hepatocitos adultos son incapaces de inducir la
expresin de COX-2 independientemente de los factores proinflamatorios
presentes. Se ha analizado el papel de la expresin heptica de COX-2 en
ratones transgnicos (hCOX-2-Tg) en un modelo de RI y de alteracin de
la homeostasis energtica inducido por una dieta rica en grasa. Nuestros
resultados muestran que la expresin de COX-2 en el hepatocito protege
frente a la RI, esteatosis heptica y obesidad, en parte debido a una mayor
sensibilidad a la insulina, un aumento en la tolerancia a la glucosa, y de la
relacin adiponectina/leptina. Asimismo, se observ una disminucin de la
esteatosis heptica, adiposidad y en el rea de los adipocitos, de los niveles
de triglicridos y cidos grasos libres tanto hepticos como plasmticos, y de
las citoquinas proinflamatorias. Los ratones hCOX-2-Tg exhibieron tambin
un incremento en el gasto energtico, induccin de la termognesis y de los
niveles de expresin de genes implicados en la oxidacin de cidos grasos,
as como un aumento en la sealizacin de insulina heptica (fosforilacin
del receptor de insulina y AKT). Resultados similares se obtuvieron en lneas
celulares hepticas murinas y humanas que sobreexpresan COX-2. Nuestros
datos demuestran que la expresin constitutiva de COX-2 en hepatocitos
protege frente a la RI heptica, la adiposidad e inflamacin en ratones hCOX2-Tg sometidos a una dieta rica en grasa.
P02r-5
Ethanol by activating the innate immune

receptors TLR4, promotes neuroinflammation,
myelin dysfunctions, synaptic alterations and
epigenetic modifications in adolescent mice with
binge ethanol drinking
Jorge Montesinos Selfa, Antoni Pla Rodrguez, Mara Pascual Mora,
Ana Isabel Gil Tebar, Clara Mar Guarch Prez, Consuelo Guerri
Sirera
Centro de Investigacin Prncipe Felipe, Valencia, ES
Adolescence is a brain maturation period from childhood to adulthood.
Plastic and dynamic processes occur in specific brain regions, such as the
prefrontal cortex (PFC) in which overproduction of axons and synapses,
followed by rapid pruning along with ongoing myelination of axons,
enhances neuronal conduction and communication. We demonstrated
that ethanol by activating the innate immune receptors toll-like receptor 4
(TLR4) induces neuroinflammation and brain damage. Considering that the
developmental brain is vulnerable to the effects of ethanol, we investigate
whether intermittent ethanol intoxication promotes TLR4-dependent proinflammatory processes, leading to myelin and synaptic dysfunctions and
epigenetic alterations. For this aim, we used wild-type (WT) and TLR4
deficient (TLR4-/-) adolescent mice exposed intermittently to ethanol (3.0
g/kg) during 2 weeks. The levels of pro-inflammatory mediators as well as
different myelin and synaptic proteins were assessed in the PFC 24h after
the last ethanol administration. Our results demonstrate that binge ethanol
treatment activated TLR4 pathway (MAPKs, p-p65) triggering the upregulation of several pro-inflammatory mediators (iNOS, COX-2), cytokines
(IL-1, TNF-) and chemokines (MCP1, MIP1). Ethanol treatment also
decreased myelin protein levels (PLP, CNPase, MAG, MBP, NG-2) and
synaptic-related proteins levels (synapsin, syntaxin, SNAP-25) in PFC of
Granada 2014
WT mice, while no differences were observed in TLR4-/- PFC. Electron
microscopy studies showed that ethanol treatment damages myelin
sheath and alters synaptic structure in WT PFC. Ethanol treatment also
increased HAT activity, the levels of acetylated H3 and H4 and upregulated
the expression of immediate-early-genes related to plasticity and drug
addiction (bdnf, c-fos, egr1, fosb) in a TLR4-dependent manner. These
findings suggest that activation of TLR4 signaling by intermittent ethanol
intoxication during adolescence alters synaptic plasticity, causes myelin
and synaptic derangements and induces epigenetic modifications, that
could be associated to addictive behaviour.
Supported by SAF-2012-33747 and ERAB (EA-13-08).
P02r-6
The innate immune receptors TLR4 participate in

ethanol-induced proteolytic dysfunctions in glial
cells
Antoni Pla Rodrguez, Mara Pascual Mora, Consuelo Guerri Sirera
Ethanol is a neurotoxic compound and its abuse induces brain damage
and can lead to neurodegeneration. Ethanol triggers inflammatory
processes in glial cells, upregulating cytokines and other inflammatory
mediators through the activation of Toll-like receptor 4 (TLR4) signaling.
Recent evidence indicates the role of protein degradation pathways in
neurodegeneration and in alcoholic liver disease, but how these processes
affect the brain remains elusive. We have recently demonstrated that
chronic ethanol consumption alters the two major protein degradation
pathways, the ubiquitin-proteasome system (UPS) and the autophagylysosome pathway (ALP), in mouse brain, and that TLR4 participates in
these proteolytic dysfunctions. With this study, we aim to evaluate the
effect of an acute dose of ethanol (50 mM) on WT and TLR4-/- mice glial
cells at different time points, through the use of techniques such as western
blot, flow cytometry or confocal and electron microscopy. Our results
show that a single dose of ethanol induces an overexpression of several
autophagy markers (ATG12, LC3-II and CTSB). Ethanol also produces an
increase in the number of autophagic vacuoles and lysosomes, which is
accompanied by a basification of the lysosomal pH. These changes in the
ALP could be in part due to a reduction of the phosphorylation levels of
the autophagy inhibitor mTOR. Ethanol also alters the UPS and induces
the formation of the immunoproteasome, by promoting the expression of
several of its inducible subunits (1i, 5i and PSME1). Interestingly, only
minor changes in the proteolytic pathways were found between control and
ethanol-treated TLR4-/- mice glial cells. Together, these results indicate
that a single dose of ethanol is capable of affecting proteolytic pathways in
the brain in a TLR4-dependent manner. These findings could provide new
insight into the mechanisms underlying ethanol-induced brain damage.
Supported by SAF2012-33747, PNSD, RTA-Network, RD12-0028-007.
Psters
estudiado las clulas madre derivadas del tejido adiposo, su conversin
a adipocito y sus propiedades como clula diferenciada, prestando una
especial atencin a los cambios en parmetros mitocondriales. La dificultad
en la preparacin de estos modelos consiste en la generacin del estado
rho0, carente de mtDNA, previo a la construccin del cbrido. Mientras
trabajamos en la resolucin de este problema, podemos simular diferentes
mutaciones en el mtDNA, preferentemente en los genes para los RNA de
transferencia, mediante el uso de distintos xenobiticos. Concretamente,
hemos elegido el linezolid, un antibitico de amplio espectro que se une
a la subunidad grande ribosomal y ha demostrado ser un potente inhibidor
de la transcripcin mitocondrial. De esta manera, es posible analizar el
efecto de una disfuncin en la expresin de genes mitocondriales sobre la
diferenciacin celular y su funcin como clula madura.
Financiacin: Instituto de Salud Carlos III (FIS-PI10/00662 y PI11/01301).
P02m-8 (R02-7)
Potenciacin de la capacidad de quemar grasas

del tejido adiposo marrn como terapia contra
la obesidad y la diabetes de tipo 2
Mara Caldern-Domnguez, Raquel Fucho, Joan Francesc Mir,
Minia Weber, Anna Orozco, Dolors Serra, Laura Herrero
Departamento de Bioqumica y Biologa Molecular, Institut de
Biomedicina de la Universitat de Barcelona-IBUB, Universitat de
Barcelona - CIBER Fisiopatologa de la Obesidad y la NutricinCIBEROBN, Instituto de Salud Carlos III, Barcelona, ES
En el 2009, cinco grupos de investigacin independientes identificaron que
el tejido adiposo marrn (TAM), que se pensaba exclusivo de roedores y
neonatos, no slo estaba presente sino tambin activo en humanos adultos.
Curiosamente observaron que su actividad disminua en pacientes obesos y
diabticos, lo que indica que este tejido participa en la termognesis inducida,
ya sea por el fro o por la dieta. Esto situ al TAM en el punto de mira, ya
que cualquier estrategia capaz de eliminar el exceso de lpidos de la obesidad
podra ser beneficioso para la salud. Por esta razn nos centramos en la
oxidacin de los cidos grasos (FAO, fatty acid oxidation), y su enzima clave,
la carnitina palmitoiltransferasa 1 (CPT1). En el presente trabajo proponemos
que un aumento de la FAO en el TAM reducira los niveles de los lpidos y
mejorara el fenotipo obeso y diabtico. Para ello sobrexpresamos en una
lnea celular de adipocitos marrones la forma mutante permanentemente
activa de la CPTIA, la CPTIAM, la cual es insensible a su inhibidor
fisiolgico, el malonil-CoA. Observamos que los adipocitos marrones que
sobreexpresan la CPT1AM muestran un aumento en la FAO, en la expresin
de la protena UCP1,y reduce el incremento del contenido de triglicridos
inducido por el palmitato. Adems de recuperar los niveles de marcadores
proinflamatorios y marcadores relacionados con el sndrome metablico, en
comparacin con las clulas GFP control. En conclusin, hemos conseguido
mejorar el fenotipo obeso y diabtico a travs del aumento de la FAO en
nuestro modelo celular, con el propsito de dar un nuevo enfoque teraputico
para el tratamiento de la diabetes inducida por la obesidad.
P02r-7
Disfuncin OXPHOS y diferenciacin celular
P02-9
Laura Llobet1, Eldris Iglesias1, Alba Pesini1, Virginia Borobia1, Sonia

Emperador1, Ester Lpez-Gallardo1, Julio Montoya1, Eduardo RuizPesini2
1
Universidad de Zaragoza - CIBERER, Zaragoza, ES, 2Universidad
de Zaragoza - CIBERER - ARAID, Zaragoza, ES
Increased serum- and glucocorticoidinducible kinase 1 (SGK1) activity potentiates

mineralocorticoid/NaCl-induced renal but not
cardiac fibrosis
Las lneas celulares transmitocondriales han sido un excelente modelo

para el estudio de las mutaciones patolgicas en el mtDNA, ya que
reproducen exactamente el ambiente genmico humano. Sin embargo
presentan algunos inconvenientes como son la aneuploida (por derivarse
generalmente de lneas celulares tumorales) y la falta de capacidad de
diferenciacin. Estos problemas pueden ser soslayados utilizando clulas
madre adultas, euploides y fcilmente diferenciables. Nosotros hemos
Catalina Sierra Ramos1, Guadalberto Hernndez Hernndez1,

Eduardo Salido2, Diego lvarez de la Rosa1
1
Department of Physiology, Centre for Biomedical Research of the
Canary Islands-CIBICAN, University of La Laguna, La Laguna, ES,
2
Centre for Biomedical Research of the Canary Islands-CIBICAN
- Centre for Biomedical Research on Rare Diseases-CIBERER,
University of La Laguna, La Laguna, ES
35
Psters
The mineralocorticoids aldosterone and deoxycorticosterone acetate
(DOCA) stimulate renal tubular salt reabsorption, increase salt appetite,
induce extracellular volume expansion, and elevate blood pressure.
Mineralocorticoid excess induces deleterious effects on cardiorenal function, including the development of fibrosis. The effects of
mineralocorticoids on renal tubular Na+ reabsorption and salt appetite
involve the serum- and glucocorticoid-inducible kinase 1 (SGK1). The
present experiments explored the involvement of excess SGK1 activity
in the development of mineralocorticoid/NaCl-induced cardiac and renal
fibrosis. To this end, transgenic mice (Tg.SGK1), made with a bacterial
artificial chromosome containing the SGK1 gene with a point mutation
rendering the kinase constitutively active, and their wild-type littermates
were uninephrectomized, treated with DOCA and given 1% NaCl in the
drinking water for 6 weeks. The treatment led to a significant increase in
blood pressure in both genotypes, slightly more pronounced in Tg.SGK1
mice. Histology after 6 weeks treatment revealed marked glomerular,
perivascular and tubulointerstitial fibrosis in the kidney cortex, which
was significantly greater in Tg.SGK1 mice. In contrast, treated animals
developed cardiac fibrosis without significant differences between
genotypes. Enhanced SGK1 activity without DOCA/NaCl treatment did
not produce fibrosis. Our results demonstrate that increased SGK1, while
not sufficient to induce fibrosis without added stimuli, plays a decisive role
in tissue-specific mineralocorticoid/NaCl-induced fibrosis.
P02-10 (R02-1)
Una dieta rica en cacao atena la resistencia

a la insulina en las ratas ZDF
Isabel Cordero-Herrera1, Mara Angeles Martn Arribas1, Fernando
Escriv Pons2, Carmen Alvarez Escol2, Luis Goya Surez1, Sonia
Ramos Rivero1
1
Departamento de Metabolismo y Nutricin. Instituto de Ciencia
y Tecnologa de Alimentos y Nutricin (ICTAN-CSIC), Madrid, ES,
2
Departamento de Bioqumica y Biologa Molecular II. Facultad de
Farmacia, Universidad Complutense de Madrid (UCM). CIBER de
Diabetes y Enfermedades Metablicas (CIBERDEM), Madrid, ES
La resistencia a la insulina es una de las principales caractersticas de la
diabetes tipo 2 (1). El cacao presenta ciertos efectos antidiabticos (2,3),
pero an no se conoce con exactitud el mecanismo molecular de accin por
el cual ejerce dichas actividades beneficiosas sobre la sealizacin de la
insulina en el hgado.
El objetivo de este trabajo fue investigar el potencial preventivo de una
dieta rica en cacao sobre la sealizacin de la insulina en una situacin
prediabtica en el hgado de las ratas ZDF (14 semanas de vida). La dieta
rica en cacao (10%) mejor la hiperglucemia y la respuesta al test de
tolerancia a la glucosa, a la vez que disminuy el peso corporal. Adems,
el cacao previno el aumento de la fosforilacin en Ser307 y Ser636/639 de
IRS-1 respecto a los animales ZDF que reciban la dieta control, mostrando
valores similares a los de las ratas control (ZLean). De manera similar, los
animales alimentados con la dieta rica en cacao mostraron unos niveles de
p-GSK3, p-GS y glucgeno comparables a los de las ratas ZLean.
Por su parte, los valores de PEPCK, aumentados en las ratas ZDF,
regresaron a niveles control en aquellos animales que reciban la dieta rica
en cacao, mientras que GK y GLUT-2 no parecieron modificarse en ningn
caso. Estos datos sugieren que una dieta rica en cacao podra mejorar la
resistencia a la insulina al prevenir la atenuacin de la sealizacin de la
hormona que se produce en el hgado en la diabetes tipo 2.
Bibliografa
Klover & Mooney: Int J Biochem Cell Biol 2004, 36: 753-8.
Cordero-Herrera et al.: Mol Nutr Food Res 2013, 57: 974-85.
Cordero-Herrera et al.: Food Chem Toxicol 2014, 64: 10-19.
Agradecimientos: AGL2010-17579, CSD2007-00063.
36

P02r-11
Identification of bone morphogenetic protein 9

(BMP9) as a novel fibrogenic factor
Jos Manuel Muoz Flix1, Nuria Perretta-Tejedor1, Cristina Cuesta1,
Jos Miguel Lpez-Novoa1, Sabine Bailly2, Carlos Martnez-Salgado3
1
Unidad de Fisiopatologa Renal y Cardiovascular, Instituto
Reina Sofa de Investigacin Nefrolgica, Departamento de
Fisiologa y Farmacologa, Universidad de Salamanca - Instituto
de Investigacin Biomdica de Salamanca-IBSAL, Salamanca,
ES, 2Inserm, U1036 - Commissariat a` lEnergie Atomique et
aux Energies Alternatives, Universit de Grenoble, Grenoble,
FR,3Unidad de Fisiopatologa Renal y Cardiovascular, Instituto
de Estudios de Ciencias de la Salud de Castilla y Len-IECSCYL,
Unidad de Investigacin, Hospital Universitario de Salamanca,
Salamanca, ES
Tubulointerstitial fibrosis, a common endpoint of chronic kidney disease,
is characterized by the presence of myofibroblasts and an excessive
accumulation of extracellular matrix (ECM) proteins in the tubular
interstitium. Transforming growth factor beta 1 (TGF-1) is a cytokine
with a relevant contribution to fibrotic development. We have previously
observed an increased expression of the TGF- type I receptor ALK1 in
fibrotic kidneys after unilateral ureteral obstruction (UUO), and the bone
morphogenetic protein-9 (BMP9) is a strong ligand for ALK1 in endothelial
cells. Thus, we aim to identify the effect of BMP9 on ECM protein
expression and its possible involvement in renal fibrosis development.
We used mouse embryo fibroblasts (MEFs) stimulated with BMP9. We
analyzed collagen I, fibronectin and connective tissue growth factor
(CTGF) expressions; we evaluated expression of phosphorylated Smads
(BMPs induced-Smad1/5/8) and TGF-1-induced Smad2/3. UUO was
performed in BMP9 deficient mice and their respective controls, and renal
tissue fibrosis was analyzed by Red Sirius and Massons trichrome staining.
BMP9 (20 ng/ml) induced an increase in collagen I, fibronectin and CTGF
expression in MEFs, as well as an increase in Smad1/5/8 phosphorylation
normally induced through ALK1/2/3/6 receptors- and in Smad2/3
phosphorylation induced by ALK4/5/7 receptors. Inhibition of both groups
of receptors with SB431542 (ALK4/5/7 inhibitor) and dorsomorphin-1
(ALK1/2/3/6 inhibitor) reduced BMP-9-induced ECM protein expression.
Obstructed kidneys from Bmp9-/- mice developed less renal fibrosis than
their respective controls.
Our data suggest that BMP9 regulates renal fibrosis stimulating ECM
protein expression by MEFS, due to an activation of receptors that
phosphorylate Smad1/5/8 and Smad2/3 proteins.
P02r-12
Deplecin del DNA mitocondrial (mtDNA) debida

a una mutacin en el gen MT-TW
Sonia Emperador1, Ester Lpez-Gallardo1, Cristina Ruiz-Ruiz2, Alba
Pesini2, Maria Dolores Fuentes2, Laura Llobet2, Eldris Iglesias2,
Eduardo Ruiz-Pesini3, Julio Montoya1
1
Universidad de Zaragoza- CIBERER, Zaragoza, ES, 2Universidad de
Zaragoza, Zaragoza, ES, 3ARAID-Universidad de Zaragoza, Zaragoza, ES
Mutaciones en genes que codifican para tRNAs mitocondriales son
responsables de causar un nmero importante de patologas en humanos.
En este trabajo presentamos el caso de un nio con sndrome de Leigh en
el que, mediante secuenciacin del mtDNA completo, encontramos una
insercin de una timina en la posicin 5537, que corresponde al gen MTTW. Esta mutacin haba sido previamente descrita en otro paciente con
enfermedad mitocondrial.
Para confirmar el efecto funcional de esta mutacin, construimos cbridos
transmitocondriales usando la lnea rho0 de carcinoma de pulmn humano
A549. En estos cbridos observamos una cada en la actividad y cantidad del
complejo respiratorio IV, disminucin de la cantidad de ATP mitocondrial
Granada 2014
y ausencia de sntesis mitocondrial de protenas. De forma ms llamativa,
detectamos una prdida completa del mtDNA. Un hallazgo similar se haba
ya descrito en cbridos NT2 portadores de la m.3243A>G en MT-TL1.
Nuestros resultados sugieren que mutaciones patolgicas de genes que
codifican tRNA mitocondriales, en fondos genticos nucleares particulares,
podran llevar a deplecin del mtDNA. Por lo que quizs se deba secuenciar
estos genes en los sndromes de deplecin.
Financiacin: ISCIII; FIS (PI10-00662; PI 11-01301); CIBERER.
P02-13
Sistema OXPHOS, nucletidos de pirimidina,

y la enfermedad de Alzheimer
Alba Pesini1, Eldris Iglesias1, Nuria Garrido2, M. Pilar BayonaBafaluy2, Laura Llobet1, Sonia Emperador1, Ester Lpez-Gallardo1,
Antonio Galarreta2, Julio Montoya1, Eduardo Ruiz Pesini2
1
Universidad de Zaragoza- CIBERER, Zaragoza, ES, 2Universidad de
Zaragoza, Zaragoza, ES
La enfermedad de Alzheimer (AD) es una enfermedad neurodegenerativa
crnica y progresiva, clnicamente caracterizada por la prdida de memoria
y el declinar cognitivo. Los sellos histopatolgicos de la enfermedad
son el acmulo de placas extracelulares de pptido beta-amiloide, los
ovillos neurofibrilares intracelulares compuestos por la protena tau
hiperfosforilada, la prdida de sinapsis y la degeneracin neuronal.
Una disfuncin del sistema de fosforilacin oxidativa (OXPHOS) ha sido
implicada en la patognesis de AD. De hecho, la cadena transportadora
de electrones podra tener un papel relevante en la formacin de las
membranas celulares y de las sinapsis entre las neuronas, ya que la cadena
respiratoria participa en la sntesis de novo de nucletidos de pirimidina,
que son claves en la produccin de fosfolpidos, principal compuesto de
todas las membranas.
Para estudiar cmo afectara la disfuncin en el sistema OXPHOS a la
produccin de neuritas y diferenciacin neuronal, utilizamos la lnea
celular de neuroblastoma SK-N-BE(2)C. Cultivamos y diferenciamos esta
lnea celular en ausencia o presencia de azida sdica (NaN3) o carbonyl
cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), que disminuiran
o aumentaran, respectivamente, el flujo de electrones a travs de la cadena
respiratoria y por lo tanto quizs podran alterar negativa o positivamente
la sntesis de nucletidos de pirimidina.
En un primer paso hemos puesto a punto las concentraciones de las drogas
que afectan el consumo de oxgeno, pero no la viabilidad celular.
Financiacin: Instituto de Salud Carlos III [FIS-PI 10/00662 y PI
11/01301], Instituto de Investigacin Sanitaria de Aragn (IIS), Asociacin
de Enfermos de Patologa Mitocondrial (AEPMI) y Universidad de
Zaragoza-Banco de Santander (UZ2012-BIO-01).
P02r-14
Efecto estimulador de la leptina sobre

la produccin de sialofosfoprotena dentinaria
(DSSP) en la pulpa dental humana
Jenifer Martin Gonzalez1, Antonio Prez-Prez2, Flora Snchez
Jimnez2, Juan Jos Segura Egea1, Vctor Snchez Margalet2
1
Departamento de Estomatologa, Facultad de Odontologa,
Universidad de Sevilla, sevilla, ES, 2Departamento de Bioqumica,
Biologa molecular e Inmunologa, Facultad de Medicina,
Universidad de Sevilla, sevilla, ES
Objetivo: La leptina, un mediador de la respuesta inflamatoria, y su receptor
(LEPR) se expresan en la pulpa dental humana. La sialofosfoproteina
dentinaria (DSPP) es una protena implicada en la odontognesis y en la
respuesta reparativa dentino-pulpar. Esta investigacin tiene como objetivo
describir la localizacin inmunohistoqumica de LEPR y estudiar el efecto
Psters
de la leptina sobre la expresin de la sialofosfoproteina dentinaria (DSPP)
por las clulas pulpares humanas.
Metodologa: Veinticinco muestras de pulpa dental humana se obtuvieron
de terceros molares recin extrados libres de caries y sin restauracin. Las
muestras de pulpa fueron procesadas y la mineralizacin producido por
odontoblastos en respuesta a la leptina se determin mediante el anlisis
de la expresin de DSPP por inmunoblot y por PCR a tiempo real (qRTPCR). La localizacin de LEPR en la pulpa dental fue examinada por
inmunohistoqumica usando anticuerpo monoclonal anti-LEPR humano.
Resultados: La immunoreactividad para los anticuerpos anti-LEPR
se localiz especialmente en el estrato odontoblstico y a nivel de la
predentina. La leptina estimul de forma dosis-dependiente la expresin de
DSPP en la pulpa humana. El anlisis del Western blot de las muestras de
pulpa estimuladas con leptina revel la presencia de una protena de peso
molecular aparente ~ 100 kDa, correspondiente al peso molecular estimado
de la DSPP. La expresin del ARNm de la DSPP se confirm por anlisis
de qRT-PCR , y el tamao de los fragmentos amplificados (280 pares de
bases) fue confirmado por electroforesis en gel de agarosa.
Conclusin: Por primera vez se ha demostrado que los odontoblastos
humanos expresan el receptor de leptina (LEPR) y que la interaccin de
la leptina con este receptor estimula la produccin por el odontoblasto
de sialofosfoprotena dentinaria (DSPP). Estos hallazgos sugieren
que la leptina juega un papel en la respuesta defensiva pulpar y en la
dentinognesis.
P02-15 (R02-5)
Obesity-related glycogen mishandling in human

adipose tissue: a key feature of metabolic stress
Sonia Fernndez-Veledo1, Victoria Ceperuelo-Mallafre1, Xavier
Duran1, Kelly Roche1, Catalina Nez-Roa1, Marta Montori-Grau2,
Lourdes Garrido-Snchez3, Francisco J. Tinahones3, Anna M
Gomez-Foix2, Joan Vendrell1
1
Hospital Universitari de Tarragona Joan XXIII - IISPV - URV
- CIBERDEM, Tarragona, ES, 2Departamento de Bioqumica y
Biologa Molecular, Institut de Biomedicina de la Universitat de
Barcelona-IBUB, Universitat de Barcelona - CIBERDEM, Barcelona,
ES, 3Hospital Universitario Virgen de la Victoria - CIBERObn,
Malaga, ES
Background: Obesity leads to excess lipid storage in adipocytes resulting
in the derangement of carbohydrate metabolism. It is well-known that
hepatic glycogen is increased under pathologic circumstances associated
with obesity such as insulin resistance. Though at low levels, studies in
murine models revealed that adipose tissue (AT) might contain glycogen
stores. However, to date the physio(patho)logical role of glycogen
metabolism in human adipose tissue remains unexplored.
Methodology: Glycogen accumulation was studied in human adipocytes
and adipose tissue from lean and obese patients by different methodological
approaches (PAS, immunofluorescence and TEM). PTG, the only glycogenassociated regulatory subunit (PP1-GTS) reported in murine adipocytes,
was overexpressed by adenoviral transfection in human adipocytes to
explore the metabolic effects of enforced glycogen deposition. Glycogen
metabolism gene expression was analyzed in subcutaneous (SAT) and
visceral (VAT) AT from lean and obese subjects in two independent cohorts.
Results: Hypoxia, which has been related to obesity-related AT dysfunction,
promotes glycogen accumulation in human adipocytes. Enforced glycogen
deposition by PTG adenoviral overexpression, modified adipokine secretion
and caused insulin resistance in human adipocytes. Human myocytes
cultured with Ad-PTG adipocyte conditioned media also shown a decrease
in insulin responsiveness. Clinical studies in two human independent
cohorts revealed that obesity is associated with changes in AT glycogen
metabolism gene expression being PPP1R3E the most predominant PP1GTS isoform in human fat depots. An increased level of glycogen synthase
protein expression that correlates with enhanced glycogen deposition was
detected in SAT from obese patients.
37
Psters
Conclusions: Increased glycogen deposition in AT in obesity modifies the
endocrine function of human adipocytes and might contribute to obesityrelated comorbidities.
P02-16
Influencia de xenobiticos ambientales

en la neuropata ptica hereditaria de Leber
Ester Lopez Gallardo1, Luis Diez2, Sonia Emperador1, Iigo
Martinez-Romero2, Laura Llobet2, Eldris Iglesias2, Alba Pesini2,
Eduardo Ruiz-Pesini2, Julio Montoya2
1
Centro de Investigacin Biomdica en Red de Enfermedades Raras
(CIBERER), Zaragoza, ES, 2Dto. Bioqumica y Biologa MolecularFacultad de Veterinaria-Universidad de Zaragoza, Zaragoza, ES
El genoma mitocondrial contiene 37 genes, todos ellos esenciales para el
buen funcionamiento del sistema de fosforilacin oxidativa. Mutaciones
en estos genes pueden causan un dficit de energa y causar enfermedades
mitocondriales. Los efectos y la severidad de las enfermedades
mitocondriales pueden variar enormemente. Las mutaciones patolgicas
son usualmente heteroplsmicas mientras que los polimorfismos neutros
son homoplsmicos. Sin embargo, hay algunas excepciones, por ejemplo,
las mutaciones que causan LHON (Neuropata ptica Hereditaria de
Leber) son homoplsmicas en la mayora de los casos. Esta enfermedad
muestra una penetrancia incompleta, por ello, es importante investigar qu
factores genticos y/o ambientales modulan la expresin del fenotipo. En
el presente trabajo hemos usado el modelo de cbridos transmitocondriales
portadores de una mutacin causante de LHON (3460/ND1) para estudiar
la combinacin de dicha mutacin con varias xenobiticos (factores
ambientales) y qu importancia puede tener en el desencadenamiento
de la patologa del paciente. Los resultados obtenidos muestran que
dicha mutacin en presencia de los xenobiticos analizados (rotenona,
capsaicina, anonacina) muestra diferencias en la funcin mitocondrial.
Financiacin: Instituto de Salud Carlos III-FIS (PI10-00662; PI 1102301), Fundacin ARAID-Programa de Apoyo a la I+D+I para jvenes
investigadores 2010, y Centro de Investigacin en Red en Enfermedades
raras (CIBERER). CIBERER es una iniciativa de ISCIII.
P02-17
AD0157, a pyrrolidinedione fungal metabolite,

inhibits angiogenesis by targeting the Akt
signaling pathway
Melissa Garca-Caballero1, Librada Caedo2, Antonio FernndezMedarde2, Miguel ngel Medina Torres3, Ana R. Quesada3
1
Universidad de Mlaga, Andaluca Tech, Departamento de
Biologa Molecular y Bioqumica, Facultad de Ciencias, and IBIMA
(Biomedical Research Institute of Mlaga), Mlaga, ES, 2Biomar
Microbial Technologies, Parque Tecnolgico de Len, Armunia
(Len), ES, 3Universidad de Mlaga, Andaluca Tech, Departamento
de Biologa Molecular y Bioqumica, Facultad de Ciencias, and
IBIMA (Biomedical Research Institute of Mlaga). Unidad 741,
CIBER de Enfermedades Raras (CIBERER), Mlaga, ES
In the course of a screening program for the inhibitors of angiogenesis
from marine sources, AD0157, a pyrrolidinedione fungal metabolite,
was selected for its angiosupressive properties. AD0157 inhibited the
growth of endothelial and tumor cells in culture in the micromolar range.
Our results show that subtoxic doses of this compound inhibit certain
functions of endothelial cells, namely, differentiation, migration and
proteolytic capability. Inhibition of the mentioned essential steps of in vitro
angiogenesis is in agreement with the observed antiangiogenic activity,
substantiated by using two in vivo angiogenesis models, the chorioallantoic
membrane and the zebrafish embryo neovascularization assays, and by the
ex vivo mouse aortic ring assay. Our data indicate that AD0157 induces
38

apoptosis in endothelial cells through chromatin condensation, DNA
fragmentation, increases in the subG1 peak and caspase activation. The
data shown here altogether indicate for the first time that AD0157 displays
antiangiogenic effects, both in vitro and in vivo, that are exerted partly by
targeting the Akt signaling pathway in activated endothelial cells. The fact
that these effects are carried out at lower concentrations than those required
for other inhibitors of angiogenesis makes AD0157 a new promising
drug candidate for further evaluation in the treatment of cancer and other
angiogenesis-related pathologies.
Our experimental work is supported by grant P12-CTS-1507 (Andalusian
Government and FEDER) and funds from group BIO-267 (Andalusian
Government). The CIBER de Enfermedades Raras is an initiative from
the ISCIII (Spain). This communication has the support of a travel grant
Universidad de Mlaga. Campus de Excelencia Internacional Andaluca
Tech.
P02r-18
Regulacin de la expresin gnica por Wnt3A

en fibroblastos de colon humano
Nria Niell, Luis del Peso, Jos Manuel Gonzlez-Sancho
Bioqumica, Universidad Autnoma de Madrid, Madrid, ES
Las vas de sealizacin por factores Wnt son esenciales en muchos
procesos del desarrollo humano, y tambin para mantener la homeostasis
del tejido adulto. La va Wnt/-catenina o cannica es la mejor conocida,
aunque tambin existen vas no cannicas, independientes de -catenina.
En el colon, la va cannica tiene una importancia fundamental y aparece
desregulada en la prctica totalidad de cnceres colorrectales. Los factores
Wnt son secretados por los fibroblastos pericriptales y actan sobre las
clulas epiteliales de la mucosa colnica. Sin embargo, no se sabe si estos
factores actan tambin de forma autocrina sobre los propios fibroblastos
que los secretan, alterando su patrn de expresin gnica. Para intentar
resolver esta incgnita, nuestro grupo se propuso estudiar el efecto de
Wnt3A, que activa la va cannica, sobre la lnea de fibroblastos primarios
de colon CCD18-Co. Para ello utilizamos una tcnica de secuenciacin
masiva de alta resolucin (RNAseq), seguida de un anlisis bioinformtico.
El anlisis estadstico nos proporcion una lista de genes regulados
diferencialmente, de los que una muestra ha sido validada mediante qPCR
y western blot. Dos de los genes validados se regulan a tiempos cortos,
lo que sugiere un posible mecanismo transcripcional directo. Tambin
aparecen genes que codifican protenas de la propia va Wnt/-catenina, o
muy relacionadas con ella. De esta lista se han seleccionado varios genes
que sern sometidos a un estudio ms profundo con el fin de analizar el
mecanismo de su regulacin por factores Wnt y el efecto de sus cambios
de expresin sobre el fenotipo, proliferacin o capacidad tumorognica de
los fibroblastos de colon.
P02-19
Cocoa enriched-diet attenuates loss of beta

cell mass and function in Zucker diabetic rats
by preventing beta cell oxidative stress and
apoptosis
Elisa Fernndez-Milln1, Isabel Cordero-Herrera2, Sonia Ramos2,
Fernando Escriv3, Carmen Alvarez3, Luis Goya2, Mara Angeles
Martn3
1
CIBER de Diabetes y Enfermedades Metablicas (CIBERDEM),
Madrid, ES, 2Dpto. de Metab. y Nutr., ICTAN (CSIC), Madrid,
ES, 3Dpto. Bioq. y Biol. Mol. II, Fac. Farmacia, UCM - CIBER de
Diabetes y Enfermedades Metablicas (CIBERDEM), Madrid, ES
Oxidative stress has been largely implicated in the pathogenesis of
pancreatic beta cell failure observed in type 2 diabetes (T2D). Consequently,
identification of natural antioxidant compounds that enhance or preserve
Granada 2014
islet beta cell mass and function is considered an emerging strategy to
prevent or treat T2D [1]. We have recently showed that flavanol epicatechin
and microbial-derived flavonoid metabolites from cocoa may have antidiabetic potential by promoting survival and function of pancreatic beta
cells in vitro [2]. However, it is unknown whether this effect can be
achieved in vivo. Therefore, in this work we investigated the effect of a
cocoa-rich diet preventing -cell deterioration in an animal model of T2D
and the mechanisms involved.
Male Zucker diabetic (ZDF) rats were fed control or 10% cocoa enriched
diets in the pre-diabetic state (from 6 to 14 weeks of life) and Zucker lean
(ZL) rats were used as control. Glucose tolerance test (GTT), beta cell
mass, beta cell apoptosis and pancreatic markers of apoptosis and oxidative
stress were evaluated. Animals fed with cocoa rich diet improved fasting
hyperglycemia, hyperinsulinemia and GTT. Besides, histopathological
study of the pancreas of these animals indicated that cocoa feeding
preserved beta cell mass and prevented beta cell apoptosis. Finally, we
showed that cocoa diet enlarged the pancreatic concentration of enzymatic
and non-enzymatic endogenous antioxidant defences preventing thus
oxidative stress in ZDF rats.
These findings provide the first in vivo evidence that a cocoa-rich diet may
delay the development of T2D in the ZDF rat by preventing pancreatic
oxidative stress and beta cell apoptosis. Therefore, cocoa flavonoids
could be considered as candidates to ameliorate hyperglycemia and delay
deterioration of beta cell mass and function in T2D.
References
[1] Robertson, RP. Theraphy Discov Med 9:132-137 (2010)
[2] Fernndez-Milln et al. Food Chem Toxicol 66:245-253 (2014)
Psters
P02-21
Las clulas madre neurales (hNSC) un modelo

para el estudio del papel de los polimorfismos
mitocondriales en la enfermedad de Parkinson
Eldris Iglesias, Laura Llobet, Alba Pesini, Sonia Emperador, Ester
Lpez-Gallardo, Julio Montoya, Eduardo Ruiz-Pesini
Universidad de Zaragoza-CIBERER, Zaragoza, ES
La enfermedad de Parkinson (PD) es una enfermedad neurodegenerativa
causada por la destruccin de las neuronas dopaminrgicas. Muchos
factores se han relacionado con esta enfermedad, tales como mutaciones
en genes nucleares (nDNA), algunos de los cuales estn implicados en
la replicacin y expresin del DNA mitocondrial (mtDNA), mutaciones
directamente en el mtDNA y, finalmente, la accin de diversos xenobiticos
que causan la disfuncin del sistema de fosforilacin oxidativa (OXPHOS)
muchos de cuyos componentes estn codificados en el mtDNA. Por lo
tanto, el mtDNA parece jugar un papel importante en la PD.
El desarrollo de un modelo de clulas neuronales dopaminrgicas estable
y fiable es especialmente necesario para el estudio de la patognesis de
esta enfermedad y el desarrollo de estrategias teraputicas. Las clulas
madre neurales (hNSC) constituyen clulas euploides con potencial para
diferenciarse en neuronas dopaminrgicas. En este trabajo presentamos la
caracterizacin gentica de genes nucleares y mitocondriales asociados a
PD y los cambios genticos moleculares y bioqumicos que se producen
tras la diferenciacin a neuronas, en particular en relacin al sistema
OXPHOS.
Financiacin: ISCIII; FIS 10-00662, 11-01301; CIBERER.
Acknowledgments: Grants AGL201017579, CSD2007-00063, BFU201125420 from Ministerio de Economa y Competitividad (MINECO) and
CIBER de Diabetes y Enfermedades Metablicas Asociadas (CIBERDEM,
ISCIII) Spain.
P02r-20
DICKKOPF-1 (DKK-1) reduce el potencial

tumorognico de clulas de sarcoma de Ewing
Lorena Paule, Nria Niell, Luis del Peso, Jos Manuel GonzlezSancho
Bioqumica, Universidad Autnoma de Madrid, Madrid, ES
El sarcoma de Ewing es la segunda causa de cncer de hueso en nios
y adultos jvenes. Se caracteriza por la presencia de translocaciones
cromosmicas, que en aproximadamente el 90% de los casos ocurren entre
los cromosomas 11 y 22 y generan la protena de fusin EWS/FLI1, que
es un factor de transcripcin aberrante con propiedades oncognicas. Los
factores Wnt activan, entre otras, la va de sealizacin Wnt/-catenina que
se encuentra desregulada en numerosos cnceres humanos. Nuestro grupo
ha demostrado previamente que esta va est constitutivamente inhibida en
sarcoma de Ewing debido a la accin de EWS/FLI1. Asimismo, la expresin
de DICKKOPF-1 (DKK-1), un inhibidor extracelular de la va Wnt/catenina y a la vez gen diana de la misma, es inhibida transcripcionalmente
por EWS/FLI1. En este trabajo hemos reexpresado DKK-1 en cuatro
lneas celulares de sarcoma de Ewing con el fin de determinar la
relevancia funcional de su silenciamiento en esta neoplasia. Experimentos
de proliferacin y supervivencia usando MTS no han mostrado un
efecto significativo de DKK-1 sobre el crecimiento y la sensibilidad a
quimioterpicos (vincristina y doxorubicina, habituales en el tratamiento
de este tumor) de las lneas A673, TC-71, A4573 y TTC-466 de sarcoma
de Ewing. Por el contrario, experimentos en ratones inmunodeprimidos
demostraron que la presencia de DKK-1 ralentiza el crecimiento de
tumores generados por clulas A673 y TC-71. En consecuencia, para
intentar determinar los genes responsables de este efecto antitumoral de
DKK-1 hemos realizado un estudio transcriptmico, mediante RNAseq,
que estamos analizando en la actualidad y que esperamos contribuya a
esclarecer el mecanismo de accin de este supresor tumoral.
P02r-22
Role of mitochondria ROS generation in ethanolinduced TLR4/NLRP3 inflammasome activation

and cell death in astroglial cells
Silvia Alfonso-Loeches, Juan Unea, M Jose Morillo, Jorge Oliver-de
la Cruz, Consuelo Guerri Sirera
Toll-like receptors (TLRs) and Nod-like receptors (NLRs) are innate
immunity sensors that provide an early/effective response to pathogenic
or injury conditions. We have reported that ethanol-induced TLR4
activation triggers signaling inflammatory responses in glial cells, causing
neuroinflammation and brain damage. However, it is uncertain if ethanol
is able to activate NLRs /inflammasome in astroglial cells, which is the
mechanism of activation, and whether there is crosstalk between both
immune sensors in glial cells. Here we show that chronic ethanol treatment
increases the co-localization of caspase-1 with GFAP+ cells, and upregulates IL-1 and IL-18 in the frontal medial cortex in WT, but not in
TLR4 knock-out mice. We further show that cultured cortical astrocytes
expressed several inflammasomes (NLRP3, AIM2, NLRP1 and IPAF),
although NLRP3 mRNA is the predominant form. Ethanol, as ATP and
LPS treatments, up-regulates NLRP3 expression, and causes caspase-1
cleavage and the release of IL-1 and IL-18 in astrocytes supernatant.
Ethanol-induced NLRP3/caspase-1 activation is mediated by mitochondrial
(m) ROS generation because when using a specific mitochondria ROS
scavenger, the mito-TEMPO (500 mM) or NLRP3 blocking peptide (4mg/
ml) or a specific caspase-1 inhibitor, Z-YVAD-FMK (10 mM), abrogates
mROS release and reduces the up-regulation of IL-1 and IL-18 induced
by ethanol or LPS or ATP. Confocal microscopy studies further confirm
that ethanol, ATP or LPS promotes NLRP3/caspase-1 complex recruitment
within the mitochondria to promote cell death by caspase-1-mediated
pyroptosis, which accounts for 73 % of total cell death (22%) and the
remaining (25%) die by caspase-3-dependent apoptosis. Suppression of
the TLR4 function abrogates most ethanol effects on NLRP3 activation
and reduces cell death. These findings suggest that NLRP3 participates,
39
Psters
in ethanol-induced neuroinflammation and highlight the NLRP3/TLR4
crosstalk in the neuropathological processes associated with alcohol abuse.
Supported by SAF2012-33747; RED-RTA, ERAB, EA 13 08.
P02r-23
Role of the mineralocorticoid receptor in mouse

skin development
Julia Boix Tarn, Elena Carceller Zazo, Lisa Sevilla, Paloma Prez
Snchez
Instituto de Biomedicina de Valencia (CSIC), Valencia, ES
The mineralocorticoid receptor (MR) is a transcription factor that plays a
key role in ion and water homeostasis. Structurally and functionally it has
a high similarity with the glucocorticoid (GC) receptor (GR). Both MR and
GR are steroid ligand-dependent intracellular receptors that belong to the
nuclear hormone receptor superfamily.
MR can bind the natural ligand aldosterone and GCs with similar affinities.
Given that endogenous GC plasma levels exceed those of aldosterone by
100-fold, one mechanism preventing continuous MR occupation by GCs
is the enzyme 11 -hydroxysteroid dehydrogenase type 2 (11HSD2).
11HSD2 converts active GCs into inactive metabolites, unable to bind
MR, thus favoring the Aldosterone-MR signaling pathway.
It is known that GCs exert a critical role in skin function through GR.
However, no much is known regarding MR in skin biology. Previous work
demonstrated that the skin phenotype of mice overexpressing either MR or
GR in epidermal keratinocytes was highly similar. Both transgenic mouse
models featured atrophic skin, a reduced number of hair follicles, and
impaired epidermal maturation, suggesting that MR may play a role in skin
development, analogously to GR.
We have analyzed MR expression during mouse skin development at
the mRNA and protein level. Our data showed a transient peak of MR
expression around embryonic (E) day 16.5 (E16.5), which decreased
thereafter. Since epidermal barrier formation starts at E16.5, the observed
peak suggested a role for MR in this process.
To evaluate the consequences of MR inactivation in developing skin, we
generated MR knock-out mice using a strategy based upon generalized
Cre-mediated recombination of MRloxP/loxP mice. We examined MR+/+,
MR+/- and MR-/- mouse skin at different embryonic and early postnatal
stages. Histopathological analysis of skin samples revealed no major
defects of MR-/- mice but rather subtle differences in the expression of
keratinocyte-specific markers. Collectively, our findings indicate that i)
MR is expressed at relatively low levels during normal skin development,
ii) its constitutive ablation does not have a major impact for skin function
at the perinatal age, and iii) GR may functionally compensate for MR loss
during mouse skin development.
P02r-24
Effects of corticosteroids in keratinocytes in vivo

and in vitro
Elena Carceller Zazo, Julia Boix Tarn, Lisa Sevilla, Paloma Prez
Snchez
Instituto de Biomedicina de Valencia (CSIC), Valencia, ES
Synthetic glucocorticoids (GCs) are effectively and largely used in therapy
for skin inflammatory diseases, however, chronic treatments or high doses
produce common undesirable side effects, such as skin atrophy.
The biological and therapeutical effects of endogenous and synthetic
GCs are mediated by binding to the glucocorticoid receptor (GR), a
ligand-dependent transcription factor of the nuclear hormone receptor
superfamily. The mineralocorticoid receptor (MR) also belongs to this
superfamily and can bind both mineralocorticoids and GCs with similar
affinities. GR and MR regulate the gene expression through binding to
identical DNA sequences located at regulatory regions of their target genes,
making difficult to discriminate the specific actions of each receptor.
40

The aim of this study was to analyze the effects of GR and MR agonists in
keratinocytes by using in vitro and in vivo approaches.
Cultured keratinocytes were treated with the synthetic GC analog
dexamethasone (Dex) and the natural mineralocorticoid aldosterone
(Aldo), individually or combined, to evaluate their effects on gene
expression. The anti-inflammatory gene Tsc22d3/Gilz, a known GR- and
MR-target in other cell types, was up-regulated to a similar extent by each
ligand but the combined treatment showed no additive effects.
We also analyzed the effects of Dex, Aldo or Dex plus Aldo after topical
application to adult mouse dorsal skin for one week. Skin samples
were analyzed by histopathology by hematoxilin/eosin staining and
immunohistochemistry using specific epidermal markers. Whereas Dex
treatment in mice resulted in epidermal thinning, as previously described,
Aldo elicited no major effects. However, combined Dex plus Aldo treatment
partially counteracted the Dex-induced epidermal hypoplasia. Since these
approaches do not clarify which receptor is mediating Dex or Aldo effects,
and to unequivocally elucidate the role of each of these transcription factors
on keratinocyte function, we have performed experiments using GR- and
MR- keratinocyte-specific knock-out mice and cell lines.
P02-25
Generacin de knock-outs de FKTN y FKRP

en la lnea muscular C2C12 de ratn mediante
la tecnologa TALEN para el estudio de
distroglicanopatas
Marcos Rubio Fernndez1, Madalina Raducu1, Miriam Aza
Carmona1, Cristina Susn Lara1, Lourdes Yuste Prez1, Laura
Rodrguez Alonso1, Jos Martn Nieto2, Jess Cruces Pinto1
1
Departamento de Bioqumica, Facultad de Medicina, Universidad
Autnoma de Madrid, Instituto de Investigaciones Biomdicas
UAM-CSIC, IdiPAZ, Madrid, ES, 2Departamento de Fisiologa,
Gentica y Microbiologa, Facultad de Ciencias, Universidad de
Alicante, Alicante, ES
Mutaciones en los genes FKTN (fukutin) y FKRP (fukutin-related protein),
que codifican dos protenas muy relacionadas, son causantes de un
conjunto de enfermedades congnitas de carcter recesivo denominadas
actualmente como distrofias musculares-distroglicanopatas (MDDG, de
sus siglas en ingls). La falta de funcin de estas protenas conduce a la
hipoglicosilacin del alfa-distroglicano (-DG). Esta protena sirve de
anclaje de la clula a la matriz extracelular (MEC) en diferentes rganos y
tejidos, sobre todo msculo, cerebro y retina. El -DG sufre una importante
y masiva O-glicosilacin postraduccional en residuos de serina/treonina
de su conocido dominio mucina, generando diferentes tipos de cadenas
tanto de O-manosilglicanos como de O-N-acetil-galactosaminilglicanos. A
travs de estos residuos se realiza la interaccin con protenas de la MEC
como laminina, agrina y perlecano, as como con las protenas sinpticas
neurexina y pikachurina. Sin embargo, hasta la fecha, la funcin de estas
protenas FKTN y FKRP en la O-glicosilacin del -DG sigue siendo
desconocida. En un intento de elucidar sus funciones, hemos generado
una serie de mutantes knock-out (KO), tanto para FKTN como para FKRP
en la lnea celular C2C12 de mioblastos de ratn, utilizando la tecnologa
TALEN (transcription activator-like effector endonuclease). Un nmero
significativo de estos mutantes KO son defectivos en la O-glicosilacin del
-DG, tras ser analizados mediante western blotting y citometra de flujo,
utilizando anticuerpos contra las cadenas glicoslicas del -DG. El objetivo
final de este estudio es analizar mediante glicmica comparada los residuos
glicoslicos afectados en el -DG por la falta de funcin de ambas protenas
frente a la lnea celular C2C12 silvestre.
Financiacin: Instituto de Salud Carlos III, proyecto PI12/0157.
Granada 2014
P02-26
Bordetella pertussis adenylate cyclase toxin:

pore formation and resealing of injured plasma
membrane by endocytosis of the pore
David Gonzlez Bulln, Asier Etxaniz Iriondo, Kepa Belloso Uribe, Aitor
Etxebarria Gallego, Csar Martn Plgaro, Helena Ostolaza Etxabe
Departamento Bioqumica y Biologa Molecular, Unidad de Biofsica
(Centro Mixto, CSIC-UPV/EHU), Leioa, ES
The adenylate cyclase toxin (ACT), the causative agent of whooping
cough in humans, is an essential virulence factor secreted by the Gram
negative bacteria Bordetella pertussis. The toxin suppresses the bactericidal
activities of phagocytic cells due to its capacity to rapidly convert cellular
ATP into cAMP. This conversion is achieved by the translocation into the
target cell cytosol of the toxin N-terminal catalytic domain (AC domain,
aminoacids 1-400). Meanwhile, the toxin C-terminal domain (RTX domain,
approximately the last 1300 residues), which mediates toxin binding to the
target cell membrane, remains in the cell membrane forming cation-selective
pores, which permeabilize cell membranes perturbing ion homeostasis
and inducing both haemolysis and cytolysis of nucleated cells. Here we
provide evidence that ACT pores formed by the toxin C-terminal portion are
internalized from the cell membrane. In sum, our findings uncover a linkage
between cell permeabilisation by ACT and how cells protect themselves
from a microbial toxin insult and may survive, and provide new valuable
insights to the current understanding of ACT action on target cells.
P02r-28
Asociacin de polimorfismos de VAV2 y VAV3 con

factores de riesgo cardiovascular inducidos por
hipertensin y diabetes
Nuria Perretta Tejedor1, Cristina Cuesta2, Jos Manuel Muoz Flix2,
Luis Garca-Ortiz3, Manuel A Gmez Marcos3, Jos Ignacio Recio
Rodrguez3, Rogelio Gonzlez-Sarmiento4, Jos Miguel LpezNovoa2, Carlos Martnez-Salgado5
1
Sofa de Investigacin Nefrolgica, Universidad de Salamanca,
Salamanca, ES, 2Unidad de Fisiopatologa Renal y Cardiovascular,
Instituto Reina Sofa de Investigacin Nefrolgica, Universidad
de Salamanca - IBSAL, Hospital Universitario de Salamanca,
Salamanca, ES, 3IBSAL, Hospital Universitario de Salamanca
- Unidad de Investigacin, Centro de Salud La Alamedilla ,
Salamanca, ES, 4Centro de Investigacin del Cncer, Universidad
de Salamanca - IBSAL, Hospital Universitario de Salamanca ,
Salamanca, ES, 5Unidad de Fisiopatologa Renal y Cardiovascular,
Instituto Reina Sofa de Investigacin Nefrolgica, Universidad
de Salamanca - IBSAL, Hospital Universitario de Salamanca IECSCYL, Unidad de Investigacin, Hospital Universitario de
Salamanca, Salamanca, ES
Hipertensin, diabetes y obesidad son factores de riesgo para el desarrollo
de enfermedades cardiovasculares, una de las principales causas de muerte
en el mundo. Hay pocos datos sobre la influencia de polimorfismos
genticos en estas enfermedades. Los factores intercambiadores de guanina
VAV2 y VAV3 juegan un papel importante en la homeostasis vascular in
vivo. Por ello, hemos evaluado la asociacin de los polimorfismos de VAV2
(rs 602990) y VAV3 (rs7528153) con la predisposicin al riesgo y dao
cardiovascular inducido por hipertensin y diabetes.
Hemos extrado DNA de sangre perifrica de 384 pacientes (152 hipertensos,
66 diabticos y 166 no diabticos no hipertensos). Los polimorfismos
fueron detectados mediante qPCR con sondas TaqMan. Hemos analizado
en esos mismos pacientes la presin sistlica y diastlica, presin de pulso,
glucemia basal, disfuncin endotelial, retinopata, hipertrofia ventricular
izquierda y riesgo cardiovascular.
El genotipo CT del polimorfismo de VAV3 Sher298Thr est asociado con
un riesgo reducido de desarrollar hipertensin, diabetes, obesidad y dao
Psters
cardiovascular (p=0.011, odds ratio (OR)=0.495, intervalo 0.313-0.784),
y el genotipo TT de este polimorfismo est asociado con un incremento de
la glucemia basal en pacientes no diabticos y no fumadores (p=0.043,
OR=3.700, intervalo 1.266-10.815). El polimorfismo de VAV2 Val584Met
est asociado con un aumento de la presin de pulso en pacientes fumadores
portadores del alelo TT (p=0.016, OR=5.538, intervalo 1.022-30.019) y con un
riesgo reducido de desarrollar sndrome metablico en pacientes no fumadores
portadores del alelo TT (p=0.05, OR=0,408, intervalo 0.180-0.927).
Nuestros resultados sugieren que el genotipo CT del polimorfismo de
VAV3 Sher298Thr est relacionado con un riesgo reducido de desarrollar
hipertensin, diabetes, obesidad y dao cardiovascular. Por otra parte,
variantes polimrficas de los genes VAV2 y VAV3 parecen estar asociados
con la presencia o ausencia de factores de riesgo cardiovascular inducidos
por hipertensin o diabetes.
P02-29
Estudio de expresin de marcadores proteicos de

la EMT en lneas celulares de cncer colorrectal
Sara Fernndez Mojn1, Rubn Lpez-Corts1, Laura Muinelo
Romay2, Emilio Gil Martn1, Almudena Fernndez Briera1
1
Grupo BB1, Departamento de Bioqumica, Gentica e Inmunologa,
Universidad de Vigo, Vigo, ES, 2Grupo de Oncologa Mdica
Traslacional, Complejo Hospitalario Universitario de Santiago de
Compostela, Santiago de Compostela, ES
La prdida del fenotipo epitelial y la adquisicin del mesenquimal, proceso
conocido como Transicin Epitelio-Mesnquima (EMT), es un evento
que lidera la progresin de los tumores epiteliales, otorgando a las clulas
tumorales un aumento de movilidad y potencial invasivo. Son muchos
los marcadores moleculares involucrados en este proceso, de entre los
cuales, nuestro inters radica en los productos de la accin de la enzima
(1,6)fucosiltransferasa [(1,6)FT], por su presumible implicacin en la
malignizacin del cncer colorrectal (CCR).
En este trabajo nos hemos propuesto monitorizar la expresin de varios
marcadores proteicos de la EMT en un panel de lneas celulares de CCR;
asimismo, hemos estudiado el efecto del silenciamiento del gen de la (1,6)
FT (FUT8) en las lneas isognicas Sw480 y Sw620. Una vez testado el xito
del silenciamiento por RT-qPCR, se ha procedido a valorar la expresin
mediante western blot de los marcadores de la EMT seleccionados.
Nuestra hiptesis reside en que la enzima (1,6)FT modula la expresin
y grado de funcionalidad de ciertas protenas de membrana vinculadas
a la EMT y, por ende, la progresin maligna de los tumores. Las lneas
Sw480 y Sw620 son un buen sistema modelo para testar esta hiptesis por
su origen respectivo en un tumor primario y una lesin metastsica del
mismo paciente.
Los resultados preliminares apuntan a que no parece haber una correlacin
entre estos clsicos biomarcadores de la EMT y el grado de malignidad
celular, lo que podra corresponderse con la posible heterogeneidad
genotpica y mecanstica de la carcinognesis colorrectal.
Estudio financiado mediante el Contrato-Programa CN 2011/024, Xunta
de Galicia.
P02-30
Anlisis de posibles protenas que interaccionan

con la fukutina
Marcos Rubio Fernndez1, Sandra Gutirrez Vindel1, Cristina Susn
Lara1, Lourdes Yuste Prez1, Jos Martn Nieto2, Jess Cruces
Pinto1
1
UAM-CSIC, IdiPAZ, Madrid, ES, 2Departamento de Fisiologa,
Gentica y Microbiologa, Facultad de Ciencias, Universidad de
41
Psters
La fukutina, una protena codificada por el gen FKTN, se ha relacionado
con un conjunto de enfermedades congnitas de carcter recesivo
denominadas actualmente como distrofias musculares-distroglicanopatas
(MDDG, de sus siglas en ingls). La falta de funcin de la fukutina conduce
a la hipoglicosilacin del alfa-distroglicano (-DG). Esta protena acta
como anclaje de la clula a la matriz extracelular (MEC) en diferentes
tejidos y rganos, sobre todo msculo, cerebro y retina. El -DG sufre
importantes y masivas O-glicosilaciones postraduccionales en residuos de
serinas/treoninas de su conocido dominio mucina, generando diferentes
tipos de cadenas tanto de O-manosilglicanos como de O-N-acetilgalactosaminilglicanos. A travs de estos residuos se realiza la interaccin
con protenas de la MEC como laminina, agrina y perlecano, as como
con las protenas sinpticas neurexina y pikachurina. Sin embargo, hasta
la fecha, la funcin de la fukutina en la O-glicosilacin del -DG es
desconocida.
En un intento de elucidar su funcin, en este trabajo hemos procedido
a sobreexpresar esta protena en la lnea celular humana HEK293T,
purificarla y analizar mediante espectrometra de masas las protenas
que coeluyen con ella. Hemos seleccionado una serie de protenas
potencialmente interesantes, centrndonos preferentemente el estudio de la
protena CMAS (citidina monofosfo-N-cido acetilneuramnico sintetasa).
Esta enzima cataliza la sntesis de CMP-Neu5Ac, un sustrato utilizado por
diferentes si aliltransferasas para la adicin de cido silico, el cual est
presente en muchas de las cadenas de O-glicanos del -DG.
Financiacin: Instituto de Salud Carlos III, proyecto PI12/0157.
P02-31 (R02-4)
Calpain-cathepsin crosstalk leads to

photoreceptor cell death in an animal model of
retinitis pigmentosa
Alberto M. Hernndez-Pinto1, Natalia Rodrguez-Muela2, Luca
Garca Ledo1, Patricia Boya1, Enrique J. de la Rosa1
1
Centro de Investigaciones Biolgicas - CSIC, Madrid, ES, 2Harvard
Department of Stem Cell & Regenerative Biology, Boston, US
Retinitis pigmentosa (RP) comprises a large group of inherited retinal
dystrophies that are clinically similar but genetically heterogeneous. At the
cellular level, loss of photoreceptor cells is frequently found in association
with the disease progression, and causes visual decline and blindness. The
cellular changes leading to photoreceptor cell death in the retina of animal
models, as well as of RP patients, are not well characterized. In this work,
we have analyzed the order of events leading to photoreceptor death in the
rd10 mouse, that carries a spontaneous mutation in the Pde6b gene coding
a misfunctional rod-specific cGMP phosphodiesterase. As a consequence,
intracellular cGMP levels are increased, inducing a sustained calcium
influx through the cGMP-gated channels.
In this study, we have measured the role of calcium influx in the activation
of caspases, calpains, and cathepsin throughout the neurodegenerative
process by flow cytometry, confocal microscopy and enzymatic assays. To
determine the role of calcium and cGMP in the photoreceptor death, we
have used a calcium ionophore (A23187) and a PDE6 inhibitor (zaprinast)
to simulate the rd10 phenotype in C57bl/6 mice (WT) retinal explants.
Rd10 retinas showed an early increase in the calcium levels followed by
an over-activation of type 2-calpains without changes in the levels of its
endogenous inhibitor calpastatin. Subsequently, exogenous activation of
cathepsin B and Lysosomal DNAse was detected in the retinas, suggesting
an impairment of the lysosomal system prior to cell death, measured
by TUNEL staining. Retinas treated with Zaprinast or A23187 partially
or totally mimicked this calcium-calpain-cathepsin pattern. Inhibition
of calpains or cathepsins in specific stages of the degenerative process
clearly rescued photoreceptors both in vitro andin vivo. Throughout all
this process, caspases remained inactive, suggesting an alternative nonapoptotic pathway to cell death in this model of RP.
42

P02-32
Desmosomal-plaque related genes and nonsmall-cell lung cancer: desciphering other

cellular functions
Joel Martn Padrn1, Laura Boyero Corral2, Miriam Yage Capilla2,
Pedro Medina Vico2, M Esther Frez Vidal3
1
University of Granada, Departement of Biochemistry and Molecular
Biology III - Centre for Genomics and Oncological Research-GENYO,
Granada, ES, 2University of Granada, Departement of Biochemistry
and Molecular Biology I - Centre for Genomics and Oncological
Research-GENYO, Granada, ES, 3University of Granada, Departement
of Biochemistry and Molecular Biology III, Granada, ES
Despite advances in early detection and standard treatment, Non-SmallCell Lung Cancer (NSCLC) is often diagnosed at an advanced stage and
carries a poor prognosis. Greater knowledge of the molecular origins and
progression of lung cancer may lead to improvements in the treatment and
prevention of the disease.
There is considerable evidence for the importance of desmosomes and their
constituents in cancer. A role has been proposed for these adhesion genes
in susceptibility to cancer and metastasis, and there is increasing evidence
that the modulation of desmosomes is an important step in the initiation
and invasiveness of human malignancies.
In addition to the well-known structural role of desmosome genes,
evidences such as their dual subcellular localization, the interaction with
single-stranded DNA and translation initiation factors, etc. suggest there is
a poorly understood role of these proteins in signaling pathways.
PKP1 is up-regulated in primary Squamous-cell lung cancer (SCC) tissues
suggesting a putative contribution in tumorigenesis. In order to gain
greater insight into the multifuntional role of some desmosomal proteins
in NSCLC tumours, we have investigated transient inhibition of the PKP1
desmosomal plaque protein in several SCC lines of lung cancer in order
to unveil role of desmosomal-plaque proteins in NSCLC carcinogenesis.
PKP1 knockdown suppressed proliferation, produced cell cycle arrest and
an increment in apoptosis. Evidence suggests potential pro-oncogenic
functions of PKP1 desmosomal-plaque protein, probably due to an
unknown regulatory role in the nucleus, where it has also been found.
P02-33
Perfil de MicroRNA en respuesta al tratamiento

con doxorubicina en cncer de mama
Eduardo Tormo1, Begoa Pineda1, Eva Serna2, Sandra Ballester1,
Octavio Burgus3, Ana Lluch4, Pilar Eroles1
1
Instituto de investigacin INCLIVA, Valencia, ES, 2Unidad Central
de Investigacin, Universidad de Valencia-INCLIVA, Valencia, ES,
3
Departamento de Patologa, Hospital Clnico Universitario de
Valencia, Valencia, ES, 4Departamento de Hematologa y Oncologa,
Hospital Clnico Universitario de Valencia, Valencia, ES
Antecedentes: El tratamiento con quimioterapia es la norma en los
cnceres de mama triple negativo (CMTN), un subgrupo de cncer que
carece de una diana especfica. Se han observado claramente dos tipos de
respuesta: respuesta patolgica completa o recada en los tres primeros
aos tras el tratamiento. Sin embargo, los mecanismos que conducen a
estas respuestas, as como los marcadores que permitan la identificacin y
la diferenciacin de estos grupos antes del tratamiento son desconocidos.
Por otra parte, existen evidencias de una expresin aberrante de microRNA
en diferentes tipos de cncer, incluyendo cncer de mama. Los microRNA,
modulan la expresin de oncogenes y supresores de tumor y tambin estn
implicados en la promocin de resistencia a los medicamentos contra el
cncer.
Objetivo: Estudiar los microRNA que se expresan diferencialmente en
respuesta al tratamiento con doxorubicina en lneas de clulas de cncer
de mama.
Mtodos: Las lneas celulares de cncer de mama MDA-MB-231 y MDAMB-468 (CMTN) y MCF7 (luminal-A) fueron expuestas a tratamiento con
Granada 2014
doxorubicina. Se realiz un anlisis de microarrays (GeneChip miRNA 2.0
array) para identificar el conjunto de microRNAs comnmente modificados
y aquellos regulados de forma diferencial. Los genes y las vas reguladas
por estos microRNA fueron analizados in silico.
Resultados: Los anlisis de PCA y Heat Maps mostraron 13 microRNAs
modificados en las tres lneas, 25 en CMTN y 69 en la lnea luminal-A.
Este patrn est relacionado con el subgrupo de cncer de mama con ms
fuerza que con el tratamiento especfico. Curiosamente, las hebras star
de microRNA fueron modificadas en un porcentaje muy alto en todos los
grupos, y el anlisis terico de los genes diana revel que los procesos y
vas relacionadas con el cncer fueron los ms afectados. Entre las vas
destacan: uniones adherentes, adhesin focal, ERBB, MAPK, PI3K y la
va WNT, y entre los procesos celulares: proliferacin, supervivencia,
angiognesis, invasin y metstasis.
Conclusin: El perfil de microRNA en lneas celulares de cncer de mama
se modifica ampliamente por el tratamiento con doxorrubicina y las hebras
star de los microRNA juegan un papel fundamental. Los genes y las vas
sobre las que actan estn relacionados con procesos de cncer, por lo que
estos microRNA podran ser relevantes en la respuesta al tratamiento y en
el desarrollo de resistencias.
P02-34
Differential localization of desmosomal plaquerelated proteins in non-small-cell-lung cancer

Laura Boyero Corral1, Mercedes Gmez-Morales2, Joel MartnPadrn1, Pedro P. Medina1, Esther Frez-Vidal3
1
Centre for Genomics and Oncological Research-GENYO, and
Department of Biochemistry and Molecular Biology 1 - School
of Sciences - Granada University, Granada, ES, 2Department of
Pathology, School of Medicine, Granada University, Granada,
ES, 3Department of Biochemistry and Molecular Biology 3 and
Immunology, School of Medicine, Granada University, Granada, ES
Squamous cell carcinomas and adenocarcinomas are the most frequent
forms of lung carcinoma, and have different prognoses and therapeutic
approaches.
During recent years, accumulating evidence has supported an important
role for several junctional proteins in carcinogenesis, tumour invasion
and metastasis. Contact between epithelial cells is mediated by several
types of cellcell junction, which are composed of complex arrays
of transmembrane and plaque proteins and are connected typically to
cytoskeletal components. Desmosomes are cellcell complexes found
primarily in epithelial tissues.
Previously, our group showed differentially expressed gene sequences
corresponding to severl desmosomal plaque-related proteins as a function
of tumour type, stage and differentiation grade in non-small-cell-lung
cancer (NSCLC).
The staining pattern of some desmosomal proteins differed between
squamous-cell carcinomas and adenocarcinomas. Expression of these
proteins marked intercellular junctions that are characteristic of the
squamous stratum of stratified flattened epithelium and of neoplasias with
this type of differentiation. We observed more intense staining of these
proteins in better differentiated areas.
In order to gain greater insight into the specific function of some
desmosomal proteins in NSCLC tumours, we have investigated the
subcellular localization of one of these proteins in lung squamous-cell
carcinomas by immunocytochemistry.
PKP1 was localized on membrane and desmosomal plaque, and mainly on
the nucleus of the cells. PKP1 knock-down caused specific inhibition of
PKP1 on immunostained cells in comparison with scramble cells.
These results may suggest other functions of PKP1 in cellular regulation
and cancer. Our evidence indicates that PKP1 is a potential target for
diagnosis and treatment of NSCLC.
Psters
P02r-35
Interaccin funcional entre las vas de TGF-

y HGF durante la transicin epitelio-mesnquima
en clulas progenitoras hepticas
Mara Garca-lvaro1, Laura Almal1, Annalisa Addante1, Daniel
Caballero1, Celia Sequera1, Hctor Romero1, Jessica Segales2,
Carlos Snchez2, Csar Roncero1, Margarita Fernndez1, Eduardo
Rial2, Isabel Fabregat3, Blanca Herrera1, Arnzazu Snchez1
1
Instituto de Investigacin Sanitaria del Hospital Clnico San Carlos
(IdISSC) - Dep Bioqumica y Biologa Molecular II, Facultad de
Farmacia, Universidad Complutense de Madrid, Madrid, ES,2Centro
de Investigaciones Biolgicas, CSIC, Madrid, ES, 3Laboratori
dOncologia Molecular, Universitat de Barcelona, Institut
dInvestigaci Biomdica de Bellvitge, Barcelona, ES
Las clulas ovales son progenitores bipotenciales de hgado adulto que
se expanden en situaciones de dao crnico y son responsables de la
reparacin del dao heptico. Sin embargo, se ha postulado que son el
origen de un subgrupo de carcinomas hepatocelulares.
Para comprender los mecanismos subyacentes a su conversin maligna,
analizamos la potencial interaccin entre las vas de TGF- y HGF en el
contexto de un proceso de EMT. Para ello, hemos utilizado un modelo
in vitro de clula oval con un receptor Met inactivo (clulas Met-/-) y su
control (clulas Metflx/flx). El fenotipo mesenquimal estable (post-EMT) se
obtuvo mediante tratamiento crnico con TGF-.
Las clulas ovales post-EMT muestran una cintica de crecimiento
similar a la de sus parentales, un fenotipo ms inmaduro y una mayor
capacidad migratoria/invasiva. Adems, presentan alteraciones en la
sealizacin disparada por TGF- resistencia a la apoptosis y al estrs
oxidativo inducidos por esta citoquina. Este fenotipo resistente est
asociado a cambios significativos en el perfil de expresin de genes pro- y
anti-oxidantes. Adems, el perfil bioenergtico muestra una disminucin
en el ndice OCR/ECAR, sugiriendo la adquisicin de un fenotipo ms
glicoltico.
Curiosamente, la ausencia de Met resulta en un proceso de senescencia tras
la induccin de EMT, lo que sugiere que Met es necesario para la expansin
de estas clulas. Por otra parte, las clulas ovales post-EMT presentan una
sealizacin y respuesta al HGF alteradas.
En conclusin, nuestros resultados muestran una profunda alteracin en
el fenotipo de la clula oval y sus propiedades funcionales despus de la
EMT inducida por TGF-. Adems, apuntan a una interaccin funcional
relevante entre las vas de HGF y TGF- en este contexto.
P02-36
El dficit de IGF-I predispone a la prdida

auditiva inducida por ruido
Adelaida Celaya1, Lourdes Rodrguez-de la Rosa1, Julio Contreras2,
Isabel Varela-Nieto1
1
Instituto de Investigaciones Biomdicas Alberto Sols CSICUAM - Centro de Investigacin Biomdica en Red de Enfermedades
Raras-CIBERER, Madrid, ES, 2Instituto de Investigaciones
Biomdicas Alberto Sols CSIC-UAM - Centro de Investigacin
Biomdica en Red de Enfermedades Raras-CIBERER - Facultad De
Veterinaria, UCM, Madrid, ES
La deficiencia del factor de crecimiento similar a insulina tipo I (IGF-I)
causa sordera neurosensorial sindrmica en el hombre (ORPHA73272,
OMIM608747), situacin que se reproduce en ratones modificados
genticamente que no expresan este gen. El IGF-I es un agente neurotrfico
durante el desarrollo y neuroprotector en el adulto. Los niveles de IGF-I
circulante descienden de forma fisiolgica con el envejecimiento lo que se
ha asociado con deterioro cognitivo en el hombre.
Nuestro objetivo ha sido estudiar si el dficit parcial en este factor
predispone a la prdida auditiva y los mecanismos moleculares
responsables de la potencial respuesta agravada al dao. Para ello se ha
estudiado la susceptibilidad de ratones Igf1+/ y Igf1+/+ al dao causado
43
Psters
por exposicin excesiva al ruido a diferentes edades, utilizando tcnicas
neurofisiolgicas (potenciales auditivos del tronco cerebral), morfolgicas
(histologa coclear, cuantificacin estereolgica de clulas ciliadas) y
moleculares (RT-qPCR, Western blotting).
Los ratones de 1-3 meses de edad de ambos genotipos resultaron
igualmente sensibles al dao, por el contrario los ratones Igf1+/ de 6
meses de edad presentaron una mayor susceptibilidad al dao inducido
por ruido que los ratones Igf1+/+. Con respecto a los animales control, los
ratones Igf1+/ presentaron un incremento de umbrales auditivos acusado e
irreversible, una mayor prdida de clulas ciliadas, as como alteraciones
en las principales vas de sealizacin del IGF-I y en la expresin gnica
de marcadores celulares y moleculares de neuroinflamacin. En su
conjunto los resultados sugieren una mayor activacin de la respuesta
inflamatoria tras el trauma acstico en la situacin de dficit parcial en
IGF-I.
Estos resultados apoyan la idea de que bajos niveles de IGF-I predisponen
a una mayor susceptibilidad al dao inducido por ruido y contribuyen a
identificar las dianas moleculares que participan en este proceso. As, las
terapias basadas en IGF-I podran contribuir a prevenir o mejorar la prdida
auditiva inducida por ruido.
Agradecimientos: Este trabajo ha recibido el apoyo del SAF2011-24391,
de la Fundacin de Investigacin Mdica Mutua Madrilea 2012 y del
proyecto AFHELO (FP7, European Union). LRR disfruta de contrato del
CIBERER y AC de AFHELO.
P02-37 (R02-8)
Increased oxidative stress and impaired

antioxidant response in Lafora disease
Carlos Rom Mateo1, Carmen Aguado2, Jose Luis Garcia Gimenez3,
Jos Santiago Ibaez Cabellos4, Marta Seco Cervera3, Federico
Pallard3, Erwin Knecht2, Pascual Sanz5
1
Instituto de Biomedicina de Valencia (CSIC) y CIBERER, Valencia,
ES, 2Centro de Investigacin Principe Felipe y CIBERER, Valencia,
ES, 3FIHCUV-INCLIVA y CIBERER, Valencia, ES,4FIHCUV-INCLIVA
y Sistemas Genmicos S.A, Valencia, ES, 5Instituto de Biomedicina
de Valencia, CSIC y CIBERER, Valencia, ES
Lafora Disease (LD, OMIM 254780, ORPHA501) is a fatal
neurodegenerative disorder characterized by the presence of glycogenlike intracellular inclusions called Lafora bodies and caused, in the vast
majority of cases, by mutations in either EPM2A or EPM2B genes,
encoding respectively laforin and malin. In the last years, several reports
have revealed molecular details of these two proteins and have identified
several processes affected in LD, but the pathophysiology of the disease
still remains largely unknown. Since autophagy impairment has been
reported as a characteristic treat in both Lafora disease cell and animal
models, and since there is a link between autophagy and mitochondrial
performance, we sought to determine if mitochondrial function could
be also altered in those models. Using fibroblasts from LD patients,
deficient in laforin or malin, we found mitochondrial alterations,
oxidative stress and a deficiency in antioxidant enzymes involved in the
detoxification of reactive oxygen species (ROS). Similar results were
obtained in brain tissue samples from transgenic mice deficient in either
the EPM2A or EPM2B genes. Furthermore, in a proteomic analysis of
brain tissue obtained from Epm2b-/- mice, we observed an increase in a
modified form of peroxirredoxin-6, an antioxidant enzyme involved in
other neurological pathologies, thus corroborating an alteration of the
redox condition. These data support that oxidative stress produced by
an increase in ROS production and an impairment of the antioxidant
enzyme response to this stress play an important role in the development
of LD.
44

P02-38 (R02-2)
Adipose tissue as a regulator of metabolic

flexibility
Sam Virtue
Institute of Metabolic Science (IMS), Addenbrookes Hospital,
University of Cambridge, Cambridge, UK
The focus of this talk is on how disruptions in the ability of organisms
to appropriately store and release lipid can impact on whole organism
metabolic health. Both healthy mice and humans preferentially use
carbohydrate in the fed state and switch to utilising a greater degree of
lipid in fasted state. The ability to perform the switch between utilising
carbohydrate and lipid can be defined as metabolic flexibility. While
metabolic flexibility has been extensively studied in humans, data from
rodent models with alterations in metabolic flexibility are relatively rare.
The transcription factor Peroxisome Proliferator-Activated Receptor
(PPAR) is essential for adipogenesis. PPAR has been implicated in the
regulation of both carbohydrate and lipid metabolism and is therefore an
excellent candidate for controlling metabolic flexibility. PPAR2 is the
adipose tissue-specific isoform of PPAR and has greater transcriptional
activity than PPAR1. Despite the known role of PPAR as a target for
the thiazolidinedione class of anti-diabetic drugs, young mice (4 months
of age) lacking PPAR2 had surprisingly normal carbohydrate metabolism
based on glucose and insulin tolerance tests.
In this talk, data from mice lacking PPAR2 will be used to demonstrate
how reductions in metabolic flexibility can be detected in mice using
indirect calorimetry. The lower metabolic flexibility of the PPAR2 KO
mouse indicated that these animals may have impaired lipid handling, a
phenotype which was subsequently confirmed; with PPAR2 KO mice
having reduced rates of lipolysis, and dramatically reduced capacity for
whole-organism lipid clearance. Finally, the relationship between reduced
adipose tissue capacity for both lipid uptake and release and human
metabolic health will be discussed.
P02r-39
Generacin de clulas madre pluripotentes

inducidas (iPSC) para el estudio de
enfermedades mitocondriales
Teresa Galera Monge, Francisco Zurita Daz, Cristina Gonzlez
Pramos, Rafael Garesse Alarcn, M. Esther Gallardo Prez
UAM-CSIC - CIBERER, Madrid, ES
Las enfermedades mitocondriales (EM) constituyen un amplio grupo
de enfermedades genticas multisistmicas cuyo nexo de unin es una
disfuncin en el sistema de fosforilacin oxidativa. La falta de modelos
experimentales adecuados para el estudio de dichas enfermedades, ha
dificultado el avance en el desarrollo de nuevas terapias. En este sentido, la
reprogramacin de clulas somticas a clulas madre pluripotentes inducidas
(iPSC) mediante la expresin ectpica de cuatro factores de transcripcin,
OCT4, SOX2, KLF4 y c-MYC, ha abierto enormes posibilidades. Por
ello, el objetivo general de este trabajo ha consistido en la generacin
de iPSC a partir de fibroblastos de pacientes con EM para el estudio y
tratamiento potencial de las mismas. Para este fin, hemos establecido una
coleccin de fibroblastos procedentes de biopsias de piel de pacientes con
EM e individuos sanos. A continuacin, se han generado lneas de iPSC
a partir de dichos fibroblastos mediante el uso de vectores retrovirales o
mtodos no integrativos tipo virus Sendai. As, hemos generado 16 lneas
de IPSC portadoras de distintas mutaciones en genes mitocondriales
codificados en el DNA mitocondrial o nuclear asociadas a distintas EM:
Sndrome de Leigh, atrofia ptica sindrmica y EM producidas por
defectos de comunicacin intergenmica. Actualmente, estamos realizando
la confirmacin, molecular y funcional, de la pluripotencia de las lneas
generadas, para posteriormente, diferenciarlas y caracterizarlas en los tipos
celulares diana. La disponibilidad de iPSC como modelo experimental
Granada 2014
de las EM nos permitir mejorar el conocimiento de los mecanismos
fisiopatolgicos de estas enfermedades y podr suponer la apertura de
nuevas vas para la identificacin de tratamientos farmacolgicos y
de terapia celular.
P02-40
La leptina revierte parcialmente el efecto

Warburg en la lnea de cncer de mama MCF-7
Mara del Mar Blanquer-Rossell, Raquel Garca-Belmonte, Jordi
Oliver, Pilar Roca, Adamo Valle
Grupo Multidisciplinar de Oncologa Traslacional - IUNICS - Universitat
de les Illes Balears - Ciber Fisiopatologa Obesidad y Nutricin
El efecto Warburg consiste en la preferencia de las clulas tumorales por
el uso de la va glucoltica en detrimento de la fosforilacin oxidativa
mitocondrial. Al tratarse de una caracterstica metablica diferencial y
dada su importancia para sostener el elevado anabolismo del tumor en
crecimiento, la reprogramacin metablica se plantea como una diana
interesante en la lucha contra el cncer. La leptina es una hormona
producida por el tejido adiposo blanco con una importante funcin en
la homeostasia energtica. Se ha observado que la leptina aumenta la
proliferacin y supervivencia de las clulas de cncer de mama tanto in
vitro como in vivo. No obstante, se desconoce la influencia que la leptina
puede tener sobre el metabolismo de estas clulas. En este estudio nos
planteamos evaluar la influencia de la leptina sobre el efecto Warburg en
la lnea de cncer de mama MCF-7. Se analiz el consumo de oxgeno
mediante un electrodo tipo Clark, la produccin de lactato mediante un
mtodo enzimtico y los niveles de ATP mediante luminometra en clulas
tratadas con leptina en respuesta a distintas dosis de 2-deoxiglucosa
y oligomicina. Se analiz tambin mediante western blot los niveles
y el grado de fosforilacin de la AMPK, protena sensora del estado
energtico; y la actividad de la PDH mediante un ensayo enzimtico
con inmunocaptura. Los resultados indicaron que la leptina revierte
parcialmente el efecto Warburg en las clulas MCF-7, induciendo un
metabolismo ms aerbico y una mayor dependencia de la mitocondria
para la sntesis de ATP. No obstante, las clulas con leptina mostraron
una mayor capacidad para activar la gluclisis al inhibir la fosforilacin
oxidativa.
Financiado por CAIB-FSE, ISCIII (PI12/01827) y FEDER-Unin Europea
Una manera de hacer Europa.
P02r-41
Mutaciones y polimorfismos mitocondriales

en pacientes mexicanos con cncer de prstata
Victoria Edwina Campos Garca1, Sandra Viridiana Salgado
Hernndez1, Jorge Ramrez Salcedo2, Patricia Ramrez Noguera2,
Sandra Daz Barriga Arceo2, Rosalba Bonilla Snchez3, Sergio Ureta
Snchez4, Jos Francisco Montiel Sosa2
1
B.Q.D. Bioqumica Diagnstica, Departamento de Bioqumica y
Farmacologa Humana, Universidad Nacional Autnoma de Mxico,
Cuautitlan Izcalli, MX, 2PhD. en Ciencias Biomdicas, Instituto
de Fisiologa Celular, Universidad Nacional Autnoma de Mxico,
Distrito Federal, MX, 3Q.F.B. Departamento de Bioqumica y
Farmacologa Humana, Universidad Nacional Autnoma de Mxico,
Cuautitln Izcalli, MX, 4MD. Mdico Urlogo, Hospital Espaol,
Distrito Federal, MX
En Mxico, el cncer de prstata es un importante problema de salud
pblica con altos costes sociales, este tipo de neoplasia tiene una tasa
de mortalidad anual de 121.57 por cada 100 mil hombres. Los factores
predisponentes de dicha patologa son la edad y antecedentes familiares
positivos pero poco se sabe acerca de la etiologa de la enfermedad. Se
ha estudiado que las mutaciones a nivel ADN mitocondrial (ADNmt) en
Psters
pacientes con cncer de prstata (CaP), son prospectos a ser herramienta
til para la deteccin de la enfermedad. Actualmente en Mxico no se
haba realizado una investigacin profunda de las mutaciones en el
mtDNA asociadas al CaP. Analizamos tres muestras de tejido prosttico
obtenidos mediante una biopsia transrectal. Se amplific todo el genoma
mitocondrial mediante la tcnica de PCR punto final, de una muestra; las
dems muestras solo se amplificaron la regin MT-ND5. Posteriormente
se recurri a la tcnica de secuenciacin bidireccional y finalmente se
examin la presencia de variantes de la secuencia entre el tejido sano y
enfermo del mtDNA. Slo una muestra mostr dos mutaciones asociadas
al CaP, en el locus MT-CO1, alelo G6261A produciendo un cambio
de aminocido de manera non-syn:A-T y en el locus MT-ND5, alelo
C12705T cuyo cambio de aminocido es syn:I-I. Esta misma muestra
mostr mutaciones en el locus MT-ATP 6, alelo A9300G con un cambio
de aminocido non-syn:A-T asociada a la miopata y en el locus MTRNR1, alelo A663G con cambio de aminocido 12S rRNA relacionada al
riesgo de arterioesclerosis coronaria. Dichas variaciones son las primera
reportadas en Mxico. Dicha investigacin es apenas un prembulo
para entender el cncer de prstata, su inicio, progreso y desarrollo de
metstasis en pacientes mexicanos.
P02-42
Metabolomic analysis of serum samples from

diabetic patients with and without cardiovascular
disease and control subjects
Beatriz Garca-Fontana1, Caridad Daz Navarro3, Pedro
Rozas-Moreno4, Olga Genilloud3, Francisca Vicente Prez3,
Jos Prez del Palacio3, Sonia Morales-Santana1,2,
Manuel Muoz-Torres1
1
Unidad de Metabolismo seo-RETICEF, Servicio de Endocrinologa,
Instituto de Investigaciones Biomdicas de Granada, Hospital
Universitario San Cecilio, Granada, ES, 2Servicio de Protemica,
Fundacin para la Inverstigacin Biosanitaria de Andaluca
Oriental Alejandro Otero FIBAO, Granada, ES, 3Fundacin
MEDINA, Centro de Excelencia en Investigacin de Medicamentos
Innovadores en Andaluca - Parque Tecnolgico de Ciencias de la
Salud, Granada, ES, 4Servicio de Endocrinologa, Hospital General
de Ciudad Real, Ciudad Real, ES
Metabolomics is an emerging and powerful discipline that provides an
accurate and dynamic image of the phenotype of mammalian systems
through the study of endogenous and exogenous metabolites in cells, tissues
and biofluids. The aim of this project is to analyze the metabolomic profiles
obtained by mass spectrometry of serum samples from type 2 diabetes
mellitus (T2DM) patients with and without cardiovascular disease (CVD)
and control subjects, in order to find new molecular pathophysiological
processes related to the onset of disease as well as new biomarkers for
CVD and diabetes.
In this study we analyzed 9 serum samples from T2DM patients with
CVD, 10 serum samples from T2DM patients without CVD and 10
serum samples from healthy subjects. These samples were analyzed by
liquid chromatography coupled with high resolution mass spectrometry
(LC-HRMS). The raw LC-HRMS data were converted into mass peak
lists which were compared across cromatographic runs and subjected
to statistical analysis (t-test, fold change analysis, principal component
analysis and partial least square-discriminant analysis). Thus, the peaks
whose intensity levels vary significantly between different groups are
detected.
The principal component analysis (PCA) successfully clustered the samples
according to the aforementioned groups. PCA loading plot revealed that
phospholipids were the main discriminatory metabolites. The role of these
molecules as biomarkers and their involvement in glucose and insulin
pathways will also be discussed in this study.
45
Psters
P02r-43 (R02-6)
Dissecting the role of the epidermal growth factor

receptor (EGFR) in diethylnitrosamine-induced
hepatocarcinogenesis in mice
Daniel Caballero Daz1, Judit Lpez-Luque1, Alba Marn-Martnez1,
Adoracin Martinez-Palacin2, Mara Garca-lvaro2, Annalisa
Addante2, Mara Garca-Bravo3, Esther Grueso4, Jose-Carlos
Segovia3, Csar Roncero2, Margarita Fernndez2, Arnzazu
Sanchez2, Isabel Fabregat5
1
Bellvitge Biomedical Research Institute-IDIBELL, Barcelona,
ES, 2Dep. of Biochemistry and Molecular Biology II, School
of Pharmacy, Complutense University of Madrid and Instituto
de Investigacin Sanitaria del Hospital Clnico San Carlos,
IdISSC, Madrid, ES, 3Cell Differentiation and Cytometry Unit
- Hematopoietic Innovative Therapies Division, Centro de
Investigaciones Energticas, Medioambientales y TecnolgicasCIEMAT and Centro de Investigacin Biomdica en Red de
Enfermedades Raras-CIBERER - Advanced Therapies Mixed
Unit. CIEMAT/IIS Fundacin Jimnez Daz, Madrid, ES, 4Division
of Medical Biotechnology - Paul Ehrlich Institute, Langen, DE,
5
Bellvitge Biomedical Research Institute-IDIBELL - Department
of Physiological Sciences II, School of Medicine, University of
Barcelona, LHospitalet de Llobregat, ES
Hepatocellular carcinoma (HCC) is one of the most common cancers and
its incidence is increasing. Hepatocarcinogenesis is a multistep process
that involves genetic and environmental factors. It is necessary to elucidate
the interaction and cross-regulation of these factors that synergistically
contributes to HCC development. In HCC there is a disruption of the
balance between cell death and survival signaling pathways. Different
physiological pro-apoptotic molecules are down-regulated, or inactivated,
due to an over-activation of anti-apoptotic signals that impairs their
functions. At the same time, some pro-survival pathways are over-activated.
One of these de-regulated survival signals is the epidermal growth factor
receptor (EGFR) pathway. On the one hand, different EGF-like ligands
are over-expressed in HCC, contributing to EGFR activation during HCC
progression. On the other hand, it has been shown that human HCC tissues
over-express EGF family receptors.
To investigate the role of the EGFR in the process of hepatocarcinogenesis our
group generated a transgenic mice that expressed specifically in hepatocytes
a truncated form of the EGFR (Alb1-EGFR). These mice were treated with
diethylnitrosamine (DEN) to induce liver tumorigenesis. By histological
techniques the livers of the mice were analyzed at different times after
treatment with DEN. Transgenic mice showed a delay in the appearance and
in the growth of tumors, as well as less inflammation and less extracellular
matrix deposition in liver parenchyma. The results suggest that the EGFR
pathway plays an important role in the process of hepatocarcinogenesis.
Further work is required to elucidate the molecular mechanisms by which
EGF contributes to liver tumor cell growth and survival.
P02r-44
Associating the decrease on neuronal viability

induced by amyloid-beta 1-40 and 1-42 with
reactive oxygen species production and the
disregulation of the expression of synaptic
proteins
Marta Domnguez-Prieto, Ana Velasco, Jos M. Medina
Universidad de Salamanca, INCYL (Instituto de Neurociencias de
Castilla y Len), Salamanca, ES
Amyloid-beta (A) accumulation together with tau protein
hyperphosphorylation are the main molecular events presumably responsible
for neurodegeneration observed in Alzheimers disease. In this work, we
studied the effect of three A peptides: A 25-35, A 1-40, A 1-42, on
cell viability and reactive oxygen species (ROS) production in rat neurons
46

in primary culture. Our results showed that the three peptides significantly
decreased neuronal viability, but only A 25-35 and A 1-42 produced
a significant increase on ROS production. In addition, we performed
immunohistochemistry against A in order to observe its localization within
the neuron, showing that the distribution pattern differed depending on the
A peptide, A 25-35 being internalized into neurons but A 1-40 and A
1-42 remaining mostly outside the cells. In order to get inside the mechanism
involved in the cell damage induced by A peptides, we analized changes in
the expression of synaptic proteins, such as synaptophysin, synaptotagmin
and PSD-95, by western-blot. Our results showed the occurrence of a
disregulation on the expression of these proteins. Since the changes in ROS
production cannot fully explain the decrease on cell viability observed in our
experiments, we propose that the disruption of synaptic structure must be
involved in neuronal death induced by amyloid-beta.
P02r-45
Bisphenol A exposure during adulthood alters the

expression of tryptophan hydroxylase type II but
not type I in the prefrontal cortex of rat: Possible
alterations in local serotonin synthesis
Beatriz Castro Bohrquez, Pilar Snchez Medina, Jess Manuel
Torres De Pinedo, Esperanza Ortega Snchez
Departamento de Bioqumica y Biologa Molecular III e
Inmunologa, Universidad de Granada, Granada, ES
Background: Bisphenol A (BPA) is able to produce neurological and
behavioral dysfunctions by mechanisms that remains unknown. Serotonin
(5-HT) system is essential for prefrontal cortex (PFC) function, an important
area for cognitive control and complex behaviors. Alterations in precortical
serotonergic signalling have been implicated in the pathogenesis of a wide
range of neuropsychiatric disorders. Tryptophan hydroxylase (Tph), which
is expressed as two isozymes (Tph1 and Tph2), catalyzes the rate-limiting
step in 5-HT synthesis and is considered one of the leading target genes for
psychiatric and behavioral disorders. Tph2 is specifically expressed in the
brain, whereas Tph1 is responsible for 5-HT synthesis in peripheral tissues.
Objective: Our objective was to evaluate the effects of adult exposure to
BPA on precortical Tph isozymes expression.
Methods: Adult male and female Wistar rats were inyected subcutaneously
during 4 days with 50 g/kg/day of BPA, the current Environmental
Protection Agency (EPA) reference dose. At 30 min after the final
administration, rats were euthanized and the brains were stored at -80C
until analysis. Quantitative RT-PCR was used to quantify mRNA levels of
Tph1 and Tph2 in the PFC. Protein levels were quantified by Western blot.
Results: BPA increased mRNA and protein levels of Tph2 in the PFC of
both male and female rats. No significant differences in Tph1 expression
were observed after BPA treatment.
Conclusion: We demonstrated for the first time that BPA, at a dose
considered safe by the EPA, modifies Tph2 expression in the PFC of adult
rats. Consequently, local synthesis of 5-HT might be altered. Given the role
of 5-HT in several psychopathologies, further studies will be required to
determine the impact of this finding.
P02-46
Dissecting the role of the epidermal growth factor

receptor (EGFR) in the molecular mechanisms
that control liver regeneration in mice
Judit Lpez-Luque1, Adoracin Martnez-Palacin2, Eva CrosasMolist1, Joaquim Moreno-Cceres1, Daniel Caballero-Daz1,
Mara Garca-Bravo3, Esther Grueso4, Jose-Carlos Segovia3, Csar
Roncero2, Margarita Fernndez2, Arnzazu Snchez2, Isabel
Fabregat5
1
Bellvitge Biomedical Research Institute-IDIBELL, Hospitalet
de Llobregat, ES, 2Dep. of Biochemistry and Molecular Biology
Granada 2014
II, School of Pharmacy, Complutense University of Madrid and
Instituto de Investigacin Sanitaria del Hospital Clnico San
Carlos-IdISSC, Madrid, ES, 3Cell Differentiation and Cytometry
Unit. Hematopoietic Innovative Therapies Division, Centro de
Investigaciones Energticas, Medioambientales y TecnolgicasCIEMAT and Centro de Investigacin Biomdica en Red de
Enfermedades Raras-CIBERER - Advanced Therapies Mixed
Unit - CIEMAT/IIS Fundacin Jimnez Daz, Madrid, ES, 4Division
of Medical Biotechnology - Paul Ehrlich Institute, Langen, DE,
5
Bellvitge Biomedical Research Institute-IDIBELL - Department
of Physiological Sciences II, School of Medicine, University of
Barcelona, Barcelona, ES
Liver regeneration (LR) is a very complex and well-orchestrated
phenomenon. The proliferation of hepatocytes is the main mechanism
during LR, in response to mitogenic signals. Among them, epidermal
growth factor (EGF) and hepatocyte growth factor (HGF) play essential
roles. However, additional studies are necessary to understand if EGF and
HGF pathways compensate one to each other in some aspects or if, on
the contrary, they play unique roles during LR. Moreover, Ttansforming
growth factor-beta (TGF-) is important at the end of LR as it inhibits
proliferation and maintains the liver in its correct size.
To elucidate the specific role of the EGFR pathway during LR after
partial hepatectomy (PH) in mice, we have generated a transgenic mouse
expressing a truncated form of the EGFR (Alb1-EGFR) specifically in
hepatocytes. The regenerative process after 2/3 hepatectomy in transgenic
mice (EGFR) was compared with that of wt mice (WT). Lack of EGFR
kinase activity correlated with a delay in proliferation in regenerating
hepatocytes, coincident with a higher activation of the TGF- pathway.
EGFR mice also showed higher basal inflammation and differences in lipid
metabolic changes during LR, being not able to induce the transient lipid
accumulation in hepatocytes which precedes the peak of the proliferative
phase. In spite of this delay in proliferation, EGFR mice fully regenerated
the liver, which means that other signaling pathways can be compensating
the lack of EGFR signaling. In this line of evidence, HGF levels and Met
(HGF receptor) phosphorylation were higher in EGFR animals after PH.
In summary, EGF plays diverse roles in liver after PH, but attenuation of its
signaling is not crucial for the final regeneration of the liver.
P02-47
Identificacin de una firma de miRNA asociada

a la recurrencia temprana en cncer de mama
Lus G. Prez-Rivas1, Jos M. Jerez2, Rosario Carmona3, Vanessa
de Luque1, Lus Vicioso4, M Gonzalo Claros3, Enrique Viguera5,
Bella Pajares1, Alfonso Snchez1, Nuria Ribelles1, Emilio Alba1,
Jos Lozano6
1
Laboratorio de Oncologa Molecular, Instituto de Biomedicina
de Mlaga-IBIMA, Mlaga, ES, 2Dpto. Lenguajes y Ciencias de la
Computacin, Universidad de Mlaga-UMA, Mlaga, ES,3Plataforma
Andaluza de Bioinformtica, Universidad de Mlaga-UMA, Mlaga,
ES, 4Servicio de Anatoma Patolgica, Instituto de Biomedicina de
Mlaga-IBIMA, Mlaga, ES, 5Dpto de Biologa Celular, Gentica
y Fisiologa Animal, Universidad de Mlaga, Mlaga, ES, 6Dpto.
Biologa Molecular y Bioqumica, Universidad de Mlaga-UMA,
Laboratorio de Oncologa Molecular, Instituto de Biomedicina de
Mlaga-IBIMA, Mlaga, ES
La aparicin de metstasis es un evento frecuente en pacientes operadas
de cncer de mama y suele estar asociado a una mayor mortalidad. El
anlisis de largas series de pacientes con amplio seguimiento ha puesto
de manifiesto que la probabilidad de recada no es constante en el tiempo,
sino que sigue un patrn bimodal con un primer pico de incidencia a los
18-24 meses tras la ciruga (recurrencia temprana), seguido de un segundo
pico de menor magnitud a los 60 meses aprox. (recurrencia tarda); pasado
este periodo, el riesgo de recada disminuye drsticamente. Por tanto, son
necesarios marcadores moleculares que permitan detectar con precisin
aquellas pacientes que presentan mayor riesgo de recurrencia temprana y
Psters
diferenciarlas de aquellas con menor probabilidad de desarrollar metstasis.
Con este propsito, hemos analizado tumores procedentes de 71 pacientes
operadas de cncer de mama atendiendo a los patrones de expresin de
1106 microRNA (miRNA), pequeas molculas de RNA implicadas en
el control de la expresin gnica y que suelen encontrarse desreguladas
en varias enfermedades, incluyendo cncer. Se ha identificado y validado
un grupo de cinco miRNA cuya expresin est disminuida en tumores
de pacientes con recurrencia temprana y menor supervivencia libre de
progresin. Notablemente, esta firma de miRNA permite discriminar
eficientemente entre pacientes con bajo y alto riesgo de recada temprana.
El anlisis de posibles mRNA dianas empleando la informacin existente
en varias bases de datos pblicas sugiere una relacin entre estos cinco
miRNA y la capacidad proliferativa y pro-angiognica del tumor.
P02-48
Maslinic acid is able to inhibit the

osteoblastogenesis and adipogenesis in two
different cell lines
Eva E. Rufino Palomares1, Amalia Prez-Jimnez2, Fernando J.
Reyes-Zurita1, Leticia E. Garca-Salguero1, Juan Peragn3, Pedro P.
Medina1, Jos A Lupiez1
1
Departamento de Bioqumica y Biologa Molecular I, Universidad
de Granada, Granada, ES, 2Biomaslinic, Enterprise, S.L, Granada,
ES, 3Departamento de Biologa Experimental, Universidad de Jan,
Jan, ES
Maslinic acid (MA) is a pentacyclic triterpene acid widely present in
dietary plants and especially abundant in olive fruit skins. Thecompound
has attracted much interest owing to its provenpharmacologic safety and
its many biologic activities, such asanti-inflammatory, antiviral, antioxidant
and antidiabetogenic activities as well as anticancer and antiastrocytoma
properties. Moreover, some studies showed that this compound increase
growth performance in animals. In this study, the MA effects on adipogenesis
and osteoblastogenesis was determined in order to analyze some of the
most important parameters related to obesity and metabolic syndrome.
Results obtained indicated that the MA had a significant inhibitory effect
on adipogenesis in the model pre-3T3-L1 adipocytes. This result coincided
with the antagonistic effect of the compound on PPAR and increased Ca2+
cytoplasmic levels in presence of MA.Taken together, these results suggest
that this compound inhibits the formation of adipose tissue.Furthermore,
it was also observed that the MA slightly increased glucose uptake in preadipocytes 3T3-L1. This suggests that this compound might exert a beneficial
effect in states of insulin resistance or hyperglycemia. The results alsoshowed
that MA, in human mesenchymal cells (hMSCs) derived from bone marrow,
inhibited the adipogenesis, confirming its potential as anti-obesity and
remodeling of body fat composition agent. Parallel, osteoblastogenesis in
hMSC cells was analyzed, finding a weak induction of molecular markers
that may be indicative of that MA positively affect bone regeneration.
This study has been supported by the Feder-Interconecta Program by means
of an investigation contract between University of Granada Foundation,
Bio-157 research group and Biomaslinic S.L. company.
P02-49
Cambios en el reclutamiento de la subunidad

ribosomal 40S por poblaciones de herogeneidad
gentica creciente del ARN del virus
de la hepatitis C
Samuel Prieto Vega1, Celia Perales2, Sunnie R Thompson3, Raquel
Romero Garca4, J.I. Esteban5, Esteban Domingo2, Jordi Gmez
Castilla6
1
Instituto de Biomedicina Lpez Neyra (CSIC), Armilla, ES,
2
CBMSO Madrid, Madrid, ES, 3Dpt. Microbiology UAB Alabama,
47
Psters
Birmingham Alabama, US, 4Hospital Universitario Puerta del Mar,
Cdiz, ES, 5Institut de Recerca Hospital Universitari Vall Hebron,
Barcelona, ES, 6Instituto de Biomedicina Lpez Neyra (CSIC),
CIBERehd, Armilla, ES
La regin genmica 5 del virus de la hepatitis C contiene una zona de
unin al ribosoma. Este dominio y sus flancos son ricos en elementos
estructurales (estructuras 1, 2 y 3) identificados in vitro por factores
bioqumicos y biofsicos: interaccin con micro ARN heptico miR-122,
sensibilidad a RNAsas dependientes de estructura (RNAsa III y P), luz
UV-c. Se obtuvieron poblaciones heterogneas de ARNin vitro por PCR
mutagnica y transcripcin in vitro y en un cultivo, creciendo el virus
en presencia de ribavirina. Evaluamos el efecto de la variabilidad en
poblaciones de ARN (HCV1-540) y de heterogeneidad gentica creciente,
en los motivos simples a los distintos niveles estructurales y evaluamos
los cambios en la unin al ribosoma. El efecto de la heterogeneidad
poblacional en el reconocimiento de los distintos factores vari a los
distintos niveles estructurales, siendo especialmente perjudicial para la
unin a la secuencia de miR-122 (estructura 1) y en el procesamiento por
la RNasa P en la estructura tipo tRNA (estructura 3). El efecto sobre la
RNasa III que reconoce ARN de doble cadena (estructura 2), fue menor,
pero permiti identificar un cambio conformacional inducido por el
incremento mutacional, validado en geles nativos y con RNasas de simple
y doble cadena T1 y V1. Se evalu la kd para la union de 40S a cada
poblacin de heterogeneidad creciente in vitro obteniendo una prdida de
la afinidad por la subunidad 40S de hasta 3 veces con el incremento de
las mutaciones. En cultivo celular, con y sin ribavirina, la accin de la
seleccin natural sobre este dominio esencial para el virus, fue la de un
incremento de la afinidad a la subunidad ribosomal 40S. Concluimos que
el reconocimiento molecular, in vitro, de las estructuras 2 es ms estable
que en las estructuras 1 y 3 y el dominio complejo RBS es el ms sensible
al incremento en la tasa de mutacin. En cultivo, el virus puede aprovechar
el incremento en la tasa de mutacin durante su replicacin para facilitar la
seleccin de mutantes ms activos en dominios esenciales como el RBS.
P02r-50

P02-51
Utilizacin de iPSC como modelo de estudio

de una nueva enfermedad rara
Pilar Mollinedo Sedano1, Jos Antonio Lpez-Escamez2, Silvia
Torices del Val1, Vernica Ramos-Meja2, Pedro J. Real-Luna2, Jos
Luis Fernndez-Luna1, Domingo Gonzalez-Lamuo3
1
FUNDACION IDIVAL, Santander, ES, 2Centre for Genomics and
Oncological Research-GENYO, Granada, ES, 3Universidad de
Cantabria, Santander, ES
Las nuevas tecnologas de anlisis genmico y los nuevos modelos
de enfermedad mediante reprogramacin celular estn siendo de gran
importancia en el estudio de las enfermedades raras. Si bien estas
enfermedades son de baja prevalencia (menos de 5 casos por cada 10.000
personas), en su conjunto (ms de 7000 patologas distintas) tienen
una notable incidencia en la poblacin (afectan al 7% de la poblacin
mundial). Nuestro grupo est estudiando el caso de una nia de 7 aos
con sospecha clnica de un sndrome de poliendocrinopata autoinmune
(sndrome APECED), asociado a mutaciones en el gen AIRE. Sin embargo,
la ausencia de mutaciones en este gen y la identificacin de un cuadro
clnico ms complejo con miopata, infecciones intestinales recurrentes y
alteraciones conductuales, indica una entidad patolgica nueva. Mediante
CGH array de alta resolucin (400K) se ha identificado una duplicacin
biallica de 4.1 Kb en el cromosoma 21 que incluye el gen LINC00315
(long intergenic nonprotein coding RNA, Gene ID: 246704), un potencial
regulador transcripcional. Con el fin de profundizar en el estudio gentico
de esta patologa analizamos el exoma completo de la paciente y de sus
padres. Se aportarn datos sobre ambos estudios. Simultneamente
estamos desarrollando un modelo de enfermedad transduciendo clulas
mononucleares de la paciente con vectores virales reprogramadores
(iPSC,induced pluripotent stem cells). Estas iPSC se diferenciarn a
distintos linajes celulares (msculo, linfocitos, epitelio, neuronas) con
relevancia en la patologa estudiada, con el fin de identificar alteraciones
en el patrn de diferenciacin o maduracin celular que permitan explicar
al menos parte del cuadro clnico de la paciente. Este modelo podra ser
utilizado para el desarrollo de estrategias teraputicas, tanto genticas
como farmacolgicas. Presentaremos datos preliminares de este modelo
celular.
Epigenetic regulation in dilated cardiomyopathy

Alicia Gonzlez, Ileana B. Gonzlez, Arturo Bujarrabal, Susana
Gonzlez
Faculty of Medicine, University of Castilla-La Mancha, Ciudad
Real, ES. Fundacin Centro Nacional de Investigaciones
Cardiovasculares, CNIC, Madrid, ES
P02-52
Dilated cardiomyopathy (DCM) is one of the most common forms

of cardiomyopathy with an estimated prevalence of 1/2500. DCM
is characterized by left ventricular enlargement (LVE) and systolic
dysfunction with an ejection fraction <50%. This disease commonly results
in congestive heart failure with signs and symptoms such as shortness of
breath, edema and increased heart rate, leading to death.
DCM is a common reason for heart transplantation in adults and children.
The mortality remains high without effective treatments. Further analysis
focusing on the development of cardiomyopathy are required in order to
improve treatment approaches and prognosis.
Recent studies in our group show that BMI1 protein, which belongs
to the epigenetic regulator Polycomb group, seems to be involved in
cardiomyopathy development. In order to study what happens at very early
stages of the disease, we have generated mice with conditional deletion
of BMI1 (cKO Bmi1) in heart. This cKO Bmi1 mice has been obtained
by crossing a cKO Bmi1 fl/fl mouse with mice bearing different cardiac
specific CRE, such as MYH6-CREERT, inducible in adult myocardium by
tamoxifen administration.
The present work focuses on the caracterization of early stages of BMI1mediated dilated cardiomyopathy and the elucidation of the most important
genes and proteins involved in cardiac dysfunction and heart failure.
Silvia Torices del Val1, Ignacio Varela Egocheaga2, Thaidy Moreno

Rodrguez2, Pilar Mollinedo Sedano1, Alejandro Balsa Criado3, Dora
Pascual Salcedo3, Lorena lvarez Rodrguez1, Marcos Lpez Hoyos1,
Jos Luis Fernndez Luna1, Vctor Martnez Taboada1
1
FUNDACION IDIVAL, Santander, ES, 2IBBTEC, Universidad de
Cantabria, Santander, ES, 3Hospital La Paz, Madrid, ES
48
Identificacin de variantes gnicas relevantes

de la va de NFB y sus consecuencias
funcionales en pacientes con artritis reumatoide
NFB es uno de los principales moduladores de las respuestas inmune

e inflamatoria. Diversos estudios en pacientes con artritis reumatoide
(AR) sugieren que NFB es esencial para la expresin de citocinas
proinflamatorias y de enzimas que promueven la destruccin articular.
Nuestra hiptesis de trabajo es que pueden existir mutaciones en los
principales genes involucrados en la regulacin de NFB que pueden tener
consecuencias funcionales relevantes y por tanto asociarse bien con la
susceptibilidad a padecer AR, o bien con el pronstico de la enfermedad.
Para comprobar esta hiptesis, hemos analizado por medio de tcnicas de
secuenciacin de nueva generacin 158 genes reguladores de la va NFB
en 66 pacientes con AR y en 30 controles sanos, obteniendo un total de
942 variantes gnicas. Seleccionamos trece de estas variantes, afectando
a 10 genes (TLR10, TLR1, TLR7, TLR8, NLRP7, NFBIZ, NOD2, TNIP1,
TRIP6 y BCL10) de acuerdo a la presencia de diferencias estadsticamente
significativas en la frecuencia del polimorfismo entre la poblacin control
y los pacientes con AR, la frecuencia allica de la mutacin en la poblacin
Granada 2014
general, el impacto del polimorfismo a nivel de la protena y la funcin
reguladora que estos genes tienen en la ruta NFB. Estas variantes fueron
confirmadas en una cohorte de 400 pacientes con AR y 200 controles
sanos mediante secuenciacin masiva. A fin de determinar las posibles
consecuencias funcionales de las variantes, se han comenzado estudios
para determinar el nivel de actividad de NFB mediante ensayos con
luciferasa como gen reportero y expresin de citocinas pro-inflamatorias
por PCR cuantitativa. Presentaremos datos preliminares sobre la posible
participacin de algunas de estas variantes gnicas en el desarrollo o
evolucin de la artritis reumatoide.
P02-53
Data mining en Drosophila para la identificacin

y caracterizacin de nuevos genes implicados
en la biognesis de la funcin OXPHOS
Sara Palacios Zambrano, Ramiro Vicente, Paula Clemente Prez,
Susana Peralta, Luca Echevarra, Rafael Garesse, Miguel Angel
Fernndez-Moreno
Autnoma de Madrid, Alcobendas, ES
La mitocondria es un orgnulo celular de origen endosimbionte que juega
un papel central en el metabolismo de la clula hospedadora destacando su
implicacin en: (i) la sntesis de la mayor parte de la energa (ATP) celular
(ii) la regulacin de la produccin de especies reactivas de oxgeno (ROS),
(iii) el control de la homeostasis del Ca2+ intracelular y (iv) el control del
proceso de muerte programada o apoptosis.
Entre estas funciones, se considera la sntesis de ATP mediante el
sistema cadena respiratoria (CR)/OXPHOS como la ms relevante y
cuya disfuncin genera las denominadas enfermedades mitocondriales.
El CR/OXPHOS est formado por numerosas subunidades estructurales,
de las que 13 estn codificadas en el propio genoma mitocondrial
(mtDNA) y ms de 80 en el DNA nuclear. Para la biognesis de este
sistema se requieren, adems, los elementos que las ensamblan y los
numerosos componentes de la maquinaria de replicacin y decodificacin
del genoma mitocondrial. Mutaciones en cualquiera de ellos pueden
provocar defectos responsables de numerosas patologas denominadas
enfermedades mitocondriales.
En ms de la mitad de las patologas mitocondriales de base gentica se
desconoce el gen o genes causantes. La identificacin de genes no descritos
implicados en defectos OXPHOS es uno de los retos actuales en este rea.
En este sentido, la utilizacin de Drosophila como modelo puede suponer
una herramienta adicional para ello. Drosophila posee una molcula de
mtDNA similar a la de mamferos y conserva los mismos elementos
responsables de su replicacin, transcripcin, mantenimiento, etc. El
rastreo de diferentes bases de datos nos han permitido identificar la
presencia de genes esenciales para la funcin OXPHOS situados sobre
bicistrones, es decir, codificados en un mismo mRNA, recordando
una organizacin procariota. Los ortlogos en mamferos de algunos
de esos genes han sido recientemente caracterizados en el laboratorio.
Para caracterizar otros nuevos candidatos procederemos a utilizar
aproximaciones de interferencia de RNA y de generacin de knock-outs
mediante el sistema CRISPR/Cas.
P02-54
Los sistemas toxina-antitoxina controlan

la infeccin por Salmonella typhimurium
Damin Lobato Mrquez1, Inmaculada Moreno Crdoba1, Viginia
Figueroa2, Francisco Garca del Portillo2, Ramn Daz Orejas1
1
Centro de Investigaciones Biolgicas (CIB-CSIC), Madrid, ES,
2
Centro Nacional de Biotecnologa (CNB-CSIC), Madrid, ES
Salmonella enterica serovar typhimurium (S. typhimurium) es una
bacteria patgena intracelular Gram-negativa que puede persistir en el
Psters
organismo infectado durante un perodo prolongado de tiempo. Es el
agente causal de enfermedades humanas de diversa severidad, desde
gastroenteritis hasta infeccin sistmica crnica. Como la mayora de las
bacterias, esta especie contiene en su genoma sistemas toxina-antitoxina
(TA). Los sistemas TA son mdulos formados, generalmente, por dos
genes adyacentes, que estn agrupados formando un opern. Uno de los
genes codifica una antitoxina, que puede ser un RNA o una protena, y
el otro una toxina que es siempre una protena estable capaz de interferir
con la proliferacin o viabilidad de la bacteria. La activacin de estos
sistemas est asociado a la respuesta a estrs, y ms recientemente se
ha visto que pueden favorecer la supervivencia del patgeno durante la
infeccin.
Nosotros hemos realizado una bsqueda sistemtica y un anlisis funcional
de los sistemas TA de S. typhimurium, determinando que la mayora de
los sistemas TA identificados son funcionales. Adems, hemos analizado,
utilizando un modelo de infeccin de fibroblastos, el papel de estos
sistemas en la persistencia intracelular. El anlisis ha permitido identificar
un subgrupo de sistemas TA que se expresan en la clula husped y cuya
presencia favorece la supervivencia y/o proliferacin limitada de S.
typhimurium en el interior de la clula infectada. Los datos demuestran un
papel activo de un grupo de sistemas TA en la persistencia de Salmonella
en fibroblastos infectados.
P02-55
Expresin, caracterizacin enzimtica e inters

biomdico de la pirrolin 5-carboxilato sintetasa
humana, una enzima clave en la biosntesis
de prolina y ornitina
Juan Manuel Escamilla Honrubia1, Clara Marco Marn1, Nadine
Gougeard2, Vicente Rubio3
1
Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, ES,
2
Centro para Investigacin Biomdica en Red sobre Enfermedades
Raras CIBERER-ISCIII, Valencia, ES, 3Instituto de Biomedicina de
Valencia (IBV-CSIC) y CIBERER-ISCIII, Valencia, ES
La 1-pirrolin-5-carboxilato sintetasa (P5CS) es un enzima bifuncional
que cataliza los dos pasos iniciales de la biosntesis de novo de prolina y
ornitina (y a travs de ornitina, arginina) en animales y plantas, siendo su
dficit un error congnito transmitido con herencia recesiva en unos casos
y dominante en otros. Con la finalidad de establecer las caractersticas
bioqumicas y funcionales de esta enzima e intentar determinar su
estructura, as como para explorar los efectos de las mutaciones clnicas,
tratando de entender su dominancia o recesividad, hemos expresado la
P5CS recombinante humana como protena de fusin con MBP, de la que
eliminamos luego la MBP por procedimientos estndar. Nuestro sistema de
expresin usa baculovirus/clulas de insecto Sf9, no habiendo tenido xito
con sistemas de expresin bacteriana y escaso rendimiento con la expresin
usando clulas HEK293. Hemos generado, estables y activas, las dos formas
de splicing alternativo de la enzima, determinando las caractersticas
cinticas de sus dos reacciones parciales (glutamato 5-quinasa, G5K; y
glutamil 5-fosfato reductasa, G5PR) y de la global, estudiando su grado de
acoplamiento, y corroborando que slo la isoforma ms corta experimenta
retroinhibicin por ornitina. Probamos que la estructura cuaternaria de la
enzima es hexamrica, de acuerdo con la dominancia negativa propuesta
para explicar los casos de herencia dominante. Los ensayos con mutantes
dominantes han demostrado tendencia a la rpida desagregacin. Los
experimentos cristalogrficos no han generado por el momento resultados
positivos.
Ayudas Prometeo 2009/051 (Generalitat Valenciana) y BFU201130407(MINECO). N. Gougeard es contratada CIBERER.
49
Psters
P02-56
deficientes en Zmpste24 en relacin al tiempo de desarrollo de los procesos

tumorales ha dificultado el abordaje de esta cuestin.
El presente trabajo, surgido de la colaboracin entre los laboratorios de
Lpez-Otn y Juan Cadianos, del Instituto de Medicina Oncolgica y
Molecular de Asturias, y enmarcado dentro del proyecto de tesis doctoral de
Jorge de la Rosa, ha permitido estudiar la implicacin de este enzima y su
sustrato en el cncer. Para ello se generaron ratones mosaico de Zmpste24,
en los que aproximadamente la mitad de las clulas del organismo carecen
de esta proteasa y, por tanto, acumulan prelamina A. Sorprendentemente,
estos ratones son frtiles y no experimentan sntomas de envejecimiento
acelerado, pese a que las clulas con prelamina A persisten en proporciones
similares a las clulas normales. En consecuencia, resultan prometedores
tanto el desarrollo de terapias gnicas y celulares como el suplemento de
factores sistmicos para el tratamiento de estas enfermedades. Asimismo,
la esperanza de vida normal de estos ratones permiti estudiar la relevancia
del sistema Zmpste24/prelamina A en el cncer. La aplicacin de distintos
protocolos de carcinognesis en ratones mosaico y ratones control permiti
observar que los primeros presentaban un menor nmero de tumores
infiltrantes. Igualmente, el silenciamiento de ZMPSTE24 en distintas clulas
tumorales humanas redujo significativamente su capacidad invasiva. Estos
resultados sugieren por primera vez que la proteasa ZMPSTE24 podra ser
una diana antitumoral.
A novel liquid chromatography coupled with a

tandem mass spectrometry (LC-MS/MS) method
to assess LpxC inhibition
Caridad Daz Navarro1, Victoria Knight-Connoni2, Kevin Barry2, Olga
Genilloud1, Francisca Vicente Prez1, Jos Prez del Palacio1
1
Fundacin MEDINA, Parque Tecnolgico de Ciencias de la Salud,
Granada, ES, 2Cubist, Lexington, MA, Lexington, US
LpxC, an essential enzyme for lipid A biosynthesis in gram-negative
bacteria, represents an attractive target for therapeutics. However,
antibacterial drug discovery efforts focused on inhibitors of LpxC have
found that the enzyme assay is not suitable for high-throughput screening
using conventional technologies such as assays with radiolabeled substrates
involving charcoal or TLC separation and fluorescence-based methods that
detect the free amine in the reaction product by coupling to fluorescamine.
These methods are cumbersome and vulnerable to false-positive hits due to
fluorescence quenching.
In this work we present a novel liquid chromatography coupled with a
tandem mass spectrometry (LC-MS/MS) method which offers a fast,
sensitive, and reliable alternative to the typical LpxC inhibition assay. It
employs mass spectrometry to directly detect product with high sensitivity.
Using this system, the data collected illustrate enzyme concentration
linearity and standard kinetics for conditions used in screening for LpxC
inhibitors. Additional validation experiments, such as DMSO tolerance and
reference inhibitor (Chir90) testing, were also carried out successfully.
This method represents the first communication of a methodology for the
separation of the enzymatic reaction product by mean of conventional
high pressure liquid chromatography (HPLC) which makes feasible the
implementation of a screening for antibacterial inhibitors of the LpxC
using a high-throughput mass spectrometry assay in many drug discovery
setups.
P02-57
El sistema Zmpste24/prelamina A en el cncer

y el envejecimiento
Jorge de la Rosa1, Carlos Lpez-Otn2, Juan Cadianos3
Instituto de Medicina Oncolgica y Molecular de Asturias (IMOMA)
- Departamento de Bioqumica y Biologa Molecular, Instituto
Universitario de Oncologa (IUOPA), Universidad de Oviedo ,
Oviedo, ES, 2Instituto de Medicina Oncolgica y Molecular de
Asturias (IMOMA), Oviedo, ES, 3Departamento de Bioqumica y
Biologa Molecular, Instituto Universitario de Oncologa (IUOPA),
Universidad de Oviedo, Oviedo, ES
Los avances producidos en los ltimos aos en torno al envejecimiento han

sido favorecidos por el estudio de los denominados sndromes progeroides
humanos, cuyos pacientes desarrollan de manera prematura y exacerbada
mltiples alteraciones caractersticas de la edad avanzada. Las conocidas
como laminopatas progeroides se engloban dentro de este grupo de
patologas y, mayoritariamente, se deben a defectos en el procesamiento de
la protena de la envuelta nuclear, denominada lamina A. Esta protena es
sintetizada como un precursor, la prelamina A, que debe sufrir una serie de
modificaciones post-traduccionales hasta dar lugar a la protena madura. En
la ltima etapa de su procesamiento interviene la proteasa Zmpste24, cuya
participacin se conoce desde hace ms de una dcada gracias al trabajo del
laboratorio de Carlos Lpez-Otn, de la Universidad de Oviedo. Entonces,
la generacin de ratones deficientes en esta proteasa puso de manifiesto
la incompatibilidad de los defectos en el sistema Zmpste24/prelamina
A con el desarrollo normal del organismo. Ms recientemente, estudios
realizados por el mismo grupo con este modelo murino han permitido el
desarrollo de nuevas terapias para combatir estas enfermedades. Dado
que el envejecimiento y el cncer son procesos ntimamente relacionados,
el sistema Zmpste24/prelamina A podra estar tambin implicado en el
desarrollo tumoral. Sin embargo, la corta esperanza de vida de los ratones
50
P03. Biologa del desarrollo

P03-1
Single-enhancer KO to study the formation

of the epaxial musculature during mouse
embryonic development
Macarena Lpez Mayorga1, Natalia Moncaut2, Maria Rosa Girldez
Perez1, Lydia Teboul3, Cristina Bernal Lozano1, Ana Castro Caal1,
Jaime Carvajal Garca-Valdecasas1
1
Centro Andaluz de Biologa del Desarrollo, Sevilla, ES, 2The
Institute of Cancer Research , London, UK, 3MRC-Harwell,
Oxfordshire, UK
The determination and specification of skeletal muscle in vertebrates is
orchestrated by the myogenic regulatory factors Myf5, Mrf4, MyoD and
Myogenin. Myf5 is the first to be expressed in the embryo, initiating and
co-ordinating the myogenic cascade. In absence of Myf5, progenitors
fail to be specified at the correct time but activation of MyoD rescues the
phenotype and myogenesis progresses. Myf5/MyoD KO animals lack all
skeletal muscles.
Myf5 transcription is controlled by over 25 regulatory elements which
drive expression in a specific spatiotemporal manner. Although their
contribution to the expression pattern is well defined we still lack an
understanding on the role of the different subpopulations of muscle
progenitor cells. Furthermore, there is still no connection between the
spatiotemporal activation of Myf5 and its function within a particular set
of myogenic precursors. The Early Epaxial Enhancer (EEE) operates in the
dermomyotomal dorsomedial lip and is the first enhancer to activate Myf5.
In order to address these questions we have generated a new enhancerspecific KO strain in which the EEE has been targeted. By RNA-seq, we
have identified three gene-networks that show clear differences between the
WT and KO embryos; two of these networks are involved in myogenesis.
qPCR has been used to validate the data and preliminary analyses of the
expression patterns of some of the genes in these networks also confirms
these findings. Crucially, we have crossed this new allele into the MyoD KO
strain and show that in the absence of MyoD rescue, specific back muscles
are lost or severely reduced, resulting in severe scoliosis of the spine.
We are now in the process of identifying all the muscle groups that may
be affected in this double KO as this will allow us to generate a map of the
musculature that originates from the dorsomedial lip of the dermomyotome.
Granada 2014
Psters
P03-2
P03-4
Adhesion to the Fibronectin synergy site could be

critical for platelet function
Dose dependent pitx2 loss of function impairs

zfhx3, wnt8a and calcium handling; novel links
to atrial arrhythmogenesis
Mara Benito Jardn, Joaqun Lilao Garzn, Mercedes Costell

Rossell
Departamento de Bioqumica y Biologa Molecular Universitat de
Valencia, Burjassot, ES
Fibronectin (FN) is a core component of many extracellular matrices
(ECM) where it regulates a variety of cell activities through direct
interactions with integrins. The fibrillar organization of FN precedes
the assembly of other ECM proteins. FN fibrillogenesis is a cell driven
process that requires integrins, which bind the RGD motif in the 10th
type III module (FNIII10). The RGD motif can bind v3 and 51 on
mesenchymal cells and IIb3 integrins on platelets. While the RGD site
is sufficient for v3 to bind and assemble FN, it has been claimed that
51 also requires residues in the flanking module (FNIII9), called synergy
site, for FN binding and fibrillogenesis. Aimed at studying the role of the
synergy site in vivo, we generated a mouse strain in which key residues of
the synergy site were mutated (FNSyn mice). Surprisingly, FNSyn mice
are born without apparent phenotype indicating that this site is not essential
for 51 binding during embryogenesis. However, a slight increase in the
tail bleeding time and a pronounced delay in skin wound closure suggest
a critical role of the synergy site for platelet and skin cell adhesion to FN.
To eliminate a potential compensatory effect of v3 integrins, we crossed
the FNSyn mice with a mouse strain lacking the expression of 3 integrins.
The 3 integrin-null mice suffer from a Glanzmann-like thrombasthenia,
a bleeding disorder that was exacerbated in the double mutants. About
87% of 3 integrin-null mice are born and of those around 20% will die
in the following weeks. However, when mice lack 3 integrins as well as
the FN-synergy site the 96,2% succumb before E17.5 (151 mice analyzed
at E17.5-P21) of severe hemorrhages and a failure to separate blood and
lymphatic vessels. These findings demonstrate that 3 integrins only
partially compensate the lack of a functional FN-synergy site. We are
currently testing how the synergy site promotes adhesion and will discuss
these results and the implications for human hemostasis at the meeting.
P03-3 (R03-19-1)
Developmental origin of the coronary

endothelium. Of elephants and blind men
Ramn Muoz-Chpuli
Universidad de Mlaga, Mlaga, ES
The developmental origin of the coronary endothelium has been the matter
of a debate in recent years. The classical view of a sprouting of the coronary
arteries from the aortic root was abolished 30 years ago by the evidence of
a coronary ingrowth, i.e. a primary coronary capillary plexus appears in
the subepicardium, surrounds the truncus arteriosus and finally connects
with the aortic lumen. The question then was the origin of the subepicardial
capillary plexus. Different hypothesis have been postulated to explain this
origin, basically angioblasts migrating from the developing liver, outgrowth
of the sinus venosus endocardium, angiogenesis from the ventricular
endocardium or cells delaminating from the epicardium. This latter option
has been particularly controversial, since experimental evidence has been
provided either supporting or ruling out an epicardial contribution to the
coronary endothelium. We have studied coronary development in a number
of murine transgenic lines, and we have concluded that the embryonic and
the neonatal coronary endothelium is a mosaic of endocardial-derived and
epicardial-derived cells, in a ratio of about 3:1, receiving later a postnatal
contribution from circulating endothelial progenitors. As in the Indian
story of the elephant and the blind men, different reports had detected only
a part of the whole picture, attributing to the coronary endothelium a single
origin. The endothelial developmental heterogeneity could be relevant in
order to explain the existence of atherosclerotic lesionprone sites in the
coronary arteries.
Estefana Lozano-Velasco1, Francisco Hernndez-Torres1, Jorge

Domnguez Macas1, Leif Hove-Madsen2, Juan Cinca3, Amelia
Arnega Jimnez1, Diego Franco Jaime1
1
Departamento Biologa Experimental, Universidad de Jan, Jan,
ES, 2CSIC-ICCC, Barcelona, ES, 3Hospital Sant Pau, Barcelona, ES
Atrial fibrillation (AF) is the most common cause of arrhythmogenesis in
humans yet the genetic cause of AF remains elusive. Recent genome-wide
association studies (GWAS) have reported risk variants in four distinct
genetic loci (4q25, 1q21, 16q22 and 16q13) which have been associated
with AF. Among them, the most significant are 4q25 risk variants, which
are located in the vicinity of the PITX2 gene. Given the key developmental
role of Pitx2 during cardiogenesis and particularly its role in pulmonary
vein deployment, it has been postulated that Pitx2 dysfunction might be the
molecular link to AF.
Experimental evidences in distinct laboratories, including ours,
have demonstrated that Pitx2 loss of function predisposes to atrial
arrhythmogenesis. However, the molecular mechanisms driven by Pitx2
in this context remain somehow elusive, proposing either embryonic
or mature gene expression defects. In order to get further insights into
the molecular mechanisms driven by Pitx2 and their putative relation
with novel AF GWAS associated genes, we have generated a new Pitx2
conditional mouse line, by intercrossing Sox2Cre and Pitx2floxed mice.
Epiblast deletion of Pitx2 leads to the generation of heterozygous and
systemic null Pitx2 null mutants, respectively. As expected, embryonic
mortality and cardiac defects were similarly observed in Sox2CrePitx2 null
mice as those previously reported for Pitx2 knock-out mice.
Molecular analyses of the left atrial appendage in heterozygous
Sox2CrePitx2 mice (20-30% reduction in Pitx2 expression) and atrialspecific NppaCrePitx2 null mice (60-70% reduction in Pitx2 expression)
demonstrate that AF GWAS associated genes such as Zfhx3, Kcnn3
and Wnt8a are severely impaired while other such as Cav1, Synpo2l or
Prrx1 are not. Surprisingly, beta-adrenergic signaling is not altered in
these models whereas multiple calcium handling genes such as Serca2a,
calsequestrin, phospholamban are severely impaired in atrial-specific
NppaCrePitx2 null mice but not in heterozygous Sox2CrePitx2 mice.
Functional assessment of calcium handling further underscores these
findings. Importantly, neither Zfhx3 nor Wnt8a gain-of-function or lossof-function experiments impairs Pitx2 expression, suggesting that Pitx2 is
upstream of these genes. Furthermore, these data suggest a dosedependent
relation between Pitx2 expression and the susceptibility to display basal
or only inducible electrophysiological defects. We are currently studying
the hierarchical between Pitx2, AF GWAS associated genes and calcium
handling, as well as to putative involvement of post-transcriptional
modulators such as microRNAs.
P03-5 (R03-19-6)
Filopodia mediate Hedgehog transport

and signalling
Isabel Guerrero Vega
CSIC - UAM, Madrid, ES
The Hedgehog (Hh) signalling pathway is a highly conserved regulator
of patterning during development and homeostatic events in adult organs.
Hh also directs tissue-patterning acting as a morphogen. To signal in a
concentration dependent manner requires a graded distribution of Hh
from producer to receptive cells. During production, Hh undergoes two
lipid modifications resulting in a highly hydrophobic molecule with a
cholesterol tag at the C-terminal and a Palmitoylation at the N-terminal.
Hh lipid modifications are needed to achieve a graded distribution, but
constrains its extracellular movement, leading to debate about how Hh is
transported to target cells. We show that Hh and most of the extracellular
51
Psters
components of the Hh signaling localize in filopodia-like structures
or cytonemes arising at the basolateral side of Drosophila wing disc
and abdomen epithelia. In vivo imaging, using actin reporters, and
membrane markers, such as the CD4 or the Hh co-receptor Ihog fused to
fluorescent tags, shows that these cytonemes are very dynamic and their
extension correlates spatially and temporally with the formation of the
Hh gradient. Mutant conditions for proteins required for actin dynamics
affect both cytoneme formation and Hh gradient extension. In addition,
in vivo vesicle-like structures are visualized moving along cytonemes
in anterograde direction. Immunoelectron microscopy also shows Hh,
the Hh co-receptor Ihog, and other Hh pathway components located in
extracellular vesicles of heterogeneous size (ranging from 50 to 250 nm)
at the Drosophila wing imaginal disc. These vesicles present Hh in the
outer side of the double membrane. Biochemical characterization of the
exosomal fraction of Drosophila cultured cells indicates the presence
of active Hh and co-receptor Ihog together with exosome markers.
Furthermore, RNA interference for genes necessary for production/
release of exovesicles has a direct effect over Hh secretion and signaling
in vivo and in vitro, indicating that Hh is transported in bonafide
exosomes. We propose that a Hh intracellular trafficking in the producing
cells allows Hh to be released in exosomes and cytonemes. In our view
this membrane anchored Hh dispersion could represent an advantage for
a lipid-modified molecules to be spatially regulated to form a gradient.
Furthermore, we show that this signaling mechanism based on cell-cell
contact is similar to the synaptic process in neuronal cells.
P03r-6
Caracterizacin del tejido linfoide asociado al

intestino en ratones SAMP8 propensos
al envejecimiento
Alba Garcia-Just1, Llusa Mir2, Anna Prez-Bosque1, Concepci
Amat1, Miquel Moret1
1
Departament de Fisiologia, Facultat de Farmcia, Institut de
Nutrici i Seguretat Alimentria, Universitat de Barcelona,
Barcelona, ES, 2APC Europe, Granollers, Barcelona, ES
El envejecimiento es un proceso caracterizado por un incremento de
la susceptibilidad a determinadas enfermedades y patologas. En este
sentido, el tejido linfoide asociado al intestino (GALT) tiene un papel
fundamental en las funciones de barrera y de defensa frente a patgenos
externos. El presente estudio tiene como objetivo caracterizar el
GALT en distintas etapas del desarrollo, en un modelo de ratones
senescentes, para su utilizacin futura en estudios de los efectos de la
dieta sobre indicadores mucosales (intestinales) de envejecimiento. Se
han utilizado ratones propensos al envejecimiento de la cepa SAMP8
y resistentes al envejecimiento acelerado (SAMR1). Se han obtenido
los ganglios linfticos mesentricos (GLM) y las placas de Peyer
(PP) del intestino delgado, a las 3 semanas, a los 4 y 9 meses. Se ha
realizado el recuento linfocitario de los dos tejidos y la cuantificacin
de la actividad SA--galactosidasa (indicador de senescencia) por
citometra de flujo. Los ratones SAMP8 presentan una disminucin del
peso de los GLM a los 4 y 9 meses, con una reduccin significativa
del nmero de leucocitos respecto a los SAMR1 (69% y 57%,
respectivamente; p<0,05). En las PP, se observa una reduccin del 35%
(p<0,05) pero slo a los 9 meses de edad. En ambos grupos, la actividad
SA--galactosidasa de los linfocitos ganglionares aumenta con la edad,
siendo mayor en los ratones SAMP8 que en los SAMR1 (p<0,05).
Estos resultados demuestran que el envejecimiento se caracteriza
por una inmunosupresin en el GALT, que podra explicar la mayor
susceptibilidad a infecciones y patologas asociadas al envejecimiento.
Asimismo estos resultados sugieren que los ratones SAMP8 son un
buen modelo para el estudio de la senescencia.
52

P03-7 (R03-19-3)
Spiracle formation and tissue remodeling

during Drosophila development: a vertex model
approach
Javier Argello, Kai Dierkes, Arturo DAngelo, Jerome Solon
Cell and Developmental Biology Group, Biomechanics of
Morphogenesis, Centre for Genomic Regulation (CRG), Barcelona,
ES
Strong efforts are being made to understand how large-scale tissue
movements emerge during morphogenesis. In this study, we analyze how
the development of spiracles in embryos of the fruit fly Drosophilacould
generate forces inducing tissue remodeling. High-resolution time-lapse
movies of tomato-E-cadherin and histone-RFP expressing embryos show
a simultaneous migration of spiracles and posterior epidermal cell layers
toward the posterior part of the embryo. To explain the mechanics of
this process, we discuss two different interplays of forces: (A) during its
development, spiracles push the tissue; (B) the tissue is originally moving
and pulls the spiracles.
The dynamics of the system is computationally simulated under both
conditions using a 2D vertex model that takes into account the surface
elasticity of cells, contractility of the acto-myosin cortex and adhesion
between neighbor cells, as well as the boundary conditions of pushing (A)
or pulling (B). Tissue movements within the simulations of the two different
cases (A and B) are then compared with the in vivo situation using Particle
Image Velocimetry (PIV). We show meaningful differences between the
simulations and the experimental evidences when the tissue is pulled, while
the pushing boundary condition generates a general movement close to in
vivo. Our work suggests that tissue flow is the consequence of pushing
forces generated during the formation of spiracles in Drosophila.
P03r-8
miRNAs 212 and 132 are involved in morphine

effects on cell proliferation through BDNF
regulation
Ada Jimnez Gonzlez, Adrin Garca Concejo, Raquel E. Rodrguez
Castilla y Len) IBSAL, Salamanca, ES
Morphine effects on cell proliferation in Central Nervous System
(CNS) have been studied over the last years. It is known that this drug
alters the proliferative cell expression patterns. In the present work, we
have analyzed whether morphine is triggering these changes in zebrafish
development through epigenetic factors such as miRNAs 212 and 132
and their involvement on BDNF expression. These miRNAs appear in the
same genetic cluster and have important roles through different pathways
on CNS development. To studied miRNA 132 levels during zebrafish
development, we performed qPCR in several developmental stages,
finding an increase and decrease of its levels along the development. We
also checked the effects of morphine and the involvement of miRNA 132 in
the opioid pathway by knocking down the mu opioid receptor, finding that
the levels of miRNA 132 changed between the control group and morphine
treated embryos. These results indicate that morphine is triggering its
proliferation effects through a different pathway than the one triggered
by the mu receptor. Futhermore, when we studied miRNA 132 in miRNA
212 morphants we found an increase in these levels, which suggests that
miRNA 132 is trying to balance the absence of miRNA 212. Besides, we
have found changes in BDNF protein expression levels in morphine treated
embryos and when the mu opioid receptor and miRNA 212 were knocked
down at 48 hpf. These results suggest that BDNF, an essential neurotrophin
for the correct development of the CNS, has an important role in the
mechanisms triggered by morphine, leading to a better understanding of the
influence of morphine in the mechanisms of pain and addiction processes.
Granada 2014
P03r-9
Changes in dopaminergic development in

zebrafish embryos after morphine exposure
mediated by miRNA-212 and miRNA-29a
Adrin Garca Concejo, Ada Jimnez Gonzlez, Raquel E. Rodrguez
Castilla y Len) IBSAL, Salamanca, ES
Several studies have shown that the levels of expression of miRNA-212
are modified after drug exposure, which may indicate a relation between
this molecule and the appearance of tolerance. In addition, miR29a has been related to Notch signaling, which is known to mediate
dopaminergic differentiation at the early stages of Central Nervous
System development. We have performed a study of the expression levels
of miR-212 during the normal development of zebrafish embryos, to
determine the stages in which this miRNA is more expressed. Our results
have shown that after morphine exposure the levels of microRNAs 29a
and 212 are altered, pointing to a relation between these two molecules.
To analyze a possible cross-talk between Notch and opioid signaling we
used DAPT, an inhibitor of the metalloproteases needed to activate this
pathway, and mindbomb mutants, where Notch pathway is disrupted, in
addition to oprm1 morphants (lacking opioid signaling). Our results show
that these two cascades are antagonic when influencing dopaminergic
differentiation. Using a luciferase assay, we have also demonstrated,
that miR-212 effectively represses oprm1 expression by interacting
with the 3UTR of the mRNA. We hence proposed a mechanism by
which morphine is regulating the expression of mu opioid receptor and
inducing the appearance of tolerance. Finally, the expression patterns of
some dopaminergic markers, such asth, pitx3 and nurr1, were modified,
leading to a totally new pattern of expression, with all these genes being
expressed in the midline. The understanding of these mechanisms that
involve both signaling pathways could lead to unravel the initial stages
of the addictive process.
P04. Biologa molecular computacional

P04-1 (R04-3)
Predicted structure and function of divergent

members of prokaryotic flavin adenine
dinucleotide synthetase (FADS) proteins
Inmaculada Yruela1, Patricia Ferreira2, Bruno Contreras-Moreira3,
Milagros Medina2
1
Estacin Experimental de Aula Dei, Consejo Superior
de Investigaciones Cientficas (EEAD-CSIC), Instituto de
Biocomputacin y Fsica de Sistemas Complejos (BIFI), Zaragoza,
ES, 2Departamento de Bioqumica y Biologa Molecular y Celular,
Universidad de Zaragoza, Instituto de Biocomputacin y Fsica de
Sistemas Complejos (BIFI), Zaragoza, ES, 3Estacin Experimental
de Aula Dei, Consejo Superior de Investigaciones Cientficas
(EEAD-CSIC), Instituto de Biocomputacin y Fsica de Sistemas
Complejos (BIFI), Fundacin ARAID, Zaragoza, ES
Flavin adenine dinucleotide synthetases (FADSs) are well-known as a
group of prokaryotic bifunctional enzymes that carry out the dual functions
of riboflavin phosphorylation to produce flavin mononucleotide (FMN)
and its subsequent adenylylation to generate FAD (hereafter FADS-type I).
An extensive bioinformatics survey using the available genomes in public
databases revealed that certain gram-positive pathogenic bacteria (i.e.
Listeria monocytonegens, Listeria welshimeri, Lactobacillus plantarum,
Bacillus cytotoxicus) and plant chloroplasts contain also variants of
FADS sequences, named FADS-type II and plant-like FADS, respectively.
Both variants of FADS proteins constitute distinct evolutionary classes
characterized by shorter and non-conserved C-terminal domains. The
Psters
putative structures and functions of the C-terminal domains of the FADStype II from Listeria monocytonegens (LmFADS-typeII) and plant-like
FADS from Glycine max. have been investigated. Previous results pointed
out that the C-terminus domain of plant-like FADS proteins could contain a
catalytic activity (i.e. hydrolase or phophatase), but different to that of their
prokaryotic counterparts [1]. On the contrary, our recent investigations
suggest that the C-terminus domain of LmFADS-type II might be related
with the pathogenic activity of gram-positive bacteria, in particular with
the defence of bacteria against the lytic action of host lysozimes [2]. This
work is a contribution to our understanding of the evolutionary history of
FADS enzymes.
References
[1] I. Yruela, S. Arilla-Luna, M. Medina, B. Contreras-Moreira.
Evolutionary divergence of chloroplast FAD synthetase proteins (2010)
BMC Evol. Biol. 10:311.
[2] S. Leysen, L. Vanderkelen, S.D. Weeks, C.W. Michiels and S.V.
Strelkov. Structural basis of bacterial defense against g-type lysozymebased innate immunity (2013). Cell. Mol. Life Sci. 70:1113-1122.
P04-2 (R04-5)
Late-replicating heterochromatin fuels genome

evolution
Daniel Rico
Centro Nacional de Investigaciones Oncolgicas - CNIO, Madrid, ES
Asynchronous DNA replication of the genome has been associated
with different mutation rates and copy number variation (CNV) in
human populations. The late replication is generally associated to
heterochromatic regions that tend to suffer high replicative stress. CNVs
typically involve intermediate to large regions, providing a potential
substrate for the generation of new genes through gene duplication.
We have elucidated the possible relevance of the association of CNV
regions with later DNA replication times on gene birth and evolution.
Our analyses show that most human genes duplicated in the Primate
lineage are located in late replicating CNV regions. We have traced
the relationship between replication timing and the evolutionary age of
duplicated genes. Strikingly, we have found that the more recently a gene
has been duplicated the later it is replicated in human and mouse cells.
These results suggests that the currently active accumulation of CNVs
in late replicating regions has being also persistently and extensively
influencing genome evolution throughout animal history. We propose
a new evolutionary model [1] based on these observations in which
chromatin structure and replication dynamics conditions gene birth and
the rising of new protein functions.
References
[1] Juan,D. Rico, D, et al. (2013) Late-replicating CNVs as a source of new
genes. Biol Open, 2, 14021411.
P04-3
The human DNA damage response network

database of proteins
Eduardo Andrs Len1, Ildefonso Cases2, Ana M. Rojas Mendoza1
Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario
Virgen del Rocio/CSIC/Universidad de Sevilla - Computational
Biology and Bioinformatics, Sevilla, ES, 2Genomics and
Bioinformatics Platform of Andalusia, Cartuja Scientific and
Technology Park INSUR Building, Sevilla, ES
The DNA damage response (DDR) is an essential signaling network that

protects the integrity of the genome. This network is built upon a repertoire
of distinct but often overlapping sub-networks, where sometimes the same
components have different roles in precise spatial and temporal scenarios.
Perturbations of this network produce genomic instability, which is
53
Psters
inherently related to aging (Fernandez-Capetillo 2010), disease (Ciccia and
Elledge 2010), and cancer, reviewed in (Lukas, Lukas et al. 2011).
Despite its importance, evolutionary studies addressing the emergence of
this network were restricted to few protein families (On, Xiong et al. 2010).
In this line, we have recently provided the largest systematic analyses of
the human DDR network and have analysed its evolutionary properties
(Arcas, Fernandez-Capetillo et al. 2014).
To complement this study, we have built a resource to explore these data,
in an evolutionary context.
From this database, it is possible to select genes according with its function
in a particular pathway or network, according with post translational
modifications (PTMs) where the gene acts as a target or a modifier and also
by sequence similarity. When searching for PTMs, affected residues and
links to Pubmed articles are provided.
In this tool, it is easy to find DDR proteins that emerged at the same age,
or involved in same networks/pathways, and also affected by similar
PTM modifiers. When a DDR protein is selected, a detailed view displays
information regarding its emergence and conservation across 47 species,
the overall agreement with the taxonomic tree, and the position of a posttranslationaly modified residue in a structure when available.
This resource is available at: http://ddr.cbbio.es.
References
Arcas, A., O. Fernandez-Capetillo, I. Cases and A. M. Rojas (2014).
Emergence and evolutionary analysis of the human DDR network:
implications in comparative genomics and downstream analyses. Mol
Biol Evol 31(4): 940-961.
Ciccia, A. and S. J. Elledge (2010). The DNA damage response: making it
safe to play with knives. Mol Cell 40(2): 179-204.
Fernandez-Capetillo, O. (2010). Intrauterine programming of ageing.
EMBO Rep 11(1): 32-36.
Lukas, J., C. Lukas and J. Bartek (2011). More than just a focus: The
chromatin response to DNA damage and its role in genome integrity
maintenance. Nat Cell Biol 13(10): 1161-1169.
On, T., X. Xiong, S. Pu, A. Turinsky, Y. Gong, A. Emili, Z. Zhang, J.
Greenblatt, S. J. Wodak and J. Parkinson (2010). The evolutionary
landscape of the chromatin modification machinery reveals lineage specific
gains, expansions, and losses. Proteins 78(9): 2075-2089.
P04-4 (R04-6)
Bioinformatics approaches for personalized

cancer therapy: from Pan-Cancer projects
to Patient Derived Xenograft (PDX) models
Elena Pieiro-Yaez, Hctor Tejero, Javier Perales-Patn, Ftima
Al-Shahrour
Centro Nacional de Investigaciones Oncolgicas - CNIO, Madrid, ES
The dawn of the age of personalized cancer treatment provides the promise
to identify the right drug for the individual that will greatly improve the
patients outcome. It is well known that cancer drugs work only in small
subset of patients. For many of these agents, there are putative markers of
response in the literature but very few are been used in clinical practice.
More importantly, new anticancer agents are targeted to specific cancer
genes and are expected to be effective only in tumors in which these genes
are mutated or otherwise abnormal.
The success of personalized treatment of cancer patients depends on
matching the most effective therapeutic regimen with the characteristics of
the individual patient, balancing benefit against risk of adverse events. The
primary challenge in achieving this goal is the heterogeneity of the disease,
recognizing that the majority of cancers are not single diseases but rather
an array of disorders with distinct molecular mechanisms.
High-throughput technologies such as next-generation sequencing have
the capacity to dissect this heterogeneity and now doing an unbiased
interrogation of the human genome, which allows strategies to search for
novel, previously unsuspected, biomarkers of drug response and afford
54

opportunities to match therapies with the characteristics of the individual
patients tumor.
In our laboratory, we have focused on developing an integrative
bioinformatics approach to demonstrate the value in integrating genomic
and clinical data with available drugs, to refine and to improve therapeutic
strategies for cancer patients.
We present a multi-disciplinary integrative effort that will convert the
information contained in multidimensional genomic data into useful
biomarkers that can classify patient tumors by prognosis and to identify the
drivers of tumor behavior that are optimal targets for therapy.
P04-5 (R04-4)
Full-LengtherNext: Una herramienta para

caracterizar transcriptomas de organismos
no modelo
Pedro Seoane Zonjic1, No Fernandez-Pozo1, Daro GuerreroFernandez2, Roco Bautista2, M. Gonzalo Claros1
1
Ciencias, Universidad de Mlaga, Mlaga, ES, 2Plataforma Andaluza
de Bioinformtica, Centro de Supercomputacin y Bioinfomtica,
Edificio de Bioinnovacin, Universidad de Mlaga, Mlaga, ES
Los estudios de transcriptomas ensamblados de novo en especies no
modelo carecen de secuencias genmicas que sirvan de referencia para
evaluar su calidad. Esta carencia impide, por ejemplo, distinguir con
claridad entre un transcrito correcto y uno artefactual, y tampoco permite
distinguir fcilmente entre polimorfismos y errores de ensamblaje. Por
otro lado, sin una referencia, tambin resulta difcil determinar el mejor
ensamblaje posible dentro de un conjunto dado, ni tampoco se puede saber
con precisin el porcentaje de transcritos encontrados respecto al total de
genes. Por otra parte, saber si se ha ensamblado un transcrito que puede
codificar una protena completa es muy til para los trabajos de laboratorio
posteriores. Por todo esto se ha desarrollado Full-LengtherNext con una
arquitectura capaz de hacer frente con eficacia y de forma escalable los
requisitos de anlisis de posensamblaje de la NGS. Con este programa (1)
se clasifican los transcritos entre secuencias completas, N- o C-terminales,
e internas), (2) se reconstruye el marco abierto de lectura del transcrito
y se corrigen los posibles cambios de fase debidos al ensamblaje, (3) se
identifica el subconjunto de transcritos sin anotar que podran codificar
protenas nuevas propias de la especie en estudio, (4) se extraen algunos
posibles RNA no codificantes, (5) se comparan distintos ensamblajes
de novo de un transcriptoma para seleccionar el mejor, (6) se obtiene el
transcriptoma representativo ms cercano al transcriptoma completo
terico del organismo de estudio. Este programa ya ha sido usado con xito
en los anlisis de los transcriptomas de, por ejemplo, pino martimo, olivo,
lenguado comn y lenguado senegals.
P04-6 (R04-2)
Supercomputing methods for macromolecular

crystallographic Ab Initio Phasing
Isabel Usn Finkenzeller
ICREA ; IBMB-CSIC, Barcelona, ES
Crystallographic analysis does not produce a direct image as in microscopy,
as only the diffracted intensities and not the phases of the scattered X-rays
are physically measurable. So far, in spite of its being computationally very
demanding, crystallography has largely turned its back on supercomputing.
Our group develops and implements crystallographic phasing methods
exploiting massive computing.
ARCIMBOLDO: from atomic resolution phasing[1] to a general ab initio
structure solution overcoming previous size and resolution barriers. Our
method enforces secondary structure rather than atomicity through a
combination of location of model fragments (alpha-helices) with PHASER
[2] and density modification with SHELXE [3]. The method has been
Granada 2014
called after the Italian painter Arcimboldo [4], who composed portraits
out of fruits and vegetables. While most collections of fragments remain a
still-life, but some are correct enough for density modification to reveal
the proteins portrait.
BORGES takes the principle ones step further, by enforcing unspecific
tertiary -rather tan secondary- structure. Like in Borges Library of Babel,
the information we need is bound to be contained already in the PDB but
how to exploiting this information while the structure is still unknown? Our
characteristic vector (CV) formalism, developed to extract folds from the
PDB allows detailed analysis of local folds.
SUBIX: from a geometric solution of nucleic acids to the analysis
and prediction of packing and the information derived from packing
interactions.
References
[1] Sheldrick, Hauptman, Weeks, Miller & Usn. International Tables for
Macromolecular Crystallography vol. F, (eds., M.G. Rossmann and E.
Arnold) 333345 (Boston, 2001).
[2] McCoy, et al. J. Appl. Crystallogr. 40, 658674 (2007).
[3] Sheldrick. Z. Kristallogr. 217, 644650 (2002).
[4] Rodrguez, Grosse, Himmel, Gonzlez, M de Ilarduya. Becker,
Sheldrick & Usn Nature Meth. 6, 651654 (2009).
[5] Sammito, Milln, Rodrguez, M de Ilarduya, Meindl, De Marino,
Petrillo, Buey, de Pereda, Zeth, Sheldrick & Usn. Nature Meth. 10, 10991101 (2013).
P04-7 (R04-7)
The exocyst 3D architecture by live cell imaging

in yeast cells
Devos Damien
CABD, Sevilla, ES
The structural characterization of macromolecular complexes is central to
understand the mechanism of action of these assemblies and to pursue in the
search of new treatments for human diseases. Macromolecular complexes
can be composed of proteins, DNA, lipid, or any other molecules and
their combination. We use integrative structure modeling to solve the
structure of macromolecular complexes with important function in a cell.
In this talk, I will introduce a new method to solve the structure of multiprotein complexes. We used fluorescent microscopy in live cell to solve
the structure of the exocyst. The structure reveals important structural,
functional and evolutionary features of this important complex in the
eukaryotic cell. This macromolecular modeling protocol has the potential
to reveal important aspects of multi-component complexes.
Psters
P05. Biomembranas y bioenergtica

P05-1
Cross-talk between R513 methylation and S516

phosphorylation of the cardiac voltage-gated
sodium channel
Pedro Beltran lvarez1, Ferran Feixas2, Slvia Osuna2, Ramon
Brugada1, Sara Pagans1
1
Institut Investigaci Biomdica de Girona Dr. Josep Trueta,
Department of Medical Sciences School of Medicine, University of
Girona , Girona, ES, 2Institut de Qumica Computacional i Catlisi
(IQCC) and Departament de Qumica, Universitat de Girona,
Campus Montilivi, Girona, ES
Introduction: we have previously provided the first evidence of arginine
methylation within the voltage-gated ion channel superfamily. R513,
R526 and R680 in the alpha subunit of the cardiac voltage-gated sodium
channel (Nav1.5) were found mono- or dimethylated [1]. The functional
relevance of Nav1.5 arginine methylation is underscored by the fact that
R513H, R526H and R680H are known Nav1.5 mutations causing sudden
cardiac death syndromes. Objectives of the current work: 1) to describe the
cross-talk between Nav1.5 arginine methylation and phosphorylation; and
2) to describe regulation of arginine methylation by mutations in residues
adjacent to R513 leading to cardiac disease.
Results: methylation and phosphorylation reactions were done in
vitro using PRMT3 and PKA, and synthetic peptides. Reactions were
monitored by MALDI-TOF. Phosphorylation of S516 completely inhibited
R513 methylation by PRMT3. Methylation of R513 reduced S516
phosphorylation rate by 4 orders of magnitude. Overall, our results suggest
an interdependence of Nav1.5 arginine methylation and phosphorylation [2].
The mutation G514C, which has been described in a patient with cardiac
conduction disease, blocked R513 methylation by PRMT3, but did not
change S516 phosphorylation rate in vitro. We speculate that G514C leads
to S516 hyperphosphorylation due to blockade of R513 methylation in vivo.
Conclusions: our work provides the first insights into how arginine
methylation of voltage-gated ion channels is regulated, the first evidence
of PKA inhibition by arginine methylation, and opens the door to
pharmaceutical intervention to balance methylation-phosphorylation
equilibria in Nav1.5 cardiopathological states.
References
[1] Beltran-Alvarez P, et al. J Proteome Res 2011.
[2] Beltran-Alvarez P, et al. In preparation.
P05r-2
P04-8 (R04-1)
DNA: When computational chemistry meets

computational biology
Modesto Orozco
Institut for Research in Biomedicine, Barcelona, ES
DNA is the key molecule of life: an exquisite combination of chemical
simplicity and biological richness that has fascinated generations of
scientists. DNA is also the meeting point of chemistry and biology, and
the discovery of the structure of the DNA fiber more than 50 years ago
by Watson and Crick can be considered as the foundation of molecular
biology. For a theoretician, DNA is one of the greatest challenges, and
a textbook example of a multi-resolution and multi-physics problem. I
will summarize in my talk how we can use theoretical methods based
on solid physical principles to gain information on the biology of
DNA, how computational chemistry meets computational biology, and
how first principles can explain not only chromatin structure, but also
functionality.
How redox proteins form transient complexes in

photosynthesis and respiration
Blas Moreno-Beltrn1, Antonio Daz-Quintana1, Katiuska GonzlezArzola1, Alejandra Guerra-Castellano1, Adrin Velzquez-Campoy2,
Pedro M. Nieto3, Marcellus Ubbink4, Miguel A De la Rosa1, Irene
Daz-Moreno1
1
Instituto de Bioqumica Vegetal y Fotosntesis, cicCartuja,
Universidad de Sevilla - CSIC, Sevilla, ES, 2Instituto de
Biocomputacin y Fsica de Sistemas Complejos (BIFI), Universidad
de Zaragoza, Zaragoza, ES, 3Instituto de Investigaciones Qumicas,
cicCartuja, Universidad de Sevilla - CSIC, Sevilla, ES, 4Institute of
Chemistry, Leiden University, Leiden, NL
Protein complex formation is at least a two-step process in which the
formation of a final, well-defined complex entails the initial formation
of a dynamic encounter complex. The lifetime of the protein complex
is determined by the dissociation rate. Highly transient complexes, with
lifetimes on the order of milliseconds, exhibit moderate or low binding
affinities, with dissociation constants in the mMmM range. Electron
55
Psters
transfer (ET) reactions mediated by soluble redox proteins exchanging
electrons between large membrane complexes in photosynthesis and
respiration are excellent examples of transient interactions.
Here, experimental approaches based on dia and paramagnetic NMR
spectroscopy are combined with NMR restraint- or charge-driven docking
simulations to study the molecular recognition processes in ET complexes,
using the cyanobacterial Cf-Cc6 interaction in photosynthesis and the plant
Cc1-Cc adduct in respiration, as physiological model systems. Both ET
ensembles exhibit optimal coupling between the redox centers although
they might differ in their dynamic behavior. Needless to say that such an
integrative methodology opens new perspectives in our understanding of
the dynamic, transient adducts formed between proteins beyond the model
systems herein analyzed.
References
Daz-Moreno et al. (2014) BBA Bioenergetics doi: 10.1016/j.
bbabio.2014.03.009.
Moreno-Beltrn et al. Under review.
P05-3
Dos estrategias en la evolucin de la

transferencia de electrones al fotosistema I:
diatomeas versus sistemas verdes
Manuel Hervs, Pilar Bernal-Bayard, Fernando P. Molina-Heredia,
Alejandro Torrado, Jos Mara Ortega, Leonor Puerto-Galn,
Mercedes Roncel, Jos A. Navarro
Instituto de Bioqumica Vegetal y Fotosntesis, Universidad de
Sevilla y CSIC, cicCartuja, Sevilla, ES
En algas verdes unicelulares y cianobacterias, la plastocianina (Pc) y el
citocromo c6 (Cc6) actan como transportadores alternativos de electrones
entre los complejos de membrana b6f y fotosistema I (PSI) en fotosntesis.
Sin embargo, en algas rojas y diatomeas el Cc6 acta en general como nico
transportador entre estos complejos. La reduccin del PSI en eucariotas implica
la formacin de un complejo transitorio inicial, que tiene que reorganizarse
hacia una configuracin optimizada antes de la propia transferencia electrnica.
Sin embargo, comparada con los sistemas verdes (algas y plantas), la diatomea
Phaeodactylum presenta una menor eficiencia, tanto en la formacin del
complejo como en la transferencia en s, debido a que la intensidad de la
interaccin electrosttica entre el Cc6 y el PSI est atenuada respecto a las
fuertes propiedades electrostticas de donadores y PSI en los sistemas verdes.
El paso de reajuste implica un conjunto preciso y especfico de interacciones,
que se ven afectadas negativamente en reacciones cruzadas entre donadores
y PSI de diatomeas, algas verdes y plantas. Nuestros anlisis cinticos y
estructurales indican que la evolucin en los dos grandes linajes fotosintticos
eucariotas implic cambios paralelos y complementarios en los donadores y
el PSI. En la lnea evolutiva que conduce a las plantas superiores, una fuerte
interaccin electrosttica determina un intercambio lento del donador, lo
que limita la transferencia de electrones al fotosistema. En diatomeas, una
interaccin electrosttica ms dbil facilita, sin embargo, un intercambio ms
rpido del Cc6, para dejar as paso a la unin de una nueva molcula donadora.

lysosome. Approximately 20 Atg (autophagy-related) proteins have
been identified in mammals that are essential in the initial stages of the
preautophagosomal structure (PAS) formation and autophagosome
elongation. Among them, the yeast ubiquitin-like system (Atg7, Atg3
and Atg8) is relatively well characterized. Several Atg8 homologues are
known in mammals, divided into the LC3 and GABARAP/GATE-16
subfamilies. It has been shown that LC3 membrane association is mediated
by the covalent attachment of LC3 to phosphatidylethanolamine (PE). The
resulting lipidated protein then drives autophagosome maturation steps
including cargo capture, growth and closure of the organelle, and lysosome
targeting or recognition. This protein-lipid covalent binding is essential
for autophagosome elongation, but its mechanism remains unknown.
In the present study, we have focused on the molecular mechanisms
by which LC3/GABARAP and GATE-16 conjugate with membranes
leading to autophagosome elongation. With this purpose, we have
performed experiments of protein-protein and protein-lipid interaction
using model membranes such as SUV (small unilamellar vesicles), LUV
(large unilamellar vesicles) and GUV (giant unilamellar vesicles). Both
mammalian ubiquitin-like system and PE-maleimide conjugated human
Atg8 homologs have been used. In order to obtain this information, circular
dichroism spectroscopy, sucrose gradient floatation, protein-lipid overlay
(lipid dot-blot), lipid monolayer experiments and vesicle aggregation
measurements have been carried out. The various proteins interact in
specific ways with membranes of different lipid compositions.
*Both authors have contributed equally in this work.
P05-5
Sphingosine induces the aggregation

of imine-containing peroxidized vesicles
Noemi Jimenez-Rojo, Ana R. Viguera, Alicia Alonso, Felix M. Goi
Unidad de Biofisica (CSIC, UPV/EHU), Leioa, ES
Lipid peroxidation plays a central role in the pathogenesis of many
diseases like atherosclerosis and multiple sclerosis. We have analyzed the
interaction of sphingosine with peroxidized bilayers in model membranes.
Cu2+ induced peroxidation was checked following UV absorbance at
245 nm, and also using the novel Avanti snoopers. Mass spectrometry
confirms the oxidation of phospholipid unsaturated chains. Our results
show that sphingosine causes aggregation of Cu2+- peroxidized vesicles.
We observed that aggregation is facilitated by the presence of negativelycharged phospholipids in the membrane, and inhibited by anti-oxidants e.g.
BHT. Interestingly, long-chain alkylamines (C18, C16) but not their shortchain analogues (C10, C6, C1) can substitute sphingosine as promoters
of vesicle aggregation. Furthermore, sphinganine but not sphingosine-1phosphate can mimic this effect. Formation of imines in the membrane
upon peroxidation was detected by 1H-NMR and it appeared to be
necessary for the aggregation effect. 31P-NMR spectroscopy reveals that
sphingosine facilitates formation of non-lamellar phase in parallel with
vesicle aggregation. The data might suggest a role for sphingosine in the
pathogenesis of atherosclerosis.
P05r-6
P05r-4
Membrane Association of Autophagy Proteins

LC3, GABARAP and GATE-16
Efecto dual de la secrecin de cardiolipinas

en el alveolo
Zurie Antn*1, Javier H. Hervs*1, Ane Landajuela1, L. Ruth

Montes1, Jos F. Rodrguez2, Flix M. Goi1, Alicia Alonso1
1
Unidad de Biofsica (CSIC, UPV/EHU) and Departamento de
Bioqumica y Biologa Molecular, Universidad del Pas Vasco, Leioa,
ES, 2Centro Nacional de Biotecnologa - CSIC, Madrid, ES
Alba de Lorenzo1, Olga Caadas2, Beln Garca-Fojeda2, Cristina

Casals2
1
Complutense de Madrid, Madrid, ES, 2Departamento de Bioqumica
y Biologa Molecular I, Universidad Complutense de Madrid - CIBER
de Enfermedades Respiratorias, Madrid, ES
Macroautophagy mediates the degradation of long-lived proteins

and organelles through the de novo formation of double-membrane
autophagosomes that sequester cytoplasm and deliver it to the vacuole/
La cardiolipina (CL) es un fosfolpido aninico de las membranas de

bacterias Gram negativas que tambin se encuentra en la membrana
mitocondrial interna de clulas eucariotas y desempea un papel clave para
56
Granada 2014
la funcin e integridad estructural mitocondrial. Sin embargo, se ha descrito
que en pacientes con neumona se produce la liberacin de cardiolipina
mitocondrial al fluido alveolar, que puede alterar la funcin pulmonar [1].
Sin embargo, estudios recientes han indicado que fosfolpidos aninicos
podran presentar una accin inmunomoduladora beneficiosa en el pulmn
[2]. Nuestra hiptesis es que las CL liberadas al fluido alveolar en procesos
infecciosos e inflamatorios podran tener un efecto perjudicial o beneficioso
en funcin de su concentracin.
Los objetivos de este estudio han sido: 1) Estudiar el efecto de la CL humana
1`,3`-Bis-(1,2-dilinolenil-sn-glicero-3-fosfo)-sn-glicerol [(18:2)4-CL] en la
funcin tensoactiva del surfactante pulmonar; y 2) Determinar el umbral de
concentracin a partir del cual este lpido presenta un posible efecto en la
respuesta inflamatoria de macrfagos al lipopolisacrido bacteriano (LPS).
Los resultados indican que concentraciones de (18:2)4-CL superiores al
6% molar relativo a los fosfolpidos del surfactante (~1 mol/ml) alteran
significativamente la actividad de adsorcin interfacial del surfactante
pulmonar. El mecanismo de inactivacin reside en que (18:2)4-CL
incrementa la fluidez de las membranas de surfactante como se demuestra
por calorimetra diferencial de barrido y anisotropa de fluorescencia. En
contraste, nuestros resultados indican que concentraciones muy bajas de
cardiolipina (0.67 nmol/ml) bloquean la secrecin de TNF-, expresin de
iNOS y la fosforilacin de Akt, MAPKs e ikB en macrfagos alveolares
estimulados con LPS. Este efecto inmunomodulador de la cardiolipina
sucede tanto a nivel extracelular como intracelular.
En conjunto, los resultados indican que las cardiolipinas secretadas por el
husped en procesos infecciosos podran tener una accin dual, beneficiosa
o daina para el husped, en funcin de la concentracin de este lpido en
el fluido alveolar.
Bibliografa
[1] Ray, N.B., L. Durairaj, B.B. Chen,., and R.K. Mallampalli. Dynamic
regulation of cardiolipin by the lipid pump Atp8b1 determines the severity
of lung injury in experimental pneumonia. Nat Med, 16(10): 1120-7, 2010.
[2] Kuronuma K, Mitsuzawa H, Takeda K, Nishitani C, Chan ED, Kuroki Y,
Nakamura M, Voelker DR. Anionic pulmonary surfactant phospholipids
inhibit inflammatory responses from alveolar macrophages and U937 cells
by binding the lipopolysaccharide-interacting proteins CD14 and MD-2.
J Biol Chem. 284(38):25488-24500,2009.
P05r-7
How phosphorylation affects Cytochrome c

structure and function
Alejandra Guerra-Castellano1, Blas Moreno-Beltrn1, Javier LpezPrados2, Francisco Rivero-Rodrguez1, Pedro M. Nieto2, Adrin
Velzquez-Campoy3, Antonio Daz-Quintana1, Miguel ngel De la
Rosa1, Irene Daz-Moreno1
1
Universidad de Sevilla - CSIC, Sevilla, ES, 2Instituto de
Investigaciones Qumicas, cicCartuja, Universidad de Sevilla - CSIC,
Sevilla, ES, 3Instituto de Biocomputacin y Fsica de Sistemas
complejos, BIFI, Universidad de Zaragoza, Zaragoza, ES
Post-translational modifications of proteins stand out as regulatory
mechanisms for the majority cell metabolic processes. One of the most
usual modifications is phosphorylation, which alters the physical features
of proteins and affects their interactions with other proteins.
Cytochrome c (Cc) phosphorylation relates to some pathological situations,
such as ischemia or cancer [1]. Cc acts as an electron carrier in the
mitochondria. Under oxidative stress conditions, it promotes Programmed
Cell Death (PCD). Both functions are regulated by phosphorylation of the
residues Thr, Ser and Tyr at positions 28, 47 and 48, respectively [2-4].
Since the specific Cc-phosphorylating kinases are still unknown, we have
made three phosphomimetic mutations. Two of them replace Thr28 and Ser47
by Asp. To mimic Tyr48 phosphorylation, we resorted to the evolved tRNA
technique by replacing the Tyr48-encoding triplet for an AMBER codon.
Then, we incorporated a non-canonical amino acid (p-carboxymethyl-L-
Psters
phenylalanine, pCMF) that emulates phosphotyrosine, with the help of a
tRNA specific for the AMBER stop codon. These mutations induce drastic
changes not only on the redox potential, dynamics and stability of Cc, so
affecting its biological function.
References
[1] Httemann M et al. Adv. Exp. Med. Biol. (2012) 748, 237-264.
[2] Yu, H et al. Biochim. Biophys. Acta (2008) 1777, 1066-1071.
[3] Garca-Heredia, JM et al. J. Biol. Inorg. Chem. (2011) 16, 1155-1168.
[4] Zhao et al. Mol. Cell. Proteomics. (2011) 10, 1-14.
Work supported by JAE Programme (JaePre_2011_01248), ESF 20072013, Andalusian Government (CVI-BIO198), Ministry of Economy and
Competitiveness (BFU2009-07190 and BFU2012-31670), European
Bio-NMR Project (2012-2013) BIO-NMR-00130 and the Centro de
Investigacin Tecnologa e Innovacin (CITIUS).
P05-8
Amyloid-membrane interactions revealed

by model lipid vesicles
Cristina Fernndez, Mercedes Jimnez, Germn Rivas, Rafael
Giraldo
We have recently reported that engineering RepA-WH1, a bacterial
DNA-toggled protein conformational switch (dWH1WH1) sharing
some analogies with nucleic acid-promoted PrPcPrPSc replication [1],
constitutes a suitable synthetic model system to study protein amyloidosis
in bacteria [2, 3]. Although amyloidogenesis has been the focus of intense
research, the origin of the amyloid toxicity remains unclear. One proposed
mechanism of cytotoxicity is lipid membrane permeabilization.
In this work we have studied the aggregation of the bacterial RepA-WH1
prionoid in the presence of cytomimetic model systems (large and giant
unilamellar lipid vesicles, LUVs, and GUVs respectively) [4]. Confocal
microscopy images of protein encapsulated into GUVs show association
and aggregation of the protein preferentially to lipid vesicles containing
acidic phospholipids. We have also observed that RepA-WH1 elicits
membrane disruption using a dye release assay on LUVs [5]. The extent
of leakage was dependent on protein concentration.We have been able
to directly measure the process of membrane permeation and leakage by
time-elapsed imaging of dye filled GUVs upon the addition of protein. This
process is fast and over the course of the experiment most of the vesicles
remain intact, suggesting the assembly of defined pores by RepA-WH1.
This model system has allowed us to test several compounds that have been
described to modulate amyloid formation. Knowledge of the effect of the
RepA-WH1 prionoid on membrane integrity, will provide insight into the
basis for cell death caused by amyloid proteins.
References
[1] Silva et al. Trends Biochem. Sci. 2008; 33(3):132-140.
[2] Giraldo, et al. Prion 2011; 5:60-64.
[3] Fernndez-Tresguerres, et al. Mol Microbiol. 2010; 77:1456-1469.
[4] Butterfield and Lashuel. Angew Chem Int Ed. 2010; 49:5628-5654.
[5] van Rooijen, et al. Biochim. Biophys. Acta 2009;1788:1271-1278.
P05-9
Permeabilizacin de membranas fosfolipdicas

por el lipopptido liquenisina
Antonio Ortiz, Jonathan R. Coronel, Jos A. Teruel, Francisco J.
Aranda
Departamento de Bioqumica y Biologa Molecular-A, Facultad de
Veterinaria, Universidad de Murcia, Murcia, ES
Se ha aislado una liquenisina de los medios de cultivo de Bacillus
licheniformis. Nuestra liquenisina consiste en un lipopptido formado
57
Psters
fundamentalmente por una porcin hidroflica con un anillo peptdico
de siete aminocidos (Gln-Leu-Leu-Val-Asp-Leu-Ile), y una porcin
hidrofbica constituida por un -hidroxicido graso de 14-16 tomos de C.
Esta estructura le confiere un marcado carcter anfiptico. Se ha observado
que liquenisina permeabiliza membranas modelo de POPC induciendo
la liberacin de la sonda fluorescente carboxifluorescena. Esta accin
est modulada por la composicin de la membrana, tanto de fosfolpidos
(como fosfatidiletanolamina), o esteroles como el colesterol. Estos efectos
son evidentes a concentraciones bastante inferiores a la cmc, y sugieren
la formacin de estructuras agregadas del lipopptido en la bicapa que
podran comportarse como poros de permeabilizacin selectiva. Por otra
parte, en estudios con eritrocitos humanos se ha visto que liquenisina es
capaz de causar la hemlisis, tambin a concentraciones por debajo de su
cmc. A partir de estudios cinticos comparativos, se ha observado que la
salida de iones K+ de los eritrocitos precede a la salida de la hemoglobina
y, adems, la hemlisis se puede evitar de modo eficaz mediante la adicin
a la solucin externa de protectores osmticos de tamao igual o superior
a 32 . Las evidencias indican que la hemlisis inducida por liquenisina
sigue un mecanismo de tipo coloide-osmtico, que implica la formacin de
poros de membrana, en el sentido ms amplio del trmino. Estos resultados
ayudan a establecer una base molecular para la funcin biolgica de
liquenisina.
P05r-10 (R05-4)
Targeting myeloid cells with liposomal

nanocarriers for therapeutic purposes
Jon Ander Nieto-Garai1, Susana Benet2, Maria Pino2, Eneritz
Bilbao1, Itziar Erkizia2, Julia G. Prado2, Javier Martinez-Picado3,
Nuria Izquierdo-Useros*2, Maier Lorizate*1
1
Bioqumica y Biologa Molecular, Universidad del Pas Vasco,
Leioa, ES, 2AIDS Research Institute IrsiCaixa, Badalona, ES,3AIDS
Research Institute IrsiCaixa and ICREA, Badalona, ES
We have recently discovered the mechanism followed by HIV-1 to
enter mature dendritic cells (mDCs) and infect bystander CD4+T cells
in lymphoid tissues, which relies on Siglec-1 (CD169) cell receptor
recognition of sialyllactose exposed on HIV-1 membrane gangliosides.
Here, we aimed to engineer stable nanoliposomes mimicking HIV-1 to
target Siglec-1 expressed on mDCs and other myeloid cells for therapeutic
compound delivery to key anatomical sanctuaries.
Fluorescent POPC nanoliposomes were prepared with increasing
concentrations of sialyllactose-containing gangliosides GM1 and GM3.
Nanoliposomes stability assays and further characterization were performed
using fluorimetric determination and quasi-elastic light scattering based
size determination. Siglec-1+ mDCs and activated monocytes were
exposed to the same concentration of nanoliposomes and capture was
analyzed with FACS or confocal microscopy. Statistical differences were
determined using a paired t-test.
Fluorescent nanoliposomes containing GM1 or GM3 specifically targeted
mDCs and activated monocytes with equal efficiency as fluorescent HIV-1
viral like particles. Ganglioside nanoliposomes were retained within the same
intracellular sac-like compartment where HIV-1 accumulates. Conversely,
nanoliposomes devoid of gangliosides where not stored. An increase of
the ganglioside content in the liposome formulation leads to an improved
capture efficiency in a dose dependent manner. Nanoliposome specificity
for Siglec-1 was demonstrated by blocking capture with two specific mAbs
against Siglec-1 (P.0007) or with soluble sialyllactose (P.004).
We have developed a stable nanoliposome formulation with a potent
and specific capacity to target Siglec-1 expressing myeloid cells. This
technology could be crucial for delivering small therapeutic molecules to
lymphoid tissues.
Funding: amfAR Mathilde Krim Fellowship 108676-55-RKRL.
*Dual Seniorship
58

P05-11
Short-chain sphingolipids preferentially insert

into tumor cell membranes and promote
chemotherapeutic drug uptake
Dalila Ciceri1, Lilia R. Pedrosa2, Flix M. Goi1, Gerben A. Koning2,
F.-Xabier Contreras1
1
Leioa, ES, 2Laboratory Experimental Surgical Oncology Section
Surgical Oncology, Department of Surgery, Erasmus Medical Center,
Rotterdam, NL
Insufficient drug delivery into tumor cells severely limits the therapeutic
efficacy of chemotherapy. Co-delivery of liposome-encapsulated drug
and synthetic short-chain sphingolipids (SCS) greatly improves drug
bioavailability by enhancing intracellular drug uptake. However, whether
SCS insert preferentially into tumor cells and the molecular mechanisms
used by those lipids to improve drug uptake is still mysterious. Here, we
show that SCS have high affinity for tumor cells and that upon insertion
those lipids have specific membrane remodeling activities that favor
drug binding and uptake. We found in a set of in vivo experiments that
external addition of new clickable SCS analogues either in free form or
in a liposomal formulation show more affinity for tumor cells than for
healthy ones. Furthermore, biophysical experiments show that SCS have
membrane-remodeling properties. Transbilayer lipid distribution of lipids
by SCS at one side of the membrane has been monitored using a pyrSM analogue. External addition of SCS in organic solution to preformed
plasma membrane like liposomes induces transbilayer lipid motion of pyrSM within the membrane. Our results demonstrate how SCS are likely to
function on drug delivery, preferentially inserting into tumor cells, and
subsequently altering the biophysical features of cell membranes leading to
transitory membrane packaging imperfections that trigger amphiphilic drug
uptake. Our assays validate the usage of SCS in liposomal chemotherapy
to promote cell-specific targeting, tumor cell membrane remodeling and
finally drug release.
P05-12
Gene knockout of the coupling proteins in

pIP501 and CloDF13 plasmids
Itziar Alkorta1, Marina Garcia-Moreno1, Itxaso lvarez-Rodrguez1,
Ines Probst2, Christina Steck2, Elisabeth Grohmann2
1
University of the Basque Country, Department of Biochemistry and
Molecular Biology (UPV/EHU) and Biophysics Unit (CSIC, UPV/
EHU), University of the Basque Country, Leioa, ES, 2Albert-Ludwigs
University Freiburg, Institute of Biology II, Microbiology, Freiburg,
DE
Type IV secretion systems (T4SSs) are bacterial multiprotein complexes
specialised in the transfer of proteins or DNA-protein complexes across
cell membranes. They are essential for conjugation, bacterial-induced
tumour formation in plant cells, toxin secretion, cell-to-cell translocation
of virulence factors, and intracellular activity of mammalian pathogens. By
enabling conjugative DNA delivery, these systems contribute to the spread
of antibiotic resistance genes among bacteria. The secretion substrates range
from single-stranded DNA/ protein complexes to multicomponent toxins.
They are assisted by integral membrane coupling factors, the multimeric
type IV coupling proteins (T4CPs), which connect the macromolecular
complexes to be transferred with the secretory conduit.
Enterococcus faecalis and the other from CloDF13, a multi-resistance
plasmid from Enterobacter cloacae. Knock-out of the T4CP gene of pIP501
will be performed by a novel gene disruption method for E. faecalis. For
pIP501, the knock-out will be tested in conjugation assays with different E.
faecalis strains, for CloDF13 between different E. coli strains. To exclude
polar effects of the knock-out on downstream genes in the T4SS operon
complementation will be performed by cloning the respective wild type gene
in trans on a vector compatible with pIP501 and CloDF13, respectively.
Granada 2014
Psters
P05-13
P05-15 (R05-8)
Modulation of the plasma membrane localization

and activation of PKCalpha by fatty acids
treatment
Perifosine modulates autophagy in human tumor

cells
Senena Corbalan Garcia, Teresa Coronado-Parra, Dolores PerezSanchez, Ruben Lopez-Nicolas, Juan C Gomez-Fernandez
Departamento de Bioqumica y Biologa Molecular-A, Facultad de
Veterinaria, Universidad de Murcia, Murcia, ES
Protein Kinase C (PKC) is a family of serine/threonine phosphotransferases
that participate in a wide variety of cellular processes that are crucial
in tumor progression. Among them, proliferation, migration, invasion
and survival of cancer cells. Many studies have demonstrated these
isoenzymes are involved in cancer due to changes in their expression
and phosphorylation levels. Unfortunately, no specific PKC modulators
to address the clinical needs have been found to date. In this work we
studied the effect of several fatty acids (OA, DHA and EPA) on cell
localization, migration and catalytic activation of PKCa. Both OA and
DHA increased the catalytic activity of the enzyme. The use of several
mutants that abolished the C2 or C1 domains function revealed that the
C2 domain was essential for activation, while the C1 domain played
a less relevant role. In cell experiments were performed in two breast
cancer cell lines: one from adenocarcinoma non-invasive (MCF-7) and
the other from a highly bone metastatic (MDA-MB-231). The three fatty
acids induced the translocation of PKCa from the cytosol to the plasma
membrane and increased its co-localization with actin filaments. In
addition, we demonstrated that OA and DHA reduced the cell migration
capacity and induced apoptosis in both cell lines, effects that increased
when PKCa was down-regulated by specific siRNA, suggesting that the
enzyme plays critical roles in the migration and survival processes in
breast cancer cells.
P05-14
Two photon fluorescence microscopy of

GUV discloses unprecedented liquid domain
segregation behavior in cholesterol-enriched lipid
bilayers
Pablo Carravilla, Flix M. Goi, Jos Luis Nieva, Jose RequejoIsidro, Nerea Huarte
Unidad de Biofsica (CSIC, UPV/EHU), Leioa, ES
Fluorescence microscopy imaging of Giant Unilamellar Vesicles (GUVs)
has been extensively used to investigate lateral segregation of liquid
microdomains. However, the general application of this approach is
hampered by artifactual light-induced domain formation. Here, we
show that this problem can be circumvented by performing two photon
fluorescence measurements of Laurdan-labeled GUVs, which also
provide quantitative information on the level of molecular order in
each domain by means of the Generalized Polarization function (GP).
To explore domain segregation in the absence of photooxidation, we
have determined Laurdan fluorescence and order degree in raft-standard
ternary mixtures of egg sphingomyelin (eSM)/ dioleoyl-sn-glycero3-phosphocholine (DOPC)/cholesterol (Chol). Our most relevant
observation is that lateral segregation into microscopic Lo-Ld domains
is never observed with eSM:Chol mole ratios lower than 1. Below this
ratio the membrane average order level depends mainly on the eSM
content and no co-existence is observed. In addition, by determining the
order levels under coexistence conditions we infer that the maximum
cholesterol content in Lo domains is attained with eSM:Chol ratios of
ca. 2:1. These observations point to eSM as the main determinant of
macroscopic lipid platform formation in these mixtures.
Jos M Jimnez-Lpez1, Pablo Ros-Marco1, Marisol FernndezOrtiz1, Miguel Garca-Gonzlez2, Josefa L Segovia1, Carmen Marco1,
Mara Paz Carrasco Jimnez1
1
Department of Biochemistry and Molecular Biology I. Faculty of
Sciences. University of Granada, Granada, ES, 2Universitary School
La Inmaculada Concepcin.University of Granada, Granada, ES
Alkylphospholipids (APLs) have been shown to inhibit the growth of
various cancer cells including human hepatoma and glioblastoma cell lines.
APLs have been evaluated in vitro as powerful cancer chemotherapeutic,
however their exact mechanisms of action on cell death and other
inhibitory pathways are unknown. In this work, we show that perifosine, as
representative APLs, produces an increase of the autophagy marker LC3II in HepG2 and U-87 MG cell lines, when compared with controls. An
increase in autophagosomes and LC3-II levels might be consistent with
both autophagy stimulation and block in the late stages of autophagy. To
discriminate whether the increase in LC3-II in perifosine-treated cells
resulted from induction of autophagy or decreased autophagic flux, we
performed assays in the presence of chloroquine to block autophagy by
inhibiting lysosomal proteases and preventing autophagosome-lysosome
fusion. We observed that perifosine treatment produces an alteration in
autophagic flux in both cell lines. We detected moreover that blockade of
autophagy at the level of the autophagosome-lysosome fusion does not
modify apoptotic cell death induced by perifosine in both cell lines. We
conclude that modulation of autophagy process is emerging as new target
for cancer therapy.
This research was aided by the Junta de Andaluca (P11-CVI-7859).
P05-16 (R05-7)
Pancreatic cancer cell glycosylation regulates cell

adhesion and invasion through the modulation of
21 integrin and E-cadherin function
Rosa Peracaula Mir1, Snia Bassagaas1, Sandra Carvalho2, Ana
M. Dias2, Joan Figueras3, Marta Prez-Garay1, M. Rosa Ortiz3, Celso
A. Reis2, Salom S. Pinho2
1
Universidad de Girona, Girona, ES, 2Institute of Molecular
Pathology and Immunology of the University of Porto (IPATIMUP),
Porto, PT, 3Hospital Universitario Dr. Josep Trueta, Girona, ES
In our previous studies we have described that ST3Gal III transfected
pancreatic adenocarcinoma Capan-1 and MDAPanc-28 cells show
increased membrane expression levels of sialyl-Lewis x (SLex) along with
a concomitant decrease in 2,6-sialic acid compared to control cells. Here
we have addressed the role of this glycosylation pattern in the functional
properties of two glycoproteins involved in the processes of cancer cell
invasion and migration, 21 integrin, the main receptor for type 1 collagen,
and E-cadherin, responsible for cell-cell contacts and whose deregulation
determines cell invasive capabilities. Our results demonstrate that ST3Gal
III transfectants showed reduced cell-cell aggregation and increased invasive
capacities. ST3Gal III transfected Capan-1 cells exhibited higher SLex and
lower 2,6-sialic acid content on the glycans of their 21 integrin molecules.
As a consequence, higher phosphorylation of focal adhesion kinase tyrosine
397, which is recognized as one of the first steps of integrin-derived signaling
pathways, was observed in these cells upon adhesion to type 1 collagen. This
molecular mechanism underlies the increased migration through collagen
of these cells. In addition, the pancreatic adenocarcinoma cell lines as
well as human pancreatic tumor tissues showed colocalization of SLex and
E-cadherin, which was higher in the ST3Gal III transfectants. In conclusion,
changes in the sialylation pattern of 21 integrin and E-cadherin appear to
influence the functional role of these two glycoproteins supporting the role of
these glycans as an underlying mechanism regulating pancreatic cancer cell
adhesion and invasion.
59
Psters
P05-17 (R05-3)
Translation of molecular geometry into membrane

fission
Anna Shnyrova1, Eva Rodrguez Hortelano1, Juha-Peka Mattila2,
Sandra L. Schmid2, Sergey A. Akimov3, Vadim A. Frolov4
1
Bioqumica y Biologa Molecular, Universidad del Pas Vasco, Leioa,
ES, 2Department of Cell Biology, UT Southwestern Medical Center,
Dallas, TX, US, 3A.N. Frumkin Institute of Physical Chemistry
and Electrochemistry, Russian Academy of Sciences, Moscow,
RU, 4Unidad de Biofsica (CSIC, UPV/EHU) and Departamento
de Bioqumica y Biologa Molecular, Universidad del Pas Vasco;
IKERBASQUE, Basque Foundation for Science, Leioa, ES
Membrane fission constitutes an essential part of the dynamic life of
cellular membranes. Proteins implicated in this process are designed
to promote high membrane curvature and localized rupture of the
membrane monolayers, both hallmarks to membrane fission. The
localized action of the fission proteins is almost without exceptions
based upon the shallow membrane insertion (membrane wedging)
resulting in disruption of the lipid monolayer continuity at protein
length scale. Such deformation produced by an individual protein,
however, is limited and do not spread far from the protein itself. Thus,
larger-scale coordination of many deformations is needed in order to
effectively remodel the lipid bilayer and induce its fission. We show
here how protein complexes implement seemingly universal and
topology independent strategy to coordinate their membrane-disrupting
action during the fission process. Briefly, creation of a protein ring-like
complex with membrane insertions leads to a local constriction of the
membrane and to a coordinated membrane wedging, that decouple
the membrane deformations on both sides of the ring. Consequently,
ring-like rupture of one monolayer of the constricted membrane creates
additional degree of freedom that critically helps the lipid bilayer to adapt
to the geometry imposed by the constricting ring. As a result, critical
membrane curvature stress can be translated into local destabilization
of lipid monolayer (far from the ring, not at the ring) leading to nonleaky physiological fission. This boundary action might constitute a
general principle of translation of molecular geometry of proteins into
bulk membrane remodeling. Thus, the proposed mechanism can be
crucially important for membrane biogenesis in cells.
P05-18
Specific interaction with cardiolipin triggers

functional activation of dynamin-related protein 1
Itsasne Bustillo-Zabalbeitia1, Sylvie Montessuit2, Etienne Raemy2,
Gorka Basaez1, Jean-Claude Martinou*2, Oihana Terrones Urio*1
1
Unidad de Biofsica (CSIC, UPV/EHU), and Departamento de
Bioqumica, Universidad del Pas Vasco, Leioa, ES, 2Department of
Cell Biology, University of Geneva, Sciences III, Ginebra, CH
Dynamin-Related Protein 1 (Drp1), a large GTPase of the dynamin
superfamily, is required for mitochondrial fission in healthy and apoptotic
cells. Drp1 activation is a complex process that involves translocation
from the cytosol to the mitochondrial outer membrane (MOM)
and assembly into rings/spirals at the MOM, leading to membrane
constriction/division. Similar to dynamins, Drp1 contains GTPase (G),
bundle signaling element (BSE) and stalk domains. However, instead
of the lipidinteracting Pleckstrin Homology (PH) domain present in
the dynamins, Drp1 contains the so-called B insert or variable domain
that has been suggested to play an important role in Drp1 regulation.
Different proteins have been implicated in Drp1 recruitment to the
MOM, although how MOM-localized Drp1 acquires its fully functional
status remains poorly understood. We previously found that Drp1 can
interact with pure lipid bilayers enriched in the mitochondrion-specific
phospholipid cardiolipin (CL). Building on our previous study, we now
explore the specificity and functional consequences of this interaction.
60

We show that a four lysine module located within the B insert of Drp1
interacts preferentially with CL over other anionic lipids. This interaction
dramatically enhances Drp1 oligomerization and assembly-stimulated
GTP hydrolysis. Our results add significantly to a growing body of
evidence indicating that CL is an important regulator of many essential
mitochondrial functions.
*Dual senior autorship.
P05-19
Structure and lipid specificity support

adaptation of pestivirus p7 C-terminal helix to
permeabilizing secretory compartments
Eneko Largo1, Antonio Alcaraz2, Nerea Huarte1, Vicente M.
Aguilella2, Manuel V. Borca3, Jos L. Nieva1
1
Biophysics Unit (CSIC-UPV/EHU) and Biochemistry and Molecular
Biology Department, University of the Basque Country (UPV/EHU),
Bilbao, ES, 2Laboratory of Molecular Biophysics, Department of
Physics, Universitat Jaume I, Castell, ES, 3Plum Island Animal
Disease Center, Greenport, US
Classical swine fever virus (CSFV) protein p7 possesses two hydrophobic
domains intervened by a short polar loop, predicted to span the membrane
as a helix-turn- helix hairpin. Here, we use a combined lipid monolayer,
planar bilayer and vesicle analysis to characterize the insertion and
permeabilization of membranes by constituent CSFV p7 helices. Structural
data and the dependence on lipid composition of these processes support
that the C-terminal helix is a poreforming protein adapted to permeabilizing
membranes of the secretory compartments or mitochondria. Together with
the observed pH-dependence and inhibition pattern, our data suggest that
CSFV p7 relies on genus-specific structures-mechanisms to perform its
viroporin function.
P05-20 (R05-6)
La protena L-plastina est implicada en el

reclutamiento de NKG2D a balsas lipdicas y en
la migracin de clulas NK mediada por NKG2D
Esther Serrano Pertierra1, Eva Cernuda-Morolln1, Tomas Brdicka2,
Vclav Horejsi2, Carlos Lpez-Larrea3
1
Servicio Neurologa, Hospital Universitario Central de Asturias,
Oviedo, ES, 2Institute of Molecular Genetics, Academy of Sciences
of the Czech Republic, Praga, CZ, 3Servicio de Inmunologa,
Hospital Universitario Central de Asturias, Oviedo, ES
Las balsas de membrana son microdominios de la membrana plasmtica
que desempean mltiples funciones biolgicas. La implicacin de estas
estructuras en la biologa de clulas T, principalmente en la transduccin
de seales mediada por TCR, ha sido ampliamente estudiada. Sin
embargo, se conoce menos sobre el papel de las balsas de membrana
en la sealizacin por receptores de clulas NK. Hemos estudiado
la distribucin del receptor activador NKG2D en las balsas lipdicas
mediante el aislamiento de DRM por gradiente de densidad de sacarosa o
mediante fraccionamiento por solubilidad selectiva a beta-octilglucsido
en la lnea celular NKL. Hemos encontrado que el complejo NKG2DDAP10 y pVav se reclutan a estas balsas tras la activacin del receptor. El
anlisis protemico cualitativo mostr que el citoesqueleto de actina est
implicado en este proceso. En particular, hemos visto que la L-plastina,
protena que une filamentos de actina, juega un papel importante en el
reclutamiento de NKG2D a las balsas lipdicas. Adems, la activacin del
receptor inhibidor NKG2A afecta parcialmente el reclutamiento a estos
dominios. Por otra parte, tambin hemos demostrado que la L-plastina
participa en la inhibicin mediada por NKG2D de la quimiotaxis de
clulas NK.
Granada 2014
Psters
P05-21 (R05-5)
showed an excess of cholesterol, resulting in a cholesterol to phospholipid

ratio 6 times higher than in NS.
Functional analysis using the Captive Bubble Surfactometer (CBS) showed
the incapacity of PAP surfactant films to reduce to low enough values the
surface tension at the air-liquid interface. NS and P were subjected to a
LH20 chromatography to obtain the protein, phospholipid and cholesterol
fractions separately. Thus, different combinations of these fractions with
the corresponding fractions of NS were analyzed in the CBS. The P Protein
fraction reconstituted into NS lipids showed an inefficient adsorption and
dynamic behavior, meaning that PAP-associated SP-B and SP-C were
dysfunctional. On the other hand, the excess of cholesterol also showed
a contribution to this impaired behavior, as seen in samples containing the
fully active protein and phospholipid complements of NS but the altered
neutral lipid fraction of P. The exacerbated amount of cholesterol in P
surfactant likely alters the structure and mechanical properties of surfactant
layers rendering them dysfunctional.
Dimerization of the transmembrane domain

regulates the function of p75 neurotrophin
receptor
Irmina Garca-Carpio1, Kirill Nadezhdin2, Sergey Goncharuk2, Konstantin
Mineev2, Eva Fernandez1, Alexander Areseniev2, Maral Vilar1
1
Neurodegeneration Unit. UFIEC-ISCIII, Majadahonda, Madrid, ES,
2
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the
Russian Academy of Sciences, Mosc, RU
p75 neurotrophin receptor (p75 NTR), is best known for its role in mediating
neuronal cell death during development or after injury but it also regulates
cell proliferation, axon guidance or survival. p75 forms disulphide-linked
dimers through the Cys257 in the transmembrane domain and that it is
essential for NGF mediated signaling. Here we demonstrate that p75 use two
different interfaces of dimerization; one promoted by Cys257 and the other
by a motif of the form AxxxG. Our functional and structural data reveals
the key role that plays the Cys257 in p75 dimer assembly, redistribution
and activation of the receptor. We propose the transmembrane domain of
p75 as a new target domain to inhibit receptor function.
P05-22 (R05-2)
The multiple faces of caveolin in the

endoplasmic reticulum: from fat storage
to the integrity of mitochondria
Albert Pol Sorolla
IDIBAPS (CELLEX), Barcelona, ES
La caveolina (CAV) es un componente esencial de las caveolas,
invaginaciones ricas en colesterol de la membrana plasmtica de la mayora
de nuestras clulas. Sin embargo, la funcin de las CAV no est restringida
a la superficie celular, las CAV se asocian dinmicamente con orgnulos
intracelulares como el retculo endoplasmtico (RE), el aparato de Golgi,
o los endosomas. Este transporte intracelular de CAV regula la distribucin
intracelular de lpidos, ya que las CAV unen colesterol y cidos grasos con
gran afinidad. En este contexto hemos demostrado dos funciones de CAV1
en el RE: i) regula los niveles de colesterol en las membranas mitocondriales,
su fluidez y la eficiencia de la cadena respiratoria y ii) regula la acumulacin
de lpidos neutros en la bicapa del RE para generar los cuerpos lipdicos
y proporcionar energa durante la regeneracin del hgado. La ausencia de
CAV1 provoca un desajuste en la distribucin intracelular de lpidos con
importantes consecuencias metablicas y patolgicas para clulas y animales.
P05-24
Molecular function of isolated Ca2+-dependent

gating rings
Roger Gimeno-Llobet, Teresa Girldez Fernndez
Departamento de Ciencias Biomedicas, Facultad de Medicina,
y Centro de Investigaciones Biomedicas de Canarias (CIBICAN),
Universidad de La Laguna, Tenerife, La Laguna, ES
In some Ca2+- regulated ion channels and transporters, ion binding is sensed by
a large C-terminal intracellular region, where eight Regulator of Conductance
for K+(RCK) domains form a gating ring. In the large conductance Ca2+
and voltage regulated channel (BK), it has been proposed that Ca2+ binding
expands the gating ring, and the large movement of the gating ring would
physically pull and open the gate located at the pore domain. Calcium binding
to this region reduces the energy required to open the channel, but the exact
mechanism underlying this process is still uncertain. Using patch-clamp
recordings and simultaneous measurements of fluorescence energy transfer
between CFP and YFP variants of the green fluorescent protein, our group
has shown that Ca2+ binding produces large structural changes that are not
obligatorily coupled to the opening of the pore of the BK channel (Miranda et
al PNAS 2013 110:5217). These results are in contrast with structural studies
of isolated gating rings, where large rearrangements are not observed after
Ca2+ binding. It is possible that the apparent discrepancy is due to involvement
of other regions of the channel in the gating ring movement. We have now
characterized the molecular function of isolated gating rings, using fluorescent
fusion proteins expressed in mammalian cells.
P05-23
Alteration of protein and lipid moieties

in a dysfunctional lung surfactant from alveolar
proteinosis
P05-25 (R05-1)
Mercedes Echaide Torreguitar, Carolina Ballester Lopez, Jesus

Perez-Gil
Complutense de Madrid, Madrid, ES
Victor Meseguer1, Yerandy Alpizar2, Sendoa Tajada3, Enoch Luis1,

Carlos Fernndez-Pea1, Tatiana Kichko4, Peter Reeh4, Mara Teresa
Prez-Garca3, Jos Ramn Lpez Lpez5, Thomas Voets2, Carlos
Belmonte1, Karel Talavera2, Flix Viana1
1
Instituto de Neurociencias de Alicante UMH-CSIC, San Juan
de Alicante, ES, 2KULeuven, Leuven, BE, 3Instituto de Biologa
y Gentica Molecular (IBGM), Universidad de Valladolid-CSIC,
Valladolid, ES, 4Friedrich-Alexander-Universitt, Erlangen-Nrnberg,
DE, 5Instituto de Biologa y Gentica Molecular (IBGM), Universidad
de Valladolid- CSIC, Valladolid, ES
Pulmonary Alveolar Proteinosis (PAP) is a rare disease characterized by an

accumulation of pulmonary surfactant at the alveoli and terminal airways.
Several studies have shown that THIS pathology is associated with an excess
of surfactant lipids and altered proteins. However, it is not clear how each of
the surfactant components contribute the most to the dysfunctional behavior of
this surfactant. In the current work, surfactant from a patient with Pulmonary
Alveolar Proteinosis (P) has been analyzed and compared with respect to
composition and functional behavior of porcine native surfactant (NS).
Compositional analysis by Western Blot revealed the presence of surfactant
protein aggregated forms. Moreover, the protein to phospholipid ratio was
26 times higher in P than in functional NS. An abnormal lipid composition
was also detected by Thin Layer Chromatography and further analysis
TRPA1 channels are membrane sensors of

bacterial endotoxins
TRPA1 is a member of the transient receptor potential (TRP) family of

cationic channels, expressed in nociceptive sensory terminals of primary
sensory neurons. TRPA1 shows polymodal activation by physical (e.g.
temperature and mechanical stretch) and chemical stimuli. Recently,
TRPA1 has emerged as a key chemosensor for many natural and industrial
chemical irritants. In addition, TRPA1 responds to reactive oxygen species
61
Psters
and reactive aldehydes. Activation of TRPA1 produces pain, release of
neuropeptides and inflammation.
Gram(-) bacterial infections are generally accompanied by inflammation and
pain. These symptoms are generally attributed to sensitization of nociceptors
by inflammatory mediators released by immune cells (e.g. monocytes and
macrophages) through activation of the Toll-like-receptor 4 (TLR4) signaling
pathway by lipopolysaccharide (LPS), a toxic byproduct of bacterial lyses.
Unexpectedly, we found that LPS exerts fast, membrane delimited, excitatory
actions on TRPA1. Activation of TRPA1 by different forms of LPS correlates
with the structure of lipid A, the lipid component of the LPS moiety.
Moreover, we found that pain and acute pathophysiological vascular
reactions, including neurogenic inflammation (CGRP release) caused by
LPS are primarily dependent on TRPA1 channel activation in nociceptor
terminals. The identification of TRPA1 as molecular determinant of LPS
effects opens novel avenues for the treatment of symptoms caused by
Gram(-) bacterial infections and offers new insights into the pathogenesis
of pain and neurovascular responses during bacterial infections.
P05-26
Perifosine modulates autophagy in human tumor

cells
J.M. Jimnez-Lpez1, P. Ros-Marco1, M. Fernndez-Ortiz1, M.
Garca-Gonzlez2, J.L. Segovia1, C. Marco1, M.P. Carrasco1
1
Department of Biochemistry and Molecular Biology I, Faculty of
Sciences, University of Granada, Granada, ES, 2Universitary School
La Inmaculada Concepcin, University of Granada, Granada, ES
Alkylphospholipids (APLs) have been shown to inhibit the growth of various
cancer cells including human hepatoma and glioblastoma cell lines. APLs
have been evaluated in vitro as powerful cancer chemotherapeutic, however
their exact mechanisms of action on cell death and other inhibitory pathways
are unknown. In this work, we show that perifosine, as representative
APLs, produces an increase of the autophagy marker LC3-II in HepG2
and U-87 MG cell lines, when compared with controls. An increase in
autophagosomes and LC3-II levels might be consistent with both autophagy
stimulation and block in the late stages of autophagy. To discriminate
whether the increase in LC3-II in perifosine-treated cells resulted from
induction of autophagy or decreased autophagic flux, we performed assays
in the presence of chloroquine to block autophagy by inhibiting lysosomal
proteases and preventing autophagosome-lysosome fusion. We observed
that perifosine treatment produces an alteration in autophagic flux in both
cell lines. We detected moreover that blockade of autophagy at the level of
the autophagosome-lysosome fusion does not modify apoptotic cell death
induced by perifosine in both cell lines. We conclude that modulation of
autophagy process is emerging as new target for cancer therapy.
This research was aided by the Junta de Andaluca (P11-CVI-7859).
P06. Bioqumica de la nutricin

P06r-1
Las semillas de tomate: una fuente oculta

de alrgenos
Laura Martn-Pedraza1, Miguel Gonzlez2, Juan Carlos LpezRodrguez1, Eva Batanero1, Rodrigo Barderas1, Miguel Blanca3,
Rosala Rodrguez1, Mayte Villalba1
1
Departamento de Bioqumica y Biologa Molecular, Universidad
Complutense de Madrid, Madrid, ES, 2Laboratorio de Investigacin,
IBIMA, Hospital Regional Universitario de Mlaga, UMA, Mlaga, ES,
3
UGC Alergia, IBIMA, Hospital Universitario de Mlaga, UMA, Mlaga, ES
62

La alergia a alimentos es un problema de salud en los pases desarrollados
que afecta a un 5% de la poblacin. Provoca sntomas graves e inesperados
y es causa frecuente de anafilaxia. En adultos los alimentos vegetales
inducen el mayor nmero de reacciones de hipersensibilidad, y la mayora
de sus alrgenos pertenecen a unas pocas familias de protenas, entre las
que destacan las cupinas y las prolaminas.
Algunos de los problemas diagnsticos de estas alergias radican en la
presencia de alrgenos especficos en ciertas partes del fruto, como las
semillas. El poder diagnstico de estas molculas se ve diluido cuando se
utiliza el extracto completo, el cual es una mezcla compleja del fruto. Este
es el caso de las semillas de tomate, que son una fuente rica en protenas de
reserva suponiendo un 80% del total.
Los objetivos del presente trabajo son detectar los alrgenos reconocidos
por las IgE de pacientes alrgicos a las semillas de tomate, con sntomas
graves como anafilaxia o angioedema, o leves como urticaria o sndrome de
alergia oral, e identificarlos mediante inmunodetecciones con anticuerpos
policlonales y con sueros de pacientes.
Los extractos se han cromatografiado en HPLC y los picos obtenidos se
han analizado en PAGE-SDS mediante tincin con Azul de Coomassie y
tras transferencia a membranas con sueros. La unin de IgE a una banda
de 10 kDa es mayoritaria en pacientes con sntomas graves de anafilaxia y
angioedema, mientras que bandas de mayor masa molecular (35-60 kDa)
son asociadas a sntomas leves.
El uso de extractos bien definidos de cada tejido y la identificacin de los
alrgenos implicados permitir determinar el patrn de sensibilizacin
de cada paciente con mayor precisin y evitar los falsos negativos que se
obtienen en las pruebas cutneas.
P06r-2 (R06-4)
Cardiotrophin-1 up-regulates apelin secretion

and gene expression in 3T3-L1 adipocytes
Miguel Lpez Yoldi1, Matilde Bustos2, Mara Asuncin Romero
Lozano2, J Alfredo Martnez1, Jess Prieto2, Mara Jess Moreno
Aliaga1
1
Department of Nutrition, Food Science and Physiology, Centre
for Nutrition Research, Faculty of Pharmacy, University of Navarra
- CIBER Fisiopatologa de la Obesidad y Nutricin (CIBERobn),
Instituto de Salud Carlos III, Pamplona, ES, 2Area of Hepatology
and Gene Therapy, Center for Applied Medical Research (CIMA),
University of Navarra, Pamplona, ES
Introduction: Cardiotrophin-1 (CT-1) is a member of the IL-6 family
of cytokines. A recent study of our group has revealed that CT-1 is a key
regulator of glucose and lipid metabolism. Apelin is an adipokine that
stimulates glucose utilization and insulin sensitivity in obese and insulinresistant mice. The aim of the present study was to analyze the potential
effect of CT-1 on apelin production in adipocytes.
Methods: Fully differentiated 3T3-L1 adipocytes were incubated with
different concentrations (1-40 ng/mL) of recombinant protein CT-1 (rCT1) or IL-6 (20 ng/mL) for 18 hours. Apelin mRNA expression levels were
determined by quantitative real-time PCR. The measurement of apelin
secretion to the medium was quantified by ELISA. The phosphorylation
levels of p-Akt (Ser-473) and p-ERK 1/2 (Thr 202/Tyr 204) as well as
total AKT and ERK protein levels were analyzed by Western blot. Specific
inhibitors were used to characterize if these pathways were involved in the
actions of CT-1 on apelin production.
Results: rCT-1 treatment (18 h) significantly increased apelin gene
expression and secretion in 3T3-L1 adipocytes in a concentration dependent
manner. In parallel, rCT-1 (20 ng/mL) induced the phosphorylation of
ERK and AKT in adipocytes. Pre-treatment with the PI3K inhibitor,
LY294002, completely blocked the stimulatory action of CT-1 on apelin
gene expression.
Conclusion: The present data demonstrate the ability of rCT-1 to stimulate
apelin synthesis and/or secretion in adipocytes and suggest that these
effects are probably mediated by PI3K/AKT pathway.
Granada 2014
P06-3 (R06-1)
Accin moduladora de marcadores

cardiovasculares y de obesidad de caf verde
y yerba mate
Laura Bravo Clemente, Sara Martnez Lpez, Raquel Mateos,
Beatriz Sarria
ICTAN-CSIC, Madrid, ES
El caf es ampliamente consumido en todo el mundo, mientras que
la yerba mate es muy popular en pases Sudamericanos, aunque su
consumo se est extendiendo. Ambas bebidas tienen un rico contenido en
compuestos fenlicos derivados de los cidos hidroxicinmicos, as como
metilxantinas, siendo la cafena la ms abundante. El caf verde presenta
una mayor concentracin de estos compuestos bioactivos en comparacin
con el clsico caf tostado. En este estudio se quiso conocer los efectos
en marcadores cardiovasculares y de obesidad del consumo regular de un
producto de caf mezcla de verde y tostado (35:65) y de yerba mate en una
poblacin normocolesterolmica (n=25) y otra hipercolesterolmica (HC,
n=27). Se llev a cabo un estudio cruzado, aleatorizado y controlado en
hombres y mujeres de entre 18-45 aos, no fumadores, no vegetarianos,
con un IMC=18-25 kg/m2, que tomaron el caf, mate o agua (control) tres
veces al da, durante 8 semanas, restringindose el consumo de ciertos
alimentos ricos en polifenoles. Al principio y final de cada intervencin
se tomaron muestras de sangre y se control la presin arterial (PA)
y frecuencia cardiaca (FC), se hizo un estudio antropomtrico y los
participantes rellenaron un registro diettico de 72 horas y cuestionario de
actividad fsica. Se analiz el perfil lipdico y la protena C reactiva (PCR)
mediante autoanalizadores, as como citoquinas pro- y anti-inflamatorias,
quimioquinas, molculas de adhesin, adipoquinas y hormonas mediante
ensayos Multiplex. Los resultados se analizaron mediante un modelo
general de medidas repetidas y test de Bonferroni dentro de cada grupo
(SPSS, v. 19.0).
Ambas bebidas, redujeron FC y PA, especialmente en los sujetos HC, en
los que tambin se observ una disminucin en los niveles de colesterol
total, VLDL-C y triglicridos. Tras la intervencin con caf los voluntarios
disminuyeron su peso corporal, as como el porcentaje de grasa corporal
(por bioimpedancia); con el mate se observaron las mismas tendencias
aunque las diferencias no fueron significativas. Con ambas bebidas se
observ una ligera menor ingesta de protena y, en el caso del mate, tambin
de grasas y energa. El consumo de caf dio lugar a un descenso en los
niveles de grelina, leptina, inhibidor de la activacin de plasmingeno-1
(PAI-1), resistina y PCR en ambos grupos de estudio.
P06-5 (R06-7)
Suplementacin dietaria con aceite de rosa

mosqueta (Rosa rubiginosa) y prevencin de la
esteatosis heptica inducida por una dieta alta
en grasa: participacin de PPAR-alfa y ACOX
Gladys Tapia Opazo1, Sergio Rodriguez1, Gladys Tapia1, Pamela
Romanque Ulloa1, Amanda Despessailles Tapia1, Alejandra Espinosa
Escalona2, Camila Dossi Muoz1
1
Facultad de Medicina, Universidad de Chile, Santiago, CL,
2
Escuela de Tecnologa Mdica, Facultad de Medicina, Universidad
de Chile, Santiago, CL
El elevado aporte dietario de cidos grasos -6 en relacin a los cidos
grasos -3, genera alteraciones en la salud cardiovascular y heptica,
dentro de las patologas crnicas no transmisibles. Por otro lado, el
pescado, rico en cidos grasos -3, es de bajo consumo en Chile, siendo
necesario buscar otras fuentes alternativas de estos cidos grasos, como
lo son aceites con un alto contenido del cido alfa linolnico, precursor
de los cidos grasos -3, eicosapentaenoico (EPA) y docosahexaenoico
(DHA). Tanto EPA como DHA son activadores del factor de transcripcin
PPAR-, el cual regula la expresin del gen que codifica para la protena
prolipoltica, ACOX. Este trabajo evalu la prevencin de la esteatosis
Psters
heptica mediante suplementacin con aceite de rosa mosqueta (RM)
rico en cido alfa linolnico, que se biotransforma en EPA y DHA. Para
ello, ratones macho C57BL/6J (n=5-9 por grupo experimental) fueron
alimentados con una dieta: a) control (10% lpidos, 20% protenas y 70%
de carbohidratos) y b) alta en grasa (60% lpidos, 10% protenas y 30%
carbohidratos) con o sin suplementacin dietaria diaria con aceite de
RM (Coesam, Chile). La dieta y la suplementacin con aceite de RM
se mantuvieron por 12 semanas. Se determin la esteatosis heptica
(histologa) y los niveles de PPAR- (RT-PCR) y de ACOX (RT-PCR).
Los resultados mostraron que los animales que fueron alimentados con
dieta alta en grasa y suplementados con aceite de RM: i) presentaron una
histologa heptica normal y una disminucin significativa en los niveles
de esteatosis heptica evaluada por el porcentaje de clulas con gotas
lipdicas, respecto al grupo que slo recibi la dieta alta en grasa (ANOVA
unifactorial, seguida del Test de Newman Keuls); y ii) disminuyeron en
forma significativa el contenido de grasa abdominal, respecto a aquellos
que slo recibieron la dieta alta en grasa. La prevencin de la esteatosis
heptica se acompa de un aumento en los niveles hepticos del mRNA
de PPAR- y ACOX. Se concluye que la administracin dietaria de un
aceite rico en cido alfa linolnico previene la esteatosis heptica lo
cual podra estar relacionado al aumento en los niveles de PPAR- y de
ACOX.
FONDECYT 1140547.
P06-6
Efecto de la suplementacin diettica con

protenas de plasma bovino en la prevencin
de alteraciones de la mucosa intestinal
en un modelo de colitis
Llusa Mir1, Anna Prez-Bosque2, Mnica Maij2, Javier Polo1,
Miquel Moret2
1
APC Europe, Granollers, Barcelona, ES, 2Departament de
Fisiologia, Facultat de Farmcia; Institut de Nutrici i Seguretat
Alimentria, Universitat de Barcelona, Barcelona, ES
En estudios previos se ha demostrado que la suplementacin diettica
con protenas plasmticas reduce la respuesta inflamatoria intestinal en
modelos de inflamacin aguda. La mucosa intestinal contiene mucinas
y TFF3, con funciones de proteccin y reparacin tisular. El objetivo de
este trabajo es analizar dichas variables en ratones KO que carecen del
gen mdr-1 y desarrollan colitis de forma espontnea, y evaluar el efecto
de la suplementacin diettica con inmunoglobulinas de plasma bovino
(IPB). Los ratones KO y los correspondientes controles recibieron una
dieta control o bien una dieta suplementada con 2% IPB desde el da 21
(destete) hasta el da 56. Se realiz un estudio histolgico para establecer
el ndice histopatolgico as como el recuento de las clulas caliciformes
de la mucosa del colon. Tambin se determin por RT-PCR la expresin
de mucinas MUC1 y MUC4 (transmembrana), MUC2 (secretora) y del
TFF3. Los ratones KO presentaron un mayor ndice histopatolgico que
los controles y la suplementacin con IPB redujo en parte estos efectos.
En los animales KO se observ un aumento de la expresin de MUC4 (4
veces) y de MUC1 (6 veces, ambas P<0,05), menor expresin de MUC2
y TFF3, que se redujo en un 80% y un 50%, respectivamente (P<0,05)
y un menor nmero de clulas caliciformes. La suplementacin con
IPB atenu los cambios en la expresin de las mucinas transmembrana
y del TFF3 sin modificar la MUC2 y restaur la poblacin de clulas
secretoras de moco. Los resultados indican que la suplementacin con
IPB restablece la funcin protectora de la mucosa intestinal, modulando
la expresin de mucinas y reduciendo las alteraciones histolgicas de la
colitis.
Financiado por el proyecto TRACE 2009 0317 (Ministerio de Economa y
Competitividad, Espaa).
63
Psters
P06r-7
Efecto del cido maslnico, un triterpeno

pentacclico natural, sobre lesiones
preneoplsicas inducidas en colon de rata
Glria Lozano-Mena, Marta Snchez-Gonzlez, M. Emlia Juan,
Joana M. Planas
Departament de Fisiologia e Institut de Recerca en Nutrici i
Seguretat Alimentria-UB, Universitat de Barcelona, Barcelona, ES
El cido maslnico es un triterpeno pentacclico presente en distintos
vegetales habituales en la dieta mediterrnea. Estudios previos han
demostrado su actividad antiproliferativa y proapopttica en la lnea
celular de cncer de colon HT-29. Con el fin de confirmar su actividad
quimiopreventiva in vivo, se ha usado un modelo de cncer de colon en
rata inducido por 1,2-dimetilhidrazina (DMH). El agente carcingeno se
inyect semanalmente por va intraperitoneal (20 mg/kg), mientras que el
cido maslnico se administr por va oral a las dosis 5, 10 y 25 mg/kg
durante 49 das. El colon se dividi en los segmentos proximal, medial
y distal, que se fijaron con formalina antes de la observacin de lesiones
preneoplsicas al microscopio ptico. La tincin de azul de metileno
permiti el recuento de focos de criptas aberrantes (FCA), mientras que
la tincin de diaminas y hierro/azul alciano se us para el recuento de
focos con deplecin de mucinas (FDM), indicativas de un mayor grado
de displasia. Adems, se determin la concentracin de cido maslnico en
contenido de colon para establecer una correlacin con el efecto observado.
El cido maslnico no redujo el nmero de FCA en colon total, que fue
de 15820, 18915, 18417 y 16018 en los grupos DMH i DMH-cido
maslnico a las tres dosis, respectivamente. En todos los grupos se observ
la misma distribucin de FCA, siendo mayor en los segmentos medial y
distal. Tampoco se observaron diferencias estadsticamente significativas
en el recuento de FDM, que fue de 285, 336, 3410 y 3512 en colon
total. En conclusin, el cido maslnico a la tres dosis ensayadas no evita la
aparicin de lesiones preneoplsicas inducidas por DMH en ratas.
Financiado por AGL2009-12866 del MEC y 2009-SGR-00471 de la
Generalitat de Catalunya.
P06-8
Preventive effect of low molecular-weight

proanthocyanidins on metabolic syndrome
parameters
Zara Pons, Maria Margalef, Susana Surez-Garca, Aleix Ribas-Latre,
Francisca Isabel Bravo, Ada Pascual-Serrano, Manuel Suarez,
Cinta Blad, Gerard Aragons, Lluis Arola, Anna Arola-Arnal,
Begoa Muguerza
Nutrigenomic Research Group, Universitat Rovira i Virgili,
Tarragona, ES
The importance of metabolic syndrome (MS) is rising due to its increasing
prevalence worldwide. For that reason, a great effort is being directed to
prevent the development of MS. In this regard, the use of functional foods
is highly recommended. A good animal model to study MS is the cafeteria
(CAF) fed rat, no only because it reproduces the MS but also because it shares
the pathogenesis of the human disease. On the other hand, proanthocyanidins
have been previously described to cause beneficial health effects in most of
the components of MS and in cardiovascular risk factors.
In this study, male wistar rats were fed CAF or standard (ST) diet for 12
weeks. Additionally, CAF fed rats were treated with different doses of low
molecular weight grape seed proanthocyanidins extract (LM-GSPE) (25,
100 and 200 mg/Kg/day; n=10) or vehicle, and the ST rats were given
vehicle (n=10). Body weight, waist perimeter, blood pressure and food
intake were measured weekly. Plasmatic parameters were checked at 7th,
10th and 12th week of experiment.
The animals fed with CAF diet presented raised body weight, waist perimeter
and blood pressure compared to ST rats. LM-GSPE decreased blood pressure
64

in a dose response manner. CAF treated rats had decreased body weight and
waist perimeter at the 7th and 8th week of experiment at doses of 100 and 200
mg/Kg/day. Differences in food and liquid intake were found for diet, but not
due to treatment. Plasma triglycerides and total cholesterol were elevated in
CAF rats compared to ST rats. A decrease in total cholesterol was found for
the highest doses of LM-GSPE in 7th and 10th weeks and a decrease in plasma
triglycerides with the lower doses at 12th week was observed.
Grape seed proanthocyanidins have a clear antihypertensive effect,
preventing the rise of blood pressure due to the CAF diet. Body weight
and waist perimeter were slightly modified by the treatments. In addition,
a clear effect on plasma cholesterol was described. Therefore, LM-GSPE
could be a promising candidate for functional foods due to the improvement
observed in most of MS components.
P06-9
Efectos de la suplementacin prolongada con

cidos grasos omega-3 en un modelo de sordera
progresiva
Raquel Martnez Vega1, Teresa Partearroyo2, Nstor Vallecillo3,
Gregorio Varela-Moreiras2, Isabel Varela-Nieto1, Mara A. Pajares1
1
Instituto de Investigaciones Biomdicas Alberto Sols
(CSIC-UAM) e IdiPAZ, Unidad 761, CIBERER , Madrid, ES,
2
Departamento de Ciencias Farmacuticas y de la Salud, Facultad
de Farmacia, Universidad CEU San Pablo, Boadilla del Monte,
Madrid, ES, 3Instituto de Investigaciones Biomdicas Alberto Sols
(CSIC-UAM) e IdiPAZ, Unidad 761, CIBERER , Madrid, ES
Los cidos grasos poli-insaturados omega-3 (PUFAs) son nutrientes
muy conocidos por sus efectos beneficiosos en el desarrollo cognitivo
y su mantenimiento, regulando la inflamacin, el estrs oxidativo y la
sensibilidad a insulina, entre otros parmetros asociados al envejecimiento
(1). Los niveles insuficientes de cido docosahexaenico (omega-3) se
han asociado con trastornos neurolgicos, vasculares, y con la prdida
auditiva asociada a la edad (ARHL) en el hombre (2). Se ha demostrado
la existencia de una relacin directa entre ARHL y niveles plasmticos
elevados de homocistena (pHcy)(3). Por el contrario, la relacin entre
ARHL y altos niveles plasmticos de PUFAs resulta ser inversa (4). En el
presente estudio hemos utilizado ratones C57BL/6J y una suplementacin
prolongada con omega-3 para evaluar su impacto en la capacidad auditiva,
los niveles de Hcy, el estrs oxidativo y la inflamacin.
Ratones de dos meses de edad fueron alimentados con dieta control o
suplementada con omega-3 durante 10 meses. La capacidad auditiva de los
animales fue evaluada mensualmente mediante ABR y DPOAE, analizando
sus umbrales auditivos. Se tomaron muestras de sangre para determinar
concentraciones de pHcy y cido flico por HPLC. La morfologa coclear
se evalu mediante inmunohistoqumica y tincin con violeta de cresylo.
Se analizaron marcadores de inflamacin y estrs oxidativo mediante
Western blotting y RT-qPCR.
El grupo control mostr umbrales auditivos significativamente elevados
en ABR (~25 dB SPL) y menores amplitudes DPOAE a frecuencias
medias-altas, cuando se compar con el grupo que recibi omega-3. No se
observaron diferencias histolgicas entre los grupos, pero s se detectaron
niveles elevados de pHcy (p=0.13) y disminuidos de cido flico srico
(p<0.05) en el grupo control comparado con el suplementado con omega-3.
Los resultados obtenidos sugieren que la suplementacin prolongada con
omega-3 podra tener un efecto protector a largo plazo en el desarrollo de
ARHL.
Referencias
[1] Da Young Oh et al., Cell, 142(5):687-98, 2010.
[2] Bamini Gopinath et al., Am J Clin Nutr, 92(2):416-21, 2010.
[3] Bamini Gopinath et al., J Nutr, 140(8):1469-74, 2010.
[4] Carla Dullemeijer et al., J Nutr Health Aging, 14(5):347-51, 2010.
Agradecimientos: Beca JAE, BFU2009-08977, SAF2011-24391, FP7AFHELO, FP7-AFHELO, FP7-TARGEAR y Lactalis-Puleva.
Granada 2014
Psters
P06-10 (R06-5)
implicados como la disminucin de endothelina-1 o el aumento de

prostaciclina. Adems, se conoce que estos compuestos tambin pueden
inhibir un gran nmero de enzimas, incluyendo la ECA, de promover
la expresin de mltiples genes reguladores de la presin arterial y de
afectar en diferentes niveles a las cascadas de sealizacin implicadas en
el control de la presin arterial.
Estudio farmacocintico del cido maslnico,

un componente bioactivo de Olea europea L.
Marta Snchez-Gonzlez1, Glria Lozano-Mena1, Tatiana
Trebolento1, Helena Colom2, M. Emlia Juan1, Joana M. Planas1
1
Departament de Fisiologia e Institut de Recerca en Nutrici i
Seguretat Alimentria-UB, Universitat de Barcelona, Barcelona, ES,
2
Departament de Farmcia i Tecnologia Farmacutica, Barcelona, ES
El cido maslnico es un componente minoritario aislado de diferentes
plantas y principalmente de las hojas y los frutos del olivo. Se han
descrito sus efectos beneficiosos en modelos animales de cncer,
diabetes y cardiopata pero se desconoce su biodisponibilidad oral. Por
lo tanto, el objetivo del presente estudio es determinar los parmetros
farmacocinticos del cido maslnico en rata tras su administracin oral.
Una dosis nica de 50 mg/kg del compuesto fue administrada mediante
sonda intragstrica a ratas Sprague-Dawley. Se obtuvieron muestras de
sangre procedentes de la vena safena a 18 tiempos distintos durante 24
horas. El plasma se proces siguiendo una doble extraccin lquidolquido con acetato de etilo antes de ser analizado por HPLC-MS.
Mediante el software Winnonlin se realiz el anlisis no compartimental
y compartimental de las concentraciones plasmticas de cido maslnico
vs tiempo, siendo el modelo bicompartimental el que mejor describi
la farmacocintica oral del compuesto. La Cmax del cido maslnico
(4,82 M) se alcanz 49,90 min despus de su administracin. El AUC
fue de 913,58 minmol/L y el CL/F de 0,116 L/min/kg. Los Vc/F y
Vp/F fueron de 13,02 y 30,56 L/kg, respectivamente. Por otro lado, la
K01 present un valor de 0,034 1/min y la K10 de 0,0088 1/min. Los
resultados obtenidos sugieren una absorcin relativamente rpida con
una buena distribucin al compartimento perifrico y una eliminacin
sostenida. En conclusin, las concentraciones plasmticas de cido
maslnico tras su administracin oral siguen una cada biexponencial
con una constante de retorno limitada y una eliminacin lenta del
organismo.
Estudio financiado por los proyectos AGL2009-12866 del MCT y 2009SGR-00471 de la Generalitat de Catalunya.
P06-11 (R06-2)
Efectos beneficiosos de ingredientes bioactivos

sobre la hipertensin arterial
Begoa Muguerza, Zara Pons, Cinta Blad, Gerard Aragones,
Manuel Suarez, Llus Arola, Anna Arola-Arnal
Nutrigenomic Research Group, Universitat Rovira i Virgili,
Tarragona, ES
La enfermedad cardiovascular es la principal causa de muerte en el mundo
y la hipertensin arterial es uno de sus principales factores de riesgo. Varios
estudios han demostrado que el consumo de alimentos o ingredientes
ricos en flavanoles, como el cacao o extracto de pepita de uva, mejoran la
funcin endotelial y disminuyen la presin arterial.
Diferentes mecanismos podran justificar las propiedades antihipertensivas
de los flavanoles. La vasodilatacin ocasionada por estos compuestos se
ha relacionado con la reduccin del estrs oxidativo, con la produccin
de xido ntrico (NO) y la inhibicin de la enzima convertidora de la
angiotensina (ECA), clave para el control de la presin arterial.
El cacao y el extracto de pepita de uva son productos ricos en flavanoles
del tipo flavan-3-ol y proantocianidinas que han demostrado gran
actividad antioxidante. Sin embargo, la concentracin real de flavonoides
que alcanzan los vasos sanguineos es tan baja que es poco probable
que estos compuestos acten por un mecanismo antioxidante directo
y posiblemente otros mecanismos relacionados con interacciones
especficas con protenas o lpidos, sern responsables de sus efectos sobre
la presin arterial. De hecho, el efecto antihipertensivo de los flavanoles
se atribuye fundamentalmente, a un aumento en la disponibilidad de NO.
Sin embargo, es posible que otros mecanismos de accin estn tambin
P06-12 (R06-6)
Apigenin K has intestinal anti-inflammatory

effect in two experimental models of rat colitis
Raquel Gonzlez Prez1, Cristina Mascaraque Molina1, Mara
Dolores Surez Ortega2, Antonio Zarzuelo Zurita1, Olga Martnez
Augustin2, Femn Snchez de Medina Lpez-Huertas1
1
Departamento de Farmacologa, Facultad de Farmacia, CIBERehd,
UGR, Granada, ES, 2Departamento de Bioqumica y Biologa
Molecular II, Facultad de Farmacia, CIBERehd, UGR, Granada, ES
Background and objectives: Flavonoids are polyphenolic compounds
which are widespread in nature and are consumed as part of the human
diet in significant amounts. Many flavonoids have been studied for their
intestinal antiinflammatory activity. The aim of this study was to test the
intestinal anti-inflammatory activity of apigenin K, a soluble form of
apigenin, in two models of rat colitis, the trinitrobenzene sulfonic acid
(TNBS) model and the dextran sulfate (DSS) model.
Methods: Apigenin K (3 mg/kg, p.o.) was administered as a pretreatment
to rats with TNBS and DSS colitis and colonic status was checked 7 and 9
days after colitis induction, respectively, by macroscopic and biochemical
examination.
Results: Apigenin K pretreatment resulted in amelioration of the
morphological signs and biochemical markers in the TNBS model. The
results demonstrate a reduction in inflamed area of 2.4 cm compared
with TNBS group, lower values of macroscopic damage (5.81.1 vs
8.61.4, p<0.05) and a slight decrease in the colonic weight/length
ratio. Myeloperoxidase and alkaline phosphatase colonic activities were
reduced by 34% (p<0,05) and 18%, respectively. Moreover, apigenin K
also ameliorated morphological signs and biochemical markers in the DSS
model, the comparable results. Thus macroscopic damage was significantly
reduced (0.50.5 vs 2.10.6, p<0.05) and the colonic weight/length ratio
was lowered by approximately 10%. Colonic myeloperoxidase and alkaline
phosphatase activities decreased 30% and 20%, respectively (p<0,05).
Apigenin K treatment additionally reduced the colonic expression of IL1,
IL6, Foxp3, TLR2 and TGF compared with the TNBS group (p>0.05 for
the latter two).
Conclusions: Apigenin K has anti-inflammatory effects in two preclinical
models of inflammatory bowel disease.
This study was supported by grants of the Centre for Technological
Industrial Development (CDTI) (CENIT SENIFOOD).
P06-13 (R06-3)
Pectinas, regulacin del peso corporal y salud

Ana Mara Rodrguez Guerrero, Francisco Javier Garca Carrizo,
Catalina Pic Segura, Andreu Palou Oliver
Laboratorio de Biologa Molecular, Nutricin y Biotecnologa
(Universitat de les Illes Balears) y CIBER Fisiopatologa de la
Obesidad y Nutricin, Palma de Mallorca, ES
Recientemente, la composicin de la microbiota intestinal ha emergido
como factor crucial en la prevencin de la obesidad (y sus complicaciones),
a su vez fuertemente influenciada por la dieta. Numerosas investigaciones
destacan el efecto de los prebiticos como moduladores de la microbiota,
en un mejor funcionamiento metablico y en la proteccin frente a la
obesidad. Un tipo particular de prebiticos, utilizados de forma tradicional
en alimentacin, son las pectinas.
65
Psters
Las pectinas son fibras hidrosolubles del tipo polisacridos no-almidn
sobre las que se ha probado su papel en la mejora del control de la glucemia
y de los niveles de colesterol, aunque su posible papel en la proteccin
frente a la obesidad y en otros aspectos relacionados con la salud metablica
est menos demostrado.
En nuestro laboratorio, hemos desarrollado un modelo de propensin
a la obesidad: descendencia de ratas sometidas a restriccin calrica
moderada durante el embarazo, modelo ms similar a la posible
situacin en humanos respecto de otros modelos ms restrictivos.
Hemos analizado el efecto protector de una suplementacin moderada
con pectinas altamente esterificadas de manzana desde el destete hasta
la edad adulta bajo una dieta control o incluyendo un factor obesognico
ms (dieta alta en sacarosa). Hemos comprobado que la suplementacin
con pectinas a largo plazo supone una proteccin significativa frente
al desarrollo de adiposidad corporal, acompaada de un mejor perfil
metablico de parmetros circulantes y parmetros de expresin gnica
en tejidos clave.
En definitiva, la suplementacin de la dieta con pectinas se perfila como
una posible herramienta eficaz en la prevencin de la obesidad y en el
mantenimiento de un perfil metablico saludable.
P06-14
Dietary squalene increases high density

lipoprotein-cholesterol and paraoxonase 1
and decreases oxidative stress in mice
Clara Gabs-Rivera1, Cristina Barranquero2, Roberto MartnezBeamonte1, Mara Angeles Navarro-Ferrando1, Joaqun Carlos SurraMuoz3, Jess de la Osada Garca4
1
Departamento Bioqumica y Biologa Molecular y Celular,
Universidad de Zaragoza, Zaragoza, ES, 2CIBER de Fisiopatologa
de la Obesidad y Nutricin, Instituto de Salud Carlos III, Zaragoza,
ES, 3Departamento de Produccin Animal, Escuela Politcnica
Superior de Huesca, Huesca, ES, 4Departamento de Bioqumica y
Biologa molecular y Celular, Facultad de Veterinaria, Universidad
de Zaragoza, Zaragoza, ES
Background and Purpose: squalene, the main hydrocarbon in the
unsaponifiable fraction of virgin olive oil, is involved in cholesterol
synthesis and it has been reported to own antiatherosclerotic and
antiesteatosic effects. However, the squalenes role on lipid plasma
parameters and the influence of genotype on this effect need to be
addressed.
Experimental Approaches: three male mouse models (wild-type, Apoa1and Apoe- deficient) were fed chow semisynthetic diets enriched in
squalene to provide a dose of 1 g/kg during 11 weeks. After this period,
their plasma parameters and lipoprotein profiles were analyzed.
Key Results: squalene administration at a dose of 1 g/kg showed
decreased reactive oxygen species in lipoprotein fractions independently
of the animal background and caused an specific increase in high density
lipoprotein (HDL)-cholesterol levels, accompanied by an increase in
phosphatidylcholine and paraoxonase 1 and no changes in apolipoproteins
A1 and A4 in wild-type mice. In these mice, the cholesterol increase
was due to its esterified form and associated with an increased hepatic
expression of Lcat. These effects were not observed in absence of
apolipoprotein A1. The increases in HDL- paraoxonase 1 were translated
into decreased plasma malondialdehyde levels depending on the presence
of Apolipoprotein A1.
Conclusions and Implications: dietary squalene promotes changes in
HDL- cholesterol and paraoxonase 1 and decreases reactive oxygen species
in lipoproteins and plasma malondialdehyde levels, providing new benefits
of its intake that might contribute to explain the properties of virgin olive
oil, although the phenotype related to apolipoproteins A1 and E may be
particularly relevant.
66

P06m-15
Marcadores diagnsticos y pronsticos

en la obesidad mrbida
JR Muoz-Rodrguez1, E Salas1, E Segura1, M Snchez1, G Lpez1,
M Palma1, P Rozas1, L Senz1, A Agarrado1, C Gonzlez-Martn2,
G Casas1, A Len1, J Martn1, LF Alguacil2
1
Hospital General Universitario de Ciudad Real, Ciudad Real, ES,
2
Hospital General Universitario de Ciudad Real, Universidad CEU
San Pablo, Boadilla del Monte, Madrid, ES
El objetivo de este estudio es evaluar la posible utilidad diagnstica de
diversos marcadores genticos y bioqumicos en la obesidad mrbida, as
como su valor pronstico y predictivo de la respuesta personalizada a la
ciruga baritrica.
Para el estudio bioqumico se seleccionaron 31 pacientes con obesidad
mrbida (IMC>40 kg/m2) que fueron sometidos a ciruga baritrica en el
HGUCR y a los que se realiz un seguimiento de 12 meses. Se reclut
el mismo nmero de controles normopesos (IMC entre 18,5 y 24,9 kg/
m2) entre el personal del hospital procurando que las proporciones de edad
y sexo resultaran homogneas. Para los estudios genticos se aument el
nmero de pacientes a 74 y el de controles a 57.
Como marcadores genticos se midieron polimorfismos de genes
relacionados con obesidad y trastornos de la conducta alimentaria (Bdnf,
Cart, Ucp2, Fto, Adra2B y Pparg). Como marcadores bioqumicos se
midieron neuropptidos (BDNF y CART), hormonas (grelina, leptina,
adiponectina, insulina) y citoquinas proinflamatorias (IL-6 y TNF).
El estudio gentico mostr diferencias significativas entre controles y
pacientes en las frecuencias de los polimorfismos rs9939609 y rs9939973
del gen Fto y rs71824147 del gen Ucp2. Los niveles de IL-6 fueron
superiores en pacientes respecto a los controles. Se detect un descenso en
BDNF un ao despus de la ciruga baritrica en los pacientes intervenidos.
El perfil hormonal, claramente distinto entre ambos grupos de inicio, se
revirti en los pacientes obesos intervenidos en lo que respecta a niveles de
adiponectina, leptina e insulina.
Los datos obtenidos, junto con otros estudios preclnicos y clnicos,
sugieren que el anlisis de los polimorfismos de Ucp2 y Fto, la citoquina
IL-6, el perfil hormonal y el neuropptido BDNF, pueden ser herramientas
valiosas bien para definir diferentes endofenotipos de obesidad, bien para
evaluar o predecir el resultado de los tratamientos contra la obesidad.
Financiado por el Instituto de Salud Carlos III (FIS PI10/00440). Los
autores agradecen a Da Amelia Gonzlez Lpez su excelente asistencia
tcnica.
P07. Bioqumica perinatal

P07-1 (R07-3)
Obesidad pregestacional. Consecuencias para

la descendencia y papel del estrs oxidativo
Martn Alcal Daz.Mor1, Sonia Claps2, Victoria E: Bolado Garca1,
Isabel Snchez-Vera Gmez-Trelles1, Franciasco Das3, Guillermo
Saez Tormo4, Mara del Pilar Ramos lvarez1, Marta Viana Arribas1
1
Facultad de Farmacia, Universidad CEU San Pablo, Madrid, ES,
2
ICBP Victoria de Girn, La Habana, CU, 3Fundacin Investigacin
Clnico de Valencia, Instituto de Investigacin Sanitaria-INCLIVA,
Valencia, ES, 4Dpto. de Bioqumica y Biol. Molecular-CIBEROBN,
Facultad de Medicina - Servicio de Anlisis Clnicos-CDB HGUV,
Universidad de Valencia, Valencia, ES
Llevamos a cabo un estudio en un modelo de rata gestante con obesidad
inducida por dieta, donde existen claras alteraciones metablicas, entre
ellas una elevada resistencia a la insulina. Se observ un incremento en
el porcentaje de malformaciones congnitas en el grupo de ratas obesas,
Granada 2014
reducido tras la administracin de vitamina E. Se observ un incremento
en el estrs oxidativo materno (mayores niveles de dao oxidativo, tanto
a protenas (PAOP) como al ADN, no siendo as en lpidos). Las mayores
concentraciones de PAOP y de 8-oxo-7,8-dihidro-2 -desoxiguanosina, se
correlacionaron positivamente con el incremento en las malformaciones
congnitas. Adems, los sistemas antioxidantes, tanto lipoflicos (vitamina
E en tejido adiposo visceral), como enzimticos (CuZnSOD, MnSOD y
GPx hepticos) se encontraron disminuidos, confirmando as la situacin
de incrementado estrs oxidativo asociado a la obesidad. Adems, la
subunidad cataltica de la glutamato cistena ligasa, enzima responsable de
la sntesis del glutation, est incrementada en obesidad como consecuencia
de la demanda de dicho antioxidante.
P07-2 (R07-4)
Early nutrition reprograms the hepatic circadian

clock: Permanent deregulation of metabolism
Cristina Garcia-Beltran1, Slvia Rib1, Susana Kalko2, Antonio
Fernndez3, Laura Martnez-Guin1, Judith Cebri1, Thais Pentinat1,
Dbora Martnez1, Carles Lern1, Mario Vallejo3, Rubn Daz1, Josep
Jimnez Chillarn1
1
Hospital Sant Joan de Du, Esplugues de Llobregat, ES, 2Hospital
Clnic de Barcelona-IDIBAPS, Barcelona, ES, 3Instituto de
Investigaciones Biomdicas Alberto Sols CSIC-UAM; Centro de
Investigacin Biomdica en Red de Diabetes y Enfermedades
Metablicas Asociadas (CIBERDEM), ISCIII, Madrid, ES
Excessive caloric intake during early stages of development increases the
risk of childhood obesity and predisposes individuals to develop late onset
of obesity, insulin resistance and type 2 diabetes.
We have developed a mouse model of neonatal overfeeding (ON) by culling
the offspring to 4 pups per dam during lactation. Control females nursed
8 pups during lactation (C). ON mice developed obesity, hyperglycaemia,
insulin resistance and glucose intolerance with ageing. The liver was the
tissue that contributed most prominently to the development of insulin
resistance. In order to underpin molecular mechanisms of insulin resistance
we analysed global gene expression profiling (Affymetrix) in livers from
ON and C male mice. The Ontology with highest significance was the
Circadian Rhythm. We next validated (qPCR) the candidate genes (Npas2,
Per1, Per3, Cry1) in adult mice. Strikingly, changes in expression (Per1,
Cry2) were already present in livers from 15-day-old ON mice. This data
suggests that altered expression of hepatic clock genes is established early
in life and do not occur as a secondary event associated to the progressive
development of obesity and/or insulin resistance.
Circadian rhythms play a major role in orchestrating daily physiological
functions, and disrupting the expression of clock genes evokes metabolic
diseases. Thus, we tested whole-body metabolism by indirect calorimetry.
We found that VO2, VCO2, energy expenditure or activity absolute
values were similar between groups during both the light and dark cycles.
In contrast, in agreement with alterations in the circadian rhythmicity,
the diurnal decrease in respiratory exchange ratio (RER) cycling started
significantly earlier in ON mice. Likewise, upon fasting, V02, VCO2, and
energy expenditure remained higher in ON mice during the night cycle.
Overall, we show that early malnutrition permanently reprograms the
hepatic circadian clock. Such reprogramming induces a sustained feedback loop that may in turn influence the physiologic behaviour of mice
leading, secondarily, to overall metabolic deregulation.
P07-3 (R07-2)
Papel de p53 en la obesidad y su posible

implicacin en la programacin del metabolismo
Ruben Nogueiras Pozo
Universidad Santiago de Compostela, Santiago de Compostela, ES
p53 es un gen supresor tumoral que desempea mltiples funciones biolgicas,
incluyendo la capacidad para modular el metabolismo a diferentes niveles,
Psters
incluyendo acciones en la grasa, el hgado o el pncreas. En este sentido
p53 parece estar involucrado en la programacin metablica de los islotes
pancreticos durante la maternidad. La funcin endgena de p53 parece estar
afectada por los diferentes estados nutricionales y los animales modificados
genticamente en los que se altera la expresin de p53 presentan importantes
cambios metablicos. Adems tambin se ha observado que la expresin de
p53 en el sistema nervioso central regula la accin metablica de algunas
hormonas que afectan a la ingesta. Futuros estudios sern necesarios para
corroborar si p53 podra ser utilizada como diana teraputica.
P07-4 (R07-5)
La obesidad previa a la gestacin induce

alteraciones metablicas en la placenta
Mnica Diez-Hochleitner Ruiz1, Julio Sevillano Fernndez1, Jimena
Pita Santibez1, Mara Haro Garca1, Marta Viana Arribas1, Gema
Medina Gmez2, M. Pilar Ramos lvarez1
1
Bioqumica y Biologa Molecular, Universidad CEU San Pablo,
Madrid, ES, 2Universidad Rey Juan Carlos, Madrid, ES
La prevalencia de la obesidad ha aumentado en los ltimos aos, por lo que
la probabilidad de que una mujer presente obesidad o sobrepeso al inicio de
la gestacin es cada vez mayor. Por estudios epidemiolgicos sabemos que
la obesidad durante la gestacin favorece un mayor riesgo de complicaciones
del recin nacido en edad adulta, aunque no se conocen en detalle los
mecanismos implicados. Por esta razn, quisimos estudiar si la obesidad
previa a la gestacin altera la homeostasis glucdica y lipdica de la madre.
Un grupo de ratas wistar recibi una dieta moderada en grasa previamente a
la gestacin (Moderate Fat Diet, MFD), y en paralelo otro grupo recibi la
dieta control (C). Tras 35 das con las dietas, la hembras MFD ya presentaban
sobrepeso, hiperlipemia, hiperinsulinemia y resistencia a la insulina. En este
momento se cruzaron los animales y se estudiaron a da 20 de gestacin. Las
madres con la dieta MFD tuvieron fetos ms pequeos que las controles,
aunque no hubo diferencias en el peso de las placentas ni en su contenido
de lpidos. Asimismo, en las placentas del grupo MFD la expresin de Glut1 y Lipina-2 estaba aumentada mientras que la de la LPL se encontraba
disminuida frente a las C. El estudio metabolmico de las placentas revel un
mayor contenido de metabolitos como carnitina, acetilcarnitina, cistationina
y serina (todos ellos relacionados con el metabolismo de los lpidos y la
glucosa) en las placentas del grupo de MFD respecto al C. Estos resultados
permiten concluir que la obesidad presente antes de la gestacin, promueve
un mecanismo adaptativo que protege a la placenta frente a una posible
lipotoxicidad, pero a pesar de ello existen alteraciones relacionadas con un
aumento en la captacin de glucosa y en el metabolismo de la cistationina
que podran tener consecuencias en el desarrollo del feto.
Financiado con SAF2010-19603 y CAM-BMD2010-2423.
P07-5 (R07-1)
Programacin metablica en el periodo prey post-natal de la predisposicin a la obesidad

en el adulto
Catalina Pic, Andreu Palou
CIBER Fisiologa de la Obesidad y Nutricin y Laboratorio de
Biologa Molecular, Nutricin y Biotecnologa (Universidad de las
Islas Baleares), Palma de mallorca, ES
Alrededor del 25% de los casos de obesidad en la edad adulta tienen su
origen en las primeras etapas del desarrollo pre- y post-natal. Son perodos
crticos en los que la restriccin materna de alimentos y la presencia o no de
determinados componentes de los mismos puede conducir a adaptaciones
permanentes de procesos metablicos responsables de estos problemas y,
segn los estudios en modelos animales, los efectos pueden ser diferentes
dependiendo del sexo, del periodo de exposicin y su severidad. Y pueden
ser de signo opuesto dependiendo de si la restriccin materna tienen lugar
67
Psters
en la gestacin (propensin a la obesidad) o en la lactancia (resistencia
a la obesidad). Entre los nutrientes que ms influyen en la programacin
metablica del control del peso corporal, en 2005 identificamos a la leptina,
un componente normal de la leche materna y ausente en las frmulas o
preparados para lactantes, que debiera ser considerado un nutriente esencial
durante el desarrollo perinatal, pues en esta etapa contribuye a la organizacin
de los sistemas de control metablico-alimentario que operarn el resto de la
vida. Aqu resumiremos datos recientes obtenidos en nuestro laboratorio que
revelan la capacidad de la leptina para corregir desajustes de la programacin
metablica del recin nacido debidos a la alimentacin materna durante la
gestacin y que, de no corregirse conducen a alteraciones metablicas en la
base del sndrome metablico y la obesidad.
P08. Bioqumica y biologa molecular

de plantas
P08-1
Anlisis de la expresin de la oxidasa alternativa

de girasol (Helianthus annuus L.) en relacin
con el estrs bitico causado por Plasmopara
halstedii
Theo Guerra Dug1, Ana Mara Fernndez Ocaa1, Jos Rafael
Pedrajas Cabrera2, Raquel Valderrama Rodrguez2, Juan Bautista
Barroso Albarracn2, Mara Victoria Gmez Rodrguez1
1
Departamento de Biologa Animal, Biologa Vegetal y Ecologa.
Universidad de Jan, Jan, ES, 2Departamento de Biologa
Experimental. Universidad de Jan, Jan, ES
La oxidasa alternativa (AOX) es una protena presente en plantas y otros
organismos que forma parte de la cadena mitocondrial de transporte de
electrones. AOX puede sustituir a la Citocromo c oxidasa como aceptor
final de electrones, con la ventaja de ser insensible al cianuro si bien su
actuacin da lugar a un menor rendimiento en trminos de ATP.
Puesto que se ha descrito que AOX desempea un importante papel frente
al estrs oxidativo y nitrosativo derivado de condiciones adversas de tipo
bitico y abitico, se ha realizado un estudio preliminar acerca del papel
que esta protena pudiera tener en la interaccin entre H. annuus y uno de
sus ms importantes patgenos (P. halstedii), as como de la posibilidad de
que AOX pudiera formar parte de los mecanismos de defensa que el BTH,
un inductor de resistencia sistmica adquirida (SAR), pudiera activar.
Se han empleado dos lneas de girasol, una susceptible HA89 y otra
resistente RHA274 y tanto a nivel de expresin como de cantidad de
protena.
Los resultados obtenidos han mostrado la existencia de diferencias en
la expresin de la AOX entre ambas lneas de girasol. Las plantas de la
lnea susceptible tienen distinto nivel de expresin de AOX que las de
la lnea resistente pero esa diferencia no parece estar relacionada con su
resistencia o susceptibilidad a P. halstedii. La infeccin da lugar en las
plantas susceptibles a un aumento de la cantidad de AOX, no debida a
una mayor transcripcin de los genes que codifican las dos isoformas,
sino a una distinta regulacin de la traduccin y/o equilibrio en la sntesis/
degradacin de la protena.
De nuestros resultados se deduce que si bien el BTH acta como protector
de la infeccin por P. halstedii, este efecto no est ligado a una activacin
de la expresin de los genes que codifican para las dos isoformas de la
AOX ni tampoco al aumento de la cantidad de protena.
Las semillas de girasol fueron amablemente cedidas por el equipo del
profesor Said Mouzeyar, Universit Blaise Pascal, Clermont-Ferrand,
Francia.
Financiado por fondos FEDER y por el MINECO (proyecto BIO201233904) y Junta de Andaluca (AGR-6374, grupo BIO286).
68

P08-2 (R08-1)
Reevaluating the involvement of plastidic

phosphoglucose isomerase in starch biosynthesis
in mesophyll cells
ngela Snchez Lpez, Abdellatif Bahaji, Francisco Jos Muoz,
Edurne Baroja-Fernndez, Jun Li, Adriana Ricarte-Bermejo,
Goizeder Almagro, Manuel Montero, Javier Pozueta-Romero
Instituto de Agrobiotecnologa (CSIC/UPNA/Gobierno de Navarra),
Nafarroa, ES
It is widely assumed that the whole starch biosynthetic process occurring
in leaf mesophyll cells resides exclusively in the chloroplast. According
to this view, transitory starch is considered the end-product of a metabolic
pathway involving plastidic phosphoglucomutase (pPGM), ADP-glucose
pyrophosphorylase (AGP) and starch synthase (SS) that is linked to the
Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase
(pPGI). In this work we isolated pgi1-3, a mutant totally lacking pPGI
activity as a consequence of aberrant splicing of intron 6 of the pPGI
encoding gene, PGI1. Starch content in pgi1-3 leaves was ca. 10-15%
of that of wild type (WT) leaves, which is similar to that of leaves of
pgi1-2, a T-DNA insertion pPGI null mutant. Unexpectedly, microscopy
analyses revealed the presence of few starch granules per chloroplast in the
mesophyll cells of pgi1-2 and pgi1-3 leaves. Both pgi1-2 and pgi1-3 leaves
accumulated WT levels of the starch precursor molecule, ADP-glucose,
and displayed reduced SS and high -amylase activities. Moreover, the
two pgi1 mutants displayed a slow growth phenotype and possessed
reduced photosynthetic capacities when cultured under continuous light
photoperiod. Importantly, pgi1-2 and pgi1-3 leaves accumulated high
starch content when plants were cultured in the presence of elevated CO2
concentration. Furthermore, introduction into pgi1-2 and pgi1-3 of a sex1
null mutation impeding -amylase-mediated starch breakdown reverted
the starch-deficient phenotype of pgi1 mesophyll cells. The overall data
(a) show that mesophyll cells of pPGI null mutants accumulate starch, (b)
provide strong evidence that the reduced starch content in pgi1 mesophyll
cells is largely the consequence of combined factors including reduced
photosynthetic capacity and pleiotropic changes in activities of enzymes
directly linked to starch metabolism, (c) support the occurrence of
important starch biosynthetic pathway(s) alternative to the classic CalvinBenson cycle-pPGI-pPGM-AGP-SS pathway, and (d) show that pPGI is an
important determinant of both photosynthetic capacity and growth.
P08-3
Anlisis protemico en plantas de Arabidopsis

thaliana deficientes en las tiorredoxinas f y m
plastidiales
Juan Fernndez Trijeque, Jos Antonio Rojas Gonzlez, Antonio
Jess Serrato Recio, Mariam Sahrawy Barragan
Departamento de Bioqumica, Biologa Celular y Molecular de
Plantas, Estacin Experimental del Zaidn, CSIC, Granada, ES
La regulacin redox es probablemente el mecanismo ms utilizado por las
plantas para controlar la activacin de enzimas o la reduccin de puentes
disulfuro de protenas implicadas en diversos procesos metablicos o de
desarrollo. Aunque existen distintas protenas capaces de llevar a cabo
intercambios tiol/disulfuro, las tiorredoxinas (Trx) son las responsables de
muchos de las funciones que ocurren en plantas. Las Trx plastidiales de
Arabidopsis thaliana (Arabidopsis) son el grupo ms numeroso de la familia
multignica a la que pertenecen, en el cloroplasto se han descrito las Trx f, m,
x, y. Hasta hace unos aos se conoca la activacin especfica de la fructosa1,6-bisfosfatasa cloroplastdica por la Trx f y de la malato deshidrogenasa
por la Trx m. Sin embargo el nmero de isoformas existentes para cada tipo,
sugiere una gran diversidad funcional lo que conlleva la dificultad de asignar
un papel fisiolgico concreto a cada una de las Trxs.
Para contribuir a la identificacin de funciones especficas a las Trx f y
m de Arabidopsis en este estudio hemos caracterizado las protenas
Granada 2014
diferencialmente expresados en mutantes de Arabidopsis deficientes
(knockout) en las Trx f1, f2, m1, m2, m3 y m4, respectivamente.
Extractos de protenas fueron preparados para comparar las protenas en
plantas mutantes y silvestres usando la tcnica de 2D-PAGE. Las protenas
diferencialmente expresadas fueron identificadas mediante MS-MS/MS
(MALDITOF-TOF) en el Servicio de Protemica (SCAI) de la Universidad
de Crdoba. Los anlisis muestran un total de 37 y 71 protenas inhibidas y
sobreexpresadas, respectivamente, en todos los mutantes. La clasificacin
de las protenas por categoras funcionales indica que varios procesos de
la planta estn afectados, entre los cuales podemos destacar el transporte
electrnico, la fotosntesis y el Ciclo de Calvin, el ensamblaje de protenas,
y la sntesis de amino cidos. Este estudio puede favorecer la asignacin de
acciones a algunas de Trx f y m analizadas.
Agradecimientos: Este trabajo ha sido financiado por el MICINN
(BIO2009-7297), MINECO (BIO2012-33292) y por la Junta de Andaluca
(P07-CVI-02795, BIO154).
P08-4 (R08-7)
Reconocimiento molecular en la interaccin

de la NADPH-tiorredoxina-reductasa cloroplstica
(NTRC) con 2-Cys peroxirredoxina
Jos A. Navarro1, Pilar Bernal-Bayard1, Francisco J. Cejudo1, Adrin
Velzquez-Campoy2, Manuel Hervs1
1
Instituto de Bioqumica Vegetal y Fotosntesis, cicCartuja, Sevilla,
ES, 2Instituto de Biocomputacin y Fsica de Sistemas Complejos
(BIFI), Zaragoza, ES
Las NADPH-tiorredoxina-reductasas (NTR) utilizan NADPH como fuente
de electrones para la reduccin de tiorredoxinas (Trx), en lo que conforma
un sistema de regulacin por oxidacin-reduccin de grupos disulfuro
que controla mltiples procesos celulares. Las NTR contienen una flavina
y un grupo disulfuro como cofactores, que operan como una cadena de
transferencia intramolecular de electrones desde el NADPH al grupo
disulfuro en el sitio activo. Adems de las NTR estndar, las plantas poseen
una NTR plastdica, llamada NTRC, con un dominio Trx fusionado a su
extremo C-terminal. NTRC, entre otras funciones, acta como un reductor
de las 2-Cys peroxirredoxinas (2-Cys Prx), que controlan los niveles
intracelulares de perxido de hidrgeno, sin embargo, las caractersticas
de la interaccin entre ambas protenas no se conoce. Hemos estudiado
los procesos de reconocimiento molecular del sistema NTRC/2-Cys Prx
mediante calorimetra de titulacin isoterma, utilizando tanto protenas
nativas como los mdulos NTR (NTRM) y Trx (TrxM) de NTRC, expresados
separadamente. Las interacciones de NTRC, TrxM y NTRM con la 2-Cys
Prx son similares en cuanto a afinidad, aunque diferentes en cuanto a la
contribucin entalpa/entropa. Nuestros datos sugieren que ambos mdulos,
NTRM y TrxM, contribuyen significativamente a la interaccin de NTRC
con la 2-Cys Prx, en concordancia con el modelo de interaccin que hemos
propuesto recientemente. Por el contrario, NTRC no interacciona con
una Trx cloroplastdica de tipo x, debido a que la presencia del mdulo
Trx impide dicha interaccin. Nuestros resultados ponen de manifiesto el
conjunto preciso y definido de interacciones que determinan la especificidad
del reconocimiento molecular en el sistema NTRC/2-Cys Prx de plantas.
P08-5 (R08-2)
Funcin y regulacin de las estrigolactonas como

fitohormonas y molculas seal en la rizosfera
Juan Antonio Lpez Rez, Roco Torres Vera, Juan Manuel Garca
Ramrez, Javier Rivero Bravo, Estefana Berrio Pozo, Mara Jos
Pozo Jimnez
Estacin Experimental del Zaidn, CSIC (EEZ-CSIC), Granada, ES
Las estrigolactonas (SL) son unas molculas multifuncionales que
estn involucradas en diferentes procesos en las plantas, donde actan
Psters
hormonas y como molculas seal en la rizosfera. Como fitohormonas,
se ha propuesto que podran jugar un papel como moduladores del
desarrollo coordinado de races y parte area en respuesta a condiciones
de estrs nutricional. De acuerdo con esta hiptesis, se ha demostrado
que las SL estn involucradas en la regulacin de la arquitectura vegetal,
formacin de races adventicias, crecimiento secundario y desarrollo
reproductivo. Adems, nuevas funciones se estn descubriendo
continuamente. Como molculas seal en la rizosfera, las SL favorecen
el establecimiento de la simbiosis con ciertos hongos beneficiosos del
suelo (simbiosis micorrcica arbuscular). Mediante el establecimiento
de este tipo de simbiosis, las plantas son capaces de sobrevivir bajo
diferentes condiciones de estrs. En la rizosfera, las SL tambin actan
como estimulantes de la germinacin de plantas parsitas de la familia
Orobancheaceae (malas hierbas), las cuales ocasionan grandes prdidas
a nivel agronmico. Debido a este papel dual de las SL en la rizosfera,
se ha propuesto que las micorrizas tambin podran ser utilizadas para el
desarrollo de nuevas estrategias de biocontrol frente a este tipo de malas
hierbas.
En nuestro grupo de investigacin, estamos interesados en estudiar cmo
diferentes condiciones de estrs, tanto de tipo abitico como bitico, afectan
a la produccin y regulacin de SL, as como analizar cmo esto afecta a
la formacin de micorrizas y a la infeccin por plantas parsitas. Por otro
lado, tambin estamos interesados en descubrir nuevas funciones de las SL,
como por ejemplo su posible implicacin en respuestas de defensa.
P08-6
HPLC-MS/MS analyses show that leaves of

the near-starchless aps1 and pgm mutants
accumulate wild type levels of ADPglucose:
further evidence for the occurrence of various
important ADPglucose biosynthetic pathway(s)
Miren Edurne Baroja Fernndez1, Abdellatif Bahaji1, ngela
Mara Snchez Lpez1, Francisco Jos Muoz1, Jun Li1, Goizeder
Almagro1, Manuel Montero1, Adriana Ricarte Bermejo1, Pablo
Pujol2, Regina Galarza2, Kentaro Kaneko3, Kazusato Oikawa3, Kaede
Wada3, Toshiaki Mitsui3, Javier Pozueta Romero1
1
Mutilva, Navarra, ES, 2Servicio de Apoyo a la Investigacin,
Universidad Pblica de Navarra, Pamplona, ES, 3Department of
Applied Biological Chemistry, Niigata University, Niigata, JP
In leaves, it is widely assumed that starch is the end-product of a
metabolic pathway exclusively taking place in the chloroplast that (a)
involves plastidic phosphoglucomutase (pPGM), ADPglucose (ADPG)
pyrophosphorylase (AGP) and starch synthase (SS), and (b) is linked to the
Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase
(pPGI). This view also implies that AGP is the sole enzyme producing the
starch precursor molecule, ADPG. However, mounting evidence has been
compiled pointing to the occurrence of important sources, other than the
pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility,
in this work two independent laboratories have carried out HPLC-MS/
MS analyses of ADPG content in leaves of the near-starchless pgm and
aps1 mutants impaired in pPGM and AGP, respectively, grown under
two different culture conditions. We also measured the ADPG content
in WT and aps1 leaves expressing in the plastid two different ADPG
cleaving enzymes, and in aps1leaves expressing in the plastid GlgC,
a bacterial AGP. Furthermore, we measured the ADPG content in ss3/
ss4/aps1 mutants impaired in starch granule initiation and chloroplastic
ADPG synthesis. We found that, irrespective of their starch contents,
pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid
ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC
accumulate WT ADPG content (0.13-0.19 nmol ADPG/g fresh weight).
In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more
ADPG than WT leaves. The overall data further provide strong evidence
that, in Arabidopsis leaves, (a) there are important ADPG biosynthetic
69
Psters
pathways, other than the pPGI-pPGM-AGP pathway, (b) pPGM and AGP
are major determinants of starch accumulation, but not of intracellular
ADPG content, and (c) most of ADPG has an extraplastidial localization
in WT leaves.

microalga, desarrollando fitoplancton con alto contenido en pptidos
antimicrobianos (AMP). La expresin del gen MitC se ha comprobado
a nivel de mensajero y a nivel de sntesis de protenas. El carcter
antimicrobiano del fitoplancton obtenido est en estudio.
P08-7 (R08-3)
P08-9
Interplay between SWR1 and NuA4 chromatin

remodelling complexes in the control of flowering
time
Induction of bioflocculation in microalgae
Pedro Crevilln Lomas, Angeles Gmez-Zambrano, Alfonso Mouriz,

Manuel Pieiro, Jos A. Jarillo
Centro de Biotecnologa y Genmica de Plantas (UPM-INIA),
Pozuelo de Alarcn, ES
Histone acetylation by the NuA4/Tip60 complex and histone H2A.Z
deposition by the SWR1/SRCAP complex are functionally linked
chromatin modifications observed across eukaryotes, regulating multiple
biological processes such as gene expression, heterochromatin boundary
maintenance, and DNA repair. In our lab, we previously characterized a
series of Arabidopsis thaliana early flowering mutants defining a major
role for SWR1-C in the regulation of flowering time, a key developmental
process in the lifecycle of plants. We are currently focused on the YEATS
domain protein YAF9/GAS41, a shared subunit between SWR1 and NuA4
complexes. There are only two Arabidopsis YEATS domain proteins:
AtYAF9a and AtYAF9b. Atyaf9a KO allele displays an early flowering
phenotype, as previously characterized SWR1-C mutants. AtYAF9A binds
to regulatory regions of master flowering genes altering their histone
acetylation levels and H2A.Z dynamics. In addition to flowering time
alterations, Atyaf9ayaf9b double mutant shows strong developmental
phenotypic defects as well as misregulation of many genes. We have
also isolated different mutant alleles for other NuA4 subunits and have
generated double mutant combinations to uncover the possible existence of
functional redundancy between homologues. We will present our current
data and speculate about their role in regulating plant development.
Encarnacin Daz Santos, Marta Vila Spnola, Marta De la Vega

Naranjo, Roco Rengel, Alicia Azogil, Rosa Len Baares, Javier
Vigara Fernndez
Laboratorio de Bioqumica y Biologa Molecular, Dpto. Qumica y
Ciencia de los Materiales, Facultad de Experimentales, Universidad
de Huelva, CEIMAR, Huelva, ES
Harvesting of microalgae biomass is a critical step, which accounts for
more than 30% of the total price of microalgal production. Reducing
its cost is essential to make economically feasible the production of bulk
products from microalgae, such as biodiesel which despite the enthusiasm
generated, is far from being competitive. Autoaggregation of flocculent
microalgae in response to stressing conditions is poorly understood, but it
is a promising approach to induce the aggregation of microalgae into flocks
and make microalgal harvesting a straightforward and cheap procedure.
The effect of the flocculent yeast strainSaccharomyces bayanus var. uvarum
and the flocculent microalgae Tetraselmis suecica on two chlorophytes:
the model freshwater microalga Chlamydomonas reinhardtii and the novel
marine microalgaPicochlorum sp HM1, was investigated. Both flocculent
microorganisms had a positive effect on microalgal cell aggregation and
improved flocculation efficiencies, which were considerably increased with
addition of yeasts grown in anaerobic conditions. In order to gain more insights
in the origin of microorganism-induced microalgal flocculation, proteins
released into the culture medium by the flocculent yeastSaccharomyces
bayanus var. uvarum were isolated and its ability to induce flocculation
was tested. The most abundant of these proteins was identified as a protein
like-glucanase and its addition in microalgal cultures caused an important
improvement of the flocculation effectiveness. The study was completed
evaluating the effect of some plant lectins on the chosen chlorophytes.
P08-8
Nuevas cepas de fitoplancton transgnico con

componentes antimicrobianos para reforzar el
sistema inmune innato de especies acuculas
Marta Vila Spinola, Encarnacin Daz-Santos, Marta de la Vega
Naranjo, Javier Vigara, Rosa Len Baares
Laboratorio de Bioqumica, Dpto. Qumica y Ciencia de Materiales,
Campus del Carmen, Facultad de Ciencias Experimentales,
Universidad de Huelva, Campus de Excelencia Internacional del
Mar-CEIMAR, Huelva, ES
El uso de antibiticos para prevenir enfermedades en muchas especies
acucolas es una prctica de riesgo que puede provocar la aparicin de
patgenos resistentes, adems los antibiticos nos son efectivos frente
a virus. La vacunacin frente a enfermedades usuales puede hacerse en
ejemplares de peces adultos, pero es imposible de aplicar a alevines o a
otras especies acucolas que carecen de un sistema inmune adaptativo,
como los bivalvos. En estos casos es necesario desarrollar alternativas de
compuestos antimicrobianos que refuercen el sistema inmunitario de los
ejemplares cultivados y mejoren sus tasas de supervivencia. Los AMP son
pequeos pptidos catinicos que pueden inactivar a un amplio espectro
de bacterias, hongos y parsitos patgenos, adems de virus y que se
encuentran muy conservados a lo largo de la evolucin, lo que hace pensar
que su papel dentro del sistema inmune innato debe de ser fundamental.
En este trabajo hemos aislado el gen que codifica un pequeo pptido
con caractersticas antimicrobianas (AMP) a partir de tejido de Mytilus
galloprovincialis (mejilln), lo hemos clonado en el vector de expresin
Phycogen high-expression vector para microalgas suministrado por
la empresa Phycogenetics SL y lo hemos insertado en el genoma de una
70
P08-10
Promotion of Arabidopsis growth and flowering

by Alternaria alternata volatile emissions is
a photocontrolled process involving dramatic
changes in the plant hormonome
Jun Li1, Nuria De Diego2, ngela Mara Snchez-Lpez1, Abdellatif
Bahaji1, Francisco Jos Muoz1, Edurne Baroja-Fernndez1, Karel
Dolezal2, Lukas Spichal2, Javier Pozueta-Romero1
1
Mutilva, ES, 2Centre of the Region Han for Biotechnological
and Agricultural Research, Department of Chemical Biology and
Genetics, Faculty of Science, Palack University, Olomouc, CZ
Plants perceive biotic stimuli by recognizing different signaling substances
originating from the interacting microorganisms. Some of them are volatile
compounds. Volatile emissions from some rhizobacterial isolates promote
growth in Arabidopsis by facilitating nutrient uptake, photosynthesis and
defense responses, and by decreasing glucose sensing and ABA levels.
Analyses of Arabidopsis mutants affected in hormone signaling have
indicated possible involvement in the reaction to rhizobacterial volatiles. We
found that the production of volatilespromoting plant growth is not restricted
to some rhizobacteria, and that volatiles emitted by different microbial
species ranging from Gram-negative and Gram-positive bacteria to different
fungi (including plant pathogens and microbes normally not interacting with
plants) promote both growth and accumulation of exceptionally high levels of
starch in leaves of both mono- and di-cotyledonous plants. This phenomenon,
initially designated as MIVOISAP (for MIcrobial VOlatiles Induced Starch
Granada 2014
Accumulation Process), involves changes in the leaf transcriptome and
metabolome. To better understand MIVOISAP, we analyzed the effect of
volatiles emitted by Alternaria alternata on Arabidopsis development. We
found that itsvolatiles induce rapid growth, development of secondary roots
and initiation of the flowering. Using different mutants we also observed
that MIVOISAP down-regulated genes involved in light signaling and in the
main hormone synthesis and signaling. We also carried out high throughput
analyses of hormone content in root and leaves of plants exposed to A.
altertata volatile emissions. The overall data showed that MIVOISAP is a
photocontrolled process involving dramatic changes in the hormonome of
the both plant organs.
This research was partially supported by the grants [BIO2010-18239]
from the Comisin Interministerial de Ciencia y Tecnologa and Fondo
Europeo de Desarrollo Regional (Spain) and by Iden Biotechnology. A-M.
S-L. acknowledges a predoctoral fellowship from the Spanish Ministry of
Science and Innovation as well as the Ministry of Education, Youth and
Sports, Czech Republic (Grant L01204 from the National Program of
Sustainability and Agricultural Research and project POST-UP, reg. No.
CZ.1.07/2.3.00/30.0004).
P08-11 (R08-5)
PpNAC1, posible candidato para articular la red

transcripcional implicada en la sntesis de pared
celular secundaria en pino
M. Beln Pascual, Blanca Craven-Bartle, Francisco M Cnovas,
Concepcin vila
Ciencias, Universidad de Mlaga, Campus de teatinos, Mlaga, ES
El conocimiento de las redes de regulacin transcripcional que controlan
procesos importantes en conferas es muy limitado. Por ello, la identificacin
y caracterizacin funcional de factores de transcripcin (TF) es esencial para
comprender la regulacin de genes implicados en procesos estrechamente
ligados a la productividad forestal. El transcriptoma de P. pinaster contiene
877 TF distribuidos en 30 familias. Este nmero es similar al descrito en
abeto blanco y menor al descrito en angiospermas (Canales y col. 2014).
Los factores de transcripcin NAC constituyen una gran familia gnica
especfica de las plantas. Usando la informacin disponible en la base de
datos Sustainpine, hemos identificado 37 posibles genes NAC en pino.
Los anlisis filogenticos y la identificacin de motivos conservados
con el programa MEME realizados en otras especies sugieren que genes
relacionados estructuralmente podran ejercer funciones similares (Shen y
col. 2009). Segn esto, hemos visto que PpNAC1 se agrupa en el mismo
clado que NST1, NST2 y SND1 de Arabidopsis, genes implicados en la
formacin de pared celular secundaria. Por tanto, PpNAC1 es un candidato
interesante para estudiar la formacin de madera en una especie forestal
como el pino donde dicho proceso es de gran importancia econmica.
Ensayos de retraso en gel y estudios de expresin transitoria en protoplastos
de pino se han utilizado con el objetivo de elucidar el papel de PpNAC1 en la
red de regulacin transcripcional que controla la formacin de pared celular
secundaria, as como el control sobre los factores R2R3-Myb, esenciales en
el metabolismo de la fenilalanina y posterior sntesis de fenilpropanoides
(Craven-Bartle y col. 2013). Resultados preliminares apuntan a que PpNAC1
regula su propia expresin as como la expresin de factores Myb implicados
en el proceso. Todo esto nos permite especular sobre su posible funcin como
regulador ro arriba de la biosntesis de fenilalanina, el aminocido precursor
de la lignina. La obtencin de lneas embriognicas de pino sobreexpresoras
de PpNAC1 y RNAi en curso, junto con su posterior caracterizacin, nos
proporcionar la informacin necesaria para demostrar la implicacin de este
factor de transcripcin en la sntesis de pared celular secundaria.
Bibliografa
Canales y col. 2014. De novo assembly of maritime pine transcriptome:
implications for forest breeding and biotechnology. Plant Biotechnology
Journal 12: 286-299.
Psters
Craven-Bartle y col. 2013. A Myb transcription factor regulates genes of
the phenylalanine pathway in maritime pine. The Plant Journal 74: 755766.
Shen y col. 2009. A Bioinformatic analysis of NAC genes for plant cell
wall development in relation to lignocellulosic bioenergy production.
Bioenergy Res 2: 217-232.
P08-12
El herbicida auxnico cido

2,4-diclorofenoxiactico induce alteraciones
del citoesqueleto de actina por carbonilacin
y S-nitrosilacin y afecta la dinmica de
peroxisomas y mitocondrias
Maria Rodrguez-Serrano1, Diana M Pazmio1, Imogen Sparkes2,
Chris Hawes2, Maria C Romero-Puertas1, Luisa M Sandalio1
1
Departamento de Bioqumica, Biologa Celular y Molecular
de Plantas, Estacin Experimental del Zaidn, CSIC, Granada,
ES, 2School of Biological & Medical Sciences, Oxford Brookes
University, oxford, UK
El cido 2,4-diclorofenoxiactico (2,4-D) es una auxina sinttica utilizada
como herbicida. Altas concentraciones de 2,4-D inducen malformaciones
en hojas (epinastia) y tallosy muerte celular. En este trabajo se han
estudiado losmecanismos moleculares implicados en la toxicidad del 2,4D mediante el anlisis de la produccin de especies de oxgeno reactivo
(ROS), xido ntrico (NO) en plantas de Arabidopsis y su efecto sobre la
estructura del citoesqueleto y la dinmica de peroxisomas y mitocondrias.
La aplicacin foliar de 2,4-D (23 mM) produca epinastia en hojas y este
fenotipo poda prevenirse por pre-tratamiento con EDTA. El anlisis de la
acumulacin de ROS mediante microscopa confocal mostr un incremento
dependiente de 2,4-D del H2O2 y O2.- asociado a pequeos orgnulos,
posiblemente peroxisomas y mitocondrias, mientras que la acumulacin
total de NO no se afectaba por el tratamiento. El anlisis del citoesqueleto
de actina mediante el uso de una lnea de Arabidopsis FABD2-GFP mostr
alteraciones el mismo dependientes del 2,4-D. El anlisis de modificaciones
postraduccionales de protenas por carbonilacin y S-nitrosilacin
demostr que la actina experimenta ambas modificaciones lo que afecta
a su polimerizacin, tal como sugiere la relacin de F-actin/G-actin. Estos
efectos se reducan por el tratamiento con EDTA, que a su vez reduca
la oxidacin de protenas y en particular de la actina. El 2,4-D tambin
reduca la velocidad y desplazamiento de peroxisomas y mitocondrias,
como consecuencia de las alteraciones observadas en el citoesqueleto.
Para determinar la fuente de ROS implicada en este proceso, se analiz
el fenotipo de distintos mutantes de Arabidospsis, entre ellos, las lneas
deficientes en xantina deshidrogenasa y enacilCoA oxidasa que mostraron
una reduccin considerable de la epinastia.Se ha analizado adems la
regulacin de la epinastia por ABA, jasmnico y etileno. Los resultados
obtenidos sugieren que la epinastia es el resultado de alteraciones del
citoesqueleto de actina dependientes de ROS y NO.
Trabajo financiado por ERDF-BIO2008-04067 y BIO2012-36742 del
MICINN y la Junta de Andaluca (BIO-337).
P08r-13
Modulacin de la capacidad antioxidante del

ciclo ascorbato-glutatin por modificaciones
post-traduccionales mediadas por xido ntrico
Juan Carlos Begara-Morales1, Beatriz Snchez-Calvo1, Mounira
Chaki1, Raquel Valderrama1, Capilla Mata-Prez1, Javier LpezJaramillo2, Mara N. Padilla1, Alfonso Carreras1, Francisco Javier
Corpas3, Juan Bautista Barroso1
1
Grupo de Bioqumica y Sealizacin Celular en xido Ntrico,
71
Psters
de Jan, Jan, ES, 2Instituto de Biotecnologa, Departamento de
Qumica Orgnica, Universidad de Granada, Granada, ES, 3Grupo
de Antioxidantes, Radicales Libres y xido Ntrico en Biotecnologa
y Agroalimentacin, Departamento de Bioqumica, Biologa
Molecular y Celular de Plantas - Estacin Esperimental del Zaidn,
CSIC, Granada, ES
Las modificaciones post-traduccionales mediadas por xido ntrico
(MPT-NO), tales como nitracin y S-nitrosilacin, pueden modular la
funcin de dianas proteicas [1]. Sin embargo, la informacin disponible
sobre su efecto en la actividad y estructura de protenas involucradas en
sistemas antioxidantes es limitada. Por esta razn, se analiz el efecto
de estas MPT-NO sobre las principales enzimas del ciclo ascorbatoglutatin, clave en la detoxificacin y regulacin de los niveles de
perxido de hidrgeno en plantas [2]. En este estudio, se utilizaron
diferentes protenas recombinantes de guisante: Ascorbato peroxidasa
citoslica (APX), monodeshidroascorbato reductasa peroxisomal
(MDAR) y glutatin reductasa (GR) citoslica y cloroplastdica. Los
resultados pusieron de manifiesto una regulacin diferente de las enzimas
del ciclo. En el caso de la APX disminuy su actividad por nitracin y
aument por S-nitrosilacin [3], mientras que las GR no se modificaron
y la MDAR fue inhibida por ambas modificaciones. Adems, en la APX
se identificaron las dianas de estas MPT-NO y su implicacin funcional.
Finalmente, utilizando plantas de guisante sometidas a estrs salino se
observ que la S-nitrosilacin de APX ocurre in vivo y que aumenta
durante esta situacin de estrs [3].
En conclusin, se evidenci que el ciclo ascorbato-glutatin presenta
regulacin por NO, sugiriendo adems una estrecha relacin entre el
metabolismo de NO y ROS en plantas.
Referencias
[1] Corpas FJ et al. Plant Sci (2011), 181:604-611.
[2] Noctor and Foyer. Annu Rev Plant Physiol Plant Mol Biol (1998),
49:249-279.
[3] Begara-Morales JC et al. J Exp Bot (2014), 65:527-538.
Financiado por Ministerio de Economa y Competitividad (proyectos
BIO2012-33904 y 20134R0569) y Junta de Andaluca (grupos BIO286 y
BIO192).
P08-14
Caracterizacin funcional de la quinasa CPK1

peroxisomal en respuesta a estrs bitico y
abitico en plantas de Arabidopsis
Katiuska Crdenas, Mara Rodrguez-Serrano, Adela Olmedilla
Arnal, Mara C. Romero-Puertas, Luisa M. Sandalio
Plantas, Estacin Experimental del Zaidn, CSIC, Profesor Albareda
1, Granada, ES
La modificacin postraduccional de protenas por fosforilacin es
uno de los mecanismos ms comunes en la regulacin de la actividad,
localizacin subcelular y degradacin de protenas. Estos procesos estn
implicados en el reconocimiento de cambios en el entorno celular y en la
regulacin de la respuesta celular frente a factores biticos y abiticos.
Los peroxisomas son fuentes importantes de seales celulares como
especies de oxgeno y nitrgeno reactivo (ROS, RNS) y hormonas,
y tienen un papel central en la regulacin de procesos metablicos
esenciales para la clula y en la respuesta celular al estrs. La presencia
de quinasas y fosfatasas en estos orgnulos ha sido recientemente
demostrada, si bien sus protenas diana y su funcin no han sido
establecidas. En este trabajo se ha llevado a cabo la caracterizacin
de la quinasa de Arabidopsis thalina CPK1 en respuesta a estrs por
metales (Cd y As), e infeccin por Pseudomonas syringae (Pst avrB).
Para ello, se ha utilizado un mutante de Arabidopsis deficiente en
esta protena, cpk1. En este mutante se ha analizado el fenotipo de la
planta en respuesta a los estreses anteriormente mencionados, as como
72

la ultraestructura de la hoja en respuesta al tratamiento con Cd. Para
establecer posibles dianas de la CPK1 se ha analizado la actividad de
protenas peroxisomales que previamente se ha demostrado que pueden
fosforilarse, la glicolato oxidasa, catalasa y superxido dismutasa.
Tambin se ha analizado el papel de esta quinasa en la regulacin de la
acumulacin de H2O2 en respuesta al tratamiento con Cd. Los resultados
obtenidos sugieren que la CPK1 podra regular la produccin de H2O2
procedente de la fotorrespiracin en situaciones de estrs por Cd. En
estas condiciones, las hojas de plantas cpk1 experimentan cambios
importantes en la ultraestructura de la clula que afectan especialmente
a los peroxisomas. Por el contrario, la deficiencia de CPK1 no afecta
a la respuesta frente al As ni a la respuesta hipersensible frente a Pst
avrB.
Trabajo financiado por ERDF-BIO2008-04067y BIO2012-36742del
MICINN y la Junta de Andaluca (BIO-337).
P08-15
PpMyb23, un nuevo factor de transcripcin Myb

R2R3 de conferas relacionado con el desarrollo
de las acculas y el metabolismo
de los flavonoides
Jose M Granados, Marina Rueda-Lpez, M Beln Pascual,
Concepcin vila, Francisco M Cnovas, Rafael A. Caas
Ciencias, Universidad de Mlaga, Campus Universitario de Teatinos,
Mlaga, ES
En un trabajo previo de nuestro laboratorio hemos caracterizado el
metabolismo anual de las acculas de rboles adultos de Pinus pinaster
(Aiton) en condiciones naturales mediante el anlisis de redes de coexpresin de genes (WGCNA). Gracias a este anlisis se pudo identificar
un nuevo factor de transcripcin Myb R2R3 de conferas, que ha sido
nombrado como PpMyb23, asociado al desarrollo de las acculas y al
metabolismo de los flavonoides. El anlisis de su secuencia muestra que
no pertenece al Subgrupo 4 de factores Myb R2R3 que han sido propuestos
como los principales responsables de la regulacin del metabolismo de
los flavonoides en conferas (Bedon et al. 2010). La caracterizacin de su
expresin gnica a lo largo del desarrollo plantular confirma que la accin
de PpMyb23 se encuentra ligada a las acculas, probablemente en respuesta
a las condiciones lumnicas. Como otros factores Myb R2R3 relacionados
con el metabolismo de los flavonoides su expresin se modifica por metiljasmonato (MeJA).
Bibliografa
Bedon F, Bomal C, Caron S, Levasseur C, Boyle B, Mansfield SD, Schmidt
A, Gershenzon J, Grima-Pettenati J, Sguin A, MacKay J. (2010) Subgroup
4 R2R3-MYBs in conifer trees: gene family expansion and contribution to
the isoprenoid- and flavonoid-oriented responses. J Exp Bot. 61(14): 38473864.
P08-16
Respuesta antioxidante y fotosinttica

de la fanergama marina Posidonia oceanica al
estrs por deficiencia lumnica
Anna Maria Ortol Garcia
Dpto. Producci Vegetal, Universitat Politcnica de Valncia,
Valncia, ES
La Posidonia oceanica (L.) Delile es una fanergama marina endmica
del mar Mediterneo, en donde forma extensas praderas cuya distribucin
est estrechamente unida a la capacidad de penetracin de la luz en el agua.
Su ecosistema est reconocido por el European Habitats Directive (92/43/
CEE) como hbitat de inters prioritario. Actualmente las praderas litorales
Granada 2014
estn en regresin como consecuencia, entre otros factores, del incremento
de la sedimentacin y turbidez del agua por vertidos urbanos e industriales,
que modifican las condiciones de iluminacin de la columna de agua bajo
la cual se asienta la pradera, generando una situacin de estrs en la planta
por la atenuacin de la luz solar.
En este trabajo se estudia la influencia de la deficiencia de luz en plantas
de posidonia de praderas naturales situadas en la costa alicantina de Denia.
Se analiza la respuesta antioxidante y fotosinttica de plantas localizadas
en praderas a 3 y a 9 metros de profundidad, de dos zonas con distinta
calidad de agua, una en las inmediaciones del emisario submarino de aguas
residuales de Denia y otra en un rea alejada de su influencia.
Se cuantifica el contenido en pigmentos fotosintticos, carbohidratos
solubles, sacarosa, almidn y la actividad del enzima sacarosa sintetasa
(SS) para analizar la respuesta fotosinttica. Se determina la actividad de
los enzimas catalasa, superxido dismutasa y ascorbato perxidasa, y se
miden los niveles de malondialdehido (MDA), como indicadores del estrs
oxidativo.
Los resultados obtenidos apuntan a una disminucin de los carbohidratos y
pigmentos fotosintticos en la planta con el incremento de la profundidad
y turbidez, y un aumento de la actividad de los enzimas antioxidantes,
en respuesta al deterioro del funcionamiento fotosinttico debido a la
limitacin de la luz solar.
P08-17
Genes encoding beta-mannanases in

Brachypodium distachyon seeds have a role not
only in cell wall softening upon germination
sensu stricto but also during post-germinative
reserve mobilization
Virginia Gonzles-Calle, Raquel Iglesias Fernndez, Victoria LlanosCasado, Pilar Carbonero
Centro de Biotecnologa y Genmica de Plantas (CBGP) - Instituto
Nacional de Investigacin y Tecnologa Agraria y Alimentaria
Universidad Politcnica de Madrid, Pozuelo de Alarcn (Madrid), ES
Brachypodium distachyon seeds are characterized by presenting low
levels of starch and high levels of (1,3; 1,4) -glucans in its endosperm
cells besides having thick mannan-rich cell walls. Upon germination
and subsequent reserve mobilization -mannanase encoding genes are
selectively induced.
Endo--mannanases (EC 3.2.1.78) are hydrolytic enzymes that catalyze
the cleavage of (1-4) bonds in the mannan polymer. In the genome of
Brachypodium, the endo--mannanase (MAN) family is represented by
six members. We have systematically explored the expression of the six
MAN genes in different organs (leaves, roots, spikes, developing seeds)
and we have found that in dry seeds BdMAN3 is the most highly expressed
gene, but its expression decreases upon germination in aleurone while
the other five are induced at 36 hours of imbibition. In the germinating
embryo the most important genes are BdMAN2 and BdMAN6. In situ
hybridization analysis shows that BdMAN2 and BdMAN6 transcripts
accumulate in the coleorhiza while BdMAN3 is mainly expressed in the
radicle tip.
Difference in sequence and expression of the MAN gene family in
Brachypodium distachyon and in other cereals, and their different putative
functions in comparison to those reported in Arabidopsis thaliana [1,2] that
are mainly involved in germination sensu stricto, are discussed.
References
[1] Iglesias-Fernndez R, Rodrguez-Gacio MC, Carbonero P, Matilla AJ
(2012) Softening-up mannan-rich cell walls. J. Exp. Botany 63: 3975-3988.
[2] Iglesias-Fernndez R, Rodrguez-Gacio MC, Barrero-Sicilia C,
Carbonero P, Matilla AJ (2011) Three endo--mannanase genes expressed
in the micropylar endosperm and in the radicle influence germination of
Arabidopsis thaliana seeds. Planta 233: 25-36
Psters
P08r-18
Microbacterium sp. 3J1 reduces the expression

of enzymes involved in ethylene pathway in roots
of pepper plants under drought conditions
Cristina Garca-Fontana, Juan Ignacio Vlchez, Jess GonzlezLpez, Maximino Manzanera
Instituto del Agua, Universidad de Granada, Granada, ES
Plant water deficit is one stress which has been extensively associated
with elevated release of ethylene. The impact of water stress on ethylene
synthesis is of interest because ethylene is responsible for senescence and
abscission induced by water deficits. Some microorganisms present in
the rhyzosphere protect plants from drought by interaction with the plant
host. The interaction of these microorganisms, that we term Drought Plant
Protecting Bacteria (DPPB), with the plant is translated in a reduction of
the ethylene produced by the plant. We have isolated a Microbacterium
sp. 3J1 strain that is desiccation tolerant soil bacterium and can interact
with plants as DPPB. The inoculation of pepper plants with this strain
reduced the ethylene produced by the plant and this in turn induced a
marked delay in senescence. Here we used proteomic to compare plants
inoculated with Microbacterium sp. 3J1 subjected to drying conditions
with non-inoculated plants. Presence of the microorganism is translated
in a reduction of S-adenosylhomocysteine hydrolase (SAHH). A
dramatic reduction of SAHH could be translated in accumulation of SAH
(S-Adenosylhomocysteine) and SAM (S-adnosylmethionine), a methyl
donor for choline production, which is used for glycinebetaine production,
a potent xeroprotectant produced in the chloroplast. SAHH also controls
biological methylation reactions by mediating the intracellular SAH/
SAM ratio. DNA and protein methylation and demethylation play
an important role in signal transduction. We do not discard additional
signal transduction effects on other pathways to increase desiccation
tolerance on the plant by the reduction in SAHH caused in presence of
Microbacterium sp. 3J1.
P08-19 (R08-4)
Differential analysis of root proteome obtained

in microbe-plant interaction as a tool for
identification of proteins involved in resistance
to drought events in pepper plants
Juan Ignacio Vlchez1, Cristina Garca Fontana2, Jess Gonzlez
Lpez1, Maximino Manzanera Ruiz1
1
Universidad de Granada, Ogjares, Granada, ES, 2Instituto del
Agua, Universidad de Granada, Granada, ES
A collection of bacterial strains isolated in our laboratory, have been
shown to reduce dehydration stress1. These strains have also been shown
to protect plants against abiotic stresses by colonizing the rhizosphere of
various plant species, and promoting plant growth by reducing ethylene
production via ACC-deaminase activity and by reducing the reduction
of S-adenosylhomocysteine hydrolase (SAHH) of the plant2. Apart from
ethynele accumulation, drought also causes damages by accumulation
of reactive oxygen species (ROS)3. Preliminary investigation on the
molecular basis of bacterial mediated drought stress alleviation was
performed by examining the mRNA levels of three important drought stress
responsive genes, DREB2A, CAT1, and DHN in inoculated plants by realtime quantitative polymerase chain reaction (qPCR)4. Here we describe
the effect of Microbacterium sp. 3J1 over the plant under water stress at
the proteomic level using two-dimensional electrophoresis assisted by
PDQuest Basic 2D Gel Analysis software. Differentially expressed proteins
were identified by mass spectrometry Mass (MALDI TOF-TOF) let us
identify a reduction in proteins such as disulfoxide isomerase, nucleoside
diphosphate kinase, Glutathione S Transferase and Annexin, involved in
response to oxidative stress. Likewise, a decrease in proteins involved in
systemic resistance as chitinases and dehydrogenases was observed as well
as in several proteins involved in the pathway of ethylene on plants such as
73
Psters
S adenosyl homocysteine hydrolase, response factor binding ethylene, beta
subunit of succinyl CoA and Caffeoil CoA.
P08-20
Identificacin de genes de referencia para

estudios de RT-qPCR en Pinus pinaster (Aiton)
Jos M. Granados, Concepcin vila, Francisco M. Cnovas, Rafael
A. Caas
Ciencias, Universidad de Mlaga, Campus Universitario de Teatinos,
Mlaga, ES
La tcnica de RT-qPCR es uno de los procedimientos experimentales ms
utilizados para el anlisis de la expresin gnica. El desarrollo experimental
requiere la normalizacin de los resultados obtenidos mediante comparacin
con otros genes de referencia cuyos niveles de expresin sean uniformes en
las condiciones estudiadas. En los primeros estudios realizados mediante
esta tcnica se usaron para la normalizacin genes implicados en el
mantenimiento de funciones celulares bsicas (housekeeping genes).
Sin embargo, los niveles de expresin de dichos genes no son invariables
y se requiere una verificacin experimental preliminar para determinar
qu genes resultan adecuados en cada organismo o tejido a estudiar. La
falta de metodologas de trabajo estandarizadas ha provocado errores
metodolgicos y experimentales en la identificacin de genes de referencia
y en la aplicacin de esta tcnica que cuestionan los resultados obtenidos.
Se han presentado diferentes propuestas para establecer una metodologa
experimental adecuada, siendo en estos momentos la ms aceptada MIQE
(Minimum Information for Publication of Quantitative Real-Time PCR
Experiments).
El pino martimo (Pinus pinaster Aiton) es una planta modelo de inters
forestal en la que se estn realizando avances en biologa molecular. En
el presente trabajo, hemos realizado la bsqueda de genes de referencia
adecuados para la normalizacin de los datos de RT-qPCR tanto en accula
adulta de P. pinaster como en los primeros estadios de desarrollo plantular
siguiendo las recomendaciones del MIQE. Nuestro objetivo es la obtencin
de resultados fiables y reproducibles en estudios de expresin gnica en P.
pinaster.
P08r-21
Deteccin y formacin endgena de cidos grasos

nitrados en el aceite de oliva
Raquel Valderrama Rodrguez, Beatriz Snchez-Calvo, Capilla MataPrez, Mara N. Padilla, Juan C. Begara-Morales, Juan B. Barroso
Departamento de Bioqumica y Biologa Molecular, Universidad de
Jan, Jan, ES
El xido ntrico (NO) y especies de nitrgeno reactivo derivadas (RNS)
reaccionan con cidos grasos insaturados originando cidos grasos
nitrados (NO2FA). Estas molculas se consideran novedosos mediadores
de sealizacin celular que abarcan un amplio conjunto de respuestas
celulares en sistemas animales, entre los que destacan una probada funcin
anti-inflamatoria y beneficios a nivel de salud cardiovascular [1]. El aceite
de oliva virgen extra (EVOO) es la fuente principal de los lpidos en la
dieta mediterrnea, y promueve respuestas anti-inflamatorias y beneficios
clnicos a travs de mecanismos pobremente definidos. El presente estudio
evalu mediante LC-MS/MS, el contenido endgeno de NO2FA, tanto en
aceituna como en EVOO procedentes de tres variedades de la provincia
de Jan. Se observ la presencia de cido nitro-linoleico conjugado (NO2CLA) en los tres aceites ensayados. El carcter electroflico de estos
NO2FA fue confirmado por la deteccin HPLC-MS/MS [2] de niveles
significativos de aductos de cistena-protena de cido nitro-oleico (NO2OA-cistena) en el fruto. Adems, la nitracin de EVOO por nitritos en
condiciones digestivas de acidez gstrica, revel que el consumo humano
de los EVOO origina un incremento de NO2-CLA y NO2-OA. Estos
74

resultados identifican por primera vez NO2FA en plantas y sugieren que el
consumo en la dieta y la generacin fisiolgica de estos lpidos electrfilos
anti-inflamatorios contribuye a los beneficios fisiolgicos de las dietas ricas
en cidos grasos insaturados.
Bibliografa
[1] Freeman BA et al., J Biol Chem. (2008), 283(23):15515-9.
[2] Trostchansky A et al., Free Radic Biol Med. (2008), 44(11):1887-96.
Financiado por fondos FEDER y por el MINECO (proyecto BIO201233904) y Junta de Andaluca (AGR-6374, grupo BIO286).
P08r-22
Bio-hydrogen production in the green algae

Chlamydomonas: effect of H2 partial pressure,
acetate and oxygen
Jose Luis Jurado Oller, David Gonzlez Ballester, Aurora Galvn
Cejudo, Emilio Fernndez Reyes
Departamento de Bioqumica y Biologa Molecular de la Universidad
de Crdoba, Crdoba, ES
Under anaerobic conditions, some unicellular algae and bacteria produce a
hydrogenase enzyme able to produce hydrogen (H2) in a reversible reaction.
H2 production in algae is a clean source of H2 that can be potentially
cheaper than any other way to produce H2 and would also contribute to the
capturing of atmospheric CO2.
Unfortunately, bio-H2 production is a low efficiency process and two of the
main limitations are: 1) the high O2 sensitivity (hydrogenases are inhibited
by O2 at transcriptional and post-translational level). 2) H2 production is a
transient process that do not last more than a few days.
We have investigated different approaches to optimize the H2 production
in the unicellular green alga Chlamydomonas reinhardtii. This alga is able
to perform three different pathways to produce H2. Two of them are linked
to photosynthesis (PSII-dependent and PSII-independent pathways) and
the main difference between these two pathways is the original source
of electrons. In the PSII-dependent pathway, the electrons come from
the photolysis of water at PSII level. Whereas in the PSII-independent
pathway, the electrons come from starch degradation. A third pathway
for H2 production is also possible through fermentative pathways that use
starch as electron donor.
As starting point of our project we screened 22,000 insertional mutants
for two different phenotypes: starch and photosynthetic mutants. Both
phenotypes can have theoretically an impact in the H2production process.
One photosynthetic deficient strain showed an improved H2 production
(PSII-linked) under low light conditions.
The effect of the H2 accumulation in the cultures headspace was also
investigated. We found that H2 partial pressure is an important factor that
can inhibit the production. By aerating the cultures we have improved H2
production by 10 times.
Finally, we studied the effect of acetate in the culture media. Cultures
that were supplemented with acetate can improve H2 production by 2
times. Moreover, continuous supplementation with acetate can result in a
sustained H2 production.
Altogether, our results show an optimization of the H2 production process
in Chlamydomonas by more than 60 times.
P08-23
Autentificacin de aceite de oliva mediante qPCR

Sonia Ramos-Gmez, Natividad Ortega Santamara, Mara D. Busto,
David Palacios, Manuel Prez-Mateos
rea de Bioqumica y Biologa Molecular, Facultad de Ciencias,
Universidad de Burgos, Burgos, ES
El elevado valor nutricional del aceite de oliva puede verse reducido
mediante adulteracin con otros aceites vegetales. Este tipo de prctica
Granada 2014
supone, adems, importantes prdidas econmicas por lo que es necesario
certificar la autenticidad del aceite de oliva. La elevada sensibilidad y
fiabilidad de las tcnicas basadas en ADN les han convertido en una
herramienta bsica en el control de calidad y seguridad alimentaria. En
este contexto, el objetivo de este trabajo fue determinar la aplicabilidad de
mtodos basados en ADN y PCR cuantitativa (qPCR) para la autentificacin
de aceite de oliva.
Se analizaron mediante qPCR nueve sistemas de amplificacin especficos
para olivo: tres previamente publicados (PIP5, G219/172H y C4-80), y
seis diseados en este trabajo (ycf1, clpP, Pre, PetN, 1post y 2post). La
aplicabilidad de dichos sistemas se determin mediante cuantificacin de
ADN de olivo, amplificacin especfica de olivo frente a otras especies
oleaginosas, cuantificacin de olivo en mezclas de ADN y amplificacin de
ADN de aceites de oliva.
De los resultados obtenidos destacar que el sistema 2post no permiti
cuantificar ADN de olivo, los sistemas ycf1, clpP y PIP5 mostraron rangos de
cuantificacin limitados, y C4-80 no present especificidad. Por el contrario,
el intervalo de cuantificacin de G219/172H y PetN oscil entre 2,5 g y
25 pg y entre 2,5 g y 2,5 pg para Pre y 1post, mostrando especificidad
para olivo frente a colza, girasol, soja y maz. La especificidad relativa de
G219/172H y 1post fue del 0,2% de ADN de olivo en ADN de ssamo, y del
0,1% para Pre y PetN. Adems, estos sistemas permitieron la deteccin de
ADN extrado de aceites de oliva. No obstante, es necesario comprobar su
especificidad en otros aceites vegetales y en alimentos elaborados.
P08-24
Efecto sobre la fotosntesis y la acumulacin de

azcares en frutos de fresa (Fragaria x ananassa)
de la reduccin de los niveles de las isoformas
cFBP1 y cyFBP de FBPasa
Antonio Jess Serrato Recio1, Jos Antonio Rojas Gonzlez1, Juan
Antonio Garca Gago2, Fernando Pliego Alfaro2, Jos ngel Mercado
Carmona2, Mariam Sahrawy Barragn1
1
Plantas, Estacin Experimental del Zaidn, CSIC, Granada, ES,
2
Departamento de Biologa Vegetal, IHSM-UMA-CSIC, Universidad
de Mlaga, Mlaga, ES
La fructosa-1,6-bifosfatasa (FBPasa) juega un papel clave en la fijacin
del CO2 atmosfrico durante la fotosntesis y en procesos gluconeognicos,
conocindose tres isoformas, localizadas en el cloroplasto (cFBP1 y
cFBP2) y el citosol (cyFBP). cFBP1 forma parte de las enzimas del ciclo
de Calvin y contiene un lazo regulador que la hace dependiente del estado
redox del cloroplasto, siendo regulada por tiorredoxina f. Sin embargo,
al contrario que con cFBP1, no se ha descrito ninguna regulacin redox
para cyFBP, la cual forma parte de la ruta de biosntesis de sacarosa. A
la isoforma cFBP2 todava no se le ha asignado una funcin fisiolgica
definida, pudiendo participar en procesos relacionados con las otras dos
isoformas, las cuales muestran un mayor nivel de expresin que cFBP2.
La fresa (Fragaria x ananassa) es un cultivo estratgico para la agricultura
espaola, siendo una de sus cualidades organolpticas ms valoradas,
por su contenido en sacarosa, el sabor dulce del fruto. Aunque en los
estados iniciales tiene cierta capacidad fotosinttica, la mayor parte de los
azcares acumulados en los frutos son transportados por el floema desde
las hojas. Para determinar el papel de las FBPasas en el contenido final
de sacarosa en fruto se realizaron construcciones antisentido para cFBP1
y sobre-expresoras para cyFBP con las que se transformaron plantas de
fresa, obtenindose una serie de lneas que contenan el marcador de
seleccin. Estas lneas fueron analizadas mediante la tcnica de westernblot para determinar el nivel de expresin de las dos isoformas estudiadas.
Asimismo, se ha relacionado la capacidad fotosinttica de estas lneas
con el contenido en FBPasas de las hojas. Para la caracterizacin de los
frutos de fresa se han analizado parmetros como el peso, la dureza o los
grados Brix (directamente relacionados con el contenido en azcares,
principalmente la sacarosa).
Psters
Agradecimientos: Este trabajo ha sido financiado por el Ministerio de
Ciencia e Innovacin (BIO2009-7297) y por la Junta de Andaluca (P07CVI-02795).
P08-25
Respuesta antioxidante al estrs hdrico en

plantas de sorgo
Magaly Prez Lara, Vctor Olalde Portugal, Silvia Edith Valds
Rodrguez
CINVESTAV Irapuato, Irapuato, MX
El sorgo es una planta gramnea que es capaz de tolerar el estrs por
deficiencia de agua, por lo que diversos estudios se han enfocado en la
respuesta bioqumica el estrs hdrico. Nuestro grupo de trabajo realiz
un estudio de protemica en hojas de sorgo, en respuesta al estrs hdrico,
haciendo uso de electroforesis en geles de 2D y la Espetrometra de masas.
Los resultados obtenidos indicaron que las protenas que aumentan, en su
mayora estn relacionadas con la respuesta antioxidante. Con el fin de validar
los cambios observados en el estudio de protemica, se plante cuantificar
la actividad antioxidante en hojas de plantas de sorgo (Sorghum vulgare
var. BJ 83 Caloro) de 58 das despus de la siembra, sometidas a estrs por
deficiencia de agua, el cual consisti en someter a las plantas a un estrs
gradual durante 7 das, llegando a suprimir el riego durante un da, mientras
que el control estuvo bajo riego adecuado. En extractos de hoja de plantas
control y sometidas a estrs hdrico, se evalu la capacidad antioxidante y
la relacin glutatin oxidado/reducido, adems se cuantific la actividad
de enzimas antioxidantes como superxido dismutasa, catalasa y ascorbato
peroxidasa. Los resultados obtenidos indican que hubo un aumento de estas
actividades en las plantas que fueron sometidas a estrs hdrico, lo cual
sugiere que esta actividad podra estar contribuyendo a la tolerancia al estrs.
En este estudio se destaca la importancia de estas enzimas en la tolerancia al
estrs hdrico estableciendo parmetros para cuantificar el nivel de estrs en
plantas de sorgo, que podran ser utilizados como referencia en otras especies
de plantas con importancia econmica y social.
P08-26 (R08-6)
La interrupcin de la funcin de las fructosa1,6-bisfosfatasas cloroplastdica y citoslica

induce cambios metablicos que afecta todos los
procesos en plantas de Arabidopsis thaliana
Jos Antonio Rojas Gonzlez1, Antonio Jess Serrato Recio1, Ina
Thormhlen2, Peter Geigenberger2, Mariam Sahrawy Barragn1
1
Consejo Superior de Investigaciones Cientficas, Estacin
Experimental del Zaidn, Granada, ES, 2Ludwig-MaximiliansUniversitt Mnchen, Martinsried, DE
Se han caracterizado tres mutantes de Arabidopsis thaliana con prdida
de funcin en las enzimas fructosa-1,6-bisfosfatasa (FBPasas) citoslica
(cyfbp) y cloroplastdica 1 (cfbp1) y de ambas (cyfbp cfbp1). Debido al
papel clave que ejerce la enzima plastidial en el Ciclo de Calvin, las plantas
mutante cfbp1 presentan un tamao ms reducido en relacin con el silvestre
afectando especialmente la tasa fotosinttica, estructura de la hoja, raz y
el desarrollo de la planta. Cuando se interrumpe la funcin de la isoforma
citoslica en la sntesis de la sacarosa provoca un acumulo de almidn en
los cloroplastos y contenido de sacarosa similar al silvestre. Interesante fue
comprobar que el doble mutante cyfbp cfbp1 puede heredar las caractersticas
de uno u otro parental, sin embargo el fenotipo es similar al del mutante cfbp1.
Los cambios fisiolgicos inducidos por la prdida de funcin de las FBPasas
en cada uno de los mutantes podran indicar modificaciones en varios
procesos metablicos importantes. Para estudiar el papel de ambas enzimas
en la sntesis y distribucin de azcares en plantas se ha suministrado, a
cada mutante, un aporte exgeno de azcares (sacarosa, glucosa, fructosa,
manitol, glucosa 1P, glucosa 6P, fructosa 6P y fructosa-1,6-bisfosfato)
observando en algunos casos la recuperacin del fenotipo silvestre. Se han
75
Psters
determinado los contenidos en hexosas, sustrato, triosas fosfato y analizado
los metabolitos en los tres mutantes mediante GS MS. Los resultados
muestran que como respuesta a la falta de una o ambas FBPasas se produce
un acumulo de sustrato y de triosas P. La prdida de la enzima cloroplastdica
cFBPasa1 provoca un significativo cambio en los niveles de los metabolitos
pertenecientes a distintas categoras, azcares, azcares alcohol, aminos
cidos, cidos orgnicos, antioxidantes, etc. Estos resultados sugieren que
en el mutante cyfbp la planta reorganiza su metabolismo carbonado, mientras
que en los mutantes cfbp1 y doble mutante, existe una respuesta general de la
planta para evitar la situacin extrema a la cual est sometida por la falta de
aporte carbonado. BIO 2012-33292. Junta de Andaluca BIO 154.
P08-27
Arabidopsis mutants in the bHLH transcription

factors SPEECHLESS and MUTE reveal
transcriptional and functional features of
stomataless plants in photosynthesis and
secondary metabolites
Marisa Prez Bueno1, Magdalena Trivio2, Alberto de Marcos2,
Carmen Fenoll2, Montaa Mena2, Matilde Barn1
1
Departamento de Bioqumica, Biologa Celular y Molecular de Plantas
- Estacin Experimental del Zaidn, CSIC, Granada, ES, 2Departamento
de Ciencias Ambientales, Facultad de Ciencias Ambientales y
Bioqumica, Universidad de Castilla la Mancha, Toledo, ES
Stomata are microscopic valves formed by guard cell pairs in the epidermis of
terrestrial plants. As they regulate gas exchange with the atmosphere, stomata
are essential for CO2 uptake and H2O loss, thus controlling photosynthesis,
transpiration and leaf temperature. Plants dynamically regulate stomatal
opening in response to environmental conditions; work in Arabidopsis during
the last years has also established that stomata abundance is also regulated
during leaf development through genetic networks whose molecular
components have been partially unveiled. For instance, loss of function of the
bHLH transcription factors SPCH and MUTE produces stomataless plants
with very limited growth even in sucrose-supplemented medium.
Using ATH1 Affymetrix microarrays we made hybridizations with RNA
from wild-type Arabidopsis, spch-3 and mute-3 mutants and identified
ca.1.800 differentially expressed transcripts (p-value with false discovery
rate correction <0.05; 2 fold change) in three pairwise comparisons.
Their functional classification identified major expression changes in
key genes. The mutant transcriptomes predict a limited photosynthesis,
but also important changes in secondary metabolism, cuticle and cell
wall composition that might modify the permeability to gases of the
mutant epidermes. The impact of the observed transcriptional changes in
photosynthesis-related genes was examined in-situ by quenching analysis of
chlorophyll fluorescence. Kinetic analyses of the red fluorescence emitted
by chlorophyll estimate photosynthetic efficiency as well as mechanisms
of energy dissipation indicative of stress. Blue and green fluorescence was
used to estimate the accumulation of pigments and secondary metabolites
suggested by the transcriptomic analysis.

Campus de Teatinos, Mlaga, ES, 3Instituto de Hortofruticultura
Subtropical y Mediterrnea La Mayora, Universidad de Mlaga,
Consejo Superior de Investigaciones Cientficas - rea de Gentica,
Facultad de Ciencias, Campus de Teatinos, Mlaga, ES
One of the most important soilborne diseases affecting avocado (Persea
americana Mill.) crops is white root rot, caused by the fungus Rosellinia
necatrix. Moreover, this fungus is considered to be an emergent threat to
crop plants worldwide. Imaging techniques applied to remote sensing are
emerging as powerful tools to be used in crop protection. In this study
we investigated the potential applications of imaging techniques, including
chlorophyll fluorescence, blue-green fluorescence and thermography, in
early detection of white root rot on leaves of infected avocado plants. The
results show that changes in certain chlorophyll fluorescence parameters
provide early indications of fungal infection prior to the development of
visual symptoms in the aerial part of the plant.
P09. Biotecnologa molecular

P09-1
Isolation and Identification of a new archaea

strain in hypersaline waters
Marta de la Vega Naranjo1, Agustn Garca Barneto2, Jos Ariza
Carmona2, Rosa Len Baares1
1
Laboratorio de Bioqumica, Dpto. Qumica y Ciencia de Materiales,
Campus del Carmen, Facultad de Ciencias Experimentales,
Universidad de Huelva, Campus de Excelencia Internacional del
Mar-CEIMAR, Huelva, ES, 2Dpto. Ingeniera Qumica, FsicaQumica y Qumica Orgnica, Campus del Carmen, Facultad de
Ciencias Experiementales, Universidad de Huelva, Huelva, ES
An extremely halophilic archea was isolated from marine salt ponds at the
Southwest of Spain. Analysis of its 16S rRNA encoding gene showed that the
isolated microorganism was phylogenetically related to species of the genus
Halorubrum within the Halobacteriaceae family. Halorubrum cells were
harvested by centrifugation and carotenoids were extracted with methanol.
Carotenoids identification was performed by HPLC and confirmed by UPLCMS. Results revealed that this Halorubrum strain produces as predominant
carotenoid, bacterioruberin. This carotenoid is a dipolar C50 carotenoid with
4 hydroxyl substitutes. It shows the typical 3-finger UV-Vis spectra with
absorption around 467, 497 and 531 nm and a molecular weight of 713. Its
biosynthesis is described as the addition of an isoprenoid-C5 unit to each of
the extremes of lycopene-C40, followed by the introduction of the 4 hydroxil
groups. Bacterioruberin contains 13 pairs of conjugated double bonds, so it can
be an effective hydroxyl free-radical scavenger; it can protect the halobacteria
from fatal injury of oxidative damage under strong light, and resist oxidative
DNA damage resulting from UV irradiation. This carotenoid could be very
useful for practical applications and the extremely halophilic archeon could be
considered as a prokaryotic candidate for the production of carotenoids.
P08-28
Fluorescence and thermal imaging techniques

allow detection of white root rot on leaves of
avocado plants infected with Rosellinia necatrix
prior to the development of symptoms
Espen Granum1, Mara Luisa Prez Bueno1, Claudia Caldern2, Cayo
Ramos3, Antonio de Vicente2, Francisco M. Cazorla2, Matilde Barn1
1
Estacin Experimental del Zaidn, Consejo Superior de
Investigaciones Cientficas, Granada, ES, 2Instituto de
Hortofruticultura Subtropical y Mediterrnea La Mayora,
Universidad de Mlaga, Consejo Superior de Investigaciones
Cientficas - Departamento de Microbiologa, Facultad de Ciencias,
76
P09-2 (R09-7)
Herramientas para el control de la expresin

durante procesos de infeccin en patgenos y
simbiontes
Carlos Medina Morillas1, Beatriz Mesa Pereira1, Eva Mara Camacho
Fernandez1, Amando Flores Daz1, Irene Jimnez Guerrero2,
Francisco Javier Lpez Baena2, Eduardo Santero Santurino1
1
Universidad Pablo de Olavide, Sevilla, ES, 2Universidad de Sevilla,
Sevilla, ES
El estudio de las interacciones entre microorganismos y sus hospedadores
presenta ciertas limitaciones que dificultan el seguimiento del proceso
Granada 2014
de infeccin en el interior de los organismos superiores. La naturaleza
del hospedador puede impedir la monitorizacin del microorganismo
durante la infeccin, por lo que se hace necesario el uso de sistemas de
microscopa adaptados a condiciones in vivo. As mismo, el limitado
nmero de herramientas que permiten el control de la expresin de
genes involucrados en las interacciones bacteria-hospedador, restringe
la posibilidad de activar o desactivar dichos genes en el momento o
lugar deseados en el transcurso de la infeccin. En nuestro laboratorio
hemos contribuido al desarrollo de un sistema de expresin de
protenas que responde a concentraciones permisivas de diferentes
inductores como el salicilato, cido acetil saliclico o 3-metil benzoato.
Dicho sistema acopla dos reguladores transcripcionales que funcionan
en cascada, el primero de los cuales controla la expresin del segundo
que finalmente regula la expresin de genes heterlogos clonados
bajo el control de un promotor inducible. Estos elementos proceden
de distintas especies bacterianas, y han demostrado su eficiencia en
la produccin de protenas heterlogas en diferentes microorganismos
tanto patgenos de animales (Salmonella) como simbiontes de plantas
(Sinorhizobium). Una de las caractersticas interesantes del sistema es
que los inductores son capaces de difundir libremente entre los distintos
tejidos del hospedador sin presentar toxicidad a las concentraciones
ensayadas. Esta propiedad junto a una correcta monitorizacin del
proceso infectivo, permite disparar la expresin de genes de inters
en el momento deseado lo cual puede ser muy til para comprender
cuando y donde actan los genes implicados en las interacciones
tanto de bacterias patgenas como simbiontes con sus hospedadores
respectivos.
P09-3 (R09-1)
Herramientas biotecnolgicas derivadas de virus

de plantas
Jos Antonio Dars Arnau
Instituto de Biologa Molecular y Celular de Plantas (CSICUniversidad Politcnica de Valencia), Valencia, ES
Los virus que infectan plantas superiores son muy sencillos
genticamente. Sin embargo, sus pequeos genomas contienen
informacin suficiente para completar complejos ciclos infecciosos
que, adems de la replicacin, movimiento y transmisin del virus,
incluyen la desactivacin de las defensas de la planta. Estos pequeos
genomas codifican un nmero limitado de protenas que suelen ser
multifuncionales y tener la capacidad de interaccionar, reclutar y
subvertir la actividad de multitud de factores del husped para que
desarrollen diversos cometidos durante la infeccin. Los viroides
son un caso extremo dentro de los agentes infecciosos de las plantas,
puesto que son RNA capaces de replicarse y moverse por el husped a
pesar de no codificar ninguna protena propia. La sencillez estructural
y riqueza funcional de los virus y viroides de las plantas facilita el
desarrollo de herramientas biotecnolgicas basadas en elementos de
su biologa. En nuestras investigaciones recientes hemos desarrollado
un vector viral desarmado, basado en el potyvirus del grabado del
tabaco, de inters en ingeniera metablica de plantas. El vector
permite coexpresar simultneamente varias protenas heterlogas en
cantidades equimolares y en la misma localizacin subcelular. As, la
coexpresin de dos factores de transcripcin de la ruta de biosntesis
de las antocianinas conlleva a la acumulacin de niveles notables de
estos compuestos antioxidantes en los tejidos de la planta invadidos por
el vector. Tambin hemos desarrollado un sistema para la produccin
de RNA recombinantes en Escherichia coli basado en la coexpresin
de un RNA derivado del viroide latente de la berenjena junto con una
protena con la que ste interacciona durante la infeccin, la isoforma
cloroplstica de la tRNA ligasa de berenjena.
Psters
P09-4 (R09-3)
Un insecticida natural interfiere con

comportamientos bacterianos clave en procesos
infectivos
Isabel M Lpez-Lara1, Joaquina Nogales2, ngel Pech-Canul1, Lydia
M Bernabu-Roda2, Nieves Calatrava-Morales2, Jos Olivares2,
Laura lvarez3, Otto Geiger1, M Jos Soto Misffut2
1
Centro de Ciencias Genmicas, Universidad Nacional Autnoma de
Mxico-UNAM, Cuernavaca, MX, 2Estacin Experimental del Zaidn,
CSIC, Granada, ES, 3Centro de Investigaciones Qumicas, Universidad
Autnoma del Estado de Morelos-UAEM, Cuernavaca, MX
En Sinorhizobium meliloti, la prdida de funcin del gen fadD que codifica
una acil-CoA ligasa especfica de cidos grasos de cadena larga, promueve
movilidad swarming e interfiere con la capacidad de establecer simbiosis
con alfalfa [1]. El anlisis de extractos lipdicos de sobrenadantes de cultivo
del mutante fadD y su cepa parental desvel la presencia en el mutante de
dos compuestos que fueron identificados como 2-tridecanona (2-TDC) y
dodecanal, ausentes en los sobrenadantes de la cepa silvestre. La aplicacin
de 2-TDC comercial en concentraciones micromolar induce movilidad en
superficie de cepas de S. meliloti, favoreciendo tanto swarming como un
desplazamiento independiente de flagelos. La 2-TDC se ha descrito como
un insecticida natural producido por variedades silvestres de tomate [2]
pero hasta ahora no se haban descrito sus efectos en bacterias. La 2-TDC
afecta comportamientos en superficie de diversas bacterias: promueve
swarming en los patovares tomato y syringae de Pseudomonas syringae,
inhibe formacin de biofilm en S. meliloti y P. syringae y es capaz de
anular el swarming de Salmonella. Interesantemente, la aplicacin de esta
metilcetona interfiere con el establecimiento de 2 interacciones plantabacteria: la formacin de ndulos en la simbiosis S. melilotialfalfa,
y el desarrollo de peca bacteriana en tomate, resultados con potencial
biotecnolgico.
Bibliografa
[1] Soto et al. 2002. Mol. Microbiol. 43:371-382.
[2] Williams et al. 1980. Science 207:888-889.
P09r-5 (R09-4)
Physical and functional standardization of

glycolytic modules for knocking-in novel
metabolic capacities in Gram-negative bacteria
Alberto Snchez-Pascuala, Vctor de Lorenzo, Pablo I. Nikel
Systems Biology Program, Centro Nacional de Biotecnologa, CSIC,
Campus de Cantoblanco, Madrid, ES
One key tenet of Synthetic Biology is the physical and functional
modularization of the components of live systems for enabling their
reuse in a different biological context. Because of their extant metabolic
network, bacteria which are appealing for biotechnological processes (e.g.,
Pseudomonas putida KT2440) often show a non-optimal energetic yield
that limit their efficiency under operation conditions. In order to overcome
this state of affairs, we took the Escherichia colis Embden-MeyerhofParnas pathway (one of the most efficient glycolytic pathways in terms of
energy balance) as the starting point for improving sugar utilization in P.
putida and other Gram-negative bacteria. To this end we engineered various
genetic/biochemical modules that are composable and interchangeable
by means of the format brought about by the Standard European Vector
Architecture (SEVA; Silva-Rocha et al., 2013, Nucleic Acids Res. 41:
D666-D675). These designs considered [i] the distribution of a minimal
set of relevant glycolytic genes from E. coli into two different catalytic
blocks, [ii] the elimination of restriction enzyme targets in the genes, while
maintaining the amino acid sequence of the polypeptides encoded, and
[iii] the creation of independent enzymatic modules with corresponding
DNA sequences flanked by appropriate restriction sites for easing their
subsequent formatting with the SEVA rules. These genetic/biochemical
77
Psters
devices allow for the re-factoring of central pathways for C consumption
in a variety of Gram-negative microorganisms, thereby increasing their
energy balance and thus developing new-to-Nature metabolic capabilities.

Herein we present our last advances in the development of nanotechnologybased strategy for delivery of therapeutic proteins into cells. For this
purpose, several chemical strategies, based on solid phase chemistry, and
basic molecular biology techniques has been optimized for the conjugation
of proteins to nanoparticles.
P09r-6 (R09-5)
Aptmeros de RNA para el estudio de la funcin

del dominio CRE del genoma del virus de la
hepatitis C
Alba Fernndez Sanls, Beatriz Berzal Herranz, Pablo Ros Marco,
Alfredo Berzal Herranz, Cristina Romero Lpez
Granada, ES
La consecucin del ciclo infectivo del virus de la hepatitis C (HCV)
depende de varios dominios genmicos altamente conservados en
estructura y secuencia, entre los que se encuentra la regin CRE (cisacting replicating element). Este elemento, esencial para la replicacin
viral e implicado en la regulacin de la traduccin, se encuentra en el
extremo 3 de la regin codificante del genoma. Su estructura presenta
tres dominios con estructura tallo lazo, denominados 5BSL3.1, 5BSL3.2 y
5BSL3.3, de los cuales los dos primeros son esenciales para la replicacin
y adems el 5BSL3.2 es importante en la regulacin de la traduccin.
Este dominio ejerce su funcin reguladora mediante la interaccin con la
protena RNA polimerasa NS5B y con regiones distantes en el genoma
como la regin 3UTR o el elemento IRES (internal ribosome entry
site) localizado en la regin 5UTR. Dada su importancia funcional, se
presenta como una potencial diana teraputica. Los mejores candidatos
para interferir con un elemento funcional estructural de RNA como el
CRE son los aptmeros de RNA. Los aptmeros son oligonucletidos
que se unen especficamente a su diana dependiendo de su secuencia y
estructura, de manera similar a los anticuerpos. Se obtienen mediante un
proceso de seleccin in vitro conocido como SELEX. Hemos aislado una
batera de aptmeros tras nueve rondas de seleccin y el anlisis de sus
secuencias ha permitido clasificarlos en cinco grupos definidos por un
motivo comn. Observamos que los motivos de secuencia cuya diana es
5BSL3.2 estn presentes en los aptmeros que inhiben en mayor medida
los niveles de RNA total (>80%) de un sistema replicativo subgenmino
de HCV. Adems, dichas secuencias consenso se encuentran en los lazos
apicales de los aptmeros. Asimismo, los ensayos de unin de NS5B al
genoma viral en presencia de los aptmeros muestran que aquellos que se
unen al motivo 5BSL3.2 compiten por la unin de la polimerasa NS5B
al extremo 3 del genoma. Los resultados obtenidos apoyan el potencial
del elemento CRE como diana teraputica, as como el potencial de los
aptmeros de RNA como agentes antivirales.
P09-7
Nanoparticles as protein carriers

Juan Diego Unciti Broceta, Victoria Cano Corts, Mara Paz Ruiz
Blas, Juan Jose Daz-Mochn, Rosario M. Sanchez-Martin
Dpto. Qumica Orgnica y Farmacutica, Universidad de Granada Centro de Genmica e Investigacin Oncolgica-GENyO, Granada, ES
The development of an efficient carrier system for delivery of functional
proteins into cells, although a challenging area of research, is a key
technology for the progress of research in the biological sciences and
medicine. Recent examples include conjugation of proteins to cell
penetrating peptides (CPP) such as those derived from HIV-1 TAT,
profection by cationic lipids and the use of several nanodevices such as
nanotubes or silica nanoparticles. Previously we have reported that aminofunctionalized, cross-linked polystyrene microspheres of highly defined
sizes (200 nm 2 m) are efficient cellular delivery devices that can enter a
broad range of cell types including adherent, suspension and primary cells.
These microspheres are inherently attractive as a carrier/delivery system
due to their lack of toxicity and highly controllable cellular loading.
78
P09r-8
A SMART chemistry for liver injury diagnosis

Antonio Delgado Gonzlez1, Mavys Tabraue Chvez2, Rosario Mara
Snchez Martn2, Salvatore Pernagallo2, Juan Jos Daz-Mochn2
1
DestiNA Genomics Ltd y GENYO - Centre for Genomics and
Oncological Research: Pfizer / University of Granada / Andalusian
Regional Government - PTS- Granada; and Dep. Medicinal and
Organic Chemistry, School of Pharmacy, University of Granada,
Granada, ES, 2DestiNA Genomics Ltd, Granada, ES
Molecular diagnostics is the process of identifying genetic variants
in biological samples. Testing for human diseases and illnesses, drug
performance and toxicities, pathogenic bacteria and viruses has created an
important, multi-billion and rapidly expanding global market. Molecular
diagnostics is increasingly being used in the cost challenged medical health
systems, animal health, food safety and pharmaceutical industries. The
limitation to its use in clinical diagnosis has been cost, testing errors, and
clinician conservatism.
DestiNA Tecnology is unique and distinguishable from ALL existing
enzymatic methods of nucleic acid analysis. It can be used to identify
any known target nucleic acid sequences, including insertion and deletion
mutations, as well as non-mutated nucleic acid sequences. DestiNA reagents
can successfully be used on the major diagnostic platforms - fluorescence
and colourimetric. This gives the technology a unique, powerful position
versus current detection methods.
In this study, DestiNA reagents are combined with two of the major
diagnostic platforms - fluorescence and colourimetric - for developing
novel tools for early detection and quantification of miRNA-122 involved
in human liver injury.
The integration of these platforms with DestiNA reagents promises to
transform and expand routine clinical diagnostic for liver injury with
benefits in terms of result consistency, time, cost, and ease of use.
P09r-9
Tumor-associated circulating microRNAs

detection by Chem-NAT
M.A. Luque-Gonzlez1, M. Tabraue-Chvez2, R.M. Snchez-Martn1,
S. Pernagallo2, J.J. Daz-Mochn1
1
GENYO - Centre for Genomics and Oncological Research:
Pfizer / University of Granada / Andalusian Regional Government
- PTS- Granada; and Dep. Medicinal and Organic Chemistry,
School of Pharmacy, University of Granada, Granada, ES, 2DestiNA
Genomica S.L, Granada, ES
Among the advanced emerging nucleic acid detection (NAT) technologies,
DestiNA Genomics has developed a unique chemistry approach for nucleic
acid testing (Chem-NAT). DestiNA core technology takes advantage of
dynamic chemistry for nucleic acid sequence specific recognition, using
aldehyde-modified natural nucleobases (so called SMART nucleobases),
and probes based on peptide nucleic acid (PNA) containing an abasic
position (DestiNA probes), which can be made complementary to any
target nucleic acid sequence [Bowler et al. in Angew. Chem. Int. Ed. 2010].
In this study the unique DestiNA reagents are combined with MatrixAssisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry
(MALDI-TOF MS) to rapidly identify tumor-associated circulating
microRNAs as biomarkers of cancer. This biological model represents
the first step in developing novel suite assays for medical diagnostic field.
Integration of DestiNA technology with MALDI-TOF MS brings a truly
Granada 2014
innovative step forward for rapidly detects circulating microRNAs with
benefits in terms of result consistency, time, cost, and ease of use.
With its potential PCR free approach, this tool promises to transform and
expand routine clinical diagnostic testing and screening for tumor-associated
circulating microRNAs in the emerging companion diagnostics market.
P09-10
Optimization of the E. coli biosynthesis of

lycopene through mathematical modelling
Nakens Nez Martn1, Julia Gallego2, Manuel Cnovas2, Nstor
Torres1
1
Departamento de Bioqumica, Microbiologa, Biologa Celular
y Gentica. Facultad Biologa, Universidad de La Laguna, San
Cristobal de La Laguna, ES, 2Departamento de Bioqumica y
Biologa Molecular e Inmunologa, Facultad de Qumica, Campus
de Excelencia Internacional Regional Campus Mare Nostrum,
Universidad de Murcia,, Murcia, ES
Isoprenoids is an ubiquitous family of lipid compounds of industrial and
commercial interest due to its key roles in cell signaling, pigmentation and
defense among others. Currently the processes used to produce isoprenoids
are based in their extraction from natural sources (mainly plants), but these
processes show poor efficiency and sustainability. Thus their biosynthesis
by microorganisms has since, long time ago be proposed as a more efficient,
cheaper and sustainable alternative.
However the bioengineering of this process have been halted by several
factors. The first one is the complexity of the metabolic pathways involved
in the biosynthesis of this family of secondary metabolites. All isoprenoids
are built from a basic block, the Dimethylallyl pyrophosphate (DMAPP)
or its isomer, the Isopentenyl pyrophosphate (IPP). This five carbon
structure is synthetized via two independent pathways, the mevalonate
and non-mevalonate pathway. The latter one is of major interest from
a biotechnological perspective since, in addition to its occurrence in
chloroplasts and protozoa, it is also present in prokaryotic microorganisms.
The goal of this project is to address the metabolic engineering of an E. coli
strain in order to optimize the production of one member of the isoprenoid
family, the lycopene. For this purpose we will built up a mathematical model
of the non-mevalonate. The model will integrate the available information
of the pathway network and regulatory features. Once it will be evaluate
in terms of stability, robustness and reliably will be used to determine the
changes, either in individual or group of enzymes or transport processes
that should be modified in order to increase lycopene productivity while
cell viability is guaranteed.
Work funded by research grants from ACIISI, Ref No. ACIISI Project ProID
2010/39 and Project BIO2011-29233-C02-02 MINECO. IMBRAIN REF.
FP7-REGPOT-2012-CT2012-31637-IMBRAIN.
P09-11
Evaluacin de la produccin
de galactooligosacridos en variantes de beta
galactosidasa de Kluyveromyces lactis
Agustn Rico Daz, Mara Esperanza Cerdn Villanueva, Manuel
Becerra Fernndez
Universidade da Corua, A Corua, ES
La beta galactosidasa de Kluyveromyces lactis (Kl--gal) es una de las
enzimas ms usadas en la industria de alimentacin, especialmente en la
lctea. Aunque la enzima tiene muchas caractersticas interesantes como
son su alta capacidad hidroltica o su seguridad (es producida por un
organismo generalmente reconocido como seguro), tiene la desventaja
de ser bastante lbil a temperaturas altas. Esta caracterstica limita su uso
en aplicaciones industriales que necesitan llevarse a cabo a temperaturas
moderadamente elevadas.
Psters
Uno de los usos ms extendidos y novedosos de las beta-galactosidasas
es su utilizacin en la sntesis de galactooligosacaridos (GOS), derivados
lcteos con propiedades prebiticas que son usados en la elaboracin
de ciertos alimentos funcionales. La enzima produce GOS mediante
transgalactosilacin, la cual se ve favorecida por altas concentraciones de
lactosa. Teniendo en cuenta que la lactosa es bastante insoluble en agua, es
necesario llevar a cabo la reaccin a temperaturas elevadas, por lo que el
uso de la Kl--gal se ve limitado.
En este trabajo se compara la capacidad de sntesis de GOS entre la
variante silvestre de la Kl--gal y una variante mutante de la misma
obtenida mediante mutagnesis dirigida, la cual posee una estabilidad
trmica mayor.
P09-12
Novel conjugation approaches for delivery

of nucleic acids mediated by nanoparticles
Victoria Cano-Corts, Juan Diego Unciti-Broceta, Mara Paz RuizBlas, Juan Jos Daz-Mochn
GENYO - Centre for Genomics and Oncological Research: Pfizer /
University of Granada / Andalusian Regional Government - PTSGranada; and Dep. Medicinal and Organic Chemistry, School of
Pharmacy, University of Granada, Granada, ES
The efficient introduction and delivery of bioactive materials into cells
(such as a drug, protein, nucleic acid, etc) to elicit, induce or control
specific biological functions is of fundamental importance throughout
many areas of biology and medicine. The ability to add a specific
plasmidic DNA into cells to modify cellular function or to deliver
RNA sequences into cells to silence a cellular function has significant
applications in Biomedicine. However, their celular access can often
be severely limited by properties such as solubility, charge or size.
Several techniques for the delivery of these therapeutic molecules are
in development such as cationic lipids, polymers and nanoparticles.
However, due to certain issues (such as poor uptake efficiencies, major
perturbations of cellular function or high cellular mortality) there is
still a need to improve transfection efficiency for specific cell lines
that are difficult to transfect. Herein we present our last advances in
the development of nanotechnology-based transfection agents. For this
purpose, several chemical strategies, based on solid phase chemistry,
and basic molecular biology techniques has been optimized for the
conjugation of DNA to nanoparticles.
P09-13
Biodiesel conversion of non-edible vegetable

oils catalyzed by magnetic cross-linked enzyme
aggregates of Candida antarctica lipase B
Iraide Bejarano1, Jone I. Otsoa-Txintxetru1, Enrique A. Pic1,
Carmen Lpez2, Mara J. Llama1, Juan L. Serra1
1
Enzyme and Cell Technology Group, Department of
Biochemistry and Molecular Biology, Faculty of Science and
Technology, University of the Basque Country (UPV/EHU), Leioa,
ES,2Ikerbasque, Basque foundation for science; Biochemistry and
Molecular Biology Department, University of the Basque Country
(UPV/EHU), Leioa, ES
In the past years, first generation biodiesel was obtained by
transesterification reaction with methanol of edible vegetable oil and
animal fats. Second-generation biodiesel can be also produced from
non-food crops such as non-edible vegetable oils. In this work magnetic
cross-linked enzyme aggregates (mCLEAs) of the lipase B from Candida
antarctica (CALB) were obtained by cross-linking covalently the
insolubilized lipase to magnetic nanoparticles (MNPs) functionalized
with amine groups, using glutaraldehyde as cross-linking reagent. The
resulting novel robust biocatalyst, denoted as mCLEA-CALB, has been
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used to catalyze the conversion to biodiesel of different non-edible
vegetable oils.
For this purpose the trans/esterification reaction catalyzed by mCLEACALB was assessed using different non-edible oils (waste-frying, unrefined
soya, Jatropha and Cameline oils) in the presence o methanol, ethanol,
2-propanol and 2-butanol as alkyl-donors. Different alcohol:triglyceride
molar ratios (1:1, 3:1, 6:1, 9:1 and 12:1) were assayed at 30C in 1 ml
reaction mixture stirred by a rotatory mixer.
Olive oil was used as control oil in all assays.Finally, the reaction mixture
was scaled up to 50 ml using control olive oil and methanol as substrates
in a magnetic reactor (Carousel 12 Plus, Radleys, UK). The biodiesel
conversion of oil to fatty acids methyl esters (FAMEs) was optimized using
different amounts of mCLEA-CALB and an alcohol:triglyceride molar ratio
of 6:1. The obtained FAMEs were analyzed by thin-layer chromatography
(TLC) in Silica Gel G-60 plates after staining with Coomassie Blue.
Then, digital images of plates were taken and analyzed using the TLC
Analyzer software (available at: http://www.sciencebuddies.org/scienceresearch-papers/tlc_analyzer.shtml). Results were exported and integrated
numerically in a spreadsheet to calculate the amount of FAMEs and
triglycerides.
Results obtained indicate that mCLEA-CALB appears as a promising novel
biocatalyst of interest to catalyze the production of second-generation
biodiesel from waste-frying oil and other non-edible vegetable oils.
Work supported by the MINECO (CTQ2011-25052), the Basque
Government (SAIOTEK S-PE12UN041) and UPV/EHU (GIU11/25).
P09-14
Inmovilizacin de la enzima -galactosidasa de

Aspergillus oryzae en membranas de quitosano
Maria del Carmen Romero Cruz1, Paulina Urrutia2, Gonzalo RuizPhilippi2, Carlos Vlchez Lobato1, Javier Vigara Fernndez1, Lorena
Wilson Soto2
1
Dpto. Qumica y Ciencia de los Materiales, Facultad de Ciencias
Experimentales, Universidad de Huelva, CEIMAR, Huelva, ES,
2
Escuela de Ingeniera Bioqumica, Pontificia Universidad Catlica,
Valparaiso, CL
La inmovilizacin de biocatalizadores persigue la mejora de las prestaciones
biotecnolgicas de los mismos con idea de aumentar sus posibilidades de
aplicacin en los distintos campos de la industria productiva. El presente
trabajo muestra un estudio de inmovilizacin de la -galactosidasa (EC
3.2.1.23) del hongo Aspergillus oryzae utilizando membranas de quitosano
activadas con glutaraldehdo, que permite la inmovilizacin covalente de la
enzima al soporte. En los estudios de optimizacin de la inmovilizacin, la
actividad enzimtica se cuantific utilizando orto-nitrofenil--galactosido
(ONPG) como sustrato. La activacin de los grupos aminos del quitosano
(xNH2) con glutaraldehdo (GT) se optimiz a una relacin de 10GT:1NH2,
obtenindose un rendimiento de la inmovilizacin prximo al 55 %, frente
al 20% obtenido con la relacin 1GT:1NH2. Para la carga enzimtica
utilizada se estableci un valor ptimo de 10 mg prot./g soporte, basada
en un compromiso entre la actividad expresada y el rendimiento de la
inmovilizacin. La membrana de quitosano activada se prepar a diferentes
grosores (2; 2,5 y 4 mm) incrementndose 3,5 veces el rendimiento de la
inmovilizacin para un grosor del 2 mm; el aumento del grosor provoc
un aumento aparente de la constante de Michaelis para el ONPG, como
consecuencia de restricciones difusionales. Independientemente del grosor
utilizado, la inmovilizacin mejor la estabilidad trmica de la enzima
respecto al sistema libre. Como inters biotecnolgico, la -galactosidasa
inmovilizada en membranas de quitosano puede utilizarse para disear un
biosensor de lactosa, acoplando la membrana a un detector que permita
cuantificar la glucosa resultante de la hidrlisis enzimtica.
Proyecto: ALGANAET (PIRSES-GA-2011-295165).
80

P09r-15
Stability and reutilization of magnetic biocatalyst

(mCLEAs) for biodiesel production from
microalgae oil
Enrique Angulo Pic1, Carmen Lpez2, lvaro Cruz-Izquierdo1,
Teresa Garca-Brcena1, Noelia Villarroel1, Mara J. Llama1, Juan L.
Serra1
1
Enzyme and Cell Technology Group, Department of
Biochemistry and Molecular Biology, Faculty of Science and
Technology, University of the Basque Country (UPV/EHU), Leioa,
ES,2Ikerbasque, Basque foundation for science; Biochemistry and
Molecular Biology Department, University of the Basque Country
(UPV/EHU), Leioa, ES
Combinatorial approaches in enzyme immobilization are increasing in
the design of novel robust immobilized biocatalysts. Catalytic functions
may improve using cross-linked enzyme aggregates (CLEAs) to obtain
more stable catalysts. Moreover, magnetic nanoparticles (MNPs) are an
attractive support for enzyme immobilization offering fast and simple
recovery of the immobilized catalyst with a magnet. The lipase B from
Candida antarctica (CALB) is one of the most used enzymes in organic
chemistry, based on its broad specificity and high selectivity. Its capability
to catalyze trans/esterification reactions makes it a very good alternative
for biodiesel production.
The aim of this work is to produce biodiesel by reactions of esterification
catalyzed by magnetic CLEAs of CALB (mCLEA-CALB), using either
free fatty acids (FFA) or oils from the microalga Scenedesmus sp, as well
as analyze the stability and possible reutilization of the biocatalyst for
the synthetic purposes. The progress of reactions was assessed by high
performance liquid chromatography (HPLC). Different experimental
conditions such as type of alcohol, alcohol:FFA molar ratio, type of
agitation, as well as different solvents were tested to find the best conditions
for obtaining biodiesel in terms of reaction rates and conversion values. In
addition, the stability of hydrolytic and biosynthetic activity of mCLEAs
was tested after incubating the immobilized enzyme for 48 h in different
organic solvents.
Using mild conditions of temperature (30C) and magnetic stirring,
biodiesel conversion of oils up to 90% was obtained with methanol as
alkyl-donor and t-butanol as solvent. The reutilization of mCLEAs was
tested using two different types of stirring methods. After 50 consecutive
cycles, the complete initial activity was maintained using mechanical
stirring.
Work supported by the MINECO (CTQ2011-25052), Basque Government
(SAIOTEK S-PE12UN041), UPV/EHU (GIU11/25) and EU (Energreen
Project EFA217/11, POCTEFA).
P09r-16
Magnetic cross-linked enzyme aggregates

of Candida antarctica lipase B. Kinetic
characterization and its use to obtain bioproducts
Teresa Garca-Brcena1, Ane Quesada1, Julen Daz1, Enrique A.
Pic1, Carmen Lpez2, Mara J. Llama1, Juan L. Serra1
1
Enzyme and Cell Technology Group, Department of Biochemistry
and Molecular Biology, Faculty of Science and Technology,
University of the Basque Country (UPV/EHU), Leioa, ES,2Enzyme
and Cell Technology Group, Department of Biochemistry and
Molecular Biology, Faculty of Science and Technology, University of
the Basque Country (UPV/EHU) - Ikerbasque, Basque foundation
for science; Biochemistry and Molecular Biology Department,
University of the Basque Country (UPV/EHU), Leioa, ES
The use of robust magnetic biocatalysts is nowadays a rapidly emerging
area because the enzyme can be easily recovered from the reaction
mixture and reused in several catalytic cycles. In this work cross-linked
Granada 2014
enzyme aggregates (CLEAs) of the lipase B from Candida antarctica
(CALB) have been covalently bound to magnetic nanoparticles (MNPs)
of magnetite to obtain a novel robust biocatalyst denoted as mCLEACALB.
In order to compare the behavior of this novel magnetically-separable
biocatalyst to that of the soluble enzyme, the main structural and
catalytic properties of mCLEA-CALB have been studied. For this
purpose the hydrolytic activity was assessed using the chromogenic
substrate p-nitrophenyl acetate (pNPA) whereas the synthetic activity
was evaluated by following the trans/esterification reaction to obtain
either biodiesel (from free fatty acids or triglycerides with methanol)
or biosurfactant (sucrose monopalmitate, SMP, from sucrose and vinyl
palmitate).
The effects of substrate concentration and temperature were studied in
the case of the hydrolytic activity. In the other hand, the kinetics of
mCLEA-CALB to obtain fatty acid methyl esters (FAMEs) was studied
using oleic, linoleic and linolenic acids at 30C, and the values of
kinetic parameters Km and Vm were determined. Also the kinetics of
the transesterification reaction was followed using non-edible vegetable
oils (unrefined soybean and jatropha oils) as well as olive oil as a
control substrate. The effect of stirring on the hydrolytic and synthetic
activity was followed using both mechanical (gyratory) and ultrasound
(sonoreactor) agitation.
Finally, the synthetic activity of mCLEA-CALB to catalyze the synthesis
of SMP was studied. The reaction was carried out at 60C with magnetic
stirring using different amount of mCLEAs and substrates, in the presence
of different organic solvents (DMSO, t-butanol and 2-methyl-2-butanol).
These results indicate that mCLEAs of CALB can be envisaged as a
promising novel biocatalyst of interest to catalyze both hydrolytic and
transesterification reactions to obtain bioproducts.
Work supported by the MINECO (CTQ2011-25052), the Basque
Government (SAIOTEK S-PE12UN041) and UPV/EHU (GIU11/25).
P09-17
DNA binding and hybridyzation on functionarized

TiO2 surfaces
Jos Antonio Mas Gutirrez1, Jos Vicente Prez-Girn2,
Ruy Sanz Gonzlez3, Miriam Jaafar Ruiz-Castellanos4, Jens Jensen5,
Oscar de Luis Jimnez6
1
Laboratorio de Genmica del Centro de Apoyo Tecnolgico,
Universidad Rey Juan Carlos, Campus de Alcorcn, Alcorcn, ES,
2
Emerging Viruses Department, Heinrich Pette Institute, Hamburg,
DE, 3CNR-IMM-MATIS, Catania, IT, 4Departamento de Fsica de la
Materia Condensada, Facultad de Ciencias, Universidad Autnoma
de Madrid, Campus de Cantoblanco, Madrid, ES, 5Thin Film
Physics Division, Department of Physics, Chemistry and Biology,
Linkping University, Linkping, SE, 6Departamento de Ciencias
Bsicas de la Salud, Facultad de Ciencias de la Salud, Universidad
Rey Juan Carlos, Campus de Alcorcn, Alcorcn, ES
Titanium dioxide (TiO2) is a versatile material with a wide range of
technological applications. The physical and chemical properties of TiO2
surfaces are influenced by morphology and crystallinity, among others.
Preparing well-ordered functional structures of TiO2, having highly active
surface areas, is of great interest due to their potential application for
biomaterials and biosensors.
One way of fabricating regular micro- and nano-patterns is by ion-beam
lithography (IBL), where a mask is used to harness an ion beam and define
the structures. Energetic ion-beams induce localized material modifications
in TiO2, which is susceptible to chemical etching. In this way a well-defined
pattern with interesting surface properties can be induced.
The applied lithography method generates a permanent and well defined
periodic structure of micrometre sized square holes having nanostructured
TiO2 surfaces, presenting different physical and chemical properties
compared to the surrounding rutile single crystal surface. On the patterned
Psters
substrates is possible to bind oligonucleotide molecules at the surfaces of
the holes without the participation of intermediate molecules as binding
functional groups.
Here we describe the direct adsorption of DNA oligonucleotides after
surface activation by UV irradiation. Effective DNA-TiO2 surface binding
was demonstrated after several astringent washes. We demonstrate also that
fixed oligonucleotides are able to hybridize specifically to its corresponding
antisense non-fixed oligonucleotide. These preliminary results allow us to
propose TiO2 as an adequate substrate for nanobiosensor applications based
on nucleic acids sequence recognition.
P09-18
Produccin y caracterizacin de la enzima

-galactosidasa de Saccharomyces cerevisiae
Mara E. lvarez Cao, Mara Esperanza Cerdn Villanueva, Manuel
Becerra Fernndez, Mara Isabel Gonzlez Siso
Universidade da Corua, A Corua, ES
La -galactosidasa de la levadura Saccharomyces cerevisiae (ScAGAL)
es una glucsido hidrolasa que cataliza la hidrlisis enzimtica de
residuos galactosa unidos por enlaces -(1,6) de galacto-oligosacridos
y galacto-mananos polimricos. La ScAGAL presenta bajo coste de
produccin, gran estabilidad a temperatura ambiente y amplio rango
de T y pH de trabajo que la identifican como una enzima robusta y de
especial importancia en numerosas aplicaciones biotecnolgicas que
van desde la industria azucarera y la alimentacin animal, pasando por
la farmacutica, hasta la produccin de etanol o biodiesel acoplado a la
degradacin de diferentes materiales (melazas, derivados de legumbres).
En este trabajo, el gen MEL1 que codifica para la ScAGAL fue clonado
y expresado en la cepa S. cerevisiaeBJ3505 generando diferentes
construcciones con el fin de favorecer la purificacin y produccin de la
enzima. Adems, la optimizacin de las condiciones de cultivo permiti
mejorar la productividad obteniendo un incremento de hasta 10 veces
ms de enzima al aumentar la concentracin de la fuente de carbono y la
aireacin del medio de cultivo, y hasta 30 veces ms si se alarga el tiempo
de cultivo hasta las 250 h. Por otro lado, la caracterizacin enzimtica
de la ScAGAL utilizando diferentes sustratos y su estudio comparativo
con otras -galactosidasas disponibles en el mercado, demuestra que la
enzima hidroliza eficientemente los sustratos naturales melibiosa, rafinosa
y estaquiosa pero no puede actuar directamente sobre galactomananos
complejos, como el galactomanano de algarrobo, pudiendo hidrolizar
los galactomano-oligosacridos sintticos, Gal1Man3 y Gal3,4Man5, en
combinacin con la enzima -manosidasa. Estas ventajas permiten que la
ScAGAL sea un buen candidato para su aplicacin en la eliminacin de
oligosacridos de la familia de la rafinosa (RFO).
P09-19
Identification of an inhibitory antibody for

imaging active urokinase plasminogen activator
(uPA)
Natalia Sevillano1, Aaron M. LeBeau2, Daniel R. Hostetter3, Henry F.
VanBrocklin2, Charles S. Craik1
1
Department of Pharmaceutical Chemistry, University of California
San Francisco, San Francisco, US, 2Center for Molecular and
Functional Imaging, Department of Radiology and Biomedical
Imaging, University of California San Francisco, San Francisco, US,
3
CytomX Therapeutics Inc, South San Francisco, US
Increased proteolytic activity is a common characteristic of highly
proliferative and invasive cancers. Proteases are involved in all stages
of cancer and represent functional biomarkers that can be targeted to
distinguish aggressive disease.
Over-expression of the plasminogen activation system (PAS) which
consists of the serine protease urokinase plasminogen activator (uPA), the
81
Psters
uPA receptor, uPAR, and uPA inhibitor, PAI-1, has been documented in
a number of primary and metastatic cancers. The expression of the PAS
directly correlates with cancers that are phenotypically aggressive, result
in poor clinical prognosis, and quickly acquire drug resistance to first line
therapies. Active uPA is a functional biomarker of aggressive cancer that
can be selectively targeted for preclinical imaging using an antagonistic
molecular approach.
Using a fully human nave Fab phage-display library we have identified
a human antibody (Fab U33) that selectively inhibits the active form
of uPA. This antibody targets and internalizes the active form of uPA
via a receptor mediated mechanism by mimicking the action of the
endogenous inhibitor of uPA, PAI-1. We have used U33 labeled with
fluorophores or radionuclides to non-invasively detect active uPA
in vivo in prostate cancer xenografts and in experimental metastasis
models.
P09-20 (R09-6)
Evolucin dirigida de una peroxigenasa

inespecfica, un biocatalizador altamente
promiscuo y selectivo
Patricia Molina-Espeja1, Eva Garcia-Ruiz2, David Gonzalez-Perez1,
Miguel Alcalde1
1
Departamento de Biocatlisis, Instituto de Catlisis y
Petroleoqumica, CSIC, Madrid, ES, 2Departamento de Ingeniera
Qumica y Biomolecular, Universidad de Illinois en UrbanaChampaign, Urbana, Illinois, US
Hace diez aos, se descubri en el hongo basidiomiceto Agrocybe
aegerita una enzima capaz de catalizar actividades que engloban aquellas
de la cloroperoxidasa de Caldaryomices fumago (CPO) y las de las
monooxigenasas del citocromo P450 [1,2]. Con el nombre de peroxigenasa
inespecfica (unspecific peroxygenase, UPO, EC 1.11.2.1), esta verstil
enzima [3] ha demostrado suplir las carencias de las anteriores, con una
elevada eficiencia y selectividad. La UPO es una enzima extracelular e
independiente de cofactores como el NAD(P)H y otras enzimas auxiliares
(slo necesita H2O2 para trabajar), al contrario que las P450, lo que reduce
el coste de su aplicacin.
Hemos sometido a esta peroxigenasa hemo-tiolada (cistena como
ligando axial) a evolucin dirigida hacia expresin funcional en
Saccharomyces cerevisiae [4]. Tras cinco ciclos, se obtuvo la variante
PaDa-I, con unos niveles de secrecin de ~8 mg/L. Tambin ha sido
sobre-expresada en Pichia pastoris, obteniendo ~230 mg/L (material
sin publicar). En ambos casos, las propiedades y comportamiento son
equivalentes a los de la enzima homloga. As, disponemos de una
plataforma de fcil uso para continuar su mejora hacia aplicaciones de
inters biotecnolgico. En nuestro caso, estamos sometiendo a PaDa-I a
evolucin hacia mejora de su actividad hidroxilativa sobre naftaleno, en
detrimento de su actividad oxidativa (material sin publicar). Esta ltima
transforma el compuesto de inters, 1-naftol, en productos no deseados.
La actividad oxidativa en la UPO parece estar muy relacionada con
su estabilidad, pero los resultados obtenidos son esperanzadores, con
una nueva variante que reduce cinco veces la actividad oxidativa
(peroxidasa) manteniendo su capacidad de transferencia de oxgeno
(actividad peroxigenasa).
Bibliografa
[1] Ullrich R, Nske J, Scheibner K, Spantzel J, Hofrichter M. Novel
haloperoxidase from the agaric basidiomycete Agrocybe aegerita oxidizes
aryl alcohols and aldehydes. Appl Environ Microb 2004; 70: 4575-81.
[3] Hofrichter M and Ullrich R. Oxidations catalyzed by fungal
peroxygenases. Curr Opin Chem Biol 2014; 19: 116-25.
82

P09-21
Identifying recombinant antibodies to

conformational states of challenging protein
targets
Natalia Sevillano1, JungMin Kim1, Hai Ta1, Kiment Verba2, David
Agard2, John Gross1, Yifan Cheng2, Charles S. Craik1
1
Department of Pharmaceutical Chemistry, University of California
San Francisco, San Francisco, US, 2Department of Biochemistry
and Biophysics, University of California San Francisco, San
Francisco, US
Monoclonal antibodies (mAbs) are ubiquitous in biomedical research
and medicine. Synthetic antibodies (recombinant antibodies, rAbs) can
be created in the laboratory, completely eliminating animals from the
antibody-production process. rAbs can be used in the same applications
as traditional mAbs such as western blotting, immunohistochemistry,
FACs and immuno-precipitation experiments. They have several
advantages over the traditional hybridoma-based antibodies, including
control over the state of the antigen that allows identification of
antibodies to conformational states of the antigen, rapid identification
of antibody binders allowing automation for high throughput production
and ability to develop antibodies to highly toxic or non-immunogenic
proteins.A fully human nave Fab phage-display library with a diversity
of 4.1 x 1010 was constructed by the Craik laboratory using methods
previously described. We have optimized the protocols for phage display
panning for fast verification of binders and initial characterization of
the epitope by semi-quantitative and competitive ELISAs without the
purification of the Fabs. Using these optimized protocols, we have
successfully generated Fabs that are useful for structural analysis against
a wide range of protein targets including proteases, membrane proteins
and protein complexes.
P09-22
Solid lipid nanoparticle composition is a key

factor for the development of a suitable drug
delivery system
Lide Arana Urbieta, Flix Mara Goi, Itziar Alkorta
Unidad de Biofsica (CSIC, UPV/EHU), and Departamento de
Bioqumica, Universidad del Pas Vasco, Leioa, ES
Development of novel drug delivery systems for the treatment of
complex diseases has become a big challenge for the last two decades.
Many drugs present low water solubility, poor absortion or rapid
elimination and suitable drug carriers are essential for the improvement
of drug bioavailability. Nanotechnology has abruptly broadened the field
of drug delivery systems and particularly, Solid Lipid Nanoparticles
(SLN) have emerged as some of the most promising nanocarriers for
controlled drug delivery. SLN have many advantages: they are able to
incorporate hydrophilic and lipophilic drugs, they present no biotoxicity
and their production can be easily scaled up. Due to their solid core,
drug release is controlled and their size facilitates drug diffusion through
some biological barriers. SLN composition must be carefully selected
depending on targeted tissue and incorporated drug. In this work we have
tested solid lipid nanoparticles (SLN) composed of long-chain fatty acids,
Epikuron 200 (largely soy bean lecithin), and bile salts. A total of five
different systems were prepared, and characterized by photon correlation
spectroscopy, transmission electron microscopy, differential scanning
calorimetry, and capacity to incorporate non-polar drugs. Our results
suggest that SLN composition is essential for particle size, polidispersity
and stability but not for drug incorporation efficiency.
Granada 2014
P09-23
Hydrolysis of mcl-polyhydroxyalkanoates
by immobilized depolymerase from
Streptosporangium roseum
Noelia Villarroel1, Alba lvarez1, Teresa Garca-Brcena1, Enrique A.
Pic1, Carmen Lpez2, Mara J. Llama1, Juan L. Serra1
1
Enzyme and Cell Technology Group, Department of Biochemistry
and Molecular Biology, Faculty of Science and Technology,
University of the Basque Country (UPV/EHU), Leioa, ES,2Enzyme
and Cell Technology Group, Department of Biochemistry and
Molecular Biology, Faculty of Science and Technology, University
of the Basque Country (UPV/EHU) - IKERBASQUE, Basque
Foundation for Science , Leioa, ES
Over the past years, the use of plastics in packaging and other products
has exacerbated the problem of disposal of solid waste. In response to
the problem and harmful effects of the plastic waste on the environment,
there is a considerable interest in the production of biodegradable plastics.
Polyhydroxyalkanoates (PHAs) provide a good fully degradable alternative
to petrochemical plastics. Among PHAs, the medium-chain length
ones (mcl-PHAs) are considered to be promising candidates for special
bioplastic applications due to properties such as elasticity, hydrophobicity,
low oxygen permeability, among others.
At the same time, PHAs have an added interest as source of R-3hydroxyalkanoic acids (R-HAs), the monomers that form the polymer,
which are scaffolds as chiral starting materials in fine chemical,
pharmaceutical and medical industries. Extracellular PHA depolymerases
are enzymes capable of degrading PHAs available in the environment after
the death or lysis of PHA-accumulating bacteria.
In our laboratory a hypothetical protein with a putative extracellular mclPHA depolymerase activity from Streptosporangium roseum DSM43021
was cloned and expressed as a hexahistidine recombinant protein in cells
of E. coli BL21 (DE3) and then purified by IMAC.
Taking into account the advantages that the immobilization can offer with
respect to the soluble enzyme in aspects such as stability or reutilization
of biocatalyst, the aim of this work was to immobilize the recombinant
protein testing different supports in order to obtain an efficient and
robust biocatalyst to hydrolyze mcl-PHAs. For this purpose, hydrophobic
adsorption to porous polypropylene (Accurel MP-1000) and covalent
linkage to magnetic nanoparticles (MNPs) were used. The biochemical
properties of the resulting biocatalysts as well as their ability to catalyze
the production of monomers or dimers of R-HAs in the hydrolytic reaction
of PHAs were compared. Also, the biocatalysts were used for several
consecutive hydrolytic cycles to demonstrate their possible reutilization in
the hydrolytic reaction of mcl-PHA which could allow a cost reduction in
future bioprocesses.
Work supported by the MINECO (CTQ2011-25052), Basque Government
(SAIOTEK S-PE12UN041) and UPV/EHU (GIU11/25). NV was the
recipient of a predoctoral scholarship from the Basque Government.
P09-24
Screening mutant libraries of versatiles

peroxidase from Pleurotus eryngii to enhance
oxidative stability
David Gonzalez-Perez1, Eva Garcia-Ruiz2, Bernardo J. Gmez
Fernndez1, Francisco Javier Ruiz-Dueas3, ngel T.Martinez3,
Miguel Alcalde1
1
Instituto de Catlisis y Petroleoqumica, CSIC, Madrid, ES,
2
Department of Chemical and Biomolecular Engineering, University
of Illinois at Urbana-Champaign, Urbana, US, 3Centro de
Investigaciones Biolgicas - CSIC, Madrid, ES
Versatile peroxidases (VP) are promiscuous biocatalyst with the
highest fragility to peroxides reported yet due to sophisticate molecular
Psters
architecture that includes three catalytic sites and several oxidation
pathways. In this work, an evolved version of VP was subjected to a
range of hybrid and evolutionary strategies in Saccharomyces cerevisiae
to study the resistance to H2O2. After 5 generations of random-,
saturation- and domain- mutagenesis together with in vivo DNA
recombination approaches, several structural determinants behind the
oxidative destabilization of the enzyme were unmasked. To keep balance
between activity and stability,selected beneficial mutations were further
relocated in novel mutational environments by in vivo sequence blocks
exchange promoting epistatic interactions. The best variant of this
process accumulated 8 mutations that increased the half-life up to 30 min
in the presence of 3,000 equivalents of H2O2 and shifted 6C upwards
the kinetic thermostability. Whilst the main heme domain was not
significantly modified, mutations in the surroundings of the Mn2+ binding
pocket and the catalytic tryptophan decreased substrates affinities for
both catalytic sites exerting a beneficial effect to the protein stabilization.
Multiple structural alignment with other H2O2 tolerant peroxidases
addressed possible roles played by the new mutations in the overall
oxidative damage process of the heme-peroxidase superfamily.
P09r-25
Screening a glycosynthase library with an

enzyme-independent assay based on a
fluorescent sensor
Hugo Aragunde Pazos, Estela Castilla, Eduardo Andrs, Xevi
Biarns, Antoni Planas
Laboratory of Biochemistry, Bioengineering Department, Institut
Quimic de Sarria, Universitat Ramon Llull, Barcelona, ES
Glycosynthases have become efficient tools for the enzymatic synthesis
of oligosaccharides, glycoconjugates and polysaccharides. They are
engineered retaining glycosidases in which the hydrolase activity is
abolished by mutation of the catalytic nucleophile but efficiently catalyze
transglycosylation when using activated glycosyl fluoride donors with
the opposite anomeric configuration to the substrate in the wild-type
enzyme. Enzyme-directed evolution approaches are applied to improve the
performance of current glycosynthases and engineer specificity for new
substrates. However, simple and general screening methods are required
since most of the reported assays are specific for each particular enzyme
[1]. Only one universal screening method has been proposed, a pH-based
assay that measures the hydrofluoric acid released as by-product of the
glycosynthase reaction, being detected by color change of a pH-indicator
[2]. However, it is not very reproducible and difficult to implement due to
sample matrix variations.
We have developed a new general screening assay independent of
enzyme specificity for the screening of glycosynthase libraries [3].
This assay is based on the use of a chemical sensor to transduce
fluoride concentration (by-product of all glycosynthase reactions using
fluoride activated donors) into a fluorescence signal. We report here
the application to a nucleophile saturation mutant library of Bacillus
licheniformis 1,3-1,4--glucanase. Beyond the expected mutations at
the nucleophile, other variants have acquired glycosynthase activity.
Surprisingly, an aspartic acid for glutamic acid replacement renders
a highly active glycosynthase, but retaining low hydrolase activity.
It appears to be an intermediate state between glycosyl hydrolase and
glycosynthase [4].
References
[1] Roman K., Withers S.G. (2010) Carbohydr. Res. 345, 1272-1279.
[2] Ben-Davis A., Shoham G., Shoham Y. (2008) Chem. Biol. 15, 546-551.
[3] Andrs E., Aragunde H., Planas A. (2014) Biochem. J. 458, 355363.
[4] Aragunde H., Castilla E., Biarns X., Faijes M., Planas A. (2014)
Carbohydr. Res. 389, 85-92.
83
Psters
P09-26
compiten de forma muy efectiva con la protena por la superficie hidroflica,

no habindose observado una dependencia significativa en su capacidad de
inhibicin con el peso molecular. Incluso en condiciones de fuerza inica
elevada, donde las interacciones electrostticas se ven minimizadas, 1 mg
de PE es capaz de provocar la desorcin de 0.5 mg de lisozima previamente
adsorbida sobre 10 mg de slice contenidos en 1 mL.
Finalmente, la capacidad de prevenir la adsorcin de protenas a superficies
de slice mediante el recubrimiento previo de estas superficies con una
monocapa de polielectrolito ha sido testada encontrndose que lavados
sucesivos de la superficie tienen un efecto prcticamente despreciable sobre
el nivel de recubrimiento de la superficie y, por tanto, sobre su proteccin
frente a la adsorcin proteica.
Identification of reciprocal adhesion genes in

pathogenic and nonpathogenic Pseudomonas
Jess de la Torre Ziga1, Maria Antonia Molina Henares1, Estrella
Duque Martin de Oliva2, Miguel Alaminos Mingorance3, Manuel
Espinosa Urgel1, Amalia Roca4, Patricia Bernal Guzman1, Matilde
Fernandez1, Sophie de Bentzmann5, Juan Luis Ramos Martin2
1
Consejo Superior de Investigaciones Cientficas-EEZ, Department
of Environmental Protection, Granada, ES, 2Abengoa Research,
Department of Biotechnology, Campus Palmas Altas, Sevilla, ES,
3
Universidad de Granada, Facultad de Medicina, Department of
Histology, Granada, ES, 4Bio-Iliberis R&D, Granada, ES, 5UPR
CNRS 9027, Marseille, FR
We used a combination of in silico and large-scale mutagenesis
approaches to expand our current knowledge of the genetic determinants
used by Pseudomonas putida KT2440 to attach to surfaces. We first
identified in silico orthologs that have been annotated in Pseudomonas
aeruginosa as potentially involved in attachment. In this search 67 pairedrelated genes of P. putida KT2440 and P. aeruginosa were identified as
associated to adhesion. To test the potential role of the corresponding
gene products in adhesion, 37 knock-out mutants of KT2440, available
in the Pseudomonas Reference Culture Collection, were analyzed with
regard to their ability to form biofilms in polystyrene microtiter plates;
of these, 7 mutants were deficient in adhesion. Since mutants in all
potential adhesion genes were not available, we generated a genomewide collection of mutants made of 15 360 independent mini-Tn5
insertions and tested them for the formation of biofilm on polystyrene
microtiter plates. Eighteen clones that exhibited a reduction of at least
3-fold in biofilm biomass formation were considered candidate mutants
in adhesion determinants. DNA sequencing of the insertion site identified
5 other new genes involved in adhesion. Phenotypic characterization of
the mutants showed that 11 of the inactivated proteins were required
for attachment to biotic surfaces too. This combined approach allowed
us to identify new proteins with a role in P. putida adhesion, including
the global regulator RpoN and GacS, the lectin-like protein LecA, PstS,
and a protein of unknown function (PP1633). The remaining mutants
corresponded to functions known or predicted to participate in adhesion
based on previous evidence, such as the large adhesion proteins LapA,
LapF, and flagellar proteins. In silico analysis showed this set of genes
to be well conserved in all sequenced P. putida strains, and that at least
9 reciprocal genes involved in attachment are shared by P. putida and
P. aeruginosa.
P09r-27
Inhibicin de la adsorcin de protenas a

superficies de slice mediante el uso de
polielectrolitos de alta densidad de carga
Felipe Hornos Adn, Roco Esquembre Tom, Javier Gmez Prez
Instituto de Biologa Molecular y Celular (Universidad Miguel
Hernndez), Elche, ES
La adsorcin es un proceso complejo que puede afectar a la estabilidad
estructural de las protenas dependiendo de factores tales como temperatura,
pH o fuerza inica. Aunque la adsorcin de protenas ha encontrado
multitud de aplicaciones biotecnolgicas, resulta necesario el desarrollo
de metodologas que permitan su modulacin para facilitar el manejo in
vitro de protenas en condiciones de alta dilucin y el almacenamiento y
manipulacin de frmacos proteicos que evite su inactivacin inducida por
la adsorcin - desorcin a superficies de vidrio o plstico y su potencial
inmunogenicidad.
Proponemos la utilizacin de polielectrolitos (PE) catinicos de alta
densidad de carga como medio eficaz para inhibir la adsorcin de protenas
a superficies de slice. En particular hemos estudiado la capacidad de
inhibicin de polielectrolitos basados en grupos amonio y en grupos amina
de distinto peso molecular. Nuestros resultados indican que estos PE
84
P09-28
El receptor de productos avanzados de glicacin

(RAGE) como una diana para la generacin de
reactivos especficos de transfeccin de clulas
eucariotas
Mara Dolores Girn Gonzlez1, Fernando Hernndez Mateo2,
Francisco Javier Lpez-Jaramillo2, Arturo Morales Portillo2, Alfonso
Salinas Castillo3, Francisco Santoyo Gonzlez2, Rafael Salto
Gonzlez1
1
Farmacia, Universidad de Granada, Granada, ES, 2Departamento de
Qumica Orgnica, Facultad de Ciencias, Universidad de Granada,
Granada, ES, 3Departamento de Qumica Analtica, Facultad de
Ciencias, Universidad de Granada, Granada, ES
En las alteraciones a largo plazo de la diabetes y en la angiognesis
asociada con la metstasis tumoral, el receptor de productos avanzados de
glicacin (RAGE) tiene un papel esencial. Este receptor se sobre-expresa
en los tejidos diana asociados a estas patologas y tras unirse a sus ligandos
se internaliza. Esto hace que este receptor constituya una diana para el
desarrollo de reactivos especficos de transfeccin.
As, derivados de polietilenimina (PEI 25 kDa) y PAMAM-G2 se
modificaron mediante la reaccin de glicacin no enzimtica de Maillard
para generar nuevos agentes de transfeccin irreversiblemente glicados
capaces de unirse al RAGE. Estos reactivos as sintetizados mantienen la
capacidad de unir DNA y protegerlo de la degradacin por endonucleasas.
Adems, mientras que los reactivos glicados mostraron una baja
capacidad de transfeccin en clulas CHO-k1 que no expresan RAGE, los
compuestos glicados actuaron como eficientes reactivos de transfeccin
en una lnea celular CHO-k1 que expresa de manera recombinante el
RAGE. La especificidad de estos reactivos quedo demostrada por el
hecho de que la pre-incubacin con albmina glicada, el ligando natural
del RAGE, o dansyl cadaverina, un inhibidor de la internalizacin del
RAGE, bloquean la transfeccin mediada por los reactivos glicados. Los
resultados obtenidos se han confirmado en las lneas celulares NRK y
RAW264.7 que expresan de manera natural el receptor. Asimismo, los
compuestos glicados retienen su eficiencia de transfeccin en presencia
de suero y son capaces de promover la transfeccin in vivo en un modelo
experimental en ratn. Por tanto, nuestros resultados muestran que
la reaccin de Maillard es un procedimiento simple y directo para la
preparacin de reactivos glicados de transfeccin especficos y que esta
tcnica es aplicable a otros reactivos de transfeccin que presenten grupos
amino libres. En conclusin, las propiedades de estos compuestos son
muy interesantes para su uso in vivo: la glicacin confiere selectividad con
un muy pequeo efecto sobre la toxicidad de los reactivos, mantienen su
actividad en presencia de suero y utiliza una va nica de internalizacin
mediada por receptores, con una alta eficiencia de transfeccin in vivoen
ratn.
Investigacin subvencionada por el Proyecto CTQ2011-29299-C02
(MEC).
Granada 2014
Psters
P09-29
P09-31
Adsorcin de protenas a superficies

hidrofbicas: caracterizacin termodinmica y
estrategias de inhibicin
Engineering biological approaches for antibiotic

detection. A new microbial biosensor based on
the Pseudomonas putida TtgR repressor
Roco Esquembre Tom, Felipe Hornos Adn, Javier Gmez Prez

Instituto de Biologa Molecular y Celular (Universidad Miguel
Hernndez), Elche, ES
Manuel Espinosa-Urgel1, Luis Serrano2, Juan Luis Ramos1,

Ana Mara Fernndez-Escamilla1
1
Department of Environmental Protection, Estacin Experimental
del Zaidn, CSIC, Granada, ES, 2European Molecular Biology
Laboratory and Centre for Genomic Regulation Systems Biology
Research Unit, Barcelona, ES
La adsorcin de protenas a superficies hidrofbicas es un proceso

complejo que puede afectar la estabilidad estructural de las protenas y
su actividad biolgica dependiendo de factores tales como temperatura,
pH o fuerza inica. Aunque la adsorcin de protenas ha encontrado
multitud de aplicaciones biotecnolgicas, resulta necesario el desarrollo
de metodologas que permitan su modulacin para facilitar el manejo in
vitro de protenas en condiciones de alta dilucin y el almacenamiento y
manipulacin de frmacos proteicos que evite su inactivacin inducida
por la adsorcin - desorcin a superficies de plstico y su potencial
inmunogenicidad.
Hemos caracterizado termodinmicamente la adsorcin de la protena
modelo lisozima a partculas coloidales de ltex (de dimetro menor de
1 m) e identificado sustancias que permiten la inhibicin efectiva de
dicho proceso. Dada su naturaleza apolar y la presencia de cargas en su
superficie para asegurar su estabilidad coloidal (evitar su aglomeracin),
la energa libre de adsorcin contiene tanto un componente electrosttico
como otro hidrofbico. Nuestros resultados indican que la adsorcin
proteica presenta cooperatividad negativa debido a la repulsin
electrosttica entre molculas individuales de protena adsorbidas sobre
la superficie hidrofbica. Como consecuencia, el nivel de recubrimiento
(y la afinidad aparente de la protena por la superficie) es mximo a un
pH cercano a su punto isoelctrico disminuyendo a medida que aumenta
la carga de la protena. Mediante calorimetra isoterma de titulacin
hemos podido caracterizar termodinmicamente las contribuciones
electrostticas e hidrofbicas del proceso de adsorcin as como
cuantificar su inhibicin mediante surfactantes y polmeros hidroflicos
como el polivinilalcohol.
P09-30 (R09-2)
Visualizando ATP intracelular en clulas vivas

Vicente Gonzlez Charro, Ivn Lpez Montero
Universidad Complutense de Madrid, Madrid, ES
La molcula de ATP (adenosn trifosfato) es la principal fuente de
energa necesaria para realizar trabajo biolgico. La mayor parte de ATP
se genera mediante el proceso de respiracin mitocondrial. Se sabe que
el ATP generado es transportado al citosol en contra de un gradiente de
concentracin, mediante el potencial de membrana actuando como fuerza
motriz. Al mismo tiempo, el ADP se transporta activamente al interior
de las mitocondrias, manteniendo su concentracin citoslica muy baja.
La relacin de concentraciones de ATP:ADP en el citosol es del orden de
200:1. Sin embargo no se conoce la distribucin de ATP en los diferentes
compartimentos intracelulares debido a que los mtodos convencionales
para la deteccin y cuantificacin de ATP, basados en anlisis de extractos
celulares, slo proporcionan niveles promediados de ATP. Por ello, la
deteccin selectiva de ATP intracelular en clulas nicas vivas se ha
convertido recientemente en un tema de investigacin muy activo. El
diseo de sensores especficos de ATP permitir entender cmo se regula
la concentracin de ATP intracelular a nivel de clula nica. En esta
contribucin se expondrn las diferentes aproximaciones, tanto qumicas
como biolgicas, que se han realizado recientemente para monitorizar
y visualizar los niveles de ATP en clulas individuales y en tiempo real.
Adems, se mostrarn resultados preliminares del uso de un sensor basado
en rodamina tanto en clulas NIH3T3 como en sistemas de membrana
artificiales.
Environmental contamination by toxic organic compounds and

antimicrobials is one of the causes for the recent surge of multidrug-resistant
pathogenic bacteria. Monitoring contamination is therefore the first step in
containment of antimicrobial resistance, and requires the development of
simple, sensitive and quantitative tools that detect a broad spectrum of toxic
compounds. The emerging approach for detecting toxic compounds is the
rational design of microbial biosensors. In this study, we have engineered
a new specific microbial biosensor based on the ttgR-regulated promoter
which controls expression of the TtgABC extrusion efflux pump, coupled
to a gfp reporter. The system was introduced in Pseudomonas putida DOTT1E, a strain characterized by its ability to survive in the presence of high
concentrations of diverse organic compounds. This whole-cell biosensor is
capable to detect a wide range of antibiotics, as well as organic compounds
such as toluene or flavonoids. Our tool opens the possibility to screen a
wide range of toxic compounds by altering the TtgR binding site to obtain
specific versions of this biosensor oriented to sense specific compounds.
P09-32
Anlisis funcional de tres acil-CoA sintetasas

implicadas en la degradacin de cidos biliares
en Pseudomonas putida DOC21
lvaro Barrientos Castaeda, Estefana Merino Garca, Joaqun
Rodrguez Fernndez, Esther Coto Alcaraz, Raquel Benavides
Serrano, Jos Mara Luengo Rodrguez, Elas Rodrguez Olivera
Dpto Biologa Molecular, Universidad de Len, Len, ES
Pseudomonas putida DOC21 es una g-proteobacteria aislada del medio
ambiente por su capacidad para utilizar cidos biliares y otros esteroides
como nica fuente de carbono. En el genoma de esta bacteria se han
identificado cinco clusters de genes que codifican funciones implicadas con
el catabolismo de estos compuestos (genes std). Tres de estos genes codifican
posibles acil-CoA sintetasas implicadas en la degradacin de cidos biliares
(stdA1, stdA2 y stdA3). Se ha procedido a la mutacin especfica mediante
delecin de cada uno de estos genes obtenindose las cepas stdA1,
stdA2 y stdA3. Dos de las cepas mutantes obtenidas, stdA1 y stdA2,
resultaron incapaces de asimilar colato y otros cidos biliares, no vindose
afectado el metabolismo de testosterona y 4-androsten-3,17-diona. Por
el contrario, el tercer mutante, stdA3, creca con dificultad en cualquier
medio en el que la fuente de carbono fuera un compuesto esteroideo.
Cuando se cultivaron dichos mutantes en medios conteniendo colato
como fuente de intermediarios metablicos y con succinato como fuente
de carbono para soportar el crecimiento bacteriano, el mutante stdA1
acumulaba en los caldos de cultivo 1/4-3-cetocolato y 1,4-3-cetocolato,
el mutante stdA2 acumulaba 7,12-dihidroxi-3-oxopregna-1,4-dien20-carboxilato (DHOPDC), y el mutante stdA3 mostraba un bloqueo
metablico a nivel del 3a-H-4(3propanoato)-7a-metilhexahidro1,5-indanodiona (HIP). Anlisis bioqumicos posteriores demostraron que
StdA1 cataliza la tioesterificacin de colato, 3-cetocolato, 1/4-3-cetocolato
y 1,4-3-cetocolato a sus respectivos derivados de CoA, la protena StdA2
cataliza la transformacin de DHOPDC a DHOPDC-CoA, mientras que la
enzima StdA3 lleva a cabo la tioesterificacin del HIP y/o sus derivados
hidroxilados a sus tiosteres de coenzima A.
85
Psters
P09-33
[3]. There is little known about the sequence of events that leads to SG
formation but, in this work, we propose that the SG assembly by TIA1 involves the nucleating trigger of dimerisation via RRM2. In addition,
post-translational modifications of TIA-1 (such as phosphorylation that
occurs in cells under stress) can also lead to the SG formation.
Fas-Activated Serine/Threonine Kinase (FAST K) is known to interact with
and phosphorylate TIA-1 [4,5]. The substitution of three serines at TIA-1
RRMs by either aspartic acid serving as a phosphoserine mimic or
alanine that can no longer be phosphorylated helps to understand the
structural effects of TIA-1 phosphorylation, as well as the TIA-1 capability
of being phosphorylated by FAST K. In fact, we have explored how such
Ser-by-Asp phosphomimetic mutants of TIA-1 trigger the conformational
changes required for RRM dimerisation and TIA-1 protein self-association
by combining Light Scattering and AU measurements with Molecular
Dynamics simulations.
Our hypothesis presents a novel paradigm for the field of neurodegenerative
research suggesting that the SG nucleation can be initiated by TIA-1 RRM
domains, rather than by the PRD module as is widely accepted.
Comparative evaluation of the antitumor

activity of new platinum based drugs on human
adenocarcioma cell lines
Enric Mil1, Roldn Corts1, Margarita Crespo2, Laia Davin2, Raquel
Martn2, Josefina Quirante3, Daniel Ruiz3, Ramon Messeguer4,
Carme Calvis4, Laura Baldom5, Josefa Badia5, Marta Cascante1
1
Department of Biochemistry and Molecular Biology, Faculty of
Biology - IBUB, Universitat de Barcelona (UB), IDIBAPS, Unit
Associated with CSIC, Barcelona, ES, 2Departament de Qumica
Inorgnica and IBUB, Facultat de Qumica, UB, Barcelona, ES,
3
Laboratori de Qumica Orgnica, Facultat de Farmcia, UB,
Barcelona, ES, 4Biomed Division LEITAT Technological Center, Parc
Cientfic de Barcelona, Barcelona, ES, 5Departament de Bioqumica i
Biologia Molecular, Facultat de Farmcia, IBUB, UB, Barcelona, ES
It is estimated that 50-70% of cancer patients are treated with platinum (II)
drugs. Despite the therapeutic benefit of the approved platinum based drugs
(cisplatin, carboplatinand Oxaliplatin), their efficacy is still limited due to
side effects and resistances. Hence, great efforts have been undertaken to
develop novel metal-based drugs. Over the last three decades thousands
of Pt-containing compounds have been designed and tested, but just a few
of them have entered clinical use (carboplatin, oxaliplatin and nedaplatin)
or are in clinical trials. Despite the therapeutic benefit of the approved
platinum drug, the efficacy of the treatments is still limited due to side
effects and intrinsic and acquired resistances. It is believed that DNA isthe
main target of platinum drug and that cisplatin and its analogues form
an intrastrand d(GpG) adduct with platinum cross-linking N7 atoms of
neighbour guanine residues of DNA. It has been also shown that carrier
amine ligands of cisplatin analogues appear to modulate the antitumor
properties of this class of drugs. Cyclometalating N-donor ligands may
offer an alternative approach to give structures quite different from that
of cisplatin and analogs, with the possibility that those could interact in
a different way with DNA and consequently show a different spectrum
of activity and toxicity profile. We have been recently involved in the
synthesis of a novel class of seven-membered platinacycles, and the lack of
information on the biological activity of these compounds prompted us to
undertake the present study.
P10. Estructura y funcin de protenas

P10-1
Effect of TIA-1 phosphorylation in dimerisation

and stress granule formation
Sofa Muoz Garca-Maurio1, Isabel Cruz-Gallardo1, Valentina
Castelli1, Antonio Daz-Quintana1, Myriam Gorospe2, Jacqueline
A. Wilce3, Irene Daz-Moreno1
1
Universidad de Sevilla CSIC, Sevilla, ES, 2Laboratory of Genetics,
National Institute on Aging-Intramural Research Program, NIH,
Baltimore, US, 3Department of Biochemistry and Molecular Biology,
Monash University, Victoria, AU
TIA-1 (T-cell restricted Intracellular Antigen-1) is an RNA-Binding Protein
(RBP) that functions as a translational repressor, sequestering target mRNA
into stress granules (SG) upon cellular stress [1]. It has been shown that the
deregulation of this system results in neurodegenerative diseases such as
Alzheimer [2].
TIA-1 is constituted by three RNA Recognition Motifs (RRMs) and
a Prion-Related Domain (PRD). Preliminary work using EMSA and
Analytical Ultracentrifugation (AU) revealed that TIA-1 RRM2 has a high
propensity to form dimers, while TIA-1 RRM23 or RRM3 alone behave as
a stable monomer, in agreement with recent NMR relaxation measurements
86
References
[1] Anderson P, Kedersha N. (2002) Visibly stressed: the role of eIF2, TIA1, and stress granules in protein translation Cell Stress Chaperones 7: 213221.
[2] Wolozin, B. (2012) Regulated protein aggregation: stress granules and
neurodegeneration Mol Neurodegener.7: 56.
[3] Wang I, Hennig J, Jagtap PK, Sonntag M, Valcrcel J, Sattler M. (2014)
Structure, dynamics and RNA binding of the multi-domain splicing factor
TIA-1 Nucleic Acids Res. 42: 5949-5966.
[4] Tian Q, Taupin J, Elledge S, Robertson M, Anderson P. (1995) Fasactivated serine/threonine kinase (FAST) phosphorylates TIA-1 during
Fas-mediated apoptosis J. Exp. Med. 182: 865-874.
[5] Izquierdo, JM, Valcarcel J. (2007) Fas-activated serine/threonine kinase
(FAST K) synergizes with TIA-1/TIAR proteins to regulate Fas alternative
splicing. J. Biol. Chem. 282: 1539-1543.
P10-2
Nucleophosmin stabilization as a strategy

to prevent its mislocalization in leukemia
Sonia Bauelos1, Igor Arregi1, Mara Sendino1, Marin AlonsoMario1, Jos Antonio Rodrguez2, Jarl Underhaug3, Aurora
Martnez3, Maria ngeles Urbaneja1
1
Unidad de Biofsica (CSIC/UPV-EHU) and Department of
Biochemistry and Molecular Biology, University of the Basque
Country, Leioa, ES, 2Department of Genetics, Physical Anthropology
and Animal Physiology, University of the Basque Country, Leioa, ES,
3
Department of Biomedicine, University of Bergen, Bergen, NO
Nucleophosmin (NPM) is a protein involved in ribosome biogenesis and
other functions affecting cell proliferation. NPM is oligomeric and built
of several domains: a compact, pentameric core, is connected through
long and flexible linkers to small, helical C-terminal domains. Albeit
continuously shuttling between nucleolus, nucleoplasm and cytoplasm,
NPM is mostly enriched in nucleoli, which probably depends on its binding
to G-rich sequences of nucleic acids. NPM deregulation has been related
to different types of cancer; in particular, NPM1 is the most frequently
mutated gene in acute myeloid leukemia (AML), and these mutations
cause the aberrant localization of the protein to the cytoplasm. It has
been shown that AML-related mutations confer NPM an additional NES
(nuclear export sequence), and impair folding of the C-terminal domain
(e.g., abolishing ability to bind nucleic acids and hence to the nucleolus),
all in all displacing the protein to the cytoplasm. Therefore, AML can be
regarded as a misfolding disease. The pharmacological chaperones
approach is an interesting strategy to restore the conformational stability
of misfolded proteins. Probing for changes in the thermal stability of
NPM C-terminal domain, we have performed a high throughput screening
of a natural compound library. 25 hits were found to increase the Tm of
Granada 2014
the domain by 6-21C. We have then tested the effect of some of these
compounds on the subcellular localization of AML-like mutants in HeLa
cells expressing fluorescent NPM constructs. One compound is able to
partially restore nucleolar localization whereas another one alleviates
the protein aggregation of misfolded mutants. We conclude that these
compounds might help to promote native localization and functionality of
mutant, oncogenic NPM.
P10-3
Recognition of nucleophosmin by the export

receptor Crm1: deciphering the basis of aberrant
traffic in leukemia
Igor Arregi Vado1, Marin Alonso-Mario1, Jos Antonio Rodrguez2,
Mara ngeles Urbaneja1, Sonia Bauelos1
1
Unidad de Biofsica (CSIC/UPV-EHU) and Department of
Biochemistry and Molecular Biology, University of the Basque
Country, Leioa, ES, 2Departamento de Gentica, Antropologa Fsica
y Fisiologa Animal. Facultad de Ciencias, EHU/UPV, Leioa, ES
Nucleophosmin (NPM) is a nucleolar protein involved in several functions
impacting cell growth, such as the assembly and export of ribosomes from
the nucleolus to the cytoplasm. NPM is an oligomeric, multidomain protein,
built of a compact core, connected to C-terminal, globular domains through
long, flexible linkers. Although NPM is normally enriched in nucleoli, its
activities require continuous shuttling between nucleolus, nucleoplasm
and cytoplasm. This traffic relies on different localization signals in the
protein: NLS for import, Crm1-dependent NES for export, and C-terminal
domains for binding to nucleolar components. NPM deregulation has
been related to different types of cancer; in particular, NPM1 gene is
frequently mutated in acute myeloid leukemia (AML), correlating with an
aberrant, cytoplasmic localization of the protein. It has been shown that
AML-related mutations of NPM impair folding of the C-terminal domain,
hampering attachment to the nucleolus, and confer an additional NES,
both factors contributing to displacement of the protein to the cytoplasm.
The molecular determinants of wild type NPM recognition by the export
receptor Crm1 are not yet understood; on the other hand, the subcellular
localization of mutant NPM in AML remains to be explained in terms of
Crm1 binding properties. To better understand the determinants of NPM
cellular traffic, both in physiological and pathological conditions, we are
characterizing NPM recognition by Crm1, and the effect of AML mutations
on the interaction. We have observed that AML-related NPM mutants
bind indeed with stronger avidity to Crm1, explaining their unbalanced
transport in leukemia.
P10-4
Identifying protein-protein interaction inhibitors

for NUPR1, a key therapeutic target in
pancreatic cancer
Mara Arruebo1, ngel Lanas2, Jos L. Neira3, Juan L. Iovanna4,
Adrin Velzquez-Campoy5, Olga Abin6
1
IIS Aragn / Instituto Aragons de Ciencias de la Salud-IACS,
Zaragoza, ES, 2IIS Aragn, Universidad de Zaragoza, Servicio
de Digestivo - Hospital Clnico Universitario Lozano Blesa de
Zaragoza, Zaragoza, ES, 3Instituto de Biologa Molecular y Celular,
Universidad Miguel Hernndez - Instituto de Biocomputacin y
Fsica de Sistemas Complejos (BIFI), Universidad de Zaragoza,
Elche, ES, 4Centre de Recherche en Cancrologie de Marseille
(CRCM), INSERM U1068, Marsella, FR, 5BIFI - Universidad de
Zaragoza - Fundacin ARAID, Diputacin General de Aragn,
Zaragoza, ES, 6IIS Aragn/Instituto Aragons de Ciencias de la
Salud (IACS) - BIFI - Universidad de Zaragoza, Zaragoza, ES
Pancreatic ductal adenocarcinoma (PDAC), the most common type of
pancreatic cancer, is one of the most aggressive human cancers and worse
Psters
prognosis because of its remarkable capacity for invasion and metastasis.
PDAC exhibits an extremely low survival rate (<3-4% at 5 years), due to its
difficult diagnosis in early stages, and its high resistance to chemotherapy
and radiotherapy. Hence, there is an urgent need to develop new and
effective therapies against this disease.Protein NUPR1 (Nuclear Protein
1), also known as p8, is a nuclear protein overexpressed in several types
of human cancers (breast, thyroid, skin...), especially in PDAC, where
NUPR1 is essential for the development and progression of the tumor.
Several studies have shown that blocking NUPR1 expression in pancreatic
cancer cells prevents the formation of tumors, thus, confirming its key role
in PDAC.
NUPR1 protein is a small (82 aminoacid residues), basic and multifunctional
protein without stable secondary and tertiary structure, belonging to the
groupof intrinsically disorderedproteins (IDPs). Its interaction with its
different partners promotes the mutual stabilization of their structures
in a particular conformation and the formation of fuzzy, but biologically
active complex. NUPR1 fuzzy complexes are involved in the regulation of
important cellular processes, such as transcription of genes, cellular cycle,
or apoptosis.
In our group, we have developed a new strategy for identifying compounds
capable of blocking NUPR1 intracellular interactions. The in vitro selected
compounds inhibited NUPR1 interactions with one of its cellular partners
and efficiently blocked NUPR1 function in cell-based studies.
Keywords: NUPR1, PDAC, IDPs, Thermal stability, Structural characterization
of proteins, HTS.
P10-5 (R10-1)
Dissecting I-DmoI endonuclease catalysis live

Rafael Molina Monterrubio1, Stefano Stella2, Pilar Redondo1, Hansel
Gomez3, Maria Jose Marcaida4, Modesto Orozco3, Jesus Prieto1,
Guillermo Montoya2
1
Structural Biology and Biocomputing Programme, Spanish National
Cancer Research Centre (CNIO), Macromolecular Crystallography
Group, Madrid, ES, 2Structural Biology Group, Novo Nordisk
Foundation Center for Protein Research, Faculty of Health and
Medical Sciences, University of Copenhagen, Copenhagen, DK,
3
Institute for Research in Biomedicine (IRB Barcelona,Joint
BSC-CRG-IRB Program in Computational Biology and Department
of Biochemistry and Molecular Biology, University of Barcelona,
Barcelona, ES, 4Department of Chemistry and Biochemistry,
University of Bern, Bern, CH
The enzymatic hydrolysis of a DNA phoshpodiester bond has been widely
studied; however, the chemical reaction has not been directly observed.
Here we watch the course of a double strand break generation by I-DmoI,
a homing endonuclease. The catalysis of this enzyme family is similar to
type II restriction enzymes, which are general tools in molecular biology
and boosted the recombinant DNA technology. By using a dynamic
crystallography approach we provide the first time-resolved view of a
two-metal ion cleavage mechanism, a central reaction to modify and edit
DNA. We developed a procedure to capture intermediates of the different
catalytic steps allowing us to analyse and watch the reaction by freezing
multiple stages. We observe the successive entrance of two metals involved
in the reaction, and finally the arrival of a third metal in a central position
of the active site. This metal plays a pivotal role triggering the consecutive
digestion of the targeted phosphodiester bonds in the non-coding and coding
strands. Finally, the central metal abandons its position after double strand
break generating a 4bp DNA overhang. We solved more than 150 crystal
structures to obtain key snapshots of different catalytic stages, showing the
orchestrated conformational changes in the amino acids, nucleotides and
metals during catalysis. Our work provides a live and visual proof of this
key biological mechanism.
87
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P10-6
P10-8 (R10-3)
The Proliferating Cell Nuclear Antigen-associated

factor p15PAF is an intrinsically disordered
protein with non-random structural preferences
at sites of interaction with other proteins
Instruct: infraestructura europea para biologa

estructural
Alfredo De Biasio1, Alain Ibez de Opakua1, Tiago N. Cordeiro2,

Maider Villate1, Nekane Merino1, Nathalie Sibille2, Moreno Lelli3,
Tammo Diercks1, Pau Bernad2, Francisco J Blanco4
1
CIC bioGUNE, Derio, ES, 2Centre de Biochimie Structurale,
Montpellier, FR, 3Institut de Sciences Analytiques, Villeurbanne,
FR, 4IKERBASQUE, Bilbao, ES
We present the first structural characterization of the p15PAF protein
showing that it is monomeric and intrinsically disordered in solution
but with non-random conformational preferences at sites of proteinprotein interactions. The Proliferating Cell Nuclear Antigen-associated
factor p15PAF is a 12 kDa nuclear protein that acts as a regulator of
DNA repair during DNA replication. The p15PAF gene is overexpressed
in several types of human cancer. The nearly complete NMR backbone
assignment of p15PAF allowed us to measure 86 N-HN residual dipolar
couplings (RDCs). Our RDC analysis reveals non-random conformational
preferences in distinct regions, including the PCNA-interacting protein
motif (PIP-box) and the KEN-box (recognized by the ubiquitin ligase that
targets p15PAF for degradation). In accordance with these findings, the
analysis of the 15N R2 relaxation rates shows a relatively reduced mobility
for the residues in these regions. The agreement between the experimental
small angle X-ray scattering curve of p15PAF and that computed from
a statistical coil ensemble corrected for the presence of local secondary
structural elements further validates our structural model for p15PAF. The
coincidence of these transiently structured regions with protein-protein
interaction and post-translational modification sites suggests a possible role
for these structures as molecular recognition elements for p15PAF.
P10-7 (R10-2)
Structural basis of regulation and oligomerization

of human cystathionine -synthase, the central
enzyme of transsulfuration
June Ereo Orbea
CICBiogune, Derio, ES
Cystathionine -synthase (CBS) controls the flux of sulfur from
methionine to cysteine, a precursor of glutathione, taurine, and H2S. CBS
condenses serine and homocysteine to cystathionine with the help of three
cofactors, heme, pyridoxal-5-phosphate, and S-adenosyl-L-methionine.
Inherited deficiency of CBS activity causes homocystinuria, the most
frequent disorder of sulfur metabolism. We present the structure of the
human enzyme, discuss the unique arrangement of the CBS domains in
the C-terminal region, and propose how they interact with the catalytic
core of the complementary subunit to regulate access to the catalytic site.
This arrangement clearly contrasts with other proteins containing the CBS
domain including the recent Drosophila melanogaster CBS structure. The
absence of large conformational changes and the crystal structure of the
partially activated pathogenic D444N mutant suggest that the rotation of
CBS motifs and relaxation of loops delineating the entrance to the catalytic
site represent the most likely molecular mechanism of CBS activation
by S-adenosyl-L-methionine. Moreover, our data suggest how tetramers,
the native quaternary structure of the mammalian CBS enzymes, are
formed. Because of its central role in transsulfuration, redox status, and
H2S biogenesis, CBS represents a very attractive therapeutic target. The
availability of the structure will help us understand the pathogenicity of the
numerous missense mutations causing inherited homocystinuria and will
allow the rational design of compounds modulating CBS activity.
88
Carlos Oscar Sorzano Snchez1, Jos Mara Carazo2

Centro Nacional de Biotecnologa, Madrid, ES, 2Centro Nacional de
Biotecnologa, CSIC, Madrid, ES
1
La biologa estructural proporciona informacin muy valiosa sobre los

mecanismos biolgicos implicados en el funcionamiento fisiolgico y
patolgico de las clulas. La obtencin de informacin experimental
de alta calidad depende en gran medida del acceso a infraestructuras
de ltima generacin para el anlisis de estructuras biolgicas desde
resolucin celular a resolucin atmica utilizando cristalografa de rayos
X, resonancia magntica nuclear, tomografa electrnica, as como otras
tcnicas biofsicas y bioqumicas. Instruct es una infraestructura europea
cuyo objetivo es proporcionar acceso mediante un proceso de revisin
por pares a un amplio abanico de tcnicas para el estudio estructural.
Instruct tiene un modelo de nodos con un nodo central. El nodo central
coordina actividades como decisiones estratgicas, acceso al portal web,
cursos, colaboraciones con la industria mientras que los nodos nacionales
coordinan el acceso de los usuarios de ese pas. En esta charla se expondrn
los recursos disponibles a travs de Instruct as como el proceso para
solicitarlos.
P10r-9
The interaction of TIA-1 with C-rich RNA

sequences is driven by its pH-dependent RRM3
domain
Isabel Cruz-Gallardo1, Jess Angulo2, Myriam Gorospe3, B. Gran
Karlsson4, Jacqueline A. Wilce5, Miguel A. De la Rosa1, Irene DazMoreno1
1
Universidad de Sevilla - CSIC, Sevilla, ES, 2School of Pharmacy,
University of East Anglia - Norwich Research Park, Norwich,
UK,3Laboratory of Genetics, National Institute on Aging-Intramural
Research Program, NIH, Baltimore, US, 4Swedish NMR Centre,
University of Gothenburg, Gothenburg, SE, 5Department of
Biochemistry and Molecular Biology, Monash University, Victoria,
AU
T-cell intracellular antigen-1 (TIA-1) is a key RNA binding protein that
regulates critical events in cell physiology by the regulation of pre-mRNA
splicing and mRNA translation [1, 2]. It possesses three RNA recognition
motifs (RRM) along with a glutamine-rich domain, with the central
domains (RRM2 and RRM3) acting as RNA binding platforms. While
RRM2 domain is primarily responsible for the RNA interaction, the RRM3
contribution to the RNA binding, as well as its targets sequences, are still
unknown. Here we combine Nuclear Magnetic Resonance (NMR), Surface
Plasmon Resonance (SPR) and pull-down assays to elucidate the sequence
specificity of TIA-1 RRM3 [3]. We demonstrate that RRM3 significantly
binds to those oligonucleotides enriched with cytosines. Notably, in
combination with RRM2 or in the context of the full length protein, the
RRM3 domain enhances the binding to C-rich sequences. In addition, the
binding of RRM3 to RNA is modified by pH conditions, having a significant
effect on the overall interaction of TIA-1 protein [4]. Our findings provide
a new insight into the role of RRM3 in regulating TIA-1 binding to C-rich
stretches, abundant at the 5 TOPs (5 Terminal Oligopyrimidine Tracts) of
translationally-repressed mRNAs under stress situations [5].
References
[1] Frch et al. (2000). Mol Cell, 6: 1089-1098.
[2] Mazan-Mamczarz et al. (2006). Mol Cell Biol, 26: 2716-2727.
[3] Cruz-Gallardo, I. et al. (2014) RNA Biol, 11.
[4] Cruz-Gallardo, I. et al. (2013). J Biol Chem, 288: 20896-20907.
[5] Damgaard and Lykke-Andersen. (2011) Genes Dev, 25: 2057-2068.
Granada 2014
Acknowledgements: Andalusian Government (P07-CVI-02896, P08CVI-3876, P11-CVI-7216 and BIO198); Bio-NMR Research Infrastructure
co-funded by EC (FP7/2007-2013, grant agreement 26186 and BIONMR-00190); NMR services at the Centro de Investigacin Tecnologa e
Innovacin de la Universidad de Sevilla (CITIUS).
P10-10 (R10-6)
Structural basis for the recruitment and

activation of the Legionella phospholipase VipD
by the host GTPase Rab5
Maria Lucas1, Andrew H. Gaspar2, Chiara Pallara3, Ander
Vidaurrazaga1, Adriana L. Rojas1, Matthias P. Machner2, Aitor
Hierro1
1
CIC bioGUNE, Derio, ES, 2Cell Biology and Metabolism
Program, National Institute of Health, Bethesda, US, 3Barcelona
Supercomputing Center, Barcelona, ES
A challenge for microbial pathogens is to assure that their translocated effector
proteins modulate only the correct host cell compartment during infection. The
Legionella pneumophila effector protein VipD localizes to early endosomal
membranes and alters their lipid and protein composition, thereby protecting the
pathogen from endosomal fusion. This process requires the phospholipase A1
(PLA1) activity of VipD that is triggered specifically upon binding to the host
cell GTPase Rab5, a key regulator of endosomes. Here, we present the crystal
structure of VipD in complex with constitutively active Rab5 and reveal the
molecular mechanism underlying PLA1 activation. An active site-obstructing
loop that originates from the C-terminal domain of VipD is repositioned upon
Rab5 binding, thereby exposing the catalytic pocket within the N-terminal
PLA1 domain. Substitution of amino acid residues located within the VipDRab5 interface prevented PLA1 activation and caused a failure of VipD
mutant proteins to target to Rab5-enriched endosomal structures within cells.
Experimental and computational analyses confirmed an extended VipDbinding interface on Rab5 that explains why this L. pneumophila effector can
compete with cellular ligands for Rab5 binding. Together, our data explain how
the catalytic activity within microbial effectors can be precisely linked to their
subcellular localization.
P10r-11
Electrostatic effects in the folding of the

SH3 domain of the c-Src tyrosine kinase: pHdependence in 3D-domain swapping and amyloid
formation
Julio Luis Bacarizo Roa1, Ana Cmara Artigas1, Sergio Martnez
Rodrguez2, Jos Luis Neira Faleiro3, Jos Manuel Martn Garca1,
Montserrat Andjar Snchez1, Emilia Ortz Salmern1
1
Universidad de Almera, Aguadulce, ES, 2Universidad de Granada,
Almera, ES, 3Universidad Miguel Hernndez, Elche, ES
The SH3 domain of the c-Src tyrosine kinase (c-Src-SH3) aggregates to form
intertwined dimers and amyloid fibrils at mild acidic pHs. In this work, we show
that a single mutation of residue Gln128 of this SH3 domain has a significant
effect on: (i) its thermal stability; and (ii) its propensity to form amyloid fibrils;
the Gln128Glu mutant forms amyloid fibrils at neutral pH but not at mild acidic
pH, while Gln128Lys and Gln128Arg mutants do not form these aggregates
under any of the conditions assayed. We have also solved the crystallographic
structures of the wild-type (WT) and Gln128Glu, Gln128Lys and Gln128Arg
mutants from crystals obtained at different pHs. At pH 5.0, crystals belong to
the hexagonal space group P6522 and the asymmetric unit is formed by a chain
of the protomer of the c-Src-SH3 domain in an open conformation. At pH 7.0,
crystals belong to the orthorhombic space group P212121, with two molecules
at the asymmetric unit showing the characteristic fold of the SH3 domain.
Analysis of these crystallographic structures shows that the residue at position
128 is connected to Glu106 at the diverging -turn through a cluster of water
molecules. Changes in this hydrogen-bond network leads to the displacement
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of the c-Src-SH3 distal loop, also resulting in conformational changes of
Leu100 that might be related to the binding of proline rich motifs. Our findings
show that electrostatic interactions and solvation of the residues close to the
folding nucleation site of the c-Src-SH3 domain might play an important role
during folding reaction and amyloid fibril formation.
Acknowledgements: This research was funded by the Spanish Ministry of
Science and Innovation and Ministry of Economy and Competitiveness and
FEDER (EU) [BIO2009-13261-C02-01/02 BIO2012-39922-C02-01/02,
CTQ2013-4493 and CSD2008-00005], Andalusian and Valentian
Regional Government (Spain) and FEDER (EU) [P09-CVI-5063 and
P10-CVI-5915; Prometeo 2013/018]. Data collection was supported
by European Synchrotron Radiation Facility (ESRF, Grenoble, France)
[BAG proposals MX-1406 and MX-1541] and ALBA (Barcelona, Spain)
[proposals 2012010072 and 2012100378]. This work has been performed
by members of the research groups BIO-328 Protein Structures of the
Andalusian Regional Government (Spain). We thank the staff at the ID14-4
beamline of the ESRF (Grenoble, France) and specially we would also like
to thank the beam line XALOC from the Spanish Synchrotron Radiation
Facility ALBA and Jordi Juanhuix, Jordi Benach and Fernando Gil for
their assistance in the measurement of the crystals.
P10r-12
Structural approaches on YIH1: a protein

involved in protein synthesis control under
stravation conditions
Sara Zamora Caballero1, Jernimo Bravo Sicilia1, Bertrand
Sraphin2
1
Instituto de Biomedicina de Valencia (CSIC), Valencia, ES,
2
IGBMC, Illkirch, FR
Protein synthesis is an essential and complex process that is susceptible to
different environmental situations. One of them might be the accumulation
of uncharged tRNAs due to starvation, leading to the activation of a cell
response that affects general protein synthesis. The proposed mechanism
requires GCN2 binding to GCN1, being this interaction mediated by the
GCN2 RWD domain. Once these two proteins have interacted, the complex
binds to the ribosome, what is essential for the detection of empty tRNAs;
since GCN1 is thought to facilitate the transfer of uncharged tRNAs from the
ribosome to the histidyl tRNA synthetase like domain of Gcn2. This signal
activates the kinase domain of GCN2, leading EIF2 phosphorylation, which
promotes the translation of some determinant transcription activators of
amino acid biosynthetic genes and also represses general protein synthesis.
YIH1 (yeast IMPACT homolog 1) also contains a RWD domain that
interacts with GCN1 through the same region as GCN2, thus competing
with GCN2 for GCN1 binding and down-regulating GCN2 activation.
Our aim is to elucidate the crystal structure of the YIH1-GCN1 complex.
Here we present the crystallization trials of this complex and also, the
C-terminal structure of a fungi YIH1 homolog. This C-terminal belongs
to the UPF 0029 group in Pfam with unknown functions described. It is
conserved in both procariotes and eucariotes. This domain is organized as
beta-alpha-beta-beta-alpha-beta, similar to a ferredoxin like domain.
P10r-13
Melanocortin receptor 1 (MC1R) variants

and human pigmentation phenotypes.
Characterization of a MC1R mutant from a family
with hypopigmentation of skin and hair
Julia Sirs-Campos, Marta Abrisqueta Gonzlez, Celia JimnezCervantes, Jos Carlos Garca-Borrn Martnez, Conchi Olivares Snchez
Universidad de Murcia, Murcia, ES
Melanocortin 1 receptor (MC1R) is a Gs protein-coupled receptor
(GPCR) expressed on the surface of melanocytes and crucial for the
89
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regulation of its proliferation and function. Upon binding melanocortins,
MC1R activates the cAMP pathway, ultimately leading to synthesis
of photoprotective eumelanins. Conversely, defective signaling is
associated with pheomelanogenesis. MC1R gene is highly polymorphic
and differences in signaling by MC1R variants result in diverse human
skin pigmentation and skin cancer susceptibility. The MC1R is sevenpass integral transmembrane protein with the canonical GPCRs
structure. The C-terminal domain may play an important role in receptor
trafficking, functional coupling and regulation of signaling. We have
analyzed for function a new natural nonsense mutation resulting in the
premature truncation of the C-terminal tail. This p.Y298* mutation was
found in human family with hypopigmentation. Deletion of the complete
cytoplasmic C-terminus in the mutant protein reduced functional
coupling to the cAMP pathway to almost undetectable levels. The
residual activity was lower for p.Y298* than for most hypomorphic red
hair color-associated alleles. The electrophoretic pattern was similar
for the p.Y298* and wildtype proteins, indicative of comparable posttranslational processing and oligomerization. Significant expression of
the p.Y298* variant on the cell surface was detected by ELISA in nonpermeabilized heterologous cells and confirmed by confocal microscopy,
thus challenging the current view of the role of the MC1R C-terminus in
forward trafficking through the biosynthetic-secretory pathway. However,
the intracellular half-life of the p.Y298* mutant in transiently transfected
cells was shorter, compared with wildtype, suggesting sensitivity to
proteolytic digestion and a faster turnover.
P10r-14
Structural characterization of the -propeller

domain of Erb1, an essential protein in
eukaryotic ribosome biogenesis
Marcin Wegrecki, Jernimo Bravo
Instituto de Biomedicina de Valencia - CSIC, Valencia, ES
Erb1 (Bop1 in mammals) is a eukaryotic protein essential in ribosome
biogenesis. Altogether with Nop7 and Ytm1 it forms a stable complex
that participates in early steps of rRNA processing and is necessary for
the proper maturation of the 60S ribosomal subunit although its exact
implication in the process remains unknown. The amino terminal part of
Erb1 harbors a poorly characterized domain (BopNt) which is directly
responsible for its function and binding to Nop7, whereas the carboxyterminus of the protein contains a big -propeller domain that is formed
by seven WD repeats. It has been postulated that this C-terminal region
is not directly involved in the main function of the protein during 60S
synthesis; nevertheless it is well conserved within the Erb1/Bop1 family
and provides an extensive surface for possible protein-protein and proteinRNA interactions. In total, 22 pre-ribosomal factors contain a -propeller
domain in their structure indicating that it is one of the most common folds
that maintain the complex network of interactions required for the 60S
assembly.
Here we present the crystal structure of the -propeller domain of Erb1
from Saccharomyces cerevisiae at 1.6 (residues 417-807) that was
obtained as a result of random proteolysis event during crystallization
trials of Erb1/Nop7 dimer. The structural information allowed us to
exactly define the boundaries of the domain and to describe its particular
features, being the presence of a long insertion within the second WD
repeat the most distinctive characteristic. This additional segment forms a
protrusion on the surface of the propeller and may play an important role
in peptide binding. We conclude that the proper folding of the insertion is
determined by the neighboring blades of the propeller. The analysis of the
electrostatic surface and conserved hot-spot residues allows us to predict
the patches that might be involved in protein-protein interactions. At last,
we show that the propeller binds to RNA through a defined surface that
can be saturated.
90

P10-15
Estudio estructural de dUTPasas dimricas

virales
Elisa Maiques1, Jordi Donderis1, Ilty Mehmedov1, Mara Angeles
Tormo-Ms2, Mara Garca2, Jos R. Penads3, Alberto Marina1
1
2
Centro de Investigacin y Tecnologa Animal (CITA-IVIA), Segorbe,
ES, 3Institute of Infection, Immunity, and Inflammation, College
of Medical, Veterinary, and Life Sciences, University of Glasgow,
Glasgow, UK
Las dUTPasas (Dut) son enzimas presentes en todos los organismos,
responsables de mantener los niveles celulares de dUTP bajos, al hidrolizar
este nucletido a dUMP y pirofosfato. Segn su estado de oligomerizacin,
las Dut se clasifican en tres grupos, monomricas, dimricas y trimricas.
Tanto las Dut monomricas como las trimricas, a pesar de no tener una
elevada homologa a nivel de secuencia, presentan un plegamiento similar
formado por hojas betas que generan un centro activo comn. En cambio,
las Dut dimricas son protenas cuya estructura secundaria est formada
por -hlices, por tanto, con un plegamiento diferente al de las otras Dut.
Adems, las propiedades bioqumicas de las enzimas pertenecientes a este
grupo tambin difieren de otras Dut, siendo capaces de hidrolizar tanto
dUTP como dUDP.
Aunque las Dut dimricas son un grupo menor dentro de la familia, son
las que presentan patgenos como Leishmania major, Trypanosoma cruzi
y Campylobacter jejuni. Diferentes bacterifagos de Staphylococcus
aureus presentan este tipo de Dut dimricas, mientras que otros presentan
las ms predominantes Dut trimricas. Las Dut trimricas han sido
ampliamente caracterizadas a nivel estructural, habiendo estructuras de
todos los reinos, desde virus hasta el hombre, incluyendo Dut de fagos
de S. aureus, donde nuestro grupo ha contribuido de forma importante.
Por el contrario, la caracterizacin estructural de Dut dimricas se reduce
a pocos ejemplos. En esta comunicacin presentaremos trabajo reciente
del laboratorio que nos ha permitido resolver la estructura tridimensional
de 3 Dut dimricas de diferentes fagos S. aureus, DI, O11 y 55,
en sus formas Apo y unidas a dUMPnPP. La comparacin de estas
estructuras entre s, y con los pocos ejemplos depositados en el PDB, nos
han permitido delimitar un mdulo mnimo compuesto por 4 -hlices
implicadas en el reconocimiento e hidrlisis del nucletido. A este ncleo
bsico se suman mltiples inserciones variables en secuencia y tamao
cuya implicacin en la catlisis y/o otras funciones ser discutida en la
presentacin.
P10-16
Structural and dynamic study of the response

regulator CheY3 from the R. sphaeroides
chemotaxis network
Lorena Varela, Christian Bell, Judith Armitage, Christina Redfield
Universidad de Oxford, Oxford, UK
The process by which bacteria bias their motility, enabling them to move
towards favourable chemical stimuli and away from unfavourable ones, is
known as chemotaxis.
The chemotaxis signalling network of E. coli. depends on
autophosphorylation of a histidine protein kinase (HPK) in response to a
signal from a sensor domain, with subsequent transfer of the phosphoryl
group to an aspartate on response regulator (RR) proteins that bind to the
flagellar motor and alter its rotation. CheY is a simple 14kDa single domain
RR that is conserved across motile species. It is formed by 5 alpha-helices
and 5 beta-strands surrounding a conserved phosphoryl accepting aspartate
residue, and once phosphorylated diffuses to the flagellar motor, binding to
its FliM component to cause switching of rotational direction and, hence, a
change from smooth-swimming to tumbling.
The photosynthetic bacterium Rhodobacter sphaeroides has multiple
chemosensory pathways formed by homologues of the E. coli chemosensory
Granada 2014
proteins. It has six homologues of the response regulator CheY and they all
have different effects on chemotaxis.
In this work we have used solution-state NMR methods to answer
important questions about the structure, dynamics and function of the
6 highly homologous CheYs from R. sphaeroides. We have performed
a detailed study on how conditions (Mg+2 and BeF3 concentrations,
pH) affect the structure and function of the CheY3 protein. Under
conditions where CheY3 is inactive (no BeF3 bound) and at low pH we
have detected one minor and one major protein conformation and upon
pH increase these populations are inverted and finally only one species
is present. We have observed that only at high pH is Mg+2required for
the protein to bind BeF3, a phosphorylation mimic, and the affinity for
Mg+2 is considerably higher than at low pH. We have evidence for the
presence of two binding sites for Mg+2, one with very high affinity and
one with lower affinity locatedclose to the BeF3 binding site. We have
also investigated backbone dynamics of the inactive and active forms
of CheY3 using heteronuclear NOE experiments and no significant
differences have been observed suggesting that the flexibility of both
conformations is similar. Finally, we have assigned the backbone
resonances of CheY3 in their inactive and active states using tripleresonance NMR methods and information about secondary structure
has been obtained from the analysis of 1H, 13C and 15N chemical shifts
using TALOS as well as CD experiments.
P10-17
Biophysical characterization of the L-isoaspartyl

O-methyltransferase from Escherichia coli
Emilio Jos Gonzlez Ramirez1, Sergio Martnez-Rodriguez2, Emilia
Ortiz-Salmern1, Montserrat Andjar-Snchez1, Ana Cmara-Artigas1
1
Departamento de Qumica y Fsica, Escuela Politcnica y Facultad
de Ciencias Experimentales, Universidad de Almera, Almera,
ES, 2Departamento de Qumica y Fsica, Universidad de Granada,
Granada, ES
The protein L-isoaspartyl O-methyltransferase (PIMT) is a highly
conserved enzyme found in a wide diversity of organisms, including
plants, insects, bacteria, and mammals. Its function is to recognize
L-Isoaspartyl residues in proteins and revert to L-aspartyl residues. This
action takes places by the transfer of a methyl group from S-adenosyl-Lmethionine (AdoMet) to the -carboxyl of the L-isoaspartyl group, which
forms a succinimide, and subsequent hydrolysis converts a fraction of
these residues to aspartyls [1].There are several studies on E.coli PIMT
(EC-PIMT) functional features, but the knowledge about its biophysical
behavior is scarce. We have performed a biophysical characterization
of this enzyme by using spectroscopic techniques. Our results shows
that E.Coli PIMT is monomeric in all the range of pH assayed (2-12)
and protein concentration (1-10 mgmL-1) at temperatures below 45oC.
We have also characterized the stability of the protein by means of
fluorescence emission. Our results show that the protein denaturizes at
urea concentrations higher than 2.5 M.
Acknowledgment: This research was funded by the Spanish Ministry of
Science and Innovation and Ministry of Economy and Competitiveness
and FEDER (EU) [BIO2012-39922-C02-01/02], Andalusian Regional
Government (Spain) and FEDER (EU) [P09-CVI-5063 and P10CVI-5915]. This work has been performed by members of the research
groups BIO-328 Protein Structures of the Andalusian Regional
Government (Spain).
References
[1] Fang, P., Li, X., Wang, J., Xing, L., Gao, Y., Niu, L., Teng, M., (2010)
Crystal Structure of the Protein L-Isoaspartyl Methyltransferase from
Escherichia coli. Cell Biochem Biophys 58, 163167.
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P10-18
Characterization of canine adipose derived

mesenchymal stem cell population: expression
of metalloproteinases MMP-2 and MMP-9
Guillermo Mejas Martnez1, Leticia G. Len2, Maria Luisa Fermn3,
Elena Merino1, Cristina Frago3, Elisabeth Kremmer4, Concepcin
Tejero Ortego1
1
Department of Biochemistry and Molecular Biology IV, Veterinary
Faculty (UCM), Madrid, ES, 2Center of Biomedical Investigations
from Canarias (CIBICAN) - Institute of Biomedical Technologies
(ITB) (ULL), La Laguna, ES, 3Department of Animal Medicine
and Surgery, Veterinary Faculty (UCM), Madrid, ES, 4Institut fr
Immunologie, GSF-Forschungszentrum Umwelt und Gesundheit,
Mnchen, DE
Adult mesenchymal stem cells (MSC) are multipotent cells able to
differentiate into adipogenic, condrogenic and osteogenic lineages.
Initially MSC source has been bone marrow, however currently adipose
tissue-derived stem cells (ADSCs) has been shown as an interesting
alternative source, making these cells particularly attractive for therapeutic
exploitation.The aim of this work was to develop an easy-to-use protocol
to isolate and characterize canine MSCs from adipose tissue. In addition,
considering the role of MMP-2 and MMP-9 in migration and facilitating
homing to the injured tissue, a study of its expression has been done.
Peritoneal fat was obtained from clinically healthy bitches at the time of
elective ovariohysterectomy, digested with collagenase I and then isolated
by centrifugation and filtration. cMSCs were cultured in IMDM medium at
7000 cell/cm2 with 10% dog serum at 38C, 5% CO2 and 90% air humidity.
In vitro growth is exponential until 25-30 days, and doubling time of all
samples studied remains constant throughout 1-4th passages. Dot plot of
stained cMSC after incubation with primary antibodies reveal that these
cells had positive results for CD90 and negative for CD34, CD45 and
MHC-II. Gelatin zymografy reveal the expression of MMP-2 and MMP-9.
In conclusion, we have isolated and characterized a canine adipose
tissue-derived mesenchymal stem cells population. As far as we know,
we provided the first evidence that canine mesenchymal derived adipose
cells constitutively express MMP-2 and MMP-9. Further investigations on
cMSC differentiation into specific tissues may lead to the development of
novel therapeutic strategies to target specific diseases.
P10r-19
Inhibition of Type IV secretion ATPase TrwD

by unsaturated fatty acids as a potential tool
to prevent wide spread dissemination of
antibiotic resistance genes
Yolanda Garca Cazorla1, Jorge Ripoll Rozada2, Cristina Machn3,
Mara Getino1, Fernando de la Cruz1, Elena Cabezn1, Ignacio
Arechaga1
1
Departamento de Biologa Molecular, Universidad de Cantabria, e
Instituto de Biomedicina y Biotecnologa de Cantabria (IBBTEC),
CSIC-UC-IDICAN, Santander, ES, 2Instituto de Biologa Molecular
e Celular (IBMC), Porto, PT, 3Instituto de Investigacin BiomdicaIRB, Barcelona, ES
Antibiotic resistance has become a major health issue [1]. Unfortunately,
the development of new antibiotics is being very slow and expensive
and we are running out of weapons to fight against the appearance
of multi-resistant bugs. Bacterial conjugation is the main mechanism
for the wide spread dissemination of antibiotic resistance genes and,
therefore, the search of specific inhibitors of conjugation (COINs)
could become a brand new strategy in this warfare. In the search of
potential COINs, previous studies have reported that unsaturated fatty
acids (uFAs) were able to inhibit conjugation [2]. Based on these
studies we have looked for the molecular targets of these compounds
and we have found that the Type IV secretion ATPase TrwD is inhibited
91
Psters
by linoleic acid and 2-alkynoic fatty acids, such as the 2-hexadecynoic
acid. These two uFAs compounds were the most effective inhibitors in
R388 plasmid conjugation [3]. The inhibitory effect is specific for the
traffic ATPase TrwD, as the remaining ATPases of the Type IV secretion
system are unaffected by both uFAs and 2-AFAs. We have characterized
the inhibition mechanism and we have found that in both cases it is
a non-competitive inhibition, as the affinities for the substrate (ATP)
and product (ADP) are not altered in the presence of the fatty acids.
Altogether, these results could open new avenues in the development
of new strategies to prevent the dissemination of antibiotic resistance
genes.
References
[1] Sommer M.O. (2014). Microbiology: Barriers to the spread of
resistance. Nature. Doi: 10.1038.
[2] Fernandez-Lopez R. et al. (2005) Unsaturated fatty acids are inhibitors
of bacterialconjugation. Microbiology. 151, 35173526.
[3] Getino M. et al. (2014) 2-alkynoic fatty acids as chemically stable
conjugation inhibitors. (Submitted)
P10-20
A triple mutant of human, Mn(II)-dependent

ADP-ribose/CDP-alcohol diphosphatase
(ADPRibase-Mn) displays cyclic ADP-ribose
(cADPR) phosphohydrolase as its major activity
Iralis Lpez-Villamizar1, Alicia Cabezas1, Jos Canales1, Ascensin
Fernndez1, Rosa Mara Pinto1, Joo Meireles Ribeiro1, Joaquim Rui
Rodrigues2, Mara Jess Costas1, Jos Carlos Cameselle1
1
Grupo de Enzimologa, Departamento de Bioqumica y Biologa
Molecular y Gentica, Facultad de Medicina, Universidad de
Extremadura, Badajoz, ES, 2Escola Superior de Tecnologia e Gesto,
Instituto Politcnico de Leiria, Leiria, PT
ADPRibase-Mn (EC 3.6.1.53) belongs to the metallo-dependent
phosphatases superfamily, where it forms a family of its own (SCOP2
ID 4002589). Rat and human ADPRibase-Mn hydrolyze ADP-ribose
(A), CDP-choline (B) and 2,3-cAMP (C) with decreasing efficiencies
(kcat/KM). They also display a unique cADPR phosphohydrolase
activity much less efficient than the activities on the other substrates [13]. Zebrafish ADPRibase-Mn has similar specificity but is inactive on
cADPR [3]. Mutagenesis of human ADPRibase-Mn showed that Phe37 is
a determinant of the preference for A, due to a hydrophobic interaction
with the nitrogenous base, not evident with B, C or cADPR in docking
simulations. The F37A mutant exhibited, versus the wild type, a 50fold decrease of efficiency on A, much less marked (only 3-5 fold) on
B, C and cADPR. The double mutant F37A+L196F showed stronger
decreases of efficiency on A, B and C, but not on cADPR. In contrast,
the C253A mutation had a weak stimulatory effect with A, Band C, much
stronger with cADPR. Finally, the triple mutant F37A+L196F+C253A
was preferentially active on cADPR. While the wild-type is 150-, 40- or
6-fold more efficient on A, B or C than on cADPR, the triple mutant
was 2.9-, 1.3- or 7.2-fold more efficient on cADPR than on A, B or C.
The change of specificity included, in absolute terms, a 7-fold increase
of the cADPR phosphohydrolase efficiency over the wild type. Further
mutagenesis could perhaps generate an even more specific cADPR
phosphohydrolase.
References
[1] Canales et al., 2008, Biochem. J. 413, 103.
[2] Canales et al., 2009, FEBS Lett. 583, 1593.
[3] Rodrigues et al., 2012, PloS ONE 7, e42249.
Support: Gobierno de Extremadura and FEDER (GR10133).
92

P10-21
Tyrosinase-catalyzed hydroxylation
of hydroquinone, a depigmenting agent,
to hydroxyhydroquinone: a kinetic study
Mara del Mar Garca Molina1, Jose Luis Munoz-Munoz2, Miguel
Angel Maria Solano2, Jose Berna1, Francisco Garcia-Canovas2
1
Universidad de Murcia, Albatera, ES, 2Universidad de Murcia Grupo GENZ, Murcia, ES
Hydroquinone (HQ) is used as a depigmenting agent. In this work we
demonstrate that tyrosinase hydroxylates HQ to 2-hydroxyhydroquinone
(HHQ). Oxy-tyrosinase hydroxylates HQ to HHQ forming the complex
met-tyrosinase-HHQ, which can evolve in two different ways, forming
deoxy-tyrosinase and p-hydroxy-o-quinone, which rapidly isomerizes
to 2-hydroxy-p-benzoquinone or on the other way generating mettyrosinase and HHQ. In the latter case, HHQ is rapidly oxidized by
oxygen to generate 2-hydroxy-p-benzoquinone, and therefore, it cannot
close the enzyme catalytic cycle for the lack of reductant (HHQ).
However, in the presence of hydrogen peroxide, met-tyrosinase (inactive
on hydroquinone) is transformed into oxy-tyrosinase, which is active on
HQ. Similarly, in the presence of ascorbic acid, HQ is transformed into
2-hydroxy-p-benzoquinone by the action of tyrosinase; however, in this
case, ascorbic acid reduces met-tyrosinase to deoxy-tyrosinase, which
after binding to oxygen, originates oxy-tyrosinase. This enzymatic form
is now capable of reacting with HQ to generate p-hydroxy-o-quinone,
which rapidly isomerizes to 2-hydroxy-p-benzoquinone. The formation of
HHQ during the action of tyrosinase on HQ is demonstrated by means
of high performance liquid chromatography mass spectrometry (HPLCMS) by using hydrogen peroxide and high ascorbic acid concentrations.
We propose a kinetic mechanism for the tyrosinase oxidation of HQ which
allows us the kinetic characterization of the process. A possible explanation
of the cytotoxic effect of HQ is discussed.
P10-22
Crystallographic structure of the first WW domain

of Human YAP2 isoform
Ana Camara-Artigas1, Julio Bacarizo1, Sergio Martnez-Rodrguez2
Department of Chemistry and Physics, University of Almera,
Agrifood Campus of International Excellence (ceiA3), Almera, ES,
2
Department of Physical Chemistry, Faculty of Sciences, University
of Granada, Granada, ES
The WW domain is a small modular domain (approximately 35-40 residues)

that takes its name from the presence of two highly conserved tryptophan
residues. These domains were first identified in the Yes tyrosine kinase
Associated Protein (YAP), and since their discovery they became focus of
attention as they have been related to several important human diseases such
as Cancer, the Liddles syndrome, Alzhehimers, Huntingtons and viral
diseases. Two major isoforms of YAP have been described in human tissues,
namely YAP1 and YAP2, containing one and two WW domains, respectively.
Up to date, most of the available WW domain structures have been
obtained in solution by NMR techniques, and very few structures of WW
domains have been solved by X-ray diffraction. These domains show an
intrinsic conformational flexibility which difficult the formation of crystal
contacts and, therefore, their crystallization. We have solved the first
crystallographic structure of the first WW domain of the h-YAP2 at 1.6
resolution in its apo form. The high quality of the data allowed us to model
residues 165-208. The Ramachandran plot shows 100% of the residues in
the preferred regions.
The interactions that stabilize this minimal modular domain have been
analyzed, showing that besides Trp177, which together with Pro202
and Phe189 form an aromatic cluster in the hydrophobic core of the
protein, some salt-bridges might play a key role in their folding reaction.
Furthermore, this structure allows us to explore for the first time the
conformational changes that take place in this domain upon binding of
Proline Rich Motifs.
Granada 2014
P10r-23 (R10-5)
A common signalosome for programmed cell

death in humans and plants
Katiuska Gonzlez-Arzola1, Jonathan Martnez-Fbregas1, Antonio
Daz-Quintana1, Simon Janocha2, Rita Bernhardt2, Adrin
Velzquez-Campoy3, Irene Daz-Moreno1, Miguel ngel De la Rosa1
1
Universidad de Sevilla - CSIC, Sevilla, ES, 2Institut fr Biochemie,
Universitt des Saarlandes, Campus B2.2, Saarbrcken,
DE,3Institute of Biocomputation and Physics of Complex Systems
(BIFI), Joint-Unit IQFR-CSIC-BIFI, Department of Biochemistry and
Molecular and Cell Biology, University of Zaragoza, Zaragoza, ES
Programmed cell death (PCD) is a fundamental event for the development
of multicellular organisms. In mammalian cells, early events in PCD
involve the release of cytochrome c (Cc) from mitochondria to the
cytoplasm to act at the first stages of the apoptotic process, playing a key
role in assembling the apoptosome. In plants, PCD is part of a general
process named hypersensitive response, where Cc is also released into
the cytosol but its role in PCD remains veiled. Such highly conserved
cytoplasmic location of Cc upon apoptotic stimuli lead to think of a
common link for PCD in evolutionarily distant species, like humans and
plants.
To better understand the role of Cc in the onset of PCD in both humans
and plants, a proteomic approach based on affinity chromatography with
Cc as bait was used. Upon combining this approach and Bimolecular
Fluorescence Complementation (BIFC), a total of 8 human and 9 plant
new proteins interacting with Cc under PCD were found [1,2]. These new
PCD Cc-partners are involved in protein folding, translational regulation,
oxidative stress, DNA damage, energetic and mRNA metabolism.
Strikingly, some of the novel human Cc-targets are closely related to those
for plant Cc, indicating that the evolutionarily well-conserved cytosolic
Cc appearing in organism from plant to mammals- interact with a wide
range of targets on PCD.
Modeling of the complexes between human and plant Cc with its
counterparts shows how the heme crevice of Cc takes part of the complex
interface in agreement with the vast majority of known redox adducts of
Cc. However, in contrast to the high turnover rate of the mitochondrial
Cc redox adducts, those occurring under PCD lead to the formation of
rather stable nucleo-cytoplasmic ensembles, as inferred from Surface
Plasmon Resonance (SPR) and Nuclear Magnetic Resonance (NMR)
measurements. On the basis of these findings, we suggest that human and
plant Cc interacts with pro-survival, anti-apoptotic proteins after its release
into the cytoplasm. Then, Cc may interfere with cell survival pathways and
unlock PCD in order to prevent the spatial and temporal co-existence of
antagonist signals.
References
[1] Martnez-Fbregas, J. et al. (2013). Mol. Cell. Proteomics, 12: 36663676.
[2] Martnez-Fbregas, J. et al. (2014). Mol. Cell. Proteomics, doi:10.1074/
mcp.M113.034322.
P10-24
Assessing the performance of ancestral protein

reconstruction
Sergio Martnez-Rodrguez, Valeria Alejandra Risso, Jos Manuel
Snchez-Ruiz
Departamento de Quimica Fisica, Facultad de Ciencias, Universidad
de Granada, Granada, ES
Ancestral sequence reconstruction is a protein engineering methodology
exploiting natural sequence diversity to obtain protein variants with
enhanced characteristics [1], and has also been suggested to allow
inferring features of the physical environment in which ancient
organisms evolved [2]. After successful evaluation of this approach
Psters
for the design of resurrected Precambrian -lactamases [3], we wanted
to further confirm the performance of this evolutionary phylogeny
method analyzing the properties of additional resurrected ancestral
-lactamases standing in the evolutionary path among Firmicutes and
Actinobacteria phyla, encoding proteins dating back between ~1.4 and
~2.9 billion years (Gyr). This alternative evolutionary reconstruction also
produced -lactamases with higher in vitro denaturation temperatures
(~25 C) and higher substrate promiscuity than modern -lactamases.
In vivo studies showed that the resurrected enzymes provided actual
Escherichia coli cells with higher resistance to different third-generation
antibiotics, providing us with plausible evolutionary scenarios for actual
microorganisms antibiotic resistance. Our results reinforce the virtues of
ancestral protein reconstruction as a biotechnological cornerstone for the
molecular design of engineered enzymes. Furthermore, our denaturation
experiments suggest a different evolutionary environment for ancestral
Gram-positive bacteria, in full agreement with the inferred colonization
of land environments by Actinobacteria/Firmicutes at some point at the
end of the Archean period [4].
References
[1] Cole MF, Gaucher EA. Utilizing natural diversity to evolve protein
function: applications towards thermostability. Curr Opin Chem Biol.
(2011) 15(3):399-406.
[2] Gaucher EA, Thomson JM, Burgan MF, Benner SA. Inferring the
palaeoenvironment of ancient bacteria on the basis of resurrected proteins.
Nature (2003) 425(6955):285-288.
[3] Risso VA, Gavira JA, Mejia-Carmona DF, Gaucher EA, Sanchez-Ruiz
JM. Hyperstability and substrate promiscuity in laboratory resurrections
of Precambrian -lactamases. J Am Chem Soc. (2013) 135(8):2899-902.
[4] Battistuzzi FU, Hedges SB. A major clade of prokaryotes with ancient
adaptations to life on land. Mol Biol Evol (2009) 26(2):335-343.
P10r-25
Caracterizacin fsico-qumica y funcional

del citocromo c6-3 de la cianobacteria Nostoc sp.
PCC 7119
Alejandro Torrado Maya, Leonor Puerto-Galn, Manuel Hervs, Jose
A. Navarro, Fernando Publio Molina-Heredia
Instituto de Bioqumica Vegetal y Fotosntesis, Universidad de
Sevilla y CSIC, cicCartuja, Sevilla, ES
En cianobacterias, las cadenas de transporte de electrones fotosinttica
y respiratoria estn localizadas en el mismo compartimento celular
y comparten algunos componentes, como son la plastocianina (Pc)
o el citocromo (Cit) c6 y el complejo b6-f. Se ha propuesto que en
estos organismos el Cit c6 y la Pc podran transportar los electrones
alternativamente desde el complejo b6-f al Fotosistema I (PSI) o a la Cit
c oxidasa. Adems de Cit c6 y Pc, en cianobacterias se han encontrado
otros posibles transportadores solubles de electrones, localizados en el
lumen tilacoidal, cuya funcin an no ha sido bien establecida, como
son el Cit c6-2 y el Cit cM. A partir de un anlisis de secuencias gnicas
en cianobacterias filamentosas formadoras de heterocistos, hemos
encontrado otra protena que podra intervenir en estos procesos: el
Cit c6-3. En estos organismos, la fijacin de nitrgeno atmosfrico
se produce en unas clulas especializadas denominadas heterocistos.
En estas clulas, debido a que la nitrogenasa se inhibe por O2, la tasa
respiratoria es muy alta, con el fin de crear un ambiente anoxignico.
En este trabajo hemos abordado la caracterizacin fsico-qumica y el
anlisis funcional del Cit c6-3. Esta protena no es capaz de interaccionar
eficientemente con el PSI, por lo que no parece actuar en fotosntesis. En
cambio, s reduce especficamente a determinadas oxidasas terminales
en los heterocistos, por lo que proponemos que podra tener un papel
relevante en respiracin.
93
Psters
P10-26
P10-28
Structural and thermodynamic characterization

of the interaction between viral late domains and
their cellular targets
Estudio estructural y funcional de la fosfatasa

MG207 de Mycoplasma genitalium
Pedro Buzn , Manuel Iglesias-Bexiga , Francisco Castillo , Andrs

Palencia1, Maria J. Macias2, Francisco Blanco3, Ana CamaraArtigas4, Irene Luque1
1
Department of Physical Chemistry, University of Granada, Granada,
ES, 2Structural and Computational Biology Programme, Institute
for Research in Biomedicine, Barcelona, ES, 3Structural Biology
Unit, CIC bioGUNE, Parque tecnolgico de Bizkaia, Deiro, ES,
4
Department of Physical Chemistry, Biochemistry and Inorganic
Chemistry, University of Almera, Almera, ES
Many viruses encode a late budding domain (L-domain) in their sequence.
These L-domains usually contain highly conserved motifs known to
mediate cellular protein-protein interactions, such as PPXY and PTAP.
These motifs are essential for the recruitment of the cellular machinery
sequestered by the virus for replication inside the infected cell. The budding
process, which is mediated by the ubiquitin-proteasome system, has been
proposed as a potential target to inhibit viral replication. We present here a
structural and thermodynamic characterization of the interactions between
a set of peptide ligands corresponding to L-domains sequences from
different viruses (including Ebola, HTLV-1, Rabies, Marburg and HIV1) and their cellular targets (the UEV domain of hTsg101 and the WW
domains of hNedd4) aimed at understanding the molecular determinants
of binding affinity and specificity in these systems. Our work reveals that
the establishment of networks of water-mediated hydrogen bonds at the
binding interface and the reorganization of the conformational distribution
in both protein and ligand upon complex formation are important features
determining the thermodynamic signature of these interactions, that need to
be taken into account for a full understanding of these systems. The work
presented suggests that combining a detailed structural and thermodynamic
analysis with screening and molecular biology techniques can provide very
valuable information for the design of specific and high affinitty inhibitors
as potential broad spectrum antivirals.
P10-27
Structural insights into the Ca+2 and PI(4,5)P2

binding modes of the C2 domains of rabphilin 3A
and synaptotagmin 1
Mara Dolores Prez Snchez1, Teresa Coronado-Parra2, Jaime
Guilln2, Juan C. Gmez-Fernndez2, Nuria Verdaguer3, Senena
Corbaln-Garca1
1
Dpto. Bioqumica y Biologa Molecular A, Universidad de
Murcia, Jumilla, ES, 2Dpto de Bioqumica y Biologa Molecular
A, Universidad de Murcia, Murca, ES, 3Department of Structural
Biology, Instituto de Biologa Molecular de Barcelona (IBMB-CSIC),
Barcelona, ES
Proteins containing C2 domains are the sensors for Ca+2 in myriad of
secretory pathways. Here, we have determined the structure of this
domain in complex with PI(4,5)P2 and IP3 at resolutions of 1.75 and 1.9
, respectively, unveiling that the polybasic cluster formed by strands 34 is involved in the interation with the phosphoinositides. A comparative
study demonstrates that the C2A domain is highly specific for PI(4,5)P2/
PI(3,4,5)P3, whereas the C2B domain cannot discrimiate among sny of the
diphosphorylated forms. Structural comparisons between C2A domains
of rabphilin 3A and synaptotagmin 1 indicated the presence of a key
glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes
the interaction with PI(4,5)P2. Together, these results provide a structural
explanation for the ability of different C2 domains to pull plasma and
vesicle membranes close together in a Ca+2-dependent manner and reveal
how this family of proteins can use subtle structural changes to modulate
their sensivity and specificity to various cellular signals.
94
Patricia Casino Ferrando1, Ana M. Martnez Martnez Mariscal2,

Alberto Marina3, Jaume Piol2, Ignacio Fita1
1
Department of Structural Biology, Instituto de Biologa Molecular
de Barcelona (IBMB-CSIC), Barcelona, ES, 2Departamento de
Bioqumica i Biologa Molecular, Institut de Biotecnologa i
Biomedicina, Universitat Autnoma de Barcelona, BellaterraBarcelona, ES, 3Departmento de genmica y protemica, Instituto
de Biomedicina de Valencia (IBV-CSIC), Valencia, ES
Mycoplasma genitalium es una bacteria parasitaria con genoma mnimo
(~580Kb) implicada en infecciones urogenitales, del endometrio y
sndrome plvico inflamatorio; carece de pared celular y por tanto no es
sensible a antibiticos que bloquean la sntesis de sta como la penicilina
y los betalactmicos [1]. La movilidad de estas bacterias se realiza por
deslizamiento gracias a una membrana que sobresale del polo de la bacteria
llamada organela terminal y que est implicada en adherencia celular.
En Mycoplasma pneumoniae, se ha correlacionado la funcin quinasa/
fosfatasa con la movilidad, de forma que mutaciones en la sern/treonina
prkC (MPN248) y su fosfatasa prpC (MPN247) producen cambios en la
movilidad as como cambios en el patrn de fosforilacin de protenas
de la organela terminal, HMW1 y HMW2 [2]. Acorde al tamao de su
genoma, M. genitalium contiene muy pocas protenas sealizadoras y tan
slo cuatro ORFs han sido anotadas como fosfatasas, la protena fosfatasa
2C (MG_108), dos serine/treonn fostatasas (MG_207 y MG_246) y
la pirofosfatasa inorgnica (MG_351). Recientemente, se demostr in
vivo que la fosfatasa MG_207 estaba implicada en virulencia, ya que la
cepa knockout en MG_207 era menos virulenta, su adherencia a clulas
eucariticas era menor y presentaba un patrn de protenas fosforiladas
diferente a la silvestre [3]. Con el fin de caracterizar la fosfatasa MG207
hemos obtenido, por cristalografa, su estructura tridimensional que
muestra un plegamiento a/ caracterstico de fosfodiesterasas, adems el
estudio funcional demuestra que MG207 presenta tanto actividad fosfatasa
como fosfodiesterasa. En el centro activo se observ la presencia de dos
metales y un sulfato y hemos confirmado que las actividades fosfatasa y
fosfodiesterasa son modulables dependiendo de la presencia de cationes
divalentes, siendo los metales de transicin Mn2+, Ni2+ y Fe2+ quienes
especialmente activan esta enzima.
Bibliografa
[1] Anagrius C, Lor B, Jensen JS. Treatment of Mycoplasma genitalium.
Observations from a Swedish STD clinic. PLoS One. 2013;8(4):e61481.
[2] Page CA, Krause DC. Protein kinase/phosphatase function correlates
with gliding motility in Mycoplasma pneumonia. J Bacteriol. 2013:1750-7.
[3] Martinez MA, Das K, Saikolappan S, Materon LA, Dhandayuthapani
S. A serine/threonine phosphatase encoded by MG_207 of Mycoplasma
genitalium is critical for its virulence. BMC Microbiol. 2013;13:44.
P10-29
Biognesis de ribosomas, the Backstage

Gisela Pll, Shuang Li, Uli Ohmayer, Thomas Hierlmeier, Philipp
Milkereit, Jorge Prez-Fernndez
Universitt Regensburg, Biochemie-Zentrum Regensburg (BZR),
Lehrstuhl Biochemie III, Regensburg, DE
La biognesis de ribosomas es un proceso celular que implica el
procesamiento y plegamiento de los RNA ribosmicos y la incorporacin
de las protenas ribosmicas. Dicho proceso es facilitado, en organismos
eucariotas, por la accin de ms de 150 protenas conocidas como factores
de ensamblaje. Algunos de estos factores, denominados Utp, son capaces
de formar subcomplejos proteicos (UTP), que participan como entidades
independientes en la biognesis de la subunidad menor del ribosoma
(40S). Dos de estos subcomplejos, tUTP/UTP A y UTP B participan en
las primeras etapas del ensamblaje y procesamiento del RNA ribosmico.
Granada 2014
Actualmente, se conoce la composicin proteica de tUTP/UTP A y UTP B
y su arquitectura ha sido analizada hasta ahora en base a las interacciones
binarias protena-protena. En este trabajo presentamos una caracterizacin
bioqumica detallada de la arquitectura de los subcomplejos tUTP/UTP
A y UTP B. Los resultados obtenidos indican la completa reconstitucin
in vitro de tUTP y UTP B. Adems, proporcionan evidencias bioqumicas
sobre la existencia de complejos binarios, ternarios o de grado superior,
en consonancia con las interacciones binarias previamente descritas.
Por ltimo, los resultados sugieren que los diferentes complejos
proteicos obtenidos constituyen diferentes mdulos de tUTP y UTP B y
proporcionan un nuevo punto de vista sobre intermediarios de ensamblaje
o desensamblaje de los subcomplejos UTP.
P10-30
N-terminal protein tails act as aggregation

protective entropic bristles: the SUMO case
Patrizia Marinelli, Ricardo Graa-Montes, David Reverter, Salvador
Ventura
Universitat Autnoma de Barcelona, Barcelona, ES
The formation of -sheet enriched amyloid fibrils constitutes the hallmark
of many diseases but is also an intrinsic property of polypeptide chains in
general, because the formation of compact globular proteins comes at the
expense of an inherent sequential aggregation propensity. In this context,
identification of strategies that enable proteins to remain functional and
soluble in the cell has become a central issue in chemical biology. We show
here, using human SUMO proteins as a model system, that the recurrent
presence of disordered tails flanking globular domains might constitute yet
another of these protective strategies. These short, disordered, and highly
soluble protein segments would act as intramolecular entropic bristles,
reducing the overall protein intrinsic aggregation propensity and favoring
thus the attainment and maintenance of functional conformations.
P10-31
Directed evolution of acyltransferases

for triglyceride production in E. coli
Omar Santn, Gabriel Moncalin
IBBTEC, Universidad de Cantabria, Santander, ES
Wax ester synthase/diacylglicerol acyltransferase (WS/DGAT) is a family
of enzymes able to perform the esterification of diacylglycerol or fatty
alcohols and acyl-CoA to produce triglycerides (TAGs) or wax esters,
respectively. Both products can be easily transformed onto biodiesel.
Although the specificity determinants of substrate recognition are still
unknown and there is no crystal structure solved from any bacterial WS/
DGATs , we recently published a study where we used limited proteolysis
and directed mutagenesis approaches to identify key folding domains
and motifs critical for the catalysis. We have also recently patented
a diacylglycerol acyltransferase (tDGAT) (ES201200967) from the
thermophilic organism Thermomonospora curvata that is able to produce
TAGs and waxes in E. coli. We have used this system for TAG production
in E.coli, but we believe it can be significantly improved through directed
evolution of tDGAT. Thus, the main goal of our work is to enhance TAG
production in E. coli by directed evolution. Moreover, this work will
provide us some information about the amino acids residues involved
in substrate recognition. For this purpose we have developed a direct
evolution protocol where we constructed mutant libraries by mutagenic
PCR in order to obtain variants of the protein. Using Nile Red, a fluorescent
dye that binds to neutral lipids we can select different variants of tDGAT
through a high throughput selection system based on fluorimetry and flow
cytometry. Mutants carrying interesting phenotypes are further selected
and sequenced. This way we were able to detect mutations that lead to a
2-fold increase in the TAG production.
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P10-32
Structural insights into the human Sigma-1

Receptor Chaperone structure and interactions
Jose Luis Ortega Roldan, Felipe Ossa, Nader Amin, Jason Schnell
Department of Biochemistry, University of Oxford, Oxford, UK
The Sigma-1 Receptor (S1R) is a ligand-regulated membrane
protein chaperone involved in the ER stress response. S1R activity is
implicated in diseases of the central nervous system including amnesia,
schizophrenia, depression, Alzheimers disease, and addiction. S1R has
been shown previously to regulate the Hsp70 BiP and the IP3R calcium
channel through a C-terminal domain. It also binds and is regulated by
a large number of small molecules such us psychostimulants (cocaine,
methamphetamine and DMT), antidepressants, antipsychotics and
steroids.
We have developed methods for bacterial expression and reconstitution
of the chaperone domain and a truncation construct lacking the first
transmembrane domain (TM) of human S1R into DPC:DPPC mixed
micelles that enable its study by solution NMR spectroscopy.
S1R is found to contain a first TM, two helices in the cytosolic loop,
followed by the second TM and a juxtaposed helix, a largely dynamic
region and a structured, helical C-terminal region that encompasses a
membrane associated domain containing four helices. The helical region
at residues ~198-206 is strongly amphipathic and proposed to anchor the
chaperone domain to micelles and membranes. Three of the helices in the
C- terminal region closely correspond to previously identified cholesterol
and drug recognition sites.
Moreover, we show experimentally using chemical shifts, spin relaxation
and H/D exchange that the second TM, whose position hasnt been
predicted accurately, spans the residues 89 to 111 and includes the entire
Sterol Binding Domain Like 1 (SBD1), partially responsible for drug
binding.
In addition, we show that the chaperone domain interacts with full- length
BiP or the isolated nucleotide binding domain (NBD) of BiP, but not the
substrate binding domain, suggesting that the NBD is sufficient for S1R
interactions.
These results, that constitute the first structural information obtained for
this protein and its interactions, advance our understanding of S1R and are
likely to be useful in refining models of the S1R drug binding sites.
P10-33
Conformational distribution of the SH3-SH2

Tandem of the Abl kinase and its modulation
by intramolecular interactions
Carles Corbi-Verge1, Fabrizio Marinelli2, Ana Zafra Ruano1, Javier
Ruiz-Sanz1, Irene Luque-Fernndez1, Jos D. Faraldo-Gmez2
1
Departamento de Quimica Fisica, Facultad de Ciencias,
Universidad de Granada, Granada, ES, 2Max Planck Institute of
Biophysics, Theoretical Molecular Biophysics Group , Frankfurt, DE
Tyrosine kinases are involved in a wide variety of key signaling processes
and are therefore tightly regulated by the cell. Indeed, numerous
pathologies, ranging from cancer to neurodegeneration, result from or
are associated with deficiencies in kinase regulation. Consequently, these
enzymes are a prominent target for novel pharmacological strategies
against human disease.
This study focuses on Abl, which is one of the most ubiquitously conserved
tyrosine kinases. Abl is present in all metazoa, where it plays an essential
role in processes as diverse as cytoskeleton reorganization, DNA repair,
and regulation of apoptosis. Accordingly, when constitutively active forms
of Abl are present in normal cells, these are transformed into cancer cells.
For example, in human white-blood cells, a chromosomal abnormality
leads to the fusion of the bcr and abl genes, which together encode for a
cytoplasm-targeted, deregulated form of Abl; Bcr-Abl interferes with the
cell cycle, resulting in uncontrolled cell proliferation, and is the principal
cause of chronic myeloid leukemia (CML).
95
Psters
Regulation of Abl can be thought as a reversible equilibrium whereby the
SH3, SH2 and catalytic domains are either dissociated or self-assembled
in one or more configurations. External factors, such as competing
interactions involving one or both regulatory domains, will bias this
equilibrium in one or other direction. For Abl in particular, the significance
of this mechanism is underscored by the fact that mutations that impair
the correct assembly of the autoinhibited complex, either in the SH3-SH2
tandem or in the domain-domain linkers, confer CML cells with resistance
against inhibitory drugs designed to target the catalytic domain of the
Bcr-Abl oncogene. This outcome has prompted considerable interest in
the mechanisms of allosteric regulation of Abl and other tyrosine kinases,
and in the development of compounds designed to interfere with these
mechanisms.
In this study, we use molecular simulations, free-energy calculations and
differential scanning calorimetry to study the determining factors of a
necessary step in the assembly of the autoinhibited form of Abl, namely the
organization of the SH3 and SH2 domains into a conformation conducive
to its association with the SH2-kinase linker. Our results show that the
conformational dynamics of the Abl SH3-SH2 tandem are clearly distinct
from those of Src and related kinases. This finding enables us to reconcile
seemingly contradictory structural and biophysical data on the mechanism
of Abl regulation. Finally, we also examine the impact of activating
mutations within the SH3-SH2 unit, particularly in the short connector
between the domains.
P10r-34
ER targeting of COPI vesicles involves cargo

recognition by tethering factors
Ana Mara Prez-Linero, Javier Manzano-Lpez, Marcos CmaraDonoso, Manuel Muiz
Departamento de Biologa Celular, Universidad de Sevilla, Sevilla, ES
Vesicular transport through the eukaryotic secretory pathway is essential
for cellular function. This process depends on cytosolic coat proteins that
form vesicles from a donor compartment and ensure their correct docking
and fusion with the acceptor compartment trough interaction with
tethering factors. Specifically, the Dsl1 tethering complex tethers Golgiderived COPI retrograde transport vesicles to the endoplasmic reticulum
(ER) membrane. In this study, we found that the Dsl1 complex interacts
physically with the p24 proteins, which are type I transmembrane
proteins assembled into heteromeric complexes. The p24 complex is an
abundant cargo passenger of COPI vesicles that facilitates COPI vesicle
budding by stabilizing the COPI coat onto the Golgi membrane. By using
genetic analysis we also show that the Dsl1 and p24 complexes interact
functionally. Indeed, the phenotypic characterization of double mutants
indicates that the Dsl1-p24 physical interaction is required for an efficient
COPI vesicle tethering to the ER. Therefore, our results strongly suggest
that a specialized cargo like the p24 complex could promote the Golgito-ER retrograde transport by coupling both budding and tethering events
of COPI vesicles.
P10r-35
Study of the Caspase-9/PP2AC interaction in the

apoptotic pathway as a potential target for cancer
therapy
Leticia Domnguez Berrocal1, Angelita Rebollo2, Jernimo Bravo
Sicilia1
1
2
INSERM UMRS 945, Hpital Piti Salptrire, Universit Pierre et
Marie Curie, Pars, FR
Apoptosis is the process of programmed cell death induced in damaged,
unnecessary or dangerous cells to signal macrophages to engulf and
degrade their corpses. This key mechanism is found to be altered in
96

most types of cancer. The apoptotic machinery depends on a family
of proteases called caspases, whose activation irreversibly triggers cell
death.
Caspase-9 is an initiator caspase that generates the active forms of
Caspase-3 and Caspase-7 by limited proteolysis, and thereby transmits
the apoptotic signal to the execution phase. PP2A is a serine/threonine
phosphatase critical to physiological processes, including apoptosis,
inducing cell death either by dephosphorylating proteins or by its own
inhibition.
Cell penetrating peptides are molecules that can translocate into cells
without causing membrane damage. Based on the characterization of
the region responsible for the interaction, we developed cell penetrating
fusion peptides specifically designed to disrupt the Caspase-9/PP2AC
binding. Using these peptides, we performed the biophysical and
biochemical characterization of the interaction, showing a direct
association between Caspase-9 and PP2AC. We are also evaluating the
therapeutical potential of these peptides to induce Caspase-9 dependent
apoptosis.
P10-36
Mutational studies on ancestral proteins

Fadia Manssour1, Valeria A Risso1, Alvaro Ingls-Prieto1, Raquel
Godoy-Ruiz2, Jose A Gavira3, Beatriz Ibarra-Molero1, Jose M
Sanchez-Ruiz1
1
Universidad de Granada, Granada, ES, 2Department of Biochemistry
and Molecular Biology, University of Maryland School of Medicine,
Maryland, US, 3Laboratorio de Estudios Cristalogrficos,
Instituto Andaluz de Ciencias de la Tierra (Consejo Superior de
Investigaciones Cientficas- Universidad de Granada), Armilla,
Granada, ES
We have carried out an extensive mutational analysis of two thioredoxins:
a laboratory resurrection of a protein of the last common bacterial ancestor
(LBCA thioredoxin), an organism estimated to have lived about 4 billion
years ago, and one of its modern descendant (E. coli thioredoxin). Both
proteins share the same overall 3D-structure but show only 55% sequence
identity[1-2].
A total of 25 variants involving exchanges between highly similar amino
acids (E/D, I/V)[3-4] were purified and their thermal stabilities were
investigated by Differential Scanning Calorimetry.
Surprisingly, our results indicate a significant correlation between the
mutation effects on the ancestral and modern backgrounds suggesting a
conservation of protein mutational energetics over billions of years.
References
[1] Ingles-Prieto A, Ibarra-Molero B, Delgado-Delgado A, PerezJimenez R, Fernandez JM, Gaucher EA, Sanchez-Ruiz JM, Gavira
A (2013) Conservation of protein structure over four billion years.
Structure21:1690-1697.
[2] Perez-Jimenez R, Ingles-Prieto A, Zhao AM, Sanchez-Romero
I, Alegre-Cebollada J, Kosuri P, Garcia.Manyes S, Kappock TJ,
Tanokura M, Holmgren A, Sanchez-Ruiz JM (2011) Single-molecule
paleoenzymology probes the chemistry of resurrected enzymes. Nat
Struct Mol Biol 18:592-596.
[3] Godoy-Ruiz R, Perez-Jimenez R, Ibarra-Molero B and Sanchez-Ruiz
JM. Relation between protein stability, evolution and structure, as probed
by carboxylic acid mutations. J Mol Biol 336:313-318.
[4] Godoy-Ruiz R, Perez-Jimenez R, Ibarra-Molero B and Sanchez-Ruiz
JM. A stability pattern of protein hydrophobic mutations that reflects
evolutionary structural optimitzation. Biophys J 89:3320-3331.
Granada 2014
P10r-37
Structure and function study of the complex that

synthesizes S-adenosylmethionine
Ben Murray1, Svetlana Antonyuk2, Alberto Marina3, Sebastiaan Van
Liempd4, Shelly Lu5, Jose Mato4, Samar Hasnain2, Adriana Rojas3
1
Molecular Biophysics group, Institute of Integrative biology, Faculty
of Health and life sciences, University of Liverpool & Structural
biology unit, CIC bioGUNE, Parque Tecnoloico de Bizkaia , Derio,
ES, 2Molecular Biophysics group, Institute of Integrative biology,
Faculty of Health and life sciences, University of Liverpool,
Liverpool, UK, 3Structural Biology Unit, CIC bioGUNE, Parque
tecnolgico de Bizkaia, Derio, ES, 4Metabolomics Unit, CIC
bioGUNE, Parque Tecnolgico de Bizkaia, Derio, ES, 5Division of
Gastroenterology and Liver Diseases, USC Research center for Liver
Disease, USC-UCLA Research center for ALPD and Cirrhosis, Keck
Scool of medicine, Los Angeles, US
S-Adenosylmethionine (SAMe) is the principal methyl donor of the cell and
is synthesized via an ATP-driven process by methionine adenosyltransferase
(MAT) enzymes. It is tightly linked with cell proliferation in liver and
colon cancer. In humans, there are three genes, mat1A, mat2A and mat2B,
which encode MAT enzymes. mat2A and mat2B transcribe MAT2 and
MAT enzyme subunits, respectively, with catalytic and regulatory roles.
The MAT2 complex is expressed in nearly all tissues and is thought to
be essential in providing the necessary SAMe flux for methylation of DNA
and various proteins including histones. In human hepatocellular carcinoma
mat2A and mat2B genes are upregulated, highlighting the importance of
the MAT2 complex in liver disease. The individual subunits have been
structurally characterized but the nature of the complex has remained elusive
despite its existence having been postulated for more than 20 years and the
observation that MAT is often colocalized with MAT2. Though SAMe can
be produced by MAT(2)4 alone, our work shows that the Vmax of the MAT2
complex is three- to fourfold higher depending on the variants of MAT that
participate in complex formation. Using X-ray crystallography and solution
X-ray scattering, the first structures are provided of this 258 kDa functional
complex both in crystals and solution with an unexpected stoichiometry of
42 and 2V2 subunits. It is demonstrated that the N-terminal regulates the
activity of the complex and it is shown that complex formation takes place
surprisingly via the C-terminal of MATV2 that buries itself in a tunnel
created at the interface of the MAT(2)2. The structural data suggest a unique
mechanism of regulation and provide a gateway for structure-based drug
design in anticancer therapies.
P10-38
The misfolding behaviour of PSD95-PDZ3 is

modified by extra-domain structures and posttranslational modifications
Javier Murciano Calles1, Marta Marin-Argany2, Eva S. Cobos1,
Sandra Villegas2, Jose C. Martinez1
1
Universidad de Granada, Granada, ES, 2Departament de Bioquimica
i Biologia Molecular, Facultat de Biociencies, Universitat Autonoma
de Barcelona, Barcelona, ES
Post-translational modifications located out from the binding pocket of the
third PDZ domain of PSD95 (PDZ3) regulate binding affinity and specificity
through an intra-domain electrostatic network. Such residues involve some
charged residues in the b2b3 loop (were a succinimide modification occurs)
and the a3 helix (an extra-structural element that links the PDZ3 domain with
the following SH3 domain in PSD95, and contains the phosphorylation target
Tyr397). We have shown that these structural elements and their related posttranslational modifications also influence the folding/misfolding pathway of
PDZ3, by using site-directed mutagenesis combined with calorimetry and
spectroscopy. We have found that, although all the assayed mutations generate
proteins more prone to aggregation than the wild-type PDZ3, those directly
affecting the a3 helix increase the population of the DSC-detected intermediate
Psters
state and the misfolding kinetics, by organizing the supramacromolecular
structures at the expense of the two b-sheets present in the PDZ3 fold.
However, those mutations affecting the b2b3 loop, included into the proneto-aggregation region composed by a single b-sheet comprising b2 to b4
chains, stabilize the trimeric intermediate previously shown in the wild-type
PDZ3 and slow-down aggregation. These results strongly suggest that the a3
helix protects to some extent the PDZ3 domain core from misfolding. This
might well constitute the first example where an extra-element, intended to
link the PDZ3 domain to the following SH3 in PSD95 and in other members
of the MAGUK family, not only regulates the binding abilities of this domain
but it also protects PDZ3 from misfolding and aggregation.
P10-39
A functional amyloid assembly controls plasmid

DNA replication in bacteria
Mara Moreno del lamo1, Ftima Gasset-Rosa1, Susana MorenoDaz de la Espina1, Elena Fernndez-Tresguerres1, Rudi Lurz2,
Rafael Giraldo1
1
Centro de Investigaciones Biolgicas-CSIC, Madrid, ES, 2Max Plant
Institute for Molecular Genetics, Berlin, DE
DNA replication is tightly regulated to constrain the genetic material within
strict spatiotemporal boundaries and copy numbers. In some plasmids
from Gram-negative bacteria, two circular DNA molecules associate
(handcuff) their replication origins (ori) through a nucleoprotein assembly
with an oligomer of a plasmid-encoded replication protein (Rep) at its
core. Handcuffing thus hampers new replication rounds and determines
plasmid incompatibility [1]. The WH1 domain in the RepA protein of the
Pseudomonas pPS10 plasmid, besides its contributions to transcriptional
repression of repA gene and to plasmid DNA replication initiation by the
full length protein [2], assembles into amyloid fibers upon binding to DNA
in vitro [3] and causes an amyloid proteinopathy when overexpressed in
Escherichia coli [4,5]. We have developed a monoclonal antibody (B3h7)
specific for an amyloid oligomeric conformation of RepA-WH1 which has
revealed here that the handcuffed RepA assemblies are amyloid by nature.
RepA amyloids are found both in handcuffed complexes reconstructed
in vitro and inside the nucleoid of bacterial cells, where plasmids with
low copy numbers are known to cluster for partition and DNA-promoted
RepA-WH1 amyloidogenesis actually occurs. A stable amyloid core would
thus provide the basis for the previously reported requirement of molecular
chaperones in dismantling handcuffs to allow for further replication
rounds. RepA is a versatile DNA binding manifold, whose abilities, beyond
transcriptional auto-repression and plasmid replication initiation, now also
include the assembly of a replication inhibitory functional amyloid.
References
[1] Giraldo R and Fernndez-Tresguerres ME (2004). Plasmid 52(2): 69-83.
[2] Gasset-Rosa F, et al. (2008). Mol Microbiol 68(3): 560-572.
[3] Giraldo R (2007). Proc Natl Acad Sci USA 104: 17388-93.
[4] Fernndez-Tresguerres ME, et al. (2010). Mol Microbiol 77: 1456-69.
[5] Gasset-Rosa F, et al. (2014). Mol Microbiol 91(6):1070-1087
P10-40
Characterization of specific amino acid changes

in rhodopsin as putative positively selected sites
in visual pigment evolution
Miguel Antonio Fernndez Sampedro, Sundaramoorthy Srinivasan,
Eva Ramon Ports, Pere Garriga Sol
Grup de Biotecnologia Molecular i Industrial, Centre de
Biotecnologia Molecular, Universitat Politcnica de Catalunya,
Terrassa (Barcelona), ES
Rhodopsin has highly conserved motifs throughout vertebrate species,
but there are differences at variable sites in residues shared among
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lower mammals (monotreme) and the therian mammals. The shared
residues among monotreme and, reptile and amphibian rhodopsins,
include amino acids at positions 7, 8, 13, 225, 346 and 348, which
are not shared with therian. Moreover, a Bayes empirical approach
predicts several positively selected sites in the therian branch: Phe37,
Gln225 and Ile290 at the transmembrane helices of the receptor; Phe13 at the N terminus; and Ser343, Ser344 and Ala346 at the C terminus
of the protein. Here, we have expressed, purified and functionally
characterized three of these rhodopsin changes: F13M, Q225R and
A346S. We find that Q225R and A346S mutants have a wild type-like
behaviour upon photobleaching/acidification and Western blot analysis
and metarhodopsin II decay rates but show different hydroxylamine
reactivity. Both Q225R and A346S mutants show increased reactivity
to hydroxylamine, reflecting lower conformational stability as observed
for echidna (most basal mammals) rhodopsins. We observe a lower Gt
activation rate for Q225R which can be explained because the mutation
is located close to the third intracellular loop important for Gt binding.
A346S shows increased Gt activation rate that could be related to the
evolutionary loss of an extra phosphorylation place at the C terminal
tail of rhodopsin. In contrast, the extracellular F13M mutant could not
bind retinal and showed a clearly different electrophoretic pattern. We
could restore chromophore binding, for F13M, by adding a cysteine
pair (N2C/D282C) that forms a stabilizing disulfide bond. This result
reveals the importance of the N-terminal domain of rhodopsin in visual
pigment evolution.
P10-41
Different retinal binding modes to visual

pigments suggest mechanism for Fine- Tuning
GPCR-ligand interactions
Sundaramoorthy Srinivasan, Miguel Fernandez, Eva Ramon, Pere
Garriga
Terrassa, ES
Cone opsins are G-protein coupled receptors localized in cone
photoreceptor cells of the retina. These receptors are activated by light
and mediate the complex process of color vision. These visual pigments
are heptahelical transmembrane proteins, bound to a light sensitive
chromophore, 11 - cis -retinal (11CR) through a protonated Schiff
base (SB) linkage at K312. Red and green opsin pigments show 95%
sequence identity and they share a similarly biophysical and biochemical
properties, yet the possible structural differences between each other
are unclear. In order that to study human red and green cone pigments
were expressed in COS-1 cells, regenerated with 11CR and purified
using immunochromatography. Were analyzed the purified pigments
using UV-vis and fluorescence spectroscopies in the dark state and after
photoactivation. Ligand binding was measured by using the analog
9-cis-retinal (9CR) in addition to 11CR. The results of the regeneration
experiments with the photoactivated receptors show that green cone
opsin can not form a pigment in the visible region of the spectrum
when regenerated with 11CR but it can band with a visible form 9CR.
The acidification of the sample regenerated 11CR showed increased
absorption at 440nm suggesting the formation of unprotonated SB linkage
activated between 11CR and green opsin. In contrast, activated red cone
opsin exhibits the formation with protonated SB both retinals. These
that results suggest the difference in aminoacid sequence between green
and red opsins not only contributes to their spectral sensitivity but also
modulates ligand binding after photoactivation. Identified the molecular
interactions these details contributing differences in ligand binding can
be a model for fine-tuning of ligand- receptor of other members of the
GPCR superfamily.
98

P10-42
Binding of activators to the kinase unit of AMPK:

theoretical study of the activation mechanism
Gleiciane Leal Moraes1, Carolina Estarellas1, Ana Castro2, Axel
Bidon-Chanal1, F. Javier Luque1
1
Departamento de Fisicoqumica, Facultad de Farmacia,
Universidad de Barcelona, Santa Coloma de Gramanet, ES,
2
Instituto de Qumica Mdica, CSIC, Madrid, ES
Mammalian AMP-activated protein kinase (AMPK) is a serine/threonine
protein kinase that acts as a key sensor of cellular energy status. Its primary
function it to sense changes in the intracellular level of ATP and to couple
the changes in ATP to phosphorylation of downstream substrates, leading
to an increase in the rate of pathways producing ATP and/or a decrease
in the rate of ATP-consuming processes. Its function in the homeostasis
of cellular energy confers AMPK a major role in numerous metabolic
disorders, such as type 2 diabetes, obesity and cancer. This explains the
progressive interest in AMPK as a therapeutic target.
AMPK is a heterotrimer composed of three different subunits: alpha, beta
and gamma. The alpha subunit carries the catalytic function and contains a
kinase domain at the N-terminus of the protein followed by an autoinhibitory
domain and a C-terminal domain required for interaction with the beta
subunit. The AMPK activity is regulated by several mechanisms, including
a number of indirect activators of AMPK (such as metformin, rosiglitazone
and resveratrol), an direct activators, such as compound A-769662 . Some
of these activators show a clear specificity for a particular AMPK subunit,
which opens they way to design and discover new compounds that could
activate AMPK in some tissues but not in others, minimizing in this way the
putative negative effects of the activation of AMPK at whole body level.
Up to now, the mechanism exerted by direct activators remained unknown.
Very recently, two X-ray structures showing the binding of direct AMPK
activators have been reported. This study reports the results of molecular
dynamics simulates carried out to gain insight into the mechanism of
AMPK activation by A-769662.
P10r-43
Macromolecular crowding modulates the

interaction of DnaK with its functional partners
Garbie Celaya, Ianire Martin, Alejandra Aguado, Yovana Cabrera,
Fernando Moro, Arturo Muga
Leioa, ES
Hsp70 proteins are central components of the cellular network of
molecular chaperones and they assist a broad range of physiological
processes, such as folding and assembly of newly synthesized proteins or
refolding of misfolded and aggregated proteins. DnaK (Hsp70) of E. coli is
an allosteric chaperone that has a nucleotide binding domain (NBD), that
binds and hydrolyzes ATP, a substrate binding domain (SBD) and a linker
that connects both domains. DnaK, together with the cochaperone DnaJ
(Hsp40) and the nucleotide exchange factor GrpE, forms the chaperone
system that is able to avoid aggregation of partially unfolded protein
conformations and to remodel and reactivate some protein aggregates by
itself or cooperating with other chaperone systems, such as ClpB (Hsp100).
DnaJ and GrpE regulate the functional cycle of DnaK, which is based
on a complex allosterical interdomain communication that promotes
transition between two main DnaK conformations that differ in substrate
affinity; when DnaK binds ATP, the linker promotes the tethering of both
domains and it acquires an open conformation with lower affinity. Binding
of substrates and DnaJ induces ATP hydrolysis and DnaK acquires a
conformation recognizable by GrpE, where the two domains separate and
behave independently. In this ADP-bound state, the SBD adopts a closed
conformation that tightly binds substrates.
The sequential association reactions that occur during the functional cycle
of the chaperone system have been characterized under dilute experimental
Granada 2014
conditions, despite the fact that in the intracellular medium the equilibrium
and transition rates between different protein conformations are most likely
affected by volume exclusion arising from macromolecular crowding. We
have studied the effect of crowding on the conformational properties,
ATPase and chaperone activities of the DnaK system.
Our data show that crowding regulates in a temperature dependent manner
the affinity of DnaK for DnaJ, but not for GrpE. The most relevant DnaJ
domain regulating this interaction is the disordered G/F rich region that can
be recognized by DnaK as a pseudosubstrate at low temperatures. Excluded
volume conditions also allow ClpB to compete with GrpE for binding to
the same DnaK region. This competition directs DnaK to reactivate stable
protein aggregates, interacting with ClpB, or to remodel substrate proteins,
interacting with GrpE. Therefore, crowding modulates the interaction of
DnaK with specific protein partners.
P10-44
Aminooxi anlogos de histidina: una

posible estrategia para modular la histidina
descarboxilasa humana
Francisca Mara Snchez Jimnez1, Rosario Castro Oropeza1,
Almudena Pino ngeles1, Maximilian A. Khomutov2, Jos Luis
Urdiales Ruiz1, Alexander Khomutov2
1
Dept. Biologa Molecular y Bioqumica. Universidad de MlagaUnidad 741 CIBERER, Campus Andaluca Tech, Mlaga, ES,
2
Instituto de Biologa Molecular Engelhardt, Mosc, RU
La histamina (Hia) es una amina bigena implicada en procesos
fisiolgicos esenciales (respuesta inmune, neurotransmisin, entre otros).
El desequilibrio de su metabolismo juega un destacado papel en muchas
patologas. El control de los efectos adversos de Hia se ejerce nicamente
modulando sus receptores (H1R-H4R). En clulas humanas, Hia se
sintetiza por descarboxilacin de la L-His, en la reaccin dependiente de
piridoxal 5-fosfato (PLP) que cataliza la histidina descarboxilasa (HDC,
EC 4.1.1.22). La baja tasa de expresin y la inestabilidad de HDC han
dificultado su caracterizacin estructural y funcional, y por tanto el
desarrollo de estrategias basadas en su inhibicin. Nuestra experiencia
derivada de estudios in silico (modelado y dinmica molecular) y
experimentales con HDC, nos permiti abordar la bsqueda de nuevas
posibilidades. Mediante tecnologa de barrido virtual dedujimos que
un inhibidor competitivo efectivo debera ser aquel capaz de enlazarse
eficientemente con el PLP formando una estructura similar al intermediario
PLP-substrato. Debera tambin conservar el grupo imidazol para reforzar
la afinidad por el sitio cataltico. Ambas caractersticas podan conseguirse
utilizando el compuesto 4(5)-aminooximetilimidazol (O-IMHA). Esta
hiptesis se ha contrastado experimentalmente sobre extractos de HDC
humana purificada. Hemos comprobado que O-IMHA es capaz de formar
una PLP-oxima en el centro cataltico de la enzima humana. Los resultados
indican que O-IMHA puede constituir una cabeza de sntesis prometedora
de inhibidores de HDC. Aprovechando el conocimiento actual sobre la
estructura de la enzima proponemos las configuraciones ms probables del
aducto en el sitio cataltico y podremos desarrollar nuevos derivados con
potencial traslacional.
SAF2011-26518 y CVI-06585. CIBERER (ISCIII).
P10r-45
Golgi assembly depends on vesicle tethering

mediated by cooperation of ER cargo receptors
Javier Manzano-Lpez, Ana Mara Prez-Linero, Laura OjedaMrquez, Marcos Cmara-Donoso, Manuel Muiz
Departamento de Biologa Celular, Universidad de Sevilla, Sevilla, ES
Endoplasmic reticulum (ER) cargo receptors are major transmembrane
constituents of the early secretory pathway that continuously cycle between
Psters
the ER and Golgi apparatus. They function individually to mediate selective
incorporation of specific cargo proteins into ER-derived vesicles by linking
the cargo with the COPII coat. Here, we show that cargo receptors also
interact with each other to form a High-Molecular-Weight complex at the
level of the ER exit sites. We also found that this complex is required for de
novo assembly of the cis-Golgi compartment by promoting COPII vesicle
tethering. Our results suggest that, in addition to their individual function
in selective ER export, cargo receptors cooperate to maintain the structural
and functional homeostasis of the early secretory pathway by forming a
multi-protein platform that stabilizes the COPII coat onto the vesicle
membrane and thereby ensures correct functioning of tethering factors.
P10-46
Unraveling GPCR-Zinc interactions and its role

in depression
Merc Tena Campos, Xiaoyun Dong, Diana Rivera Rodrguez, Eva
Ramon Portes, Pere Garriga Sol
Terrassa, ES
Zinc supplementation has been widely reported to improve treatment against
major depressive disorder. It is also well known that G-protein-coupled
receptor (GPCRs) are involved in this disorder, among these 5-HT1A, GalR1
and GPR39, all three potentially connected to zinc modulation. Our work
has been focused on the study and characterization of these receptors and
its relationships with zinc both in vitro and in cell culture. To this aim, we
have designed a strategy to purify the receptors in a conformationally active
state. We have used receptors tagged with the monoclonal 1D4 antibody
epitope, and employed ligand assisted purification. We have purified all
three receptors properly folded, active and in a monomeric state as shown
by native gel electrophoresis.
In the case of GPR39, it is still under debate whether zinc is the orthosteric
ligand or not, but it is clear that there is an interaction between the receptor
and the metal ion. According to our results, the receptor appears to be
purified bound to zinc, even when cell culture and purification media
are not supplemented with the cation, meaning that there is a specific
interaction between zinc and GPR39 under physiological conditions. This
interaction is confirmed by Trp fluorescence increase upon EDTA addition
to a GPR39 purified sample.
5-HT1A, GalR1 have been described to heterodimerize triggering a nonphysiological state that would underly depression. Using FRET we have
demonstrated that zinc is able to disrupt this interaction. Both purified
receptors are analyzed by surface plasmon resonance in order to determine
the kinetics of this interaction. Further investigations should be carried out
to fully uncover the relationships between these receptors, zinc and major
depressive disorder.
P10-47
Gliceraldehdo-3-fosfato deshidrogenasa,
endonucleasa IV y uracil DNA glicosilasa forman
parte de un complejo proteico implicado en
mantener la integridad del genoma en E. coli
Maria Alexandra Caas Pacheco, Laura Aguilera Gil, Elaine Ferreira
Melo, Carina S. Alvarez, Rosa Gimnez Claudio, Josefa Bada
Palacin, Laura Baldom Llavins
Departamento de Bioquimica i Bilogia Molecular, Facultat de
Farmcia, Universitat de Barcelona. Institut de Biomedicina de la
UB (IBUB), Barcelona, ES
Gliceraldehdo-3-fosfato deshidrogenasa (GAPDH) es una protena
multifuncional. A parte de su funcin en la glicolisis presenta otras
actividades. Estudios previos realizados por nuestro grupo con mutantes
gapA y clulas silenciadas con RNA antisentido sugeran la implicacin
99
Psters
funcional de GAPDH en procesos de reparacin del DNA en E. coli.
Las clulas deficientes en GAPDH presentan menor supervivencia que
las clulas control frente al tratamiento con agentes genotxicos, as
como un mayor nmero de centros apurnicos/apirimidnicos (centros
AP) espontneos en su genoma. En este estudio se han realizado ensayos
pull down seguidos de Western blot con varias protenas de los sistemas
de reparacin del DNA. Los resultados muestran interaccin de GAPDH
con: (i) SSB (single strand DNA binding protein), protena esencial en los
procesos de reparacin por recombinacin homloga y respuesta SOS (ii)
endonucleasa Endo IV, implicada en la reparacin de centros AP y (iii)
uracil DNA glicosilasa (UDG) que genera un centro AP al eliminar el
uracilo presente en el DNA. El estudio de estas interacciones en mutantes
deficientes en estas protenas indica que GAPDH, EndoIV y UDG forman
parte de un complejo de reparacin de DNA en E. coli. La morfologa
filamentosa observada en cepas silenciadas o deficientes en GAPDH a las 3
horas de recuperacin despus de un tratamiento con bleomicina o agentes
alquilantes es compatible con la inhibicin de la divisin celular por
acmulo de lesiones no reparadas en el genoma y confirma la implicacin
de GAPDH en procesos de reparacin del DNA en E. coli.

3
Estacin Experimental del Zaidin, CSIC (EEZ-CSIC), Granada,

ES, 4Departamento de Quimica Fisica, Facultad de Ciencias,
Parkinsons disease is a neurodegenerative disorder with a high

impact in our society. This is one of several diseases related to the
oligomerization and aggregation of -Synuclein (Syn). The interaction
of Syn with surfactants, lipids and membranes appears to play a key
role in its physiological function. The Syn found in cells presents a posttranscriptional modification consisting of the acetylation of its N-terminus,
which appears to affect its behavior and therefore also its function.
Sodium dodecyl sulfate (SDS) has been widely used as a simple model
for membrane environment and has been reported to accelerate amyloid
fibrillation of Syn in vitro. In this study, we compared N-acetylated and
unacetylated Syn in their mode of interaction with SDS and related their
different properties with their amyloidogenic propensity. We found that
in the presence of sub-CMC concentrations of SDS Syn forms SDSassociated partially folded oligomers. These oligomers appear to constitute
optimal species for spontaneous and efficient formation of amyloid nuclei,
which further drive rapid, lag-free amyloid fibrillation. N-acetylation
reduces the extent of Syn oligomerization favoring its interaction with
micelles, thereby reducing the rate of formation of amyloid nuclei.
P10-48
Docosahexaenoic acid effect on the conformational

stability of the rhodopsin mutants G90V and N55K
associated with retinitis pigmentosa
Xiaoyun Dong, Eva Ramon Portes, Pere Garriga Sole
Terrassa, ES
Rhodopsin is a prototypical seven-transmembrane helix receptor belonging
to the G-protein coupled receptor (GPCR) superfamily. It functions as a
visual photoreceptor and serves as the photosensitive pigment for scotopic
vision under dim light conditions. As a GPCR, its structural and functional
features are modulated by lipids. Docosahexaenoic acid (DHA) provides a
physiologically relevant milieu for stabilizing rhodopsin but the Influence
of DHA on rhodopsin mutants conformational properties have not been
previously explored. We have studied the structural features of two
thermosensitive mutants, G90V and N55K, which are associated with the
retinal degenerative disease retinitis pigmentosa and show thermal unstability
when solubilized in the classical neutral detergent dodecyl maltoside (DM).
We have used 1,2-didocosa-hexaenoyl--sn-glycero-3-phosphocholine
(DDHA-PC) liposomes for reconstituting the purified rhodopsin mutants.
G90V mutant reconstituted in DDHA-PC liposomes showed a 4-fold thermal
stability increase than in DM. In turn, N55K mutant showed a slight increase
on its thermal stability under the same conditions. The active conformation
metarhodopsin II decayed faster, as measured by retinal release fluorescence
spectroscopic measurements, for both mutants in DDHA-PC liposomes
compared with DM. DDHA-PC liposomes also preserved G90V and N55K
opsin conformations allowing more efficient binding of exogenously added
11-cis-retinal ligand. DDHA-PC liposomes provide an environment can
preserve the conformation of the pathogenic mutations by providing higher
thermal stability when compared with the DM-purified receptors. These
effects highlight the potential stabilizing role of DHA on the structural
features of rhodopsin mutants associated with retinal disease.
P10-49
N-terminal acetylation of -Synuclein decreaces

the population of partially folded oligomers and
reduces amyloid aggregation
David Ruzafa Ruiz1, Yuriko Hernndez G.2, Giovanni Bisello2,
Bertrand Morel3, Francisco Conejero-Lara4
1
Universidad de Granada, Granada, ES, 2Dipartimento di Science
del Farmaco, Universit degli Studi di Padova, Padova, IT,
100
P10-51
El dominio -hairpin, implicacin en el proceso

sealizador de dUTPasas trimricas
Jorge Donderis Martnez1, Elisa Maiques1, Ilty Mehemedov1, Mara
ngeles Tormo Ms2, Mara Garca Caballer2, Jos Penads3, Alberto
Marina1
1
Instituto de Biomedicina de Valencia, CSIC, Valencia, ES,
2
Instituto Valenciano de Investigaciones Agrarias, CITA-IVIA,
Segorbe, ES, 3Institute of Infection, Immunity, and Inflammation,
College of Medical, Veterinary, and Life Sciences, University of
Glasgow, Glasgow, UK
Las dUTPasas (DUT) son enzimas presentes en todos los organismos que
hidrolizan el dUTP a dUMP y pirofosfato inorgnico. Diversos trabajos
han descrito la actividad sealizadora de las DUT independientemente
de su actividad cataltica, como es el caso de la DUT monomrica del
Epstein-Barr virus, capaz de inducir la expresin de citoquinas proinflamatorias, o la DUT trimrica de la rata que une a PPAR y regula la
expresin gnica mediada por el complejo PPAR-RXR; o, recientemente
nuestro grupo demostr que en Staphylococcus aureus las DUT trimricas
de bacterifagos podan inducir la transferencia horizontal de islas de
patogenicidad (SaPI) en estas bacterias [1,2].
La movilidad de las SaPI depende de la unin de la DUT a la protena
Stl que reprime la isla. Dicha unin est vinculada a una conformacin
catalticamente activa de la DUT adems de la presencia de un dominio
extra de entre 20-30 residuos muy variable en secuencia y longitud entre
las diferentes DUT fgicas.
Para comprender mejor la capacidad sealizadora de estas protenas, en
este trabajo hemos resuelto la estructura tridimensional de 3 DUT con
capacidad inductora de las SaPI de diferentes fagos. Sorprendentemente,
comparando estas estructuras, junto con la DUT del fago 80, previamente
resuelta por nuestro grupo, observamos que dentro de este dominio extra
no conservado a nivel de secuencia, existe una regin central de 9 residuos
que ocupa una disposicin espacial idntica en todas ellas as como la
misma conformacin, un -hairpin, pese a la diversidad de la secuencia.
Nuestros estudios mutacionales muestran que este motivo estructural juega
un papel clave en el reconocimiento de la protena Stl y por lo tanto en la
actividad sealizadora de estas DUT.
Bibliografa
[1] Penads et al., 2013. dUTPases, the unexplored family of signalling
molecules. Curr. Opin. Microbiol. 16, 163170.
[2] Tormo-Ms et al., 2013. Phage dUTPases Control Transfer of Virulence
Genes by a Proto-Oncogenic G Protein-like Mechanism. Mol. Cell 49, 947-958.
Granada 2014
P10-52
Investigando la funcin de CutA en bacterias:

estudios estructurales y funcionales
Lorena Tremino-Agull1, Javier Espinosa2, Carles Palanca1, Asuncin
Contreras2, Vicente Rubio1
1
Instituto de Biomedicina de Valencia (IBV-CSIC) y CIBERERISCIII, Valencia, ES, 2Divisin de Gentica, Universidad de
Nuestro laboratorio ha aclarado sobre base estructural la funcin de la
protena PII, sealizador homotrimrico de 112 residuos, muy conservado
y ampliamente distribuido, que interacciona con enzimas, canales y
reguladores de expresin gnica. Ahora intentamos lo mismo con la
protena CutA, de secuencia aparentemente no homloga a PII, tambin
homotrimrica, de 112 residuos, muy conservada y ampliamente distribuida.
Su nombre se debe a que su gen se identific en una regin de dos genes
de E.coli cuya delecin sensibilizaba a cobre, sin evidencia slida de su
implicacin en proteccin a metales. Suponindole una funcin sealizadora
anloga a la de PII, realizamos un estudio de doble hbrido en levadura con
una librera del genoma y de candidatos prometedores de la cianobacteria
Synechococcus elongatus, el mismo organismo y estrategia que llevaron a
identificar las dianas de PII. Los resultados con CutA, presentados aqu, han
sido poco informativos. Tambin presentaremos la estructura cristalina de
CutA de S. elongatus a 2 de resolucin, esencialmente idntica a la de PII
excepto por faltar los largos lazos flexibles T, sin que hayamos tenido xito
en generar estructura de complejos con metales de esta protena. Finalmente,
presentaremos experimentos en que reconsideramos desde el inicio la
funcionalidad de CutA, utilizando un mutante nulo de E. coli, exclusivo de
CutA, en el que investigamos la sensibilidad a metales y otras caractersticas
fenotpicas. Confiamos en que estos experimentos proporcionen informacin
funcional slida sobre el papel de CutA y sobre las razones de su amplia
distribucin y alta conservacin.
Financiado por ayudas Prometeo 2009/051 (Generalitat Valenciana) y
BFU2011-30407 (MINECO). L. Tremino becaria FPI del MINECO.
P10-53 (R10-4)
Crystal structures of human carbamoyl phosphate

synthetase 1 (CPS1) shed light on domains
functions, substrate tunnels and allosteric
activation, and allow rationalization of inborn
CPS1 deficiency
Sergio de Cima Martn1, Luis Mariano Polo1, Carmen DezFernandez1, Javier Cervera1, Ignacio Fita2, Vicente Rubio1
1
Instituto de Biomedicina de Valencia of the CSIC, and Group
739 of the Centro de Investigacin Biomdica en Red sobre
Enfermedades Raras-CIBERER del Instituto de Salud Carlos III,
Valencia, ES, 2Institut de Biologia Molecular de Barcelona (CSIC),
Barcelona, ES
CPS1 is a large six-domain protein that constitutes the first step of the urea
cycle: the synthesis of carbamoyl phosphate from bicarbonate, ammonia
and two ATP molecules. A paramount feature of this enzyme is its absolute
requirement for N-acetyl-L-glutamate (NAG), an allosteric activator without
which it is inactive. The report of CPS1 regulation by lysine deacylation
by NAD-dependent sirtuin 5 connected the urea cycle with the age-control
machinery (Nakagawa et al. Cell 2009; 137:560). CPS1 deficiency (CPS1D)
is an inborn disorder that cause severe neonatal hyperammonemia leading
to mental retardation or even to death. More than 300 mutations have been
reported in CPS1D patients, of which the majority are missense mutations
showing little recurrence and having unproven disease-causing potential. The
structure of the E. coli homologous CPS has been known for >15 years, but
differences with CPS1 (40% identity; use of glutamine instead of ammonia;
insensitivity to NAG) rendered essential to obtain the structure of CPS1 for
proper understanding of its functioning, and for evaluating disease causation
Psters
by CPS1D mutations. Using a baculovirus/insect cell system we have finally
succeeded in producing recombinant human CPS1 in large amount and pure
form, allowing us to experimentally examine the effects of reported mutations
and ascertain its disease-causing potential (Dez-Fernndez et al. Human Mut
2013; 34:1149). In addition we have determined the structure of CPS1, in
both apo and ligand-bound (NAG and ADP/Pi) forms. The liganded structure
revealed how NAG binds in a pocket of the C-terminal domain and has
identified elements stabilized by ADP binding and conformational changes that
lead to define the carbamate tunnel, which in the apo form is heavily branched
and open to the environment. Our structures decipher the CPS1 inability to use
glutamine and reveal a potential channel for ammonia intake. Furthermore,
they help rationalize the disease-causing role of most clinical CPS1 mutations.
Supported by Fundacin Alicia Koplowitz and Valencian (Prometeo
2009/051) and Spanish (BFU2011-30407; FPU to CD-F) governments.
P10-54
Unveiling the mechanism of transcriptional

auto-regulation of parD system
Srdja Drakulic, Ana Mara Hernndez-Arriaga, Carlos FernndezTornero, Ramn Daz Orejas
Centro de Investigaciones Biolgicas (CIB/CSIC), Madrid, ES
Survival of extrachromosomal genetic material in bacterial population
involves the activity of toxin-antitoxin systems (TA). In particular, the
maintenance of R1 plasmid requires the employment of three stability
systems: parA, parB and parD. The focus of our work is on the parD system,
a type II toxin-antitoxin system, consisting of stable protein toxin Kid, a
ribonuclease which activity leads to cell growth arrest, and of proteolyses
prone Kis antitoxin, which through protein-protein interactions abolishes
the toxic effect of Kid. Interestingly, parD operon exhibits a transcriptional
auto-regulation. Depending on their relative molar ratio and the presence
of promoter DNA, Kis and Kid form differentially sized complexes, with
variable capacity of parD operon transcriptional regulation.Our preliminary
results indicate that KisKid complexes coexist with RNAP at the promoter,
raising questions of how KisKid system and RNAP interact, and how
would this lead to parD operons transcription decrease. In order to provide
the answers to previously raised questions, we have combined structural,
in a first place negative-staining EM, with biochemical/functional analysis
of the RNAP core-enzyme (subunits composition: , , , , ), RNAP
core in complex with the sigma70 factor (RNAP holoenzyme), KisKid
repressor with its promoter DNA (PO), and RNAP holoenzyme:KisKid:PO
complex. The 3DEM structure of the repressor-DNA complex indicates
that two KisKid-heterooctamers, bound respectively at the operator sites
I and II, establish protein-protein interactions to form a supercomplex in
which the RNAP binding site seems to be exposed through the extensive
DNA bending. Our current work is dedicated to the determination of the
structural and biochemical properties of the RNAP holoenzyme-KisKidDNA complex, with the goal of deciphering the transcriptional regulation
mechanism of the KisKid repressor complex on the parD system.
P10-55
The why of linkers in structural biology: the case

of Galectin-4
Mnica lvarez Prez1, Eliza Buzamet1, Rubn M. Buey2, Sabine
Vrtesy3, J. Fernando Daz2, Sabine Andr3, Hans-Joachim Gabius3,
Margarita Menndez1, Dolores Sols1
1
Instituto de Qumica Fsica Rocasolano, CSIC and Centro de
Investigacin Biomdica en Red de Enfermedades RespiratoriasCIBERES, Madrid, ES, 2Centro de Investigaciones Biolgicas,
CSIC, Madrid, ES, 3Institut fr Physiologische Chemie,Tierrztliche
Fakultt, LMU Mnchen, Munich, DE
Cell surface glycans are versatile biochemical signals decoded and translated
into responses by endogenous lectins [1]. Among them, the members of the
101
Psters
galectin family with ability to crosslink glycans of cellular glycoconjugates
fulfil special tasks, e.g. in growth control and glycoprotein routing as
galectin-4 (Gal-4) does [2, 3]. To address the issue on the functional
significance of the 42-amino-acid linker connecting the two lectin domains,
we engineered variants and performed structural analysis in solution.
Analytical ultracentrifugation and gel filtration revealed an impact of linker
truncation on quaternary structure, further studied by additional variants.
This type of structural analysis was flanked by precipitation analysis with a
pan-galectin counterreceptor, i.e. the glycoprotein asialofetuin. The obtained
results disclose new information on linker presence and encourage respective
analysis using this strategy on homodimeric Gal-1, engineered to have the
Gal-4 linker, and the chimera-type Gal-3.
References
[1] Gabius et al. (2011) From lectin structure to functional glycomics:
principles of the sugar code. TIBS 36, 298-316.
[2] Ledeen et al. (2012) Beyond glycoproteins as galectin counterreceptors:
tumor-effector T cell growth control via ganglioside GM1. Ann N Y Acad
Sci 1253, 206-221.
[3] Velasco et al. (2013) Neuronal galectin-4 is required for axon growth
and for the organization of axonal membrane L1 delivery and clustering. J
Neurochem 125, 49-62.
P10-56
Tubulin dimer dissociation by chaperones TBCE

and TBCB is a mechanically driven process
Gerardo Carranza1, Marina Serna2, Jaime Martn-Benito3, Jose Mara
Valpuesta3, Juan Carlos Zabala1
1
Universidad de Cantabria, Santander, ES, 2Imperial College,
London, UK, 3Centro Nacional de Biotecnologa (CNB-CSIC),
Madrid, ES
A group of molecular chaperones termed tubulin cofactors (TBCs)
participate in tubulin proteostasis inside the cell playing an important role
in the spatial and temporal regulation of the microtubule cytoskeleton.
While tubulin heterodimer formation is well characterized, its dissociation
pathway is not well understood.
In the study reported here we showed the structural determination by electron
microscopy and image processing of the complex between human TBCE,
TBCB and -tubulin (EB), which is formed upon dissociation of the tubulin heterodimer by both chaperones, as well as its interpretation through
the molecular fitting of the atomic structures of the protein domains.
Docking of the atomic structures of the different domains of the three
proteins, including the UBL domain of TBCE, solved in this study by X-ray
crystallography, has allowed us to construct an atomic model of the EB
complex with important functional consequences regarding the molecular
mechanism of the tubulin dimer dissociation by both tubulin cofactors. This
process, which is not energy-dependent, takes place through the distortion of
the /-tubulin interface caused by a steric clash between the TBCE CAPGly and LRR domains and -tubulin. Finally, the protruding arrangement of
both UBL domains in the EB complex suggests a direct interaction of this
complex with the proteasome, mediating -tubulin degradation.
P10-57
Insights into the human carboxypeptidase D

structure deduced from single-particle electron
microscopy
Javier Garcia Pardo1, Sebastian Tanco1, Pablo Gallego1, Daniela
Lufrano2, David Reverter1, Jos L. Carrascosa3, Julia Lorenzo1,
Francesc X. Avils1
1
Institut de Biotecnologia i Biomedicina and Departament de
Bioquimica i Biologia Molecular, Universitat Autnoma de
Barcelona, Bellaterra, ES, 2Laboratorio de Investigacin de
Protenas Vegetales, Facultad de Ciencias Exactas, Universidad
102

Nacional de La Plata, La Plata, AR, 3Department of Macromolecular
Structure, Centro Nacional de Biotecnologa, Consejo Superior de
Investigaciones Cienfcas-CSIC, Madrid, ES
Carboxypeptidase D (CPD) is a 180 kDa membrane-bound
metallocarboxypeptidase that contains three carboxypeptidase (CP)
domains. The biological function of this enzyme is not well known
but it is thought to participate in the maturation of hormones and other
bioactive peptides in the trans-Golgi network and functions as a docking
receptor for duck hepatitis B virus. Only the first and second domains
have detectable carboxypeptidase activity whereas the third domain seems
to be implicated in the virus recognition. Previous reports described the
three-dimensional structures of the first and second CP domains solved
by X-ray crystallography. Nonetheless, nothing is known about the three
dimensional structure of the full-length enzyme and the implications for
its biological function.
To study the structure of full-length human CPD we cloned, recombinantly
expressed and purified a soluble form of this enzyme, lacking the
transmembrane domain. For this purpose we used a mammalian cell-based
expression system that allows us to purify milligrams of soluble and active
human CPD.
The oligomeric state of the enzyme was determined by molecular exclusion
chromatography and dynamic light scattering (DLS), showing that the
protein self-associates to a homotrimeric complex with a molecular weight
of approximately 450 kDa. The CPD trimeric complex was stable up to 1
M salt concentrations, suggesting that complex association is driven by
hydrophobic interactions.
By single particle EM reconstructions we confirmed the trimeric native
state of this soluble human CPD enzyme. In the trimer each CPD monomer
has almost triangular appearance with dimensions of approximately 80 x
80 . The oligomeric structure has a 3-fold rotational symmetry with a
high structural flexibility.
Finally, using the interactions observed in the tridimensional structure of
duck CPD dII previously solved, we propose a three-dimensional structure
model of human CPD to explain the obtained single particle EM 2D
reconstructions.
P10-58
La asociacin de las GTPasas pequeas RagA

y Rab3 al motor de dinena tiene lugar a travs
de su unin a DYNLT
Javier Merino Gracia1, Ruth A. Valero2, M Flor Garca-Mayoral3,
Marta Bruix3, Ignacio Rodrguez-Crespo2
1
Universidad Complutense de Madrid, Madrid, ES, 2Departamento
de Bioqumica y Biologa Molecular I, Universidad Complutense de
Madrid, Madrid, ES, 3Instituto de Qumica-Fsica Rocasolano, CSIC,
Madrid, ES
Las cadenas ligeras de dinena, DYNLT, DYNLL y DYNLRB, son pequeas
protenas dimricas que forman parte del motor de dinena, una estructura
macromolecular de ~1.2 MDa encargada del transporte retrgrado por
los microtbulos. Estas tres cadenas se asocian al extremo N-terminal de
la cadena intermedia de dinena, DYNC1I. Aunque DYNLT y DYNLL no
son homlogas entre s, adoptan un plegamiento casi idntico, con lminas
beta antiparalelas como superficies de unin del dmero, flanqueada por dos
alfa hlices en cada monmero. Para DYNLL se ha descrito su asociacin
a numerosas protenas celulares y virales que poseen una de las dos
secuencias consenso de unin: KSTQT y KDTGIQVDR, las cuales adoptan
una disposicin de cadena beta que se inserta de forma antiparalela en el
homodmero previamente formado. Sin embargo, DYNLL posee un nico
sitio de unin para polipptidos, implicando que si se asocia a la cadena
intermedia de dinena no puede unirse simultneamente a una protena diana.
Nuestro trabajo muestra que DYNLT, al contrario que DYNLL, funciona
como una protena de acoplamiento de protenas diana al motor de dinena,
presentando dos sitios de unin. RagA y Rab3D son dos pequeas GTPasas
que se asocian con DYNLT. Por medio de dobles hbridos de levaduras y
Granada 2014
mutagnesis dirigida de las GTPasas, hemos identificado el sitio de unin
de DYNLT, y los requerimientos de la secuencia para la interaccin. Con
tcnicas de NMR hemos localizado los aminocidos de DYNLT implicados
en la unin a la cadena intermedia de dinena y los implicados en su unin
a las GTPasas pequeas. En experimentos de inmunoprecipitacin hemos
confirmado que DYNLT funciona como protena puente capaz de asociar
RagA y Rab3D a microtbulos. As, establecemos un modelo que describe
un nuevo mecanismo de asociacin de protenas al motor de dinena, para su
transporte por los microtbulos.
P10-59
Psters
fungal laccases are capable to promote complex hetero-polymerizations of
great significance for the synthesis of dyes [1].
The present work describes a laboratory evolution protocol to engineer
efficient fungal laccases for the synthesis of polymeric dyes. The departure
point for this study was the laccase IG-88 mutant from of Myceliophthora
thermophila, which was previously evolved through 21 rounds of
directed evolution for secretion in yeast, organic co-solvent tolerance and
alkalophilicity [2-4]. With the help of a HTS-assay based on the oxidative
cross-coupling (heterocoupling N-C) of 2,5-diaminobenzenesulfonic acid
(2,5-DABSA) and policatechol, laccase mutant libraries were screened and
new variants with improved turnover rates under operational conditions
(alkaline pH, high temperature) were identified.
La prdida funcional de VAMP2 en clulas

plasmticas humanas provoca una disminucin
de la secrecin de inmunoglobulinas
Laura Gmez Jaramillo, Raquel Romero Garca, Ana B. Ramos
Amaya, Gema Jimnez Gmez, Jos A. Brieva, Antonio Campos Caro
Hospital Universitario Puerta del Mar, Cdiz, ES
Objetivo: Las clulas plasmticas (CP) son clulas secretoras por
excelencia que presentan una maquinaria exoctica muy desarrollada,
donde se engloban las protenas SNARE. Estas participan en todo proceso
de fusin de membrana que tenga lugar en la clula, gracias a la formacin
de un complejo constituido por tres miembros, uno de cada una de las
tres familias descritas de estas protenas; SNAP25, Syntaxinas y VAMP.
Resultados previos implican a SNAP-23 y STX4 en la secrecin de Igs. El
objeto de este estudio es definir qu miembro de la familia VAMP completa
el complejo que lleva a cabo la secrecin de Ig en CPs humanas.
Metodologa: Usamos la lnea celular U266. Caracterizamos la
expresin y distribucin de VAMP2 mediante ensayos de western blot
e inmunofluorescencia, respectivamente. Como estudios de prdida de
funcin realizamos tres estrategias; 1) Silenciamos su expresin con ARNi,
2) Sobreexpresamos la cadena ligera de la toxina tetnica, neurotoxina
que degrada VAMP2 y VAMP3 pero no afecta a la integridad de los
dems miembros de la familia y 3) Expresamos varias construcciones
de VAMP2 que consisten en distintas deleciones del fragmento
transmembrana de la protena con el fin de que compitan con la forma
normal endgena provocando la formacin de complejos no funcionales.
En los sobrenadantes resultantes de todos estos ensayos hemos medido la
secrecin de IgE mediante ELISA.
Resultados: La disminucin de los niveles de VAMP2 o su prdida de
funcin en las clulas provoca una disminucin en la secrecin de Igs. El
mismo efecto observamos cuando introducimos en la clula la protena
VAMP2 delecionada en el fragmento trasmembrana que se distribuye
deslocalizada por todo el citoplasma. Adems observamos que las vesculas
que transportan la IgE en clulas U266 colocalizan parcialmente con VAMP2.
Conclusin: VAMP2 es un claro candidato a ser el tercer SNARE que,
junto con SNAP23 y STX4, forman el complejo que lleva a cabo la
secrecin de Igs en CP. Si bien, la disminucin es parcial, indicando que
existen otras protenas SNARE o reguladoras implicadas en dicho proceso.
P10-60
Synthesis of polymeric dyes through oxidative

cross-coupling by directed laccase evolution
Ana Isabel Vicente Martn1, Susana Camarero Fernndez2, Miguel
Alcalde Galeote1
1
Instituto de Catlisis y Petroleoqumica, CSIC, Madrid, ES, 2Centro
de Investigaciones Biolgicas, CSIC, Madrid, ES
Fungal laccases (EC1.10.3.2) are blue-multicopper containing enzymes
capable of oxidizing phenols, polyphenols, aromatic amines and many
other compounds with the concomitant reduction of molecular oxygen
to water. The versatility of these enzymes can be expanded by the use of
redox mediators (from natural or synthetic origin) thereby finding plenty of
applications ranging from bioremediation to novel green processes. Besides,
P11. Genmica y protemica

P11-1 (R11-1)
Where proteomics is the approach in basic and

translational plant research, but how?
Jesus V. Jorrin Novo
Agroforestry and Plant Biochemistry and Proteomics; Dpt. of
Biochemistry and Molecular Biology, Crdoba, ES
Biological investigation and knowledge at the molecular level, whether
the experimental systems are plants or other living organisms, models or
orphans, is based and the result of data generated by the use of different and
complementary methodological approaches. Historically we moved from the
classical and in vitrotechniques (e.g. protein chemistry/biochemistry) to the
in vivo and the most recent holistic, omics, ones (e.g. proteomics), that with
mathematical and bioinformatics tools build the so-called Systems biology.
Nowadays, biological research should not be just the result of data
accumulation and blind acceptance of the huge amount of data generated
by the potent omics equipments and algorithms, as the result of that could
be, in the best of the cases, merely descriptive and speculative.
As indicated in the title of this presentation, and based on my own research
experience and the current literature, it is pretended to discuss how is or
could be the contribution of proteomics to the knowledge in these living
organisms. Why, when, and how to use proteomics should be the questions
to be answered. Finally, the challenges, power and limitations of the
approach will be evaluated.
P11-2 (R11-2)
La protena quinasa DYRK1A regula transcripcin

va su reclutamiento a promotores
Susana de la Luna
ICREA y Centro de Regulacin Genmica-CRG, Barcelona, ES
DYRK1A es una protena quinasa que participa en procesos celulares
relacionados con proliferacin, diferenciacin y supervivencia. La actividad
biolgica de esta quinasa es extremadamente dependiente de la dosis gnica
ya que su sobreexpresin se ha asociado a alteraciones del neurodesarrollo
asociadas al sndrome de Down, mientras que mutaciones en uno de los
alelos del gen codificante son responsables de un nuevo sndrome asociado
al locus MRD7 (Mental Retardation Autosomal Dominant 7).
DYRK1A est presente tanto en el ncleo como en el citoplasma de clulas de
mamfero, si bien la mayora de sus actividades nucleares pueden ser explicadas
por fosforilaciones que ocurren en el citosol. Estas observaciones han planteado
dudas sobre si esta quinasa posee funciones especficamente nucleares.
Mediante una aproximacin protemica no sesgada, hemos identificado
que la fraccin nuclear de DYRK1A interacciona con componentes de la
maquinaria basal de transcripcin. El anlisis a nivel genmico de la presencia
de DYRK1A en cromatina, mediante experimentos de ChIP-Seq, ha revelado
que la quinasa es reclutada a regiones proximales de promotores dependientes
103
Psters
de la RNA polimerasa II caracterizados por la presencia de una secuencia
palindrmica muy conservada, y enriquecidos en categoras funcionales
relacionadas con traduccin y procesamiento de RNA. La presencia de
DYRK1A en genes dependientes de la RNA polimerasa III tambin ha sido
detectada. Nuestros datos indican que la expresin de un grupo de genes diana,
dentro de estas categoras, depende de la actividad quinasa de DYRK1A.
Adems, la reduccin en los niveles de DYRK1A provoca una reduccin en el
tamao de las clulas. Los resultados permiten proponer un modelo por el que
DYRK1A podra regular directamente la expresin de genes diana mediante la
fosforilacin, en regiones reguladoras promotoras, de diferentes componentes
de la maquinaria basal de la transcripcin, contribuyendo a la coregulacin de
programas de expresin implicados en la homeostasis celular.
P11-3
Caracterizacin de la expresin de haptoglobina

en cncer colorrectal
scar Mario Crespo1, Elisa Cuevas lvarez2, Emilio Gil Martn1,
Almudena Fernndez Briera1
1
Departamento de Bioqumica, Gentica e Inmunologa, Universidad
de Vigo, Vigo, ES, 2Servicio de Anatoma Patolgica, Complejo
Hospitalario Universitario de Ourense, Ourense, ES
La haptoglobina (Hp) es una protena de fase aguda conocida por unir
hemoglobina durante la hemlisis, evitando as la prdida de hierro y el dao
renal. En un estudio reciente detectamos esta protena en especmenes de
pacientes con cncer colorrectal (CCR) como potencial portadora de CDw75,
un epitopo glucdico relacionado con la progresin de esta neoplasia.
Para proseguir con este trabajo nos hemos propuesto certificar la expresin
de la Hp en CCR mediante un estudio inmunohistoqumico utilizando
especmenes de tejido sano y tumoral (12 y 55, respectivamente). Adems,
hemos pretendido caracterizar su expresin y relacin con el CDw75
mediante western blot de fracciones enriquecidas en protenas citoslicas
o de membrana procedentes de especmenes de tejido sano y tumoral de
algunos de los pacientes empleados en el estudio histolgico.
Ninguno de los especmenes de tejido sano analizados por inmunohistoqumica
present expresin de Hp, mientras que en el tejido tumoral nicamente
se detect en 9 de los 55 ensayados (16 %). Cuando estaba presente, la
Hp apareci de forma tenue, focal y restringida a las clulas del epitelio
glandular. Adems, hemos comprobado que la expresin de esta protena se
asocia significativamente con la edad del paciente, el estadio de Dukes y la
presencia de metstasis.
Mediante western blot hemos acreditado la presencia preferencial de la Hp
en la fraccin citoslica y comprobado que su patrn de bandas coincide en
masa con el del epitopo CDw75. Por otro lado, no parece haber diferencias
de expresin de la Hp entre el tejido sano y el tumoral.
Estudio financiado mediante el Contrato-Programa CN 2011/024, Xunta
de Galicia.
P11-4
Nueva funcin del GEF C3G como regulador

del secretoma plaquetario: implicacin en la
patologa trombtica
Vctor Martn Granado1, Sara Gutirrez Herrero1, Sara Alonso
Alvarez2, Sara Ortiz Rivero1, Jos Ramn Gonzlez Porras2, Neibla
Priego Bendeck3, Almudena Porras Gallo3, Carmen Guerrero Arroyo1
1
Centro de Investigacin del Cncer, IBMCC (USAL-CSIC), IBSAL,
Salamanca, ES, 2Hospital Universitario de Salamanca, IBSAL,
Salamanca, ES, 3Departamento de Bioqumica y Biologa Molecular
II, Facultad de Farmacia, UCM, IdISSC, Madrid, ES
C3G es un factor intercambiador de nuclotidos de guanina (GEF) de Rap1,
una protena de la familia Ras muy abundante en plaquetas. Especficamente,
la isoforma Rap1b participa en aspecto crticos de la funcin plaquetaria a
104

travs de la activacin de la integrina IIb3. Adems, se sabe que C3G
juega un papel fundamental en la agregacin plaquetaria, tanto in vitro como
in vivo, a travs de la activacin de Rap1b. En base a todo esto, nuestra
hiptesis es que C3G puede influir en la secrecin durante la activacin
plaquetaria. Para estudiar esta cuestin, hemos utilizado lneas de ratones
transgnicos para C3G (tgC3G) y C3GCat (un mutante con delecin del
dominio cataltico) donde ambos transgenes se expresan bajo el promotor
del gen PF4, exclusivo de megacariocitos y plaquetas. La evaluacin de la
cantidad de protenas secretadas, tras la activacin por trombina, mostr que
las plaquetas tgC3G secretaron mayor cantidad que los controles, mientras
que las plaquetas transgnicas para C3GCat secretaron una menor cantidad.
El perfil de secrecin proteica de las plaquetas tgC3G tambin fue diferente
al encontrado en los dems grupos hallndose una mayor variedad de
protenas. Un estudio protemico preliminar revel que entre las protenas
diferencialmente secretadas por las plaquetas tgC3G se encontraban
apolipoprotenas, inhibidores de proteasas, fibringeno, hemoglobina y
trombospondina-1, las cuales son de gran inters por estar involucradas en el
desarrollo de diversas patologas cardiovasculares. Estos resultados permirn
profundizar en el papel de C3G como regulador de la funcin plaquetaria
y abren nuevas perspectivas sobre sus posibles efectos fisio-patolgicos,
especialmente en el contexto de patologas trombticas.
P11-5
Efecto del clima en las comunidades bacterianas

asociadas al suelo rizosfrico en el Parque
Nacional de Sierra Nevada
Javier Pascual1, Silvia Blanco1, Juan Luis Ramos2, Pieter van
Dillewijn1
1
Departamento de Proteccin Ambiental, Estacin Experimental del
Zaidn, CSIC, Granada, ES, 2ABENGOA RESEARCH, Sevilla, ES
Teniendo en cuenta que los microorganismos presentes en el suelo, y
especialmente en el suelo asociado a las races de las plantas, la rizosfera,
ejercen un papel clave en la dinmica y el funcionamiento de los
ecosistemas, es necesario conocer cmo las comunidades bacterianas se
ven influenciadas por los cambios climticos que se producen en el medio
ambiente. Uno de los lugares que puede ser utilizado como modelo para
evaluar el efecto del clima es el Parque Nacional de Sierra Nevada, debido
a la clara estratificacin termoclimtica que existe en el macizo montaoso.
El objetivo del presente trabajo ha consistido en el anlisis de la diversidad
taxonmica y funcional de las comunidades bacterianas asociadas a la
rizosfera del tomillo salsero (Thymus zygis L.) y al suelo suelto, presente
en diferentes pisos termoclimticos de dos regiones de Sierra Nevada,
Capileira y Puerto de la Ragua.
Para llevar a cabo el estudio se ha analizado el ADN metagenmico de las
comunidades bacterianas presentes en las diferentes muestras de suelo: (i)
la diversidad taxonmica se ha analizado mediante pirosecuenciacin de
amplicones del gen ARNr 16S, mientras que (ii) la diversidad funcional se
ha estudiado utilizando GeoChips.
Los resultados obtenidos hasta la fecha muestran que el clima ejerce un papel
clave en la estructura y funcionalidad de las comunidades bacterianas del
suelo. Este trabajo forma parte de un estudio ms amplio donde se analizar
a su vez la dinmica de las comunidades bacterianas a escala temporal.
P11r-6
Anlisis transcriptmico mediante RNA-seq de

genes de respuesta a cido linolnico implicados
en situaciones de estrs abitico en Arabidopsis
Capilla Mata Prez1, Beatriz Snchez-Calvo1, Juan Carlos BegaraMorales1, Mara N Padilla-Serrano1, Raquel Valderrama1, Francisco
Luque2, Ana Fernndez-Ocaa1, Francisco J Corpas3, Juan B Barroso1
1
Granada 2014
de Jan, Jan, ES, 2Departamento de Biologa Experimental,
Universidad de Jan, Jan, ES, 3Grupo de Antioxidantes, Radicales
Libres y xido Ntrico en Biotecnologa y Agroalimentacin,
Departamento de Bioqumica, Biologa Molecular y Celular de
Plantas, Estacin Esperimental del Zaidn, CSIC, Granada, ES
El cido linolnico (LnA) es un cido graso poliinsaturado esencial de
la serie omega-3, precursor de la fitohormona cido Jasmnico (JA),
implicada en diversas situaciones de estrs abitico que cursan con
la sobreproduccin de especies de oxgeno reactivo (ROS) [1,2]. Con
el objetivo de tener un conocimiento ms amplio acerca de los genes
implicados en la respuesta a LnA, se realiz un anlisis transcriptmico en
cultivos celulares de A. thaliana, mediante la tecnologa de secuenciacin
a gran escala Illumina-mRNA-Seq. Se analiz el comportamiento
de genes que responden a la sealizacin por LnA implicados en la
generacin de un fenmeno de estrs oxidativo durante situaciones de
estrs abitico, identificndose un total de 3275 genes que respondan
a LnA. La mayora estaban relacionados con la ruta biosinttica del
JA, como la Lipoxigenasa (LOX) o Aleno xido Ciclasa (AOC) [3].
Sin embargo, una parte importante de ellos estaba relacionada con el
mecanismo de respuesta frente a diversas situaciones de estrs abitico,
como la galactinol sintasa 1 (GOLS1), diferentes enzimas detoxificadoras
de grupos carbonilo y diferentes deshidrogenasas. En definitiva, el
anlisis funcional mediante tecnologa RNA-seq permite ampliar nuestro
conocimiento sobre la conexin de ROS y la ruta de sealizacin del JA,
evidenciando que una parte significativa de estos genes codifica protenas
relacionadas con la respuesta de la planta a diferentes situaciones de
estrs y estmulos abiticos.
Bibliografa
[1] Suza WP et al. (2010) Plant Physiol Biochem 48:337-50.
[2] Hu X et al. (2009) Plant Signal Behav 4:696-7.
[3] Wasternack C, Hause B (2013) Ann Bot.
Financiado por Ministerio de Economa y Competitividad (proyectos
BIO2012-33904, AGR-6374, AGR-6038, 20134R0569) y Junta de
Andaluca (grupos BIO286 y BIO192).
P11-7 (R11-4)
MicroRNAs in tumor development

Pedro Medina Vico
MiRNAs are a class of small RNA molecules that regulate gene expression
at the posttranscriptional level. Initially discovered in Caenorhabditis
elegans, they were considered an oddity of nematodes until it was
realized that some of them were phylogenetically conserved in a wide
variety of animals including humans. Today, miRNAs are increasingly
seen as important regulators of gene expression, ushering in a renewed
appreciation of the regulative capabilities of ncRNA. At the cellular level,
miRNAs are significant in the establishment and maintenance of cell
identity. Aberrant levels of miRNAs often result in loss of differentiation,
a hallmark of cancer. Not surprisingly, therefore, dysfunctions of the
miRNA pathway affect many cellular processes that are routinely altered
in cancer, such as differentiation, proliferation, apoptosis, metastasis, and
senescence.
Although the miRNA era started only a few years ago, it has brought
great promise for diagnosis, prognosis, and therapy of cancer. The quick
development of powerful techniques such as miRNA microarrays, shortRNA deep sequencing, specific quantitative polymerase chain reaction
(PCR) of miRNAs, and antisense technologies is expected to have a
significant impact on clinical oncology in the next decade.
Psters
P11-8
Bsqueda de termoenzimas hidrolticas en una

metagenoteca de aguas termales
Juan Jos Escuder Rodrguez, Kamila Knapik, Mara Isabel
Gonzlez Siso, Manuel Becerra Fernndez
Universidad de A Corua, A Corua, ES
La metagenmica funcional, es decir, el estudio del genoma colectivo de una
comunidad microbiana presente en una muestra ambiental, es una poderosa
herramienta para explotar la gran diversidad de microorganismos no
cultivables en la bsqueda de nuevas enzimas con aplicacin industrial. La
tcnica se basa en la extraccin del material gentico presente en la muestra
ambiental y su clonacin en un hospedador heterlogo para la obtencin
de una biblioteca metagenmica (metagenoteca) en la que identificar
actividades enzimticas de inters aadiendo los sustratos adecuados al
medio de cultivo. Esta metodologa, en comparacin con la metagenmica
basada en secuencia, permite encontrar nuevos productos gnicos que
no presentan homologa significativa con las secuencias conocidas y
depositadas en las bases de datos. Determinados procesos industriales se
llevan a cabo en condiciones adversas para la mayora de microorganismos,
tales como elevadas temperaturas, pH extremo, alta salinidad o alta
concentracin de solventes. En este sentido, es comn la bsqueda de
enzimas procedentes de comunidades microbianas de ambientes extremos,
en los que se dan condiciones similares. En este trabajo se ha construido
una metagenoteca a partir de una muestra de aguas termales para la
bsqueda de enzimas hidrolticas estables a elevadas temperaturas. Debido
a que los fenmenos de transcripcin, traduccin, plegamiento y secrecin
del producto gnico pueden ser incorrectos al emplear como hospedador la
bacteria mesfila Escherichia coli, se ha empleado un vector lanzadera que
permite la transferencia de la metagenoteca a la bacteria termfila Thermus
thermophilus para incrementar los productos gnicos detectables.
P11m-9
Identificacin de biomarcadores y nuevas

protenas relacionadas con la enfermedad
aterosclertica en pacientes con diabetes
mellitus tipo 2
Beatriz Garca-Fontana1, Sonia Morales-Santana2, Victoria
Longobardo3, Antonia Garca-Martn1, Rebeca Reyes-Garca1, Pedro
Rozas-Moreno4, Jos Antonio Garca-Salcedo5, Manuel MuozTorres1
1
Unidad de Metabolismo seo (RETICEF), Servicio de
Endocrinologa, Instituto de Investigaciones Biomdicas de
Granada, Hospital Universitario San Cecilio, Granada, ES, 2Unidad
de Metabolismo seo (RETICEF), Servicio de Endocrinologa,
Instituto de Investigaciones Biomdicas de Granada, Hospital
Universitario San Cecilio - Servicio de Protemica, Fundacin
para la Investigacin Biosanitaria de Andaluca Oriental -Alejandro
Otero- (FIBAO) , Granada, ES, 3Servicio de Protemica, Instituto
de Parasitologa y Biomedicina Lpez Neyra (CSIC), Granada,
ES, 4Servicio de Endocrinologa, Hospital General de Ciudad Real,
Ciudad Real, ES, 5Unidad de Enfermedades Infecciosas, Instituto
de Investigaciones Biomdicas de Granada, Hospital Universitario
San Cecilio, Granada, ES
La prevalencia de diabetes mellitus est aumentando rpidamente
alcanzando proporciones epidmicas. Esta enfermedad se asocia a
numerosas complicaciones, siendo la enfermedad aterosclertica (EA) una
de las ms relevantes debido a su morbimortalidad. Por ello, la identificacin
precoz de pacientes diabticos con alto riesgo de padecer EA es relevante
para reducir el impacto de esta enfermedad y sus complicaciones. El
objetivo de este estudio fue comparar el proteoma srico de pacientes con
diabetes mellitus tipo 2 (DM2) con o sin EA con el de sujetos sanos para
identificar biomarcadores o protenas esenciales que intervengan en la
patognesis de la DM2 y la EA.
105
Psters
Se realiz un estudio protemico incluyendo 18 hombres divididos en tres
grupos: i) 6 pacientes con DM2 y EA, ii) 6 pacientes con DM2 sin EA; iii)
6 controles sanos. Se realiz una deplecin proteica de las 14 protenas
mayoritarias para mejorar la sensibilidad de deteccin en suero. Los perfiles
proteicos fueron separados mediante 2D-DIGE y las imgenes obtenidas
fueron analizadas mediante el software especfico DeCyder 7.0. Las
protenas expresadas diferencialmente se identificaron por espectrometra
de masas-masas (MALDI TOF-TOF) o cromatografa lquida acoplada a
espectrometra de masas (LC-MS/MS).
Nuestros resultados muestran una sobreexpresin de la protena de unin
a retinol plasmtico (RBP) y de la glutation peroxidasa 3 (GPx -3) y una
disminucin de los niveles sricos de Transtirretina (TTR) en pacientes con
DM2 y EA en comparacin con pacientes diabticos sin EA y controles
sanos.
El anlisis protemico revela cambios en protenas relacionadas con
procesos inflamatorios en pacientes con DM2 y EA sugiriendo que el
proceso inflamatorio juega un papel muy importante en el desarrollo de
complicaciones vasculares en la DM2. Estas protenas podran actuar como
biomarcadores o dianas teraputicas de la enfermedad aterosclertica en
pacientes con DM2 mejorando la supervivencia de estos pacientes.
P11r-10 (R11-5)
Fucosylation increase in -1 acid glycoprotein

(AGP) in pancreatic cancer
Meritxell Balmaa1, Estela Gimnez2, ngel Puerta3, Esther Llop1,
Carme de Bols4, Slvia Barrabs1, Mercedes de Frutos3, Rosa
Peracaula1
1
Biochemistry and Molecular Biology Unit, Department of Biology,
University of Girona, Girona, ES, 2Department of Analytical
Chemistry, University of Barcelona, Barcelona, ES, 3Institute of
Organic Chemistry (CSIC), Madrid, ES, 4Institute Hospital del Mar
of Medical Investigations (IMIM), Barcelona, ES
Pancreatic cancer (PDAC) is the fourth leading cause of cancer death
[1]. Its low survival rate (around 5% in 5 years) is due to the intrinsic
aggressiveness of this tumour and also to a late diagnosis. The only
biomarker available for PDAC is CA19-9, but it is currently only used to
monitor the pathology because of its false positive results.
Previously, our group performed N-glycan analysis of major serum acute
phase proteins, including -1 acid glycoprotein (AGP), in pancreatic
cancer, chronic pancreatitis and healthy control sera and concluded that
increase in core fucosylation in AGP could be cancer associated [2].
In this study, AGP from serum samples from a larger cohort (N=31) was
purified using an anti-AGP immunoaffinity column. Different ELISAs
using specific fucose recognizing lectins and antibodies against fucosylated
epitopes were performed to determine differences in AGP glycoepitopes
between the groups. In addition, relative quantitation of AGP glycosylation
variants by stable isotope labelling of AGP released N-glycans was
carried out using [12C]/[13C] aniline and ZIC-HILIC-ESI-TOF-MS [3].
Furthermore, AGP isoforms were analysed by capillary electrophoresis
(CE-UV) [4]. An increase in fucosylation levels using Aleuria aurantia
lectin was found in PDAC patients compared to chronic pancreatitis and
healthy controls and this increase was more marked in the advanced
PDACs. The sialofucosylated antigen (sialyl-Lewis x) also tended to be
increased in the AGP of the PDAC group. Complementarily, MS results
showed an increase in the levels of certain fucosylated glycan structures
between chronic pancreatitis and PDAC. Moreover, significantly different
profiles by CE-UV, which separates AGP isoforms, were obtained between
AGP from controls and PDACs. Altogether these results suggest that AGP
fucosylation levels could be useful as a PDAC marker.
References:
[1] Siegel R, et al. CA Cancer J Clin 2014.
[2] Sarrats A, et al. Proteomics Clin Appl 2010.
[3] Gimenez E, et al. Anal Bioanal Chem 2013.
[4] Puerta A, et al. Methods Mol Biol 2013.
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P11-11
Identificacin de protenas celulares de unin

al elemento CRE del genoma del virus de la
hepatitis C
Pablo Ros Marco, Cristina Romero Lpez, Alba Fernndez Sanls,
Alfredo Berzal-Herranz
Armilla, ES
El genoma del virus de la hepatitis C (HCV) es un RNA monocatenario
de polaridad positiva con una longitud aproximada de 9,6 Kb. Existen
diversos dominios estructurales en su genoma que destacan por su
capacidad para regular procesos esenciales para el virus tales como su
traduccin y replicacin. Dos de estos dominios son el lugar de entrada
interna del ribosoma (IRES) o la regin 3X-tail, situados en las regiones
no traducibles 5UTR y 3UTR respectivamente. Otro dominio de gran
inters en el genoma del virus es el denominado cis-replicating element
(CRE), presente en el extremo 3 de la regin codificante del virus. CRE
tiene capacidad de interactuar con IRES o 3X-tail y as regular cambios
necesarios entre las etapas de traduccin y replicacin del ciclo infectivo del
virus. En este trabajo hemos tratado de identificar protenas celulares que
muestren asociacin con el elemento CRE y puedan afectar a su interaccin
con otros dominios del genoma viral. As, experimentos de incubacin del
fragmento CRE con lisados celulares en condiciones de competicin con
IRES, con el propio CRE o con un RNA no relacionado, fueron utilizados
para demostrar la especificidad de unin de CRE a protenas celulares. Las
protenas obtenidas tras los ensayos de pull-down se analizaron mediante
electroforesis desnaturalizante (SDS-PAGE) 1D y cromatografa lquida
acoplada a espectrometra de masas (LC-MS/MS). Estas tcnicas nos han
permitido identificar un conjunto de protenas que interactan con la regin
CRE del HCV entre las que destacan miembros de la familia hnRNP,
tubulinas, helicasas y factores relacionados con la traduccin. Asimismo,
ensayos de unin de CRE a subunidades ribosomales demuestran que CRE
se une especficamente a la subunidad 40S. Todos estos resultados son de
gran importancia para investigar la relacin de CRE con diferentes factores
celulares y otros dominios del virus y as poder comprender cmo dichas
interacciones afectan a procesos biolgicos del virus.
P11-12
Efecto del probitico Shewanella putrefaciens

Pdp11 en el proteoma heptico del lenguado
senegals (Solea senegalensis) cultivado a
diferentes densidades de biomasa
Juan Jurado1, Trnsito Garca-Garca1, Miguel ngel Morigo2,
Mara Jos Prieto-lamo1
1
Dpto. Bioqumica y Biologa Molecular, Universidad de Crdoba,
Crdoba, ES, 2Dpto. Microbiologa, Universidad de Mlaga, Mlaga, ES
La introduccin de nuevas especies es uno de los desafos pendientes para
desarrollar una acuicultura productiva, viable y sostenible. En este sentido,
el lenguado senegals (Solea senegalensis) es una especie con alto potencial
en la acuicultura marina espaola. Sin embargo, la escasez de herramientas
veterinarias constituye una seria limitacin para la implantacin del cultivo
intensivo del lenguado, muy susceptible a patologas infecciosas por
Photobacterium, Vibrio y Tenacibaculum, sobre todo cuando se cultiva a
densidades elevadas de biomasa. Los probiticos (PB) constituyen una opcin
interesante ya que en los peces aumentan tanto la eficiencia de la alimentacin
como la resistencia a las enfermedades y a otras situaciones de estrs.
Con el objeto de investigar las bases moleculares de este efecto beneficioso
a nivel de expresin de protenas, en este trabajo hemos estudiado el
efecto del PB Shewanella putrefaciens Pdp11 en el proteoma heptico
del lenguado senegals cultivado a dos densidades diferentes: (i)
densidad normal (DN: 7 Kg/m2), y (ii) densidad alta (DA: 30 Kg/m2).
Este estudio se llev a cabo mediante la separacin de protenas por
electroforesis bidimensional en geles desnaturalizantes de poliacrilamida
Granada 2014
(2-DE) utilizando tiras de amplio rango de pH (3-11NL). Las protenas
diferencialmente expresadas se identificaron mediante el uso combinado
de huella peptdica y espectrometra de masas en tandem (PMF + MS/MS).
El tratamiento de los peces con el PB provoc cambios en la expresin de
6 protenas a DN y de 2 a DA de cultivo. Por otra parte, el estrs generado
por el cultivo de los peces a DA en ausencia de PB, afect a la expresin de
11 protenas, produciendo mayoritariamente incrementos de expresin. Por el
contrario, en presencia de PB, la DA provoc principalmente disminuciones
de expresin. Entre las protenas afectadas, hay protenas implicadas en el
metabolismo de glcidos (PGAM1, GAPDH y F16P1), en el metabolismo de
aminocidos y protenas (LAP, PHGDH, FAAA, y SPYA), en la detoxificacin
de xenobiticos (GSTR y GSTT) o en el transporte de hierro (TF).
Financiacin: AGL2011-30381-CO3-03.
P11-13
Nueva funcin del GEF C3G como regulador

del secretoma plaquetario: implicacin en la
patologa trombtica
Victor Martin Granado1, Sara Gutirrez Herrero1, Sara Alonso
Alvarez2, Sara Ortiz Rivero1, Jos Ramn Gonzlez Porras2, Neibla
Priego Bendeck3, Almudena Porras Gallo3, Carmen Guerrero Arroyo1
1
Centro de Investigacin del Cncer, IBMCC (USAL-CSIC), IBSAL,
Salamanca, ES, 2Hospital Universitario de Salamanca, IBSAL,
Salamanca, ES, 3Departamento de Bioqumica y Biologa Molecular
II, Facultad de Farmacia, UCM, IdISSC, Madrid, ES
C3G es un factor intercambiador de nuclotidos de guanina (GEF) de Rap1,
una protena de la familia Ras muy abundante en plaquetas. Especficamente,
la isoforma Rap1b participa en aspectos crticos de la funcin plaquetaria
a travs de la activacin de la integrina IIb3. Adems, se sabe que C3G
juega un papel fundamental en la agregacin plaquetaria, tanto in vitro como
in vivo, a travs de la activacin de Rap1b. En base a todo esto, nuestra
hiptesis es que C3G puede influir en la secrecin durante la activacin
plaquetaria. Para estudiar esta cuestin, hemos utilizado lneas de ratones
transgnicos para C3G (tgC3G) y C3GCat (un mutante con delecin del
dominio cataltico) donde ambos transgenes se expresan bajo el promotor
del gen PF4, exclusivo de megacariocitos y plaquetas. La evaluacin de la
cantidad de protenas secretadas, tras la activacin por trombina, mostr que
las plaquetas tgC3G secretaron mayor cantidad que los controles, mientras
que las plaquetas transgnicas para C3GCat secretaron una menor cantidad.
El perfil de secrecin proteica de las plaquetas tgC3G tambin fue diferente
al encontrado en los dems grupos hallndose una mayor variedad de
protenas. Un estudio protemico preliminar revel que entre las protenas
diferencialmente secretadas por las plaquetas tgC3G se encontraban
apolipoprotenas, inhibidores de proteasas, fibringeno, hemoglobina y
trombospondina-1, las cuales son de gran inters por estar involucradas en el
desarrollo de diversas patologas cardiovasculares. Estos resultados permirn
profundizar en el papel de C3G como regulador de la funcin plaquetaria
y abren nuevas perspectivas sobre sus posibles efectos fisio-patolgicos,
especialmente en el contexto de patologas trombticas.
P11-14
Efecto del probitico Shewanella putrefaciens

Pdp11 sobre la expresin transcripcional del
lenguado senegals (Solea senegalensis) en
respuesta a factores de estrs fisiopatolgico
Mara-Jos Prieto-lamo1, Laura Maldonado-Escudero1, Silvana T
Tapia-Paniagua2, Miguel ngel Morigo2, Juan Jurado1
1
Cordoba, ES, 2Dpto. Microbiologa, Universidad de Mlaga, Mlaga, ES
El cultivo del lenguado senegals (Solea senegalensis), un organismo no
modelo de inters para la acuicultura espaola, se ve dificultado por su
Psters
susceptibilidad a diversas patologas y situaciones de estrs, sobre todo
cuando se cultiva a altas densidades de biomasa, condiciones necesarias
para la rentabilidad de las explotaciones. Actualmente, la acuicultura
europea carece de herramientas veterinarias suficientes lo que supone una
limitacin para el desarrollo de este sector. Los probiticos son una opcin
interesante ya que parecen aumentar la eficiencia de la alimentacin, la
tolerancia a situaciones de estrs, y la resistencia a enfermedades. El
anlisis de los patrones de expresin transcripcional aporta una valiosa
informacin sobre el funcionamiento coordinado de los genes en respuesta
a distintas variables. Hemos analizado, mediante qRT-PCR en tiempo real,
los perfiles hepticos de expresin transcripcional de S. senegalensis, en
respuesta a estrs por sobrepoblacin y a una infeccin por bacterias del
genero Vibrio. En ambos casos se ha investigado el efecto del probitico
Shewanella putrefaciens Pdp11. Se han cuantificado 28 transcritos que
codifican protenas implicadas en la respuesta inmune, respuesta a estrs,
metabolismo central y crecimiento, y proteasas relacionadas con varios
procesos biolgicos. Los resultados muestran que los lenguados cultivados
a alta densidad presentan niveles disminuidos de varios transcritos de
la respuesta inmune, lo que explicara su mayor susceptibilidad a sufrir
infecciones. Por el contrario la infeccin indujo la expresin de la mayora
de los genes relacionados con la respuesta inmune. En las condiciones
analizadas, el probitico no tuvo un efecto apreciable sobre los cambios de
expresin debidos a la sobrepoblacin, pero produjo una mayor resistencia
a la infeccin en estos lenguados, lo que se relaciona con la recuperacin de
niveles normales de los transcritos de la respuesta inmune. Los resultados
respaldan el efecto beneficioso de S. putrefaciens Pdp11 como tratamiento
profilctico en situaciones de susceptibilidad a patgenos durante el cultivo
del lenguado.
Financiacin: AGL2011-30381-CO3-03.
P11-15
iTRAQ analysis of hepatic proteins in free-living

Mus spretus mice to assess the contamination
status of areas surrounding Doana National Park
Nieves Abril Daz, Eduardo Chicano-Galvez, Carmen Michn Doa,
Carmen Pueyo de la Cuesta, Juan Lpez-Barea
Crdoba, ES
Doana National Park (DNP), a Biosphere Reserve located at SW
Spain, includes some of the most environmentally valuable ecosystems
in Europe. Nevertheless, DNP is threatened by pesticides, metals and
contaminants carried out by local streams and Guadalquivir River. A
follow-up study started by combining conventional biomarkers and 2-DE
proteomic approaches that resolved near 3,000 protein spots. Based on
these pioneering studies, we have combined several omics approaches
for the identification of molecular biomarkers of pollution exposure,
using the aboriginal species Mus spretus as bioindicator. This mouse is
phyllogenetically related to Mus musculus, thus facilitating the use of its
gene/protein sequence databases for massive identification of proteins and
transcripts with altered expression. Here we report the results of a second
generation proteomic study in free-living M. spretus from neighboring
areas of Doana National Park (DNP) with different pollution levels.
Liver proteins from mice captured at the Matochal rice fields (MAT),
the Rocina stream (ROC), two sites of the Partido streams (PAR,
AJO) and the reference site Lucio del Palacio (LP), were labeled
with iTRAQ-8plex tags to quantify differentially expressed proteins. A
previous isoelectric focusing step was included to improve separation of
the highly complex peptide mixture. Mass spectrometry was carried out in
a LTQ Orbitrap system for protein dentification and iTRAQ reporter ion
quantitation using the Mus musculus database as reference. Of the near
100,000 peptides sequenced, 1032 proteins were identified and quantified
by iTRAQ reporter ions, leading to the identification of 118 proteins that
were significantly altered (> 2.5 fold variation) in at least two problem areas
(PAR, AJO, ROC or MAT) compared to the reference (LP). The results
107
Psters
of iTRAQ analysis were confirmed by Western blotting. The identified
proteins provide insight about the different defense strategies in free-living
mice affected by pollutants in DNP and its surrounding areas (CTM201238720-C03-02, CVI-3829, CTM2009-12858-CO2-02, BIO1675).
P11r-16
Anlisis de la expresin de la protena

CD26/DPPIV en diferentes lneas celulares
de cncer colorrectal
Marta Rodrguez Quiroga1, Lorena Vzquez-Iglesias1, Leticia Barcia
Castro1, Andrea Daz Daz1, Oscar J Cordero2, Fco.Javier Rodriguez
Berrocal1, Mara Pez de la Cadena1
1
Dpto.Bioqumica, Gentica e Inmunologa, Universidad de Vigo,
Vigo, ES, 2Dpto.Bioquimica y Biologa Molecular, Ed. CIBUS,
Universidad de Santiago de Compostela, Santiago de Compostela,
ES
La protena CD26 (dipeptidil peptidasa IV, DPPIV; EC 3.4.14.5) es una
glicoprotena transmebrana exopeptidasa, localizada en la superficie
celular de linfocitos, clulas endoteliales y epiteliales. Sus funciones,
dependientes e independientes de la actividad enzimtica parecen estar
relacionadas con la regulacin de respuestas inflamatorias e inmunolgicas,
la transduccin de seales y la apoptosis, sugiriendo su implicacin en
la progresin tumoral. Tambin se encuentra en forma soluble en varios
fluidos biolgicos y hemos propuesto su utilidad como biomarcador en
cncer colorrectal (CCR). Se ha descrito como supresor tumoral tanto en
cncer de pncreas como en melanoma y hay indicios de que cumple esta
funcin en CCR.
Numerosos estudios muestran que las clulas tumorales reactivan lo que
se conoce como Transicin Epitelio-Mesnquima (EMT). En su intento
por liberarse de su confinamiento habitual, las clulas sufren una serie de
cambios que conllevan a una mayor capacidad migratoria, invasividad y
una elevada resistencia a apoptosis. Las clulas que estn sufriendo esta
transicin se pueden identificar generalmente por la presencia o ausencia
de determinadas molculas que las dotan de una estructura ms flexible,
capacidad de viajar, y por una forma diferente a la de la clula epitelial.
En este trabajo nos propusimos analizar la expresin de la protena CD26
y de dos protenas implicadas en la EMT: E-cadherina y vimentina en 8
lneas celulares derivadas de cncer colorrectal de diferentes estadios:
SW1116, SW480, SW620, DLD-1, COLO205, T84, HT-29 y Caco-2
mediante tcnicas de western blot y citometra de flujo.
Los resultados muestran que las lneas SW480 y SW620 presentan
un fenotipo mesenquimal ya que tienen niveles elevados de la protena
vimentina mientras que los niveles de E-cadherina son significativamente
menores a los de las otras lneas celulares analizadas que muestran un
fenotipo de tipo epitelial. Los resultados obtenidos tanto por citometra
como por western blot indican que la protena CD26 se expresa casi
exclusivamente en clulas de fenotipo epitelial.
Financiacin: AECC (GCB13131592CAST), INBIOMED (CN2012/273),
REGICC (CN2012/217).
P11m-17
Estudio del interactoma de Ixr1 en Saccharomyces

cerevisiae
Aida Ins Barreiro Alonso, ngel Vizoso Vzquez, Mnica Lamas
Maceiras, Mara Esperanza Cerdn Villanueva
Universidad de A Corua, A Corua, ES
Ixr1 es una protena HMG de la levadura Saccharomyces cerevisiae. Dicha
protena se identific por su unin a DNA platinado. El cisplatino es una
droga antitumoral empleada principalmente para el tratamiento de tumores
slidos como los presentes en el cncer de ovario, testculo o cuello [1].
Ixr1 es, adems, un factor transcripcional implicado en la adaptacin
108

a condiciones de normoxia e hipoxia. As, se ha visto que reprime la
expresin aerobia de genes como COX5B [2], TIR1 [3] y HEM13 [4]
as mismo tambin se ha observado que en el caso de HEM13 activa su
expresin en condiciones de anaerobiosis. Por otra parte Ixr1 participa en
la respuesta a estrs oxidativo, ya que se ha determinado que su ausencia
incrementa la sensibilidad a la presencia de perxidos [5]. Estructuralmente
Ixr1 se caracteriza por la presencia de dos dominios HMG dispuestos en
tndem, y varias regiones de poliglutaminas. Diversos estudios han puesto
de manifiesto que Ixr1 interacciona con DNA platinado a travs de sus
dominios HMG [1].
Para poder profundizar en el conocimiento de las diferentes funciones en las
que est implicado Ixr1 es necesaria la bsqueda de nuevas interacciones
de esta protena. El sistema TAP (Tandem Affinity Purification) y el sistema
de doble hbrido son dos tcnicas de gran utilidad a la hora de identificar
nuevas interacciones entre protenas. En este trabajo se han empleado
estos dos mtodos para encontrar posibles interacciones de Ixr1 con
otras protenas en Saccharomyces cerevisiae. Para ello se ha optimizado
el mtodo TAP empleando diferentes etiquetas/eptopos y mejorando las
condiciones de purificacin.
Bibliografa
[1] M.M . McANulty et al., Mutat. Res. (1996), 75-86.
[2] J.R Lambert et al., Proc. Natl. Acad. Sci. USA (1994), 91:7345-9.
[3] J.P. Bourdineaud et al., Mol. Microbiol. (2000), 38:879-890.
[4] R. Castro-Prego et al., FEMS Yeast Res. (2010), 10:309-321.
[5] R. Castro-Prego et al., Biochem. J. (2010), 425(1):235-43.
P11r-18
Niveles de protena y actividad enzimtica

de MMP-9 en cncer de pulmn no microctico
Leticia Barcia Castro1, Sonia Blanco Prieto1, Lorena Vzquez
Iglesias1, F.J. Rodriguez-Berrocal1, M. Isabel Botana Rial2, M. Paez
de la Cadena1
1
Dpto. Bioqumica, Gentica e Inmunologa, Universidad de
Vigo, Vigo, ES, 2Servicio de Neumologa, Complexo Hospitalario
Universitario de Vigo, Vigo, ES
Las enzimas Metaloproteasas de Matriz Extracelular (MMP) juegan un
papel esencial en la progresin del tumor en distintos tipos de cncer. En
particular la MMP9 o gelatinasa B degrada componentes de la matriz como
el colgeno tipo IV por lo que se la relaciona con procesos de invasin
y metstasis. Aunque la funcin reside en su actividad enzimtica, la
mayora de los estudios sobre su valor como marcador tumoral se realizan
cuantificando los niveles de protena.
El objetivo de nuestro estudio es determinar en el suero de pacientes con
cncer de pulmn no microctico (CPNM), los niveles de protena y de
actividad enzimtica de MMP-9 mediante tcnicas de ELISA y ensayos
de zimografa. En las zimografas se cuantific la actividad atendiendo a
sus diferentes isoformas: proMMP9 (92 kDa), el conjunto de la proMMP9
(92 kDa) y la pro-MMP9-NGAL (130 kDa) y finalmente, la MMP9 total
(proMMP9, proMMP9-NGAL y Homodmero proMMP9).
Los resultados muestran que los niveles de MMP-9 (tanto de protena como
de actividad) se incrementan en las muestras de pacientes con CPNM en
comparacin con el grupo control. La media de los niveles de protena en el
grupo control es de 209,04148,6 (ng/mL) frente a 392,29241,61 (ng/mL)
en CPNM. En el caso de la actividad, considerando el conjunto constituido
por la proMMP9 y proMMP9-NGAL, los valores para el grupo control
son de 16,5711,38 (u.a.) frente a 25,3913,9 (u.a.) de las muestras con
CPNM. El estudio de correlacin entre los niveles de MMP-9 obtenidos
mediante zimografa y los obtenidos por ELISA muestran correlaciones
diferentes dependiendo de la banda de actividad considerada siendo el
conjunto proMMP9 y proMMP9-NGAL el que presenta mejor correlacin
con los niveles sricos.
Financiacin: Agrupamento Inbiomed 2012/73, Proyecto PS09-00405,
Instituto de Salud Carlos III y fondos FEDER.
Granada 2014
Psters
P11-19
P11-21
Anlisis mediante pirosecuenciacin del grado

de metilacin del gen SEPT9 en suero para el
diagnstico de cncer colorrectal
Anlisis protemico de vesculas de membrana

externa aisladas de cultivos de la cepa probitica
Escherichia coli 1917 en medio DMEM
Loretta De Chiara, Olalla Otero-Estvez, Mara Pez de la Cadena,

Francisco Javier Rodrguez Berrocal, Vicenta Soledad Martnez Zorzano
Departamento de Bioqumica, Gentica e Inmunologa, Facultad de
Biologa, Universidad de Vigo, Vigo, ES
Lorena Toloza Maturana, Laura Aguilera Gil, Mara Jos Fbrega

Fernndez, Rosa Gimnez Claudio, Laura Baldom Llavins, Josefa
Bada
Departament de Bioqumica i Biologia Molecular, Facultat de
Farmcia, Universitat de Barcelona, Institut de Biomedicina de la
UB (IBUB), Barcelona, ES
Recientemente, tras analizar cualitativamente el estado de hipermetilacin

del gen SEPT9, ste ha sido propuesto como marcador diagnstico
para cncer colorrectal (CCR). El objetivo de este trabajo es cuantificar
mediante pirosecuenciacin el porcentaje de metilacin de SEPT9 en
suero de individuos sometidos a colonoscopia. Adicionalmente se evalu
su valor diagnstico para detectar casos de CCR y de neoplasia avanzada
(adenomas avanzados o CCR).
Por pirosecuenciacin se estudiaron 7 sitios CpG incluidos en la regin
promotora del gen, en muestras de suero de: 21 individuos sin hallazgos, 15
con patologas benignas, 19 con adenomas no avanzados, 7 con adenomas
avanzados y 22 casos de CCR. Para evaluar la capacidad diagnstica se
construyeron curvas ROC y se calcularon las reas bajo la curva (AUC)
para cada sitio CpG y sus combinaciones realizadas mediante regresin
logstica binaria. Fijando la especificidad al 90%, se estim la sensibilidad
del marcador en cada supuesto.
Para el diagnstico de CCR, los valores de AUC obtenidos para cada uno
de los sitios CpG estudiados estuvieron comprendidos entre 0,51-0,60,
con valores de sensibilidad de 4,5-18,2%. La mayor sensibilidad (36,4%)
se alcanz combinando las posiciones 1 a 6 (AUC: 0,74). En cuanto a la
deteccin de neoplasia avanzada, el AUC result entre 0,50-0,59 para los
7 sitios analizados, con sensibilidades de 10,3-24,1%. La combinacin
ptima (posiciones 1 a 4 y 7) mostr un AUC de 0,70 (sensibilidad 41,4%).
En resumen, a pesar de la baja sensibilidad que presenta la metilacin de
SEPT9 para la deteccin tanto de CCR como de neoplasia avanzada, el
anlisis por pirosecuenciacin permiti identificar los sitios CpG que ms
contribuyen, lo que permitir optimizar su capacidad diagnstica.
Financiacin: Fundacin Cientfica de la Asociacin Espaola contra
el Cncer (GCB13131592CAST) y Fondo de Investigacin Sanitaria del
Instituto de Salud Carlos III - FEDER (PI12/00117).
P11-20
Anlisis protemico para el estudio de los

mecanismos de ensamblaje y desensamblaje de
las RNA polimerasas eucariticas
Ana Isabel Garrido-Godino, Vernica Martnez-Fernndez, Abel
Cuevas-Bermdez, Francisco Navarro Gmez
Departamento de Biologa Experimental, Universidad de Jan, Jan, ES
Las RNA polimerasas son complejos multiproteicos encargados de la
transcripcin. A pesar de que existe una amplia informacin de cmo
estos complejos funcionan e interactan en el ncleo celular de eucariotas,
poco se sabe sobre los mecanismos de su ensamblaje citoplasmtico y
su posterior entrada al ncleo, as como de los procesos de reciclado y
desensamblaje nucleares. Nuestra investigacin ha permitido ahondar en
estos procesos y describir nuevos mecanismos y protenas implicados
en los mismos. Mediante tcnicas de anlisis protemicos, combinadas
contcnicas genticas, bioqumicas y de biologa molecular hemos
determinado la existencia de dos nuevas protenas, Bud27 y Rtr1 que
median estos procesos y que tienen un efecto directo en el ensamblaje de las
RNA polimerasas eucariticas y en el proceso transcripcional. Continuar
con este tipo de abordaje, ayudar a clarificar y profundizar en estos
mecanismos que tienen un inters crucial para entender la transcripcin,
no solo desde el punto de vista funcional en el ncleo, sino desde un punto
de vista dinmico que implica, adems, los procesos de ensamblaje y
desensamblaje de la maquinaria de transcripcin.
Escherichia coli Nissle 1917 (EcN) es un probitico utilizado en el

tratamiento de patologas intestinales. EcN favorece el equilibrio de
la microbiota y mejora la homeostasis intestinal. Sin embargo, existe
poca informacin sobre cmo las molculas efectoras liberadas por este
probitico acceden a las clulas del husped. Las bacterias gram-negativas
producen vesculas de membrana externa (OMV). Estas estructuras
desempean un rol importante en la interaccin bacteria-husped. Por
ello, las OMV generadas por microbiota comensal y probiticos gramnegativos se vislumbran como agentes importantes en la sealizacin
a nivel intestinal. Los estudios protemicos pueden aportar informacin
relevante en este contexto.
Aqu se presenta el proteoma de las OMV aisladas de cultivos de la cepa EcN
en DMEM, medio habitual para el cultivo de lneas de epitelio intestinal.
Las protenas de las OMV fueron separadas en geles de poliacrilamida-SDS,
extradas del gel y analizadas por espectrometra de masas. Las protenas
identificadas fueron clasificadas en grupos funcionales segn las bases
UniProt y GenProtecEc y los resultados comparados con los obtenidos
del proteoma de las OMV aisladas de cultivos en LB, recientemente
publicado por nuestro grupo de investigacin. Se identificaron un total
de 148 protenas, 114 eran comunes al proteoma de las OMV en LB. La
distribucin en grupos funcionales fue similar en ambos proteomas. En
cuanto a su localizacin subcelular las OMV en DMEM presentan una
mayor proporcin de protenas de membrana interna y de citoplasma. As,
el medio de cultivo determina la composicin de las OMV. En conjunto
los resultados demuestran que las OMV de probiticos contienen protenas
capaces de interaccionar con clulas del husped y modular su funcin.
* Estos autores han contribuido por igual.
P11-22
c-Myc como predictor de respuesta a la

radioquimioterapia en el cncer de recto
Marta Cuadros Celorrio1, Raquel Conde Muio2, Pablo Palma2,
Victoria Snchez3, Sofa Boyero4
1
Departamento de Bioqumica y Biologa Molecular III e
Inmunologa, Universidad de Granada - Centro Pfizer-Universidad
de Granada-Junta de Andaluca de Genmica e Investigacin
Oncolgica (GENYO), Granada, ES, 2Seccin de Ciruga Colorrectal,
Servicio de Ciruga General, HUVN Granada, Granada, ES, 3Centro
Pfizer - Universidad de Granada - Junta de Andaluca de Genmica
e Investigacin Oncolgica (GENYO), Granada, ES, 4Universidad de
Granada, Granada, ES
Introduccin: El tratamiento actual del cancer de recto localmente
avanzado (CRLA) incluye regmenes de radioquimoterapia (RQPT)
antes de la exresis quirrgica. Hasta la fecha, no existe ningn marcador
que pueda predecir la respuesta a esa RQPT, sin poderse detectar el alto
ndice de pacientes que no se benefician de esta actuacin, no exenta de
morbilidad y de un alto coste econmico.El objetivo de este estudio es
evaluar la expresin de c-Myc como posible marcador de respuesta a la
RQTP en pacientes afectos con CRLA.
Material y mtodos: Para determinar si la sobreexpresin del ARNm
de c-Myc se correlacionaba con una amplificacin del ADN se realiz
hibridacin in situ con fluorescencia (FISH). Adems, se realiz la
inmunohistoqumica de c-Myc. Se analizaron mediante curvas ROC la
109
Psters
sensibilidad y especificidad de la sobreexpresin de c-Myc para predecir
la respuesta al tratamiento.
Resultados: Identificamos mediante microarrays 257 genes cuya expresin
era distinta y significativa entre respondedores y no respondedores a la
RQTP. En la red de interconexin ms importante destacaban 24 genes
relacionados directa o indirectamente con c-Myc. Ninguno de los tumores
amplific c-Myc. El 100% de las muestras presentaron un elevado nmero
de clulas tumorales positivas (>90%), independientemente de su respuesta
al tratamiento.El anlisis de curvas ROC indica que la sobreexpresin de
c-Myc tiene una especificidad del 93.8% y una sensibilidad del 70% para
determinar la respuesta al tratamiento.
Conclusiones: La sobreexpresin de c-Myc a nivel de ARNm muestra
una buena correlacin con la respuesta a la QRTP en pacientes afectos de
CRLA.
P11-23 (R11-6)
Differentiation of mouse embryonic stem cells

towards pancreatic beta-cell surrogates through
regulation of Pdx1 by nitric oxide
Carmen Salguero Aranda1, Rafael Tapia Limonchi1, Gladys Cahuana
Macedo2, Ana Beln Hitos Prados1, Irene Daz Contreras1, Karim
Hmadcha1, Mario Fernndez Fraga3, Franz Martn Bermudo1, Bernat
Soria Escoms1, Juan R. Tejedo Huamn1, Francisco Bedoya Bergua1
1
Centro Andaluz de Biologa Molecular y Medicina RegenerativaCABIMER, Sevilla, ES, 2Universidad Pablo de Olavide, Sevilla, ES,
3
Hospital Universitario Central de Asturias, Asturias, ES
Pdx1 (Pancreatic and duodenal homeobox 1) is a transcription factor
that regulates the pancreatic development and the differentiation and
functionality of beta cells. Our group has shown that exposure of mouse
embryonic stem (mES) cells to high concentrations of NO donors such
as diethylenetriamine nitric oxide adduct (DETA-NO) increases the
expression of Pdx1. In that study we report that the increase of Pdx1
expression after NO treatment is associated with the release of Polycomb
Repressive Complex 2 (PRC2) and the histone acetyltransferase P300
from its promoter. Moreover, it is observed some changes in bivalent
marks of histone H3k27me3 and H3K4me3, site specific changes of DNA
methylation, and no changes in H3 acetylation of Pdx1 promoter. The
knowledge of the regulation of the gene Pdx1 has allowed us develop a
protocol to differentiate mES cells towards insulin producing cells. The
protocol consists of adding to the culture medium 0.5 mM DETA-NO
for 19 hours, 100 M valproic acid for 6 days, 50 M P300 inhibitor for
20 hours and a final step of suspension culture to form aggregates for 3
days. This simple and cost effective differentiation protocol allows the
generation of cells stably expressing markers of beta-cells such as Pdx1,
Nkx6.1, GcK, Kir6.2, Glut-2 and insulin.
Subvencionado por Ministerio de Economa y Competitividad-Secretara
de Estado de Investigacin Desarrollo e Innovacin (IPT-2011-1615900000) y Junta de Andaluca (CTS- 7127/2011).

particular, la nefropata diabtica (ND) es la principal causa (44,3%) de ERC
terminal en el mundo. Estos pacientes presentan un riesgo cardiovascular
(CV) aadido y se estima que el 40% de la poblacin espaola con
enfermedad renal oculta (no diagnosticada) fallecer principalmente
de problemas cardiovasculares. Si bien es conocida la existencia de un
sndrome cardio-renal, o desorden por el cual la disfuncin del corazn
o rin afecta al otro rgano, no se comprenden del todo los procesos que
operan especficamente en este contexto.
En este trabajo, perseguimos un doble objetivo: a) la identificacin de
nuevos marcadores de ERC, ND y riesgo CV, y b) la aplicacin de nuevas
tecnologas como la Espectrometra de Masas de Imagen (MSI) al estudio
de la aterosclerosis como patologa de base en el sndrome cardio-renal.
En un abordaje multidisciplinar empleando RMN, DIGE y SRM-LC-MS/
MS en modelos animales tempranos de ND y aterosclerosis y en muestras
humanas, hemos analizado el proteoma y el metaboloma de tejido renal,
tejido arterial, orina y exosomas aislados de orina. En paralelo, hemos
desarrollado y aplicado con xito un protocolo MSI de anlisis de arterias
que permite identificar, in situ, mapas moleculares especficamente
asociados a regiones arteriales concretas (zona de placa, capa ntima o capa
media) y su implicacin en el desarrollo de la lesin aterosclertica.
P11-25
Aislamiento de una nueva hidrolasa a partir de

un manantial geotermal mediante metagenmica
funcional
M Eugenia de Castro1, Esther Rodriguez-Belmonte2, Maria Isabel
Gonzlez-Siso2
1
Universidad de A Corua, A Corua, ES, 2rea de Bioqumica y
Biologa Molecular, Facultad de Ciencias, Universidad de A Corua,
A Corua, ES
Las beta-galactosidasas (EC 3.2.1.23) constituyen una gran familia
de protenas con capacidad para catalizar la hidrlisis de enlaces -D
(1,4)-galactosdicos y participar en reacciones de transgalactosilacin.
Por ello, son ampliamente utilizadas en la industria alimentaria como
intermediarias para la produccin de leche sin lactosa, la revalorizacin
de los sueros o la obtencin de galacto-oligosacridos, constituyentes
fundamentales de los prebiticos, entre otros. En los ltimos aos, la
posibilidad de obtener beta-galactosidasas termoestables y el creciente
mercado de alimentos funcionales han incrementado su inters industrial.
La metagenmica funcional permite analizar el genoma colectivo de una
comunidad microbiana expresndolo de manera heterloga y constituye un
campo emergente de la biotecnologa que ha permitido la caracterizacin
de enzimas procedentes de microorganismos de ambientes difciles de
aislar por sus condiciones extremfilas.
En el presente estudio se recoge la purificacin y caracterizacin
enzimtica de una hidrolasa obtenida mediante bsqueda funcional en una
metagenoteca de aguas termales procedentes del manantial del Riocaldo
(Lobios, Ourense), no descrita hasta el momento y que presenta actividad
hidrolasa estable a 65C.
P11-24 (R11-3)
P11-26
Estrategias micas aplicadas al estudio de la

patologa renal y vascular
Recombinant glycoproteins gp125 and gp350 for

immunodiagnostic of EBV
Irene Zubiri, Marta Martn-Lorenzo, Laura Gonzlez-Calero, Mara

Posada-Ayala, Aroa S. Maroto, Fernando Vivanco, Gloria lvarezLlamas
Departamento de Inmunologa, IIS-Fundacin Jimnez Daz, UAM,
Madrid, ES
Ivn Iglesias Baena, Encarnacin Campos Gutierrez, Fernando

Morcillo de Amuedo, Julian Lpez-Viota, Jos Rojas, Ainel Alemn
Vircell SL, Granada, ES
La enfermedad renal crnica (ERC) es un problema emergente en todo

el mundo. En Espaa, en pacientes seguidos en atencin primaria con
enfermedades tan frecuentes como hipertensin arterial (HTA) o diabetes
mellitus (DM), la prevalencia de ERC puede alcanzar el 35-40%. En
110
Serodiagnosis of Epstein-Barr virus (EBV) gain relevance for

its relationship with nasopharyngeal carcinoma and other EBVassociated diseases. Viral capsid glycoproteins gp125 and gp350 has
been described as good antigens for early infections, which proteins
combined with EBNA and p18 could improve the sensitivity. The yeast
and mammalian overexpression of recombinant proteins is an important
Granada 2014
tool to obtain post-translational modifications, which can be essential
in its antigenicity, and higher yield than the native form obtained from
virus culture.
We have purified the tagged gp125 from Baculovirus and Pichia
pastoris system, and the N-terminal gp350 and C-terminal gp350 from
Saccharomyces cerevisiae and Pichia pastoris system. The improvement of
the expression conditions in a bioreactor were carried out with the control
of parameters in order to reduce degradation and to obtain higher biomass.
After yeast cell disruption and clarification, the purification strategy were
carried out by affinity chromatography in FPLC system and analyzed using
the specific monoclonal antibodies.
Both enzyme-linked immunosorbent assay (ELISA) and magnetic
nanoparticules (MNP) have been used to get an antigen characterization of
the different fussion proteins, developing a new method of diagnostic with
high sensitivity and specificity. The gp125 obtained from Pichia pastoris
showed similar activity than the commercial native antigen, and contribute
to distinguish between possitive and negative serum samples for IgM. We
have observed the crucial role of the expression in eukaryotic system of
early antigens from the capsid of EBV in order to conserve the relevance
glycosilations into the conformational epitopes.
P11-27
P1 recombinant antigen for the Mycoplasma

pneumoniae diagnosis
Anabel Lpez Ramos1, Ana Leticia Jimnez Escobar2, Ivan Iglesias
Baena1, Encarnacin Campos Gutierrez1, Fernando Morcillo1, Julian
Lpez Viota1, Jose Rojas1
1
Vircell SL, Granada, ES, 2Universidad de Granada, Granada, ES
Mycoplasma pneumoniae is a human pathogen that causes atypical
bacterial pneumonia with high prevalence in children and young adults.
Other pathogens produce pneumonia with a nonspecific clinical, so to
develop an immunological method with high sensitivity and specificity. A
kit for the diagnostic of M. pneumoniae by ELISA has been developed by
Vircell S.L. for detection of the infection in the early state (IgM).
The adhesin P1 has been described as a good antigen for serological test
for its immunogenicity. This membrane protein has been obtained from
the bacterial extract, but in order to improve the relation yield-cost, we
have designed the cloning and overexpression of P1 in eukaryotic and
prokaryotic system.
The C-terminal fragment of P1 has been cloned into expression vector for
Baculovirus and Escherichia coli, obtaining a tagged proteins that were
purified by affinity chromatography in FPLC system and analyzed using
specific monoclonal antibodies. We have developed the expression and
purification process to obtain the purified protein.
The obtained recombinant P1, conserve the crucial epitopes for the
discrimination between positive and negative serum samples. Indeed,
we have develop a new platform for mycoplasma diagnosis, using
nanoparticles labelled to the antigen.
P11-28
Efecto de la aeroplisinina-1 sobre protenas

involucradas en el proceso angiognico e
inflamatorio
Javier A. Garca-Vilas, Ana R. Quesada, Miguel ngel Medina Torres
Universidad de Mlaga, Andaluca Tech, Departamento de Biologa
Molecular y Bioqumica, Facultad de Ciencias, and IBIMABiomedical Research Institute of Mlaga - Unidad 741, CIBER de
Enfermedades Raras-CIBERER, Mlaga, ES
La aeroplisinina-1 metabolito secundario sintetizado por esponjas como
mecanismo de defensa, ha sido caracterizada por nuestro grupo como
un potente agente antiangiognico por modular protenas involucradas
estrechamente en este proceso, tanto en clulas endoteliales de cultivos
Psters
primarios como en clulas endoteliales inmortalizadas. Estudios
posteriores que hemos realizado con la aeroplisinina-1, han dilucidado que
este compuesto tambin presenta capacidad antiinflamatoria al disminuir
los niveles de protenas a las que se les atribuyen importantes actividades
inflamatorias por mediar algunos estadios como puede ser la atraccin de
clulas inflamatorias, proteasas para facilitar la trasvasacin de las clulas
desde el torrente sanguneo hasta el foco inflamatorio, adems de disminuir
los niveles de COX-2 responsable de sintetizar molculas de carcter
inflamatorio.
Tanto el proceso angiognico como el proceso inflamatorio, no slo
se encuentran regulados por las protenas a las que se les asocian
directamente, sino que est descrito que en ambos procesos ejerce
un importante papel las protenas responsables del metabolismo
redox. Mediante aproximacin protemica hemos comprobado que
el tratamiento con aeroplisinina-1 modifica los niveles de protenas
relacionadas con el metabolismo redox, sugiriendo que el efecto
antiinflamatorio y antiangiognico de la aeroplisinina-1 demostrado
en estudios anteriores, se debe al efecto multidiana sobre diversos
mdulos del metabolismo permitiendo engrosar las evidencias de
este compuesto como un potente agente modulador de los procesos
asociados a los tumores.
Supported by grant P12-CTS-1507 (Andalusian Government and FEDER)
and funds from group BIO-267 (Andalusian Government). The CIBER de
Enfermedades Raras is an initiative from the ISCIII.
P11-29
Actividad proteosomal en pacientes mexicanos

con cncer de prstata
Jos Francisco Montiel-Sosa1, Victoria Edwina Campos Garcia2,
Sandra Daz-Barriga Arceo1, Jorge Ramirez Salcedo3, Sergio Ureta4,
Patricia Ramrez Noguera1
1
PhD. en Bioqumica, Departamento de Bioqumica y Farmacologa
Humana, Universidad Nacional Autnoma de Mxico, Cuautitln
Izcalli, MX, 2B.Q.D. Bioqumica Diagnstica, Departamento
de Bioqumica y Farmacologa Humana, Universidad Nacional
Autnoma de Mxico, Cuautitlan Izcalli, MX, 3PhD. en Ciencias
Biomedicas. Instituto de Fisiologia Celular, Universidad Nacional
Autnoma de Mxico, Mxico, MX, 4M.D.Mdico Urlogo, Hospital
Espaol, Distrito Federal, MX
Dentro de las seales celulares involucradas en proceso de cncer de
prstata CaP, el sistema ubiquitina proteosoma y protenas similares
a ubiquitina (SUMO) juegan un papel muy importante. Con objeto de
conocer el estado de ubiquitinacin proteca y sumoilacin in vitro en
biopsias de pacientes con cncer de prstata se estim, la capacidad de
formacin in vitro de aductos-Ub y aductos-SENP especficos utilizando
las protenas de fusin recombinantes HA-Ub-Vinilsulfona y HA-SUMO
-1 y HA-SUMO 2/3-vinilsulfona (VS). Las sondas HA-Ub-VS y HASUMO-1-VS y -2/3-VS producto de la generacin de unaprotena de
fusin Tag-HA, reaccionaron covalentemente con los sitios nucleoflicos
activos de las enzimas DUB y SENP respectivamente. Estas enzimas estn
presentes en el tejido prosttico y dependiendo de su estado y actividad,
se pudieron unir covalentemente con el grupo VS de cada sonda. Los
resultados al momento muestran que la actividad similar a quimotripsina
estimada por medio de ensayos fluorognicos y utilizando sustratos
especficos acoplados a coumarina est aumentada significativamente
un 10 % respecto al control negativo analizado. Estas deducciones son
importantes desde el punto de vista funcional del sistema ubiquitina
proteosoma en la clulas. Este sistema multienzimtico, es capaz de
reconocer sustratos protecos intracelulares necesarios en la modulacin
de seales enfocadas en el control de la funcin y degradacin de
protenas sealizadoras en diversos procesos de transformacin celular
y cncer. El aumento en la actividad proteoltica similar a quimotripsina
sugiere una activacin significativa en los pacientes de cncer analizados
quiz con este fin.
111
Psters
P11-30
Hemos abordado un exhaustivo estudio mutacional de la regin de 56

aminocidos C-terminal de la GS de Synechocystis tanto in vitro (mediante
ensayos de retardo en gel e inactivacin con protenas purificadas) como
in vivo (expresando en Synechocystis las versiones mutantes de la GS).
Hemos identificado tres residuos (glutamato 419, asparragina 456 y
arginina 459 de la GS de Synechocystis) fundamentales para la interaccin
GS/IFs y la inactivacin de la enzima. Analizando los resultados a la luz
de la estructura de la GS de Synechocystis (PDB ID 3NG0), obtenida
recientemente, observamos que estos tres residuos se localizan prximos y
expuestos al solvente, constituyendo un ncleo para la interaccin GS/IFs
responsable de la inactivacin de la enzima.
Distinct serum proteome profiles associated

with collagen-induced arthritis, and complete
Freunds adjuvant-induced inflammation in
CD38/ mice
Antonio Rosal-Vela1, Sonia Garca-Rodrguez1, Jorge Postigo2,
Marcos Iglesias2, Jess Merino2, Ramn Merino3, Mercedes
Zubiaur1, Jaime Sancho1
1
Instituto de Parasitologa y Biomedicina Lpez-Neyra, CSIC,
(IPBLN-CSIC), Armilla (Granada), ES, 2Departmento de Biologa
Molecular, Instituto de Formacin e Investigacin Marqus de
Valdecilla, Universidad de Cantabria, Santander, ES, 3Instituto de
Biomedicina y Biotecnologa de Cantabria/CSIC-Universidad de
Cantabria-SODERCAN, Santander, ES
Collagen-type-II-induced arthritis (CIA) is milder in CD38-/- than in WT
mice. Their serum samples were treated with ProteoMiner-beads to equalize
protein concentrations resulting in a significant enrichment in proteins from
extracellular vesicles (blood microparticles, exosomes). ProteoMinerequalized samples were subjected to 2D-DiGE and MS-MALDI-TOF/TOF
analyses to identify proteins that were differentially expressed in CD38-/versus WT mice either with arthritis (Col.II+/CIA+), with no arthritis (Col.
II+/CIA-), or with inflammation (CFA-treated). Altered proteins included
those involved in acute phase response (SAA1), inflammation (B2MG,
HABP2, Hemoglobin), complement activation (Ficolins, C4-B fragments,
C1qb, C3-b-chain), polyclonal B-cell activation (Ig-kappa-light-chain) and
lipid metabolism (APOE, APOAI, APOAII, APOAIV, APOJ/Clusterin). Of
the proteins that changed in abundance, Hemoglobin and APOE could be
indicative of the milder pathological process found in CIA+CD38-/- mice,
whereas another set of proteins such as HABP2, Ig-kappa-light-chain,
SAA1, Ficolin-1, and Ficolin-2 were clearly associated to the stronger
response of WT mice to either collagen immunization or CFA-treatment.
Multivariate analyses revealed that the differential protein abundance
of 28 distinct protein spots was able to discriminate between CIA+ and
CIA- mice, independently of their genetic background. This approach is
suitable for the identification of arthritis, or inflammation serum proteome
signatures in mice with distinct genetic backgrounds.
P12. Metabolismo del nitrgeno

P12-1 (R12-3)
Anlisis mutacional de la interaccin GS-IFs en

cianobacterias
Roco Robles Rengel, Lorena Saelices Gmez, Francisco Javier
Florencio Bellido, Mara Isabel Muro Pastor
Universidad de Sevilla-CSIC, Sevilla, ES
La glutamina sintetasa de tipo I (GS) de Synechocystis sp. PCC 6803se
regula a nivel transcripcional y postraduccional en funcin del balance
carbono/nitrgeno celular. La regulacin postraduccional se lleva a cabo
mediante la interaccin reversible protena-protena con dos factores
inactivantes, IF7 e IF17 [1]. En el caso de Anabaena sp. PCC 7120,
existe un nico factor inactivante denominado IF7A, homlogo a IF7
de Synechocystis. La GS de Synechocystis es inactivada por IF7, IF17 e
IF7A, mientras que la GS de Anabaena slo es inactivada por su propio
factor, IF7A. Basndonos en esta observacin, hemos construdo cuatro
protenas quimricas entre la GS de Synechocystis y la de Anabaena.
El estudiocomparado de estas protenas nos ha permitido localizar una
regin carboxilo terminal de 56 residuos de aminocidos responsable de
la interaccin GS/IFs. Por otra parte conocemos la naturaleza electrosttica
de dicha interaccin y se han identificado tres residuos de arginina de los
factores inactivantes, claves para la funcionalidad de estas protenas [2].
112
Bibliografa
[1] Garca-Domnguez M, Reyes JC, Florencio FJ (1999) Prot Natl Acad
Sci USA 96, 7161-7166.
[2] Saelices L, Galmozzi CV, Florencio FJ, Muro-Pastor MI. Mol Microbiol.
2011 Nov; 82 (4):964-75.
P12-2 (R12-1)
Anlisis global de la degradacin de residuos

cianurados industriales por Pseudomonas
pseudoalcaligenes CECT5344
Conrado Moreno Vivin1, Vctor M. Luque-Almagro1, Isabel Manso1,
Isabel Ibez1, Lara P. Sez1, M. Paz Escribano1, Purificacin
Cabello2, Francisco Castillo1, M. Dolores Roldn1
1
Crdoba, ES, 2Dpto. Botnica, Ecologa y Fisiologa Vegetal,
Universidad de Crdoba, Crdoba, ES
Diversas actividades industriales, como la minera y el electropulido del
oro y otros metales preciosos en la joyera, generan residuos que contienen
una elevada concentracin de cianuro, un compuesto muy txico para la
mayora de los seres vivos. As, la industria joyera de Crdoba genera
anualmente varias toneladas de un residuo alcalino que contiene hasta 1,5
M de cianuro y diversos metales, como cobre, hierro y zinc, el cual debe
ser tratado mediante procedimientos fsico-qumicos. La bacteria alcalfila
Pseudomonas pseudoalcaligenes CECT5344 puede utilizar cianuro,
cianato, diferentes nitrilos y complejos cianuro-metlicos como nica
fuente de nitrgeno, por lo que es un buen candidato para la biodegradacin
de los residuos cianurados industriales. Para optimizar la degradacin del
residuo de la joyera en biorreactores, se ha estudiado el efecto del pH, la
aireacin, la fuente de carbono y la cantidad de inculo inicial, entre otros
parmetros, y se ha comprobado que esta estirpe puede consumir hasta 12
mM de cianuro total cuando crece a pH 9, lo que evita la volatilizacin
del cianuro como HCN. La secuenciacin completa del genoma de esta
estirpe (1,2) ha permitido realizar estudios protemicos y genmicos que
revelan que el cianuro provoca en esta bacteria una respuesta global de
adaptacin a esta fuente de nitrgeno txica. La induccin de una oxidasa
alternativa asociada a una malato:quinona oxidorreductasa permite, no
slo la respiracin insensible al cianuro, sino tambin la formacin de
oxalacetato, que reacciona con el cianuro formando una cianhidrina. La
nitrilasa NitC inducible por cianuro utiliza esta cianhidrina para formar
amonio, que es asimilado por la ruta GS/GOGAT (3,4). El cianuro tambin
induce sistemas de captacin de hierro, ya que la biodisponibilidad del
hierro disminuye por la formacin de complejos estables con el cianuro.
Adems, el residuo cianurado induce una respuesta de estrs oxidativo
y de defensa frente a metales. Finalmente, se han obtenido mutantes en
los genes implicados en la biosntesis de polihidroxialcanoatos de cadena
corta y media identificados en el genoma, lo que ha permitido comprobar
que, segn la estirpe empleada (silvestre o mutantes), es posible producir
material bioplstico de diferente naturaleza durante el crecimiento con
el residuo cianurado, lo que confiere un valor aadido al proceso de
biorremediacin de este residuo industrial txico.
Bibliografa
[1] Luque-Almagro et al. Environ. Microbiol. 15: 253-270 (2013).
Granada 2014
[2] Wibberg et al. J. Biotechnol. 175: 67-68 (2014).
[3] Luque-Almagro et al. Microbiology 157:739-746 (2011).
[4] Estepa et al. Environ. Microbiol. Rep. 4: 326-334 (2012).
Financiado por MICINN (BIO2011-30026-C02-01) y Junta de Andaluca
(Proyecto Excelencia CVI-7560).
P12-3
Proteolytic control of the Bradyrhizobium

japonicum transcription factor FixK2
Juan J. Cabrera1, Noem Fernndez1, Mariette Bonnet2, Monika
Stegmann2, Zeljka Maglica3, Eilika Weber-Ban3, Hauke Hennecke2,
Eulogio J. Bedmar1, Socorro Mesa1
1
Department of Soil Microbiology and Symbiotic Systems,
Estacin Experimental del Zaidn, CSIC, Granada, ES, 2Institute
of Microbiology, ETH Zurich, Zrich, CH, 3Institute of Molecular
Biology and Biophysics, ETH Zurich, Zrich, CH
FixK2 is a key regulator that controls a large number of genes required for
the anoxic and microoxic, endosymbiotic and nitrogen-fixing life styles of
the -proteobacterium Bradyrhizobium japonicum [1]. FixK2is an unusual
member of the CRP/FNR family of bacterial transcription factors, because
it is active in vitro without an additional effector molecule and is regulated
posttranslationally by the oxidation of its singular cysteine residue [2].
In addition to its oxidation-mediated control, FixK2 is also regulated by
proteolysis. Despite the induction of fixK2 gene expression at low-oxygen
concentrations, the steady-state levels of the FixK2 protein remains constant
regardless of the growth conditions [2]. Further, the B. japonicum ClpAP1
proteolytic system degrades FixK2 in vitro [3]. Remarkably, the recently
solved FixK2structure in complex with DNA revealed that the C-terminus
of FixK2 is surface-exposed, and therefore a target for proteolysis [4].
We also observed that a truncated variant is always co-purified together
with N-terminally His6-tagged FixK2 proteins. Mass spectrometric analysis
revealed that this form a C-terminally cleaved derivative (between V220
and L221), which lacks the last twelve amino acids. Likewise, this shorter
FixK2 species is also present in cells of B. japonicum. In this work, we
have constructed a series of protein variants with modifications within
the C-terminus of FixK2. Our results showed that the C-terminal stretch
of twelve amino acids, and particularly L221, play a crucial role in FixK2
proteolysis, protein folding, DNA binding and in vitro activity. In order to
find out the function of the carboxy-terminal part of FixK2in vivo, we are
currently testing different proteins derivatives with regard to their ability to
complement a fixK2 mutant strain.
Bibliografa
[1] Mesa et al. (2008) J. Bacteriol. 190:6568-79.
[2] Mesa et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106:21860-5.
[3] Bonnet et al. (2013) FEBS Lett. 587:88-93
[4] Bonnet et al. (2013) J. Biol. Chem. 288:14238-46.
This work was supported by the ERDF-co-financed grant AGL2011-23383
from MINECO.
P12-4
Caracterizacin de la asparraginasa de pino:

implicaciones en el desarrollo del sistema vascular
Sonia H. E. Van Kerckhoven, Rafael A. Caas, Fernando de la Torre,
Concepcin Avila, Francisco M. Cnovas, Francisco R. Cantn
Universidad de Mlaga, Dpto Biol. Molecular y Bioqumica, Mlaga, ES
La asparragina es un metabolito clave en plantas para el transporte y reserva
temporal de nitrgeno. La principal va de movilizacin del nitrgeno
contenido en el grupo amido de la asparragina implica a la enzima
asparraginasa (ASPG, EC 3.5.1.1), especialmente en tejidos que demandan
altas cantidades de este elemento, tales como semillas en desarrollo y hojas
Psters
jvenes. Durante el desarrollo y crecimiento del tronco de los rboles, stos
afrontan un consumo masivo de carbono y nitrgeno para proveer la sntesis
de celulosa y lignina. Estudios previos han demostrado que la expresin
del gen de asparraginasa en el pino est asociada al intensivo desarrollo del
sistema vascular que se produce en el tallo de las plntulas una vez que sta
ha agotado las reservas de la semilla, y que su expresin est confinada a
las clulas de la regin del cambium [1]. La observacin de que este gen
se expresa tambin en clulas de xilema secundario en diferenciacin de
rbol adulto sugiere que la ASPG podra jugar un papel importante en el
desarrollo vascular y ser de gran relevancia en la produccin de biomasa.
Aunque conocemos, en lneas generales, la implicacin de la asparraginasa
en el transporte y movilizacin de nitrgeno, desconocemos los mecanismos
moleculares que controlan espacial y temporalmente su actividad. En
nuestro grupo seguimos diferentes aproximaciones para dilucidar el control
transcripcional y post-transcripcional de esta enzima y su implicacin en el
desarrollo del sistema vascular. Nuestros objetivos especficos son determinar
las caractersticas del procesado y activacin de la asparraginasa de pino y
los factores que regulan la expresin gnica asociada al desarrollo vascular.
Bibliografa
[1] Rafael A. Caas, Fernando de la Torre, Francisco M. Cnovas,
Francisco R. Cantn. (2007). Planta, 225:1205-1219.
Este trabajo ha sido financiado con ayudas del Ministerio de Economa y
Competitividad (BIO2012-33797, AGL9-12139C0202) y una ayuda F.P.U.
del Ministerio de Educacin a SVK (AP2010-5434).
P12-5 (R12-9)
Control de RegR sobre la expresin de los genes

nor de Bradyrhizobium japonicum: micro-oxia
versus anoxia
Ana Salas Huertas, Mara Jess Torres Porras, Mara Jess Delgado
Igeo, Eulogio J. Bedmar Gmez
Departamento de Microbiologa del Suelo y Sistemas Simbiticos Estacin Experimental del Zaidn, CSIC, AP 419, Granada, ES
Bradyrhizobium japonicum es una bacteria del suelo que establece una
asociacin simbitica con plantas de soja y es capaz de desnitrificar tanto
en vida libre como en simbiosis. Los genes napEDABC, nirK,norCBQD y
nosRZDYFLX, que codifican las enzimas nitrato-, nitrito-, xido ntricoy xido nitroso-reductasa, respectivamente, son necesarios para llevar
a cabo dicho proceso. En B. japonicum, se han descrito dos cascadas
reguladoras, FixLJ-FixK2 y RegSR-NifA, de respuesta a oxgeno. Estudios
recientes de nuestro grupo de investigacin han demostrado que la protena
reguladora RegR es necesaria para la mxima induccin de los genes nor
en condiciones de cultivo anaerbicas con nitrato y que este control es
independiente de RegS [1]. En este trabajo, se ha investigado si el control
que ejerce RegR sobre los genes nor ocurre tambin en micro-oxia o si
es exclusivo de condiciones anxicas. Para ello, se han cultivado la cepa
parental USDA110spc4 y las mutantes en los genes regS y regR, en medio
mnimo con succinato como fuente de carbono, con nitrato como aceptor
final de electrones y en condiciones micro-xicas (2% de oxgeno en la
atmsfera gaseosa). Tras el cultivo de las clulas, se han realizado estudios
de expresin de los genes nor mediante la determinacin de la actividad
-galactosidasa de una fusin transcripcional norC-lacZ y estudios de
deteccin de la protena NorC mediante tincin del grupo hemo c.Los
resultados obtenidos han confirmado que la protena reguladora RegR
est implicada en la induccin de los genes nor en respuesta a anoxia y
nitrato. Sin embargo, en condiciones de micro-oxia los niveles de actividad
-galactosidasa de la fusin norC-lacZ, as como la expresin de la protena
NorC en la cepa mutante regR, fueron superiores a los detectados en la
cepa parental. Esto sugiere que, a diferencia de lo observado en anoxia,
la protena RegR podra tener un papel represor sobre la expresin de los
genes nor en condiciones micro-xicas.
113
Psters
Bibliografa
[1] Torres et al., 2014. PLOS one, en prensa.
Este trabajo se ha subvencionado con fondos cofinanciados por FEDER del
proyecto AGL2010-18607 del Ministerio de Economa y Competitividad.

durante la germinacin y desarrollo postgerminativo temprano de juda. El
gen PVN1, que corresponde con la protena purificada, fue el que mostr
mayor valor de expresin en ejes en desarrollo. La inclusin de nitrato en
el medio de imbibicin redujo la expresin del gen, lo que sugiere una
relacin con el estado nutricional de plntulas de juda.
El gen nosZ como marcador molecular de la

desnitrificacin en ecosistemas naturales
Bibliografa
Cabello-Daz JM, Quiles FA, Lambert R, Pineda M y Piedras P (2012).
Plant Physiol. Biochem., 53, 54-60.
Quiles FA, Raso MJ, Pineda M y Piedras P (2009). Physiol. Plant., 135,
19-28.
David Correa-Galeote, German Tortosa, Eulogio J. Bedmar

Departamento de Microbiologa del Suelo y Sistemas Simbiticos Estacin Experimental del Zaidn - Agencia CSIC, Granada, ES
Financiacin: Ministerio de Economa y Competitividad (AGL201234230) y el Plan Andaluz de Investigacin (BIO-115).
P12-6 (R12-8)
La relacin entre el contenido en nitratos, la emisin de N2O y las distintas

comunidades desnitrificantes en diferentes muestras medioambientales an no
ha sido establecida. En este estudio se determin la abundancia relativa genes
de la desnitrificacin narG, napA, nirK, nirS y nosZ con el fin de correlacionar
los distintos genes tanto entre s, como con el substrato inicial y el producto
final de la reaccin de la desnitrificacin y a su vez, tratar de establecer un gen
de la desnitrificacin como marcador molecular de dicho proceso.
Para ello se seleccionaron dos sitios de muestreo dentro del Parque Nacional
de Doana uno con escaso contenido en nitratos (Laguna del Acebrn, S1)
y otro con elevado contenido en nitratos (Arroyo de la Caada, S2). La
recogida de sedimentos se realiz en los meses de abril y octubre de los
aos 2008, 2009 y 2010. El aislamiento, purificacin y amplificacin de los
genes 16S rRNA as como de los genes de la desnitrificacin se realiz de
la manera habitualmente realizada en el laboratorio.
De acuerdo con el test de Spearman, la abundancia relativa de los genes de
la desnitrificacin no present correlacin ni con el contenido en nitratos
ni con la emisin de N2O en S1. Por el contrario, para S2 existe correlacin
significativamente positiva entre el contenido en nitratos y cada uno de los
genes de la desnitrificacin, si bien el gen nosZ fue el gen que present
una mayor correlacin. La emisin de N2O y la abundancia relativa
de los genes de la desnitrificacin en S2 fue negativa, encontrndose la
mayor correlacin negativa para el gen nosZ. Por otra parte, tanto en S1
como en S2, todos los genes de la desnitrificacin se correlacionaron
estadsticamente entre s, observndose la mayor correlacin entre el gen
nosZ y el resto de genes analizados.
Por tanto, se propone el gen nosZ como un posible marcador molecular
de las comunidades desnitrificantes en muestras medioambientales
contaminadas con nitratos.
P12-7 (R12-7)
Identificacin de una nucleasa en ejes

embrionarios de juda regulada por nitrato
Roco Lambert Rodrguez, Juan Miguel Cabello Daz, Cristina
Caballo Linares, Francisco Antonio Quiles Luque, Pedro Piedras
Montilla
Universidad de Crdoba, Crdoba, ES
Las actividades nucleasas de plantas estn implicadas en la degradacin
de cidos nucleicos asociada a procesos de muerte celular programada y a
los de restriccin, reparacin y recombinacin del ADN. Sin embargo, el
conocimiento sobre la funcin de las nucleasas de plantas es muy limitado.
En ejes en desarrollo de juda se ha detectado una actividad nucleasa
mayoritaria que utiliza ARN y ADNmc como sustratos, cuyos valores de
actividad incrementaron tras la emergencia radicular coincidiendo con
el incremento de actividad nucleotidasa (Cabello-Daz et al, 2012) y la
acumulacin de ureidos (Quiles et al, 2009), lo que podra sugerir una
implicacin en la movilizacin de reservas. Esta enzima se ha purificado
y el gen que la codifica (PVN1) se ha identificado mediante MALDI TOF/
TOF. Las caractersticas de la enzima purificada as como la secuencia
del gen indican que pertenece a la familia S1/P1 de nucleasas. Utilizando
la base de datos del genoma de juda (www.phytozome.com), se han
identificado 5 miembros de esta familia, cuya expresin se ha analizado
114
P12-8 (R12-10)
NRT1 y el transporte de compuestos

nitrogenados en Chlamydomonas
Victoria Calatrava, Jos Higuera, Zaira Gonzlez, Emilio Fernndez
Reyes, Aurora Galvn Cejudo
Crdoba, ES
El nitrato es una seal positiva que activa la ruta de asimilacin de nitrato/
nitrito. Para la activacin de la ruta son necesarios dos factores, 1) la
entrada de nitrato al interior de la clula, y 2) la presencia de un NIT2
funcional. NIT2 es un factor de transcripcin del tipo RWP-RK y es el
principal gen regulador de la ruta que necesita adems de una protena
dedo de zinc (NZF1) para la ptima funcionalidad de NIT2, en respuesta
a nitrato. Los mutantes nit2 ni sienten el nitrato ni crecen en medios con
nitrato [1, 2].
NRT2 y NRT1 son transportadores de nitrato que pertenecen a familias de
protenas no relacionadas. A la familia NRT2 pertenecen transportadores
de nitrato/nitrito de alta afinidad y algunos de ellos esenciales para el
crecimiento con nitrato como nica fuente de nitrgeno. A la familia
NRT1 pertenecen transportadores de nitrato, aminocidos, oligopptidos.
Recientemente esta familia se ha denominado NPF (NRT1/PTR Family).
En el presente trabajo se ha estudiado el papel de NRT1 de Chlamydomonas
(NPF7) en la sealizacin por nitrato y el efecto sobre la utilizacin de
fuentes de nitrgeno orgnica, urea, aminocidos, pptidos.
Bibliografa
[1] Camargo A, Llamas A, Schnell RA, Higuera JJ, Gonzlez-Ballester
D, Lefebvre PA, Fernndez E, Galvn A (2007) Nitrate signaling by the
regulatory gene NIT2 in Chlamydomonas. Plant Cell 19: 3491-3503.
[2] Higuera JJ, Fernandez E, Galvan A (2014) Chlamydomonas NZF1,
a tandem-repeated zinc finger factor involved in nitrate signalling by
controlling the regulatory gene NIT2. Plant Cell Environ. doi: 10.1111/
pce.12305.
Financiado por JA-P08-CVI-042157 y MINECO-BFU2011-29338 (EU
FEDER Programa).
P12-9 (R12-2)
25 aos de estudios sobre metabolismo

de nitrgeno y carbono en haloarqueas
Mara Jos Bonete, Mnica Camacho, Carmen Pire, Rosa Mara
Martnez-Espinosa, Julia Esclapez, Vanesa Bautista, Anna Vegara,
Susana Daz, Francisco Prez-Pomares, Basilio Zafrilla, Laia PedroRoig, Gloria Bravo-Barrales, Francisco I. Llorca
Divisin de Bioqumica y Biologa Molecular Universidad de
Los estudios sobre microorganismos extremfilos han aumentado
exponencialmente en los ltimos aos. Estos organismos se han adaptado
Granada 2014
a vivir en medios inhspitos caracterizados por temperaturas, presiones,
valores de pH y fuerza inica extremos. Investigaciones recientes
muestran que sus maquinarias celulares son nicas, ofreciendo una fuente
valiosa de nuevos biocatalizadores y compuestos de alto valor aadido
en el mercado.
Nuestro grupo de investigacin ha centrado sus esfuerzos en el
estudio y caracterizacin del metabolismo del carbono y del nitrgeno
de haloarqueas. Como es bien sabido, el N es uno de los elementos
bsicos de los seres vivos, y la disponibilidad de una fuente adecuada
del mismo a menudo limita la productividad primaria en algunos
ambientes naturales y en la agricultura. En relacin a ello, cabe resaltar
que las publicaciones sobre la regulacin a nivel transcripcional y
post-transcripcional de la asimilacin del nitrato/amonio son escasas
en el Dominio Archaea. Este hecho ha motivado que recientemente
nuestro objetivo se centre en comprender los mecanismos que subyacen
en la regulacin de este metabolismo y sus diferencias con los otros
Dominios.
Hasta el momento se ha aislado, caracterizado y expresado enzimas como
glutamato, isocitrato y glucosa deshidrogenasas, glutamina sintetasa,
ciclodextrin glucanotransferasa, nitrato y nitrito reductasas y protenas
reguladoras como GlnK (PII). Estos estudios se han complementado
con otros de carcter fisiolgico para conocer con detalle cmo estas
haloarqueas se adaptan a condiciones cambiantes del medio o a medios
extremos en los que adems existen compuestos txicos. En este sentido,
la va de desnitrificacin se presenta como una de las ms destacadas para
explorar posibles usos de las haloarqueas en biorremediacin de aguas
y suelos salinos. Para comprender mejor esta ruta metablica se han
purificado y caracterizado enzimas implicadas en ella y se han realizado
anlisis informticos para identificar los genes involucrados en esta ruta en
haloarqueas desnitrificantes.
Psters
P12-11
Transcriptmica de la deficiencia en glutamina

sintetasa plastdica en la leguminosa modelo
Lotus japonicus
Marco Betti, Carmen M. Prez-Delgado, Margarita Garca-Caldern,
Antonio J. Mrquez
Departamento de Bioqumica Vegetal y Biologa Molecular, Facultad
de Qumica, Universidad de Sevilla, Sevilla, ES
En las plantas existen diferentes isoformas de glutamina sintetasa
(GS) cuya funcin fisiolgica precisa est an por esclarecer. En este
trabajo se muestra un resumen de todos los estudios de transcriptmica
llevados a cabo hasta la fecha con los mutantes Ljgln2-2 deficientes en
GS plastdica (GS2) de L. japonicus, que fueron previamente aislados
en nuestro laboratorio, y que constituyen los primeros mutantes de su
clase que han sido identificados y caracterizados en plantas leguminosas.
Dichos estudios han puesto de manifiesto la importancia de la GS2 en
distintos procesos tales como la respuesta a sequa [1] y la reasimilacin de
amonio fotorrespiratorio [2], estableciendo las mltiples interconexiones
tanto de la GS2, como de la sequa y la fotorrespiracin, con distintos
procesos metablicos de la clula. El hecho de que muchos de los cambios
observados sean comunes para los distintos procesos examinados sugiere
que la GS2 media en la respuesta a diferentes situaciones de estrs como
el producido por la sequa por la existencia de un ciclo fotorrespiratorio
incompleto en las plantas. Otros estudios ms recientes utilizando redes de
coexpresin han identificado posibles factores de transcripcin que pueden
tener papeles clave en la respuesta de las plantas [3].
P12r-10
Bibliografa
[1] Daz P, Betti M, Snchez DH, Udvardi MK, Monza J, Mrquez AJ
(2010) New Phytol. 188, 1001-1013.
[2] Prez-Delgado CM, Garca-Caldern M, Snchez DH, Udvardi M,
Kopka J, Mrquez AJ, Betti M (2013) Plant Physiol. 162, 1834-1848.
[3] Prez-Delgado CM (2014) Tesis Doctoral, Universidad de Sevilla.
Estudio funcional de promotores de genes

de la asimilacin de nitrgeno en Haloferax
mediterranei
Agradecemos la financiacin del proyecto P10-CVI-6368 y BIO-163 de

la Consejera de Economa, Innovacin y Ciencia, Junta de Andaluca, y
Plan Propio Universidad Sevilla.
Rebeca Gonzlez Guerrero, Anna Vegara Luque, Gabriel Felipe

Rodrguez Lozano, Julia Mara Esclapez Espliego, Carmen Luca
Pire Galiana, Mara Jos Bonete Prez
Grupo Biotecnologa de Extremfilos, Universidad de Alicante, San
Vicente del Raspeig, ES
Haloferax mediterranei es una arquea halfila extrema capaz de adaptar
su metabolismo en funcin de la fuente de nitrgeno disponible. Puede
crecer en presencia de amonio, nitrato, nitrito o aminocidos como nica
fuente de nitrgeno. Se han llevado a cabo anlisis mediante microarrays
y por qPCR que indican cules son los genes cuya transcripcin se ve
afectada en funcin de la disponibilidad y de la fuente de nitrgeno.
Entre estos genes se encuentran los que codifican la glutamina sintetasa
(glnA), glutamato sintasa (gltS) y NAD-glutamato deshidrogenasa
(gdh-1). Se han clonado los promotores de estos genes fusionados con
el gen de la -galactosidasa de Haloferax lucentensis (bgaH) como gen
reportero en el vector pVA-513 con el que se ha transformado Haloferax
mediterranei, midindose la actividad -galactosidasa de las clulas
transformadas en medios con diferentes fuentes de nitrgeno. Adems
se han realizado mutantes en el promotor del gen gltS que permiten la
identificacin de sitios clave en la regulacin de la actividad de este
promotor.
Por ltimo con el objetivo de identificar los posibles reguladores
transcripcionales que se unan al promotor del gen gltS, se han realizado
ensayos de cromatografa de afinidad DNA-protena, mediante la unin
del promotor biotinilado a una matriz magntica con estreptavidina, e
incubando con extractos de Haloferax mediterranei obtenidos en diferentes
condiciones de cultivo.
P12-12 (R12-11)
THB1, una hemoglobina truncada regulada por

nitrgeno, modula los niveles de xido ntrico
(NO) y la actividad NR en Chlamydomonas
Francisco Javier Ocaa Calahorro, Emanuel Sanz Luque, Aurora
Galvn Cejudo, Emilio Fernndez Reyes
Departamento de Bioqumica y Biologa Molecular de la Universidad
de Crdoba, Crdoba, ES
La gran familia de hemoglobinas est presente en todos los organismos
vivos. Durante mucho tiempo, se ha relacionado a estas protenas con el
transporte y almacenamiento de oxgeno. Sin embargo, su participacin
en diferentes procesos fisiolgicos y una emergente variedad de funciones,
le confieren una gran importancia, aunque la mayora de ellas son todava
poco conocidas. En los ltimos aos, se ha relacionado a las hemoglobinas
con la degradacin del NO, que est implicado en multitud de procesos de
sealizacin celular, de esta manera se evitaran posibles efectos txicos
del NO debido a su acumulacin. En el alga verde Chlamydomonas
reinhardtii, encontramos la mayor familia de hemoglobinas truncadas
descritas hasta la fecha (THB1 -12). En este trabajo, se describe cmo
dos de ellas (THB1 y THB2) estn reguladas por la fuente de nitrgeno
y el factor de transcripcin NIT2, que controla gran parte de los genes
implicados en la asimilacin de nitrato en el alga (transportadores y
reductasas especficas). Tambin se muestra cmo THB1 se sobreexpresa
en presencia de NO y cmo es capaz de convertir in vitro el NO en nitrato.
Adems, se caracteriza cmo la nitrato reductasa est implicada de manera
115
Psters
muy eficiente en la reduccin de la THB1 necesaria para su actividad, y
cmo su represin produce un aumento de la actividad NR. Por lo tanto,
sugerimos en este trabajo, que esta hemoglobina controla los niveles de NO
e inhibe la actividad NR simultneamente.
P12-13 (R12-6)
Estudio mediante tcnicas micas del papel

sealizador de la glutamina sintetasa plastdica
en el metabolismo de la leguminosa modelo
Lotus japonicus
Margarita Garca-Caldern, Jess Girldez, Carmen M. PrezDelgado, Marco Betti, Antonio J. Mrquez
Departamento de Bioqumica Vegetal y Biologa Molecular, Facultad
de Qumica, Universidad de Sevilla, Sevilla, ES
El mutante fotorrespiratorio Ljgln2-2 de la leguminosa modelo Lotus
japonicus es deficiente en la enzima glutamina sintetasa plastdica
(GS2), siendo incapaz de reasimilar el amonio fotorrespiratorio cuando
se cultiva en condiciones de aire (fotorrespiracin activa) [1]. Cuando
el mutante se cultiva bajo una atmsfera de alto CO2, la planta no realiza
la fotorrespiracin, suprimiendo el efecto de la deficiencia en GS2 en
este proceso. Para analizar si la GS2 pudiera estar implicada en otros
aspectos del metabolismo de la planta adems de en la fotorrespiracin,
se utilizaron tcnicas micas en el estudio de plantas silvestres y
mutantes bajo condiciones de alto CO2. En plantas en simbiosis con
Mesorhizobium loti, genes correspondientes al metabolismo del
almidn y sacarosa y metabolismo de aminocidos mostraron menor
expresin en plantas mutantes respecto a WT. Los resultados sugieren
que en plantas noduladas, la deficiencia en GS2 conlleva alteraciones
en la expresin de genes del metabolismo del C y N, sugiriendo un
papel de la GS2 en la sealizacin del balance C/N en plantas de L.
japonicus. Estudios de co-expresin han permitido establecer la
relacin de la GS2 con el metabolismo del C, compatible con otros
resultados anteriormente obtenidos sobre la deficiencia en nodulacin
de mutantes Ljgln2-2 [2].
Bibliografa
[1] Prez-Delgado CM, Garca-Caldern M, Snchez DH, Udvardi MK,
Kopka J, Mrquez AJ, Betti M. 2013. Plant Physiol. 162, 1834-48.
[2] Garca-Caldern M, Chiurazzi M, Espuny MR, Mrquez AJ. 2012.
MPMI 25, 211-9.
Agradecemos la financiacin de la Consejera de Economa, Innovacin y
Ciencia de la Junta de Andaluca (proyecto P10-CVI-6368 y BIO-163) y
Plan Propio Universidad de Sevilla.
P12-14 (R12-4)
Biosntesis de fenilalanina en conferas:

caracterizacin de la familia arogenato
dehidratasa de Pinus pinaster
Jorge El-Azaz Ciudad, Fernando de la Torre, Concepcin vila,
Francisco M. Cnovas
Departamento de Biologa Molecular y Bioqumica, Universidad de
Mlaga, Mlaga, ES
En las plantas, la biosntesis de los aminocidos aromticos tirosina (Tyr)
y fenilalanina (Phe) tiene lugar en los cloroplastos a travs de la ruta del
prefenato, que tiene su origen en la ruta del siquimato. En la ruta del prefenato,
la biosntesis de Phe tiene lugar mediante dos reacciones consecutivas:
la primera de ellas consiste en la conversin de prefenato en arogenato a
travs de una reaccin de transaminacin (actividad prefenato-arogenato
aminotransferasa, PAT) y posteriormente la transformacin del arogenato en
Phe (actividad arogenato dehidratasa, ADT). Muy recientemente, dos nuevas
publicaciones han aportado evidencias respecto a la existencia de una va
116

alternativa de biosntesis de Phe, que no dependera de arogenato (Yoo et
al., 2013; de la Torre et al., 2014). En esta ruta alternativa el prefenato sera
transformado en fenilpiruvato a travs de la enzima prefenato dehidratasa
(PDT). El fenilpiruvato producido en esta reaccin sera posteriormente
convertido en Phe a travs de una transaminasa de aminocidos aromticos.
Una particularidad de especial inters es el hecho de que las actividades ADT
y PDT se encuentran en las mismas protenas en Arabidopsis thaliana (Cho
et al., 2007).
En los ltimos aos, nuestro grupo de investigacin ha estado trabajando
en la ruta del prefenato, centrados principalmente en la enzima PAT de
Pinus pinaster, y ms recientemente en la caracterizacin de familia de
las ADT en esta misma especie. La presente comunicacin desarrolla el
trabajo de caracterizacin que estamos realizando en la familia de genes
ADT/PDT de P. pinaster, integrada por al menos 9 genes candidatos
presentes en el transcriptoma de esta especie (Canales et al., 2013).
Estos genes candidatos presentan patrones de expresin caractersticos
y dependientes del rgano y el estadio de desarrollo de la planta. De
estos 9 genes, 3 de ellos forman un grupo filogentico caracterstico
de gimnospermas. Los objetivos de nuestro trabajo se encuentran
actualmente enfocados en la caracterizacin funcional de este grupo de 3
genes ADT/PDT caractersticos de conferas.
Bibliografa
Canales J, et al. (2013). Plant Biotechnol J. 12(3):286-99.
Cho MH, et al. (2007)J Biol Chem. 282(42):30827-35.
De la Torre F, El-Azaz J, vila C and Cnovas FM. (2014). Plant Physiol.
164(1):92-104.
Yoo H, et al. (2013). Nat Commun. 4:2833.
P12-15 (R12-5)
Efectos de elevados niveles de CO2 y nitrato

durante el desarrollo de las hojas primarias
de girasol
Francisco Jos Canales Castilla, Purificacin De la Haba, Elisabeth
Barrientos, Lourdes de la Mata, Elosa Agera
Departamento de Botnica, Ecologa y Fisiologa Vegetal, Facultad
de Ciencias, Universidad de Crdoba, Crdoba, ES
Se ha estudiado el efecto de elevados niveles de CO2 y nitrato sobre el
desarrollo de las hojas primarias de girasol. Las plantas se cultivaron 42
das con CO2 ambiental (400 mL L-1) y 10 mM de nitrato (C10), CO2
ambiental y 25 mM de nitrato (C25) y elevado CO2 (800 mL L-1) y 25 mM
de nitrato (T), en condiciones controladas de luz, temperatura y humedad
relativa, recolectndose el primer par de hojas a los 16, 22, 32 y 42 das.
Los resultados indican que algunos procesos metablicos son sensibles a
la alta concentracin de CO2 y nitrato durante la ontogenia de las hojas
primarias de girasol. As, las plantas cultivadas con elevada concentracin
de CO2 mostraron un mayor crecimiento que las plantas cultivadas con CO2
ambiental, como refleja la determinacin del peso seco (PS) de la planta
completa, PS y rea foliar as como la determinacin de la masa foliar
especfica (SLM). La elevada concentracin de CO2 y nitrato (T) aument
significativamente la fijacin fotosinttica de CO2 en las hojas en relacin
con la concentracin de CO2 ambiental (C10 y C25), principalmente en
hojas de jvenes. El contenido en pigmentos fotosintticos disminuy
con el desarrollo de la hoja en todos los tratamientos, especialmente con
elevados CO2 y nitrato (T), producindose la mayor disminucin entre
los 28 y 42 das. Estos resultados sugieren que el elevado CO2 acelera
la degradacin de pigmentos fotosintticos y posiblemente tambin la
senescencia de la hoja. En los tres tratamientos, se genera un importante
estrs oxidativo durante la senescencia de hojas primarias de girasol,
como se revela por la acumulacin de H2O2, siendo esta acumulacin ms
elevada en plantas crecidas con elevado CO2, y elevado nitrato. En hojas
senescentes el incremento en los niveles de H2O2 ocurri en paralelo con
una disminucin en la actividad de las enzimas antioxidantes (catalasa
y ascorbato peroxidasa). Nuestro resultados indican que una elevada
exposicin a CO2 puede provocar estrs oxidativo debido a la disminucin
Granada 2014
Psters
en la actividad de enzimas antioxidantes posiblemente como consecuencia

de un incremento de la carbonilacin de protenas (Qui et al. 2008.
Photosynth Res 97: 155-66).
sea mnimo o rico. Entre ellos destacan algunas de las oxidasas terminales y
otros genes que podran proporcionar pistas acerca de la ruta de asimilacin
del cianuro.
Agradecimientos: Investigacin financiada por la Junta de Andaluca

Grupo de Investigacin BIO-0159).
Los autores agradecen la financiacin de este trabajo por parte del

Ministerio de Ciencia e Innovacin (BIO2011-30026-C02-01), del
Gobierno de Extremadura (GR10165) y FEDER 2007-2013. D. Macas
agradece la beca concedida por el Programa de captacin y formacin de
recursos humanos de excelencia en investigacin, desarrollo e innovacin
2010 de la UEX cofinanciado a su vez por la Junta de Extremadura. Los
autores agradecen la ayuda tcnica de G. Gutirrez.
P12-16
Regulacin de enzimas esenciales en el

metabolismo de H. mediterranei en funcin
de la fuente de nitrgeno
Anna Vegara, Catalina Ruz, Mnica Camacho, Julia Esclapez,
Vanesa Bautista, Mara Jos Bonete
Divisin de Bioqumica y Biologa Molecular, Universidad de
Haloferax mediterranei es una arquea halfila extrema que puede utilizar
diferentes fuentes de nitrgeno inorgnicas u orgnicas para su crecimiento.
Este microorganismo posee dos genes (glnK1 y glnK2) que codifican
protenas de transduccin de seales altamente conservadas, ambas
pertenecientes a la superfamilia de las PII. Estas protenas reguladoras
actan en la clula para que pueda adaptarse a las diferentes condiciones
del medio; perciben seales del balance entre carbono y nitrgeno,
modificando la actividad de las principales enzimas implicadas en los
ciclos de dichos elementos. Con la generacin de mutantes knockout en
H. mediterranei deficientes en los genes glnK1 y glnK2, mediante estudios
fisiolgicos y ensayos enzimticos, se ha estudiado cmo afecta la ausencia
de estas protenas reguladoras a la actividad de enzimas clave como son
glutamina sintetasa, isocitrato deshidrogenasa y glutamato deshidrogenasa.
Los resultados sugieren que la actividad isocitrato deshidrogenasa regula la
ruta de asimilacin de amonio, ya que un aumento de su actividad produce
una disminucin de la de glutamato deshidrogenasa. Adems, las dos
protenas PII ejercen la misma funcin de activacin sobre la actividad de
glutamina sintetasa.
P12-17
Efecto del cianuro sobre la expresin gnica

de Pseudomonas pseudoalcaligenes CECT 5344
Gracia Becerra, Mara Isabel Igeo, Mara Isabel Carmona, Mara
Isabel Guijo, Faustino Merchn, Rafael Blasco Pl
Departamento de Bioqumica y Biologa Molecular y Gentica,
Facultad de Veterinaria, Universidad de Extremadura, Cceres, ES
Pseudomonas pseudoalcaligenes CECT 5344 es una bacteria que se
aisl por su capacidad de utilizar cianuro como fuente de nitrgeno. El
anlisis trascriptmico (RNA-seq) de la respuesta global a cianuro de la
cepa CECT 5344 en medio mnimo ha puesto de manifiesto que la mayora
de los genes regulados positivamente por cianuro estn relacionados con
procesos que ya han sido previamente descritos en el mismo contexto,
pero utilizando otras aproximaciones experimentales. As, la presencia de
cianuro induce la expresin de operones implicados en el metabolismo
del nitrgeno (cianato, aminocidos y nitrilos), en la respuesta al estrs
oxidativo y en la respiracin insensible a cianuro (oxidasas terminales).
El cianuro induce tambin la expresin de un nuevo grupo de genes
probablemente involucrados en la degradacin de compuestos aromticos.
Los genes regulados negativamente codifican protenas pertenecientes a
tres categoras: protenas ribosmicas, transportadores (fosfato y sulfato)
y protenas implicadas en el metabolismo energtico (ciclo de Krebs y
fosforilacin oxidativa). Estos resultados se han validado por q-PCR. El
papel de los nuevos genes implicados en el metabolismo del cianuro, y no
descritos hasta la fecha, se analizar mediante mutagnesis dirigida.
La mayora de los genes cuya expresin vara en respuesta a cianuro en
medio mnimo tambin lo hace, y en el mismo sentido, en medio LB. Sin
embargo, existen genes cuya expresin vara dependiendo de que el medio
P12-18
Papel de Trk1 y de la fuente de nitrgeno en

la regulacin del transportador de K+ Hak1 en
levaduras
Norberto Escudero*1, Mara C. lvarez2, Jos Ramos2, Jos M.
Siverio*1
1
Departamento de Bioqumica, Universidad de La Laguna,
La Laguna, ES, 2Departamento de Microbiologa, Universidad de
Crdoba, Crdoba, ES
La levadura Hansenula polymorpha presenta dos transportadores de K+;
Hak1, de alta afinidad y regulado por los niveles de K+; y Trk1, de baja
afinidad y constitutivo. HAK1 es inducido a bajas concentraciones de K+.
La fuente de nitrgeno tambin afecta los niveles de Hak1. La presencia de
concentraciones elevadas de K+ desencadena la endocitosis y degradacin
vacuolar de la permeasa. La delecin del gen TRK1 desregula HAK1. En
una cepa trk1, los niveles tanto del gen (HAK1) como de la permeasa
(Hak1) son ms altos que en la cepa silvestre. Los mutantes trk1 son
hipersensibles a Li+ y Na+; y los elevados niveles de Hak1 son regulados
a la baja slo en concentraciones superiores a 50 mM K+. Nuestros
resultados sugieren que Trk1 podra actuar como un sensor de K+ en la
levadura por mecanismos que desconocemos. La sobreexpresin de TRK1
podra aportar pistas a este respecto. Para ello hemos creado una cepa en la
que TRK1 se expresa bajo el promotor del gen de la nitrato reductasa YNR1.
Resultados muy preliminares indican que los niveles de Hak1 son bajos en
medios con bajo K+.
P13. Neurobiologa molecular

P13-1 (R13-3)
APC/C-Cdh1 coordinates neurogenesis

and cortical size during development
Mara Delgado Esteban1, Irene Garca-Higuera2, Sergio Moreno2,
ngeles Almeida1
1
Instituto de Investigacin Biomdica de Salamanca-IBSAL,
Hospital Universitario de Salamanca, Fundacin IECSCYL Instituto de Biologa Funcional y Genmica-IBFG, CSIC/Universidad
de Salamanca, IBSAL , Salamanca, ES, 2Instituto de Biologa
Funcional y Genmica-IBFG, CSIC/Universidad de Salamanca,
IBSAL, Salamanca, ES
The morphology of the adult brain is the result of a delicate balance
between neural progenitor proliferation and the initiation of neurogenesis
in the embryonic period. Here we assessed whether the anaphasepromoting complex/cyclosome (APC/C) cofactor, Cdh1 which regulates
mitosis exit and G1-phase length in dividing cellsregulates neurogenesis
in vivo. We use an embryo-restricted Cdh1 knockout mouse model and
show that functional APC/C-Cdh1 ubiquitin ligase activity is required for
both terminal differentiation of cortical neurons in vitro and neurogenesis
in vivo. Further, genetic ablation of Cdh1 impairs the ability of APC/C to
117
Psters
promote neurogenesis by delaying the exit of the progenitor cells from the
cell cycle. This causes replicative stress and p53-mediated apoptotic death
resulting in decreased number of cortical neurons and cortex size. These
results demonstrate that APC/C-Cdh1 coordinates cortical neurogenesis
and size, thus posing Cdh1 in the molecular pathogenesis of congenital
neurodevelopmental disorders, such as microcephaly.
This work was funded by FEDER (European regional development fund)
and Instituto de Salud Carlos III (PI12/00685; RD06/0026/1008 and
RD12/0014/0007).
P13r-2
Mitochondrial supercomplexes assembly explains

differences in reactive oxygen species production
between neurons and astrocytes
Irene Lpez Fabuel, Juan Pedro Bolaos
Instituto de Biologa Funcional y Genmica, Salamanca, ES
Understanding the physiological roles of reactive oxygen species (ROS) in
the brain requires dissecting out the contribution of each of the four different
neural cell types to ROS formation. Here, the abilities of mitochondria
isolated from rodent neurons and astrocytes, the most abundant brain cell
types, to spontaneously form ROS were characterized. We found that ROS
production is about one order of magnitude higher in astrocytes than in
neurons. This observation is intriguing in view of the widely held notion
that astrocytes, which express high levels of antioxidants, are efficient ROS
detoxifiers. We also found that the main source of ROS in both cell types
is the mitochondrial electron transport chain, but the difference is ROS
production cannot be accounted by the relative abundance of total complex
I, a major superoxide anion source within mitochondria. Furthermore, the
high ROS production by astrocytes is conserved amongst several rodents,
including Wistar rat and C57BL6 mice. In view that the mitochondrial
supercomplex assembly factor, SCAFI, is not functional in C57BL6 mice,
we conclude that the high ROS production in astrocytes is not due to a
putative differential expression of this assembly protein. Interestingly, we
found that in astrocytes a large proportion of complex I protein occurs free,
whereas in neurons most complex I is part of supercomplexes. We conclude
that this differential complex I assembly into supercomplexes explains the
high mitochondrial ROS production by astrocytes. These results suggest
cell-specific physiological roles for mitochondrial ROS in the brain.
P13r-3
Understanding ApoD neuroprotective function:

modulation of glial ApoD subcellular traffic upon
metabolic and oxidative stress
Raquel Pascua-Maestro1, Mara Dolores Ganfornina2, Diego
Snchez2
1
Estudiante de doctorado, Dpto. Bioqumica y Biologa Molecular y
Fisiologa / IBGM Universidad de Valladolid / CSIC, Valladolid, ES,
2
Profesor titular, Valladolid, ES
Apolipoprotein D (ApoD) is expressed in the nervous system, increases
with aging and neurodegeneration, and is induced in response to
serum deprivation or oxidative stress. To understand how ApoD traffic
through different subcellular compartments affects glial cells protecting
mechanisms, we analyze the time course of ApoD subcellular traffic upon
exposure to low-serum and paraquat stimuli in a human astroglioma cell
line.
During treatment, the spatial pattern of ApoD reorganizes from large
intracellular perinuclear organelles to a dotted surface pattern, indicative of
engagement in intracellular vesicles followed by a second phase of action
on the membrane. A small fraction of ApoD locates in the endoplasmic
reticulum and Golgi apparatus. Both signals are independent of the
118

experimental conditions. We also detect ApoD in the extracellular medium,
indicating secretion to the extracellular space.
Culture growth and metabolic or oxidative stress alters caveolae and
endosome distribution. The intracellular localization of ApoD indicates
that secretion is followed by interaction with the plasma membrane,
caveolin-dependent endocytosis, and location in endosomes. No ApoD is
immunodetected in mitochondria. Interestingly, an important fraction of
ApoD is located in lysosomes. Large LAMP2-ApoD-positive organelles
indicate ApoD presence in autophagolysosomes which is confirmed
by co-localization with LC-3. ApoD presence inside lysosomes and
autophagolysosomes is stable over time, suggesting an active role in
autophagy.
Previous hypotheses on ApoD neuroprotective roles in glial cells must be
refined: control of plasma membrane and of the lysosome/autophagosome
function must be key elements in the function of this lipid-binding protein.
Support: MICINN (BFU2011-23978), JCyL (VA180A11-2).
P13-4 (R13-1)
Molecular mechanisms underlying Parkinsons

disease: LRRK2 takes centerstage
Sabine Hilfiker
CSIC, Granada, ES
Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene are the
most common cause of familial Parkinsons disease (PD), and risk
factor variants increase the risk for sporadic PD. However, despite the
importance of LRRK2 for the pathogenesis of both familial and sporadic
versions of the disease, its biological function, and the mechanism(s)
by which pathogenic mutations cause neurodegeneration in PD remain
largely unknown. LRRK2 is a large multi-domain containing protein
with various protein-interaction domains, a GTPase and a protein kinase
domain. Altered catalytic activity correlates with neurotoxicity, indicating
that targeting those activities may provide clues as to novel therapeutic
strategies for LRRK2-linked PD. However, the cellular readout(s) of such
altered catalytic activities remain largely unclear. Recent cell biological
studies have started to highlight possible early cellular events which
are altered in the presence of pathogenic LRRK2. These events include
alterations in endocytosis, autophagy, retromer-mediated trafficking
from late endosomes to the Golgi, and Golgi integrity. How mutant
LRRK2 may induce such divergent effects on intracellular membrane
trafficking pathways remains unclear, and may involve a large variety of
distinct molecular mechanisms. Alternatively, since membrane trafficking
pathways are highly interconnected and interdependent, a small number of
LRRK2-mediated cellular alterations may give rise to the wide spectrum
of vesicular trafficking alterations. I will present our data on the role of
pathogenic LRRK2 in regulating Rab protein activities which can account
for the majority of the observed cellular alterations.
P13r-5
The human Tp53 Arg72Pro polymorphism

modulates neuronal vulnerability to amyloid-beta
neurotoxicity
Rebeca Lapresa Ruiz de Gauna, Mara Delgado Esteban, ngeles
Almeida Parra
Instituto de Investigacin Biomdica de Salamanca-IBSAL, Hospital
Universitario de Salamanca, Fundacin IECSCYL - Instituto
de Biologa Funcional y Genmica-IBFG, CSIC/Universidad de
Salamanca, IBSAL, Salamanca, ES
Alzheimers disease (AD) is the most common form of dementia in the
elderly. AD is a neurodegenerative disorder of the Central Nervous System
characterized by an altered amyloid-beta (Ab) metabolism, leading to
deposition of Ab peptides into neuritic plaques, tau hyperphosphorylation,
Granada 2014
which forms neurofibrillary tangles, neuroinflammation and the
degeneration of synapses and neurons in the hippocampus and cortex,
regions implicated in learning and memory.
It has been described that p53 plays an essential role in this neurodegeneration
process [1]. The p53 protein naturally occurs in humans in two variants
with single nucleotide polymorphism (SNP) resulting in Arg or Pro at
residue 72. This SNP occurs in the proline-rich domain of this protein,
which regulates its apoptotic activity [2]. Recently, we have demonstrated
that the Arg72-p53 genotype increases neuronal susceptibility to ischemiainduced apoptosis [3].
The objective of this study was to determine the role of p53 Arg72Pro
polymorphism on the susceptibility of neurons to Ab-induced
neurodegeneration and its potential role as a genetic biomarker for AD risk.
We used cortical neurons in primary culture from wild-type (wt) mice and
both knockout p53 mice (KOp53) and Knock-in human p53, expressing
either p53Pro or p53Arg. Neurons were then incubated in culture medium
containing either 10 M Ab(25-35), the active fragment of Ab, or 10 M
Ab(35-25), the inactive form, for 24 hours. We analysed protein expression
levels by Western Blot and mitochondrial function and apoptosis by flow
cytometry.
We first found that Ab promoted p53 stabilisation in cortical neurons.
Moreover, genetic depletion of p53 prevented Ab-induced mitochondrial
depolarization and neurodegeneration, suggesting that p53 might play an
important role in AD. Furthermore, we observed that the polymorphic
variant p53Arg increased the susceptibility of neurons to mitochondrial
depolarization and the subsequent apoptosis caused by Ab, in comparison
to the p53Pro one. Thus, p53 Arg72Pro polymorphism modulates the
susceptibility of neurons to Ab neurotoxicity and could determine the
extent of brain damage in AD. These results pose Tp53 Arg72Pro SNP as a
good biomarker candidate for AD risk.
This work was funded by FEDER, ISCIII (PI12/0685, RD12/0014/0007)
and Junta de Castilla y Len. R. Lapresa was funded by MECD (Ministerio
de Educacin, Cultura y Deporte, becas FPU).
Bibliografa
[1] Lanni C, Racchi M, Memo M, Govoni S and Uberti D (2012) Free
Radic Biol Med 52:1727-1733.
[2] Pietsch EC, Humbey O, and Murphy ME (2006) Oncogene 25:16021611.
[3] Gomez-Sanchez JC, Delgado-Esteban M, Rodriguez-Hernandez I,
Sobrino T, Perez de la OssaN, Reverte S, Bolaos JP, Gonzalez-Sarmiento
R, Castillo J and Almeida A (2011) J Exp Med 208:429-437.
P13-6
Cdh1 is essential for neurite outgrowth and

synaptic plasticity in the adult brain in vivo
Vernica Bobo Jimnez, Mara Delgado Esteban, Juan Pedro
Bolaos Hernndez, ngeles Almeida Parra
Instituto de Investigacin Biomdica de Salamanca-IBSAL Instituto de Biologa Funcional y Genmica-IBFG, Centro Superior
de Investigaciones Cientficas-CSIC/Universidad de SalamancaUSAL, Salamanca, ES
The E3 ubiquitin ligase APC/C (Anaphase Promoting Complex/Ciclosome)
is a multi subunit complex that regulates progression from metaphase to
anaphase, exit from mitosis and G1 maintenance in proliferating cells.
To be active, APC/C requires the binding of either one of two activators:
Cdc20 or Cdh1. Neurons are postmitotic cells that undergo an active
downregulation of cell cycle-related proteins to survive. However,
neurons retain the ability to activate the cell cycle machinery in response
to pathological circumstances, including both acute injury and chronic
neurodegenerative disorders. Recently, we described that Cdh1 is the main
activator of APC/C in cultured cortical neurons. Moreover, APC/C-Cdh1
is essential for neuronal survival in vitro, as it prevents the activation of
the cell cycle machinery and the subsequent neuronal apoptosis. Here we
Psters
assessed whether Cdh1 regulates neuronal survival in vivo by generating
mice lacking Cdh1 from excitatory neurons of the adult forebrain.
These animals were viable but exhibited a severe impairment in neurite
outgrowth and arborisation, leading to synapse loss and neurodegeneration
in the adult cerebral cortex and hippocampal CA1 region. Moreover,
Cdh1 loss induced basal anxiety and spatial learning and memory deficits.
Our results demonstrate that Cdh1 is essential for neurite outgrowth and
synaptic plasticity in the adult brain and supports the role of APC/C-Cdh1
in neuronal integrity and survival.
Funded: Instituto de Salud Carlos III (PI12/0685 and RD12/0014/0007),
Junta de Castilla y Len (GRS244/A/08 and GREX206), Ministerio de
Ciencia e Innovacin (SAF2010-20008) and Fundacin Miguel Casado
San Jos.
P13r-7
Papel regulador de Wrap53 en la isquemia

cerebral
Irene Snchez Morn, Cristina Rodrguez, ngeles Almeida
Instituto de Investigacin Biomdica de Salamanca-IBSAL, Hospital
Universitario de Salamanca, Fundacin IECSCYL, Salmanca Instituto de Biologa Funcional y Genmica-IBFG, CSIC/Universidad
de Salamanca, IBSAL, Salamanca, ES
Durante la isquemia cerebral se interrumpe el aporte de glucosa y oxgeno
a la regin cerebral afectada, lo que desencadena una compleja cascada de
sealizacin celular, denominada cascada isqumica, que culmina en la
muerte neuronal. Se ha descrito que la protena supresora de tumores p53
est implicada en la muerte neuronal isqumica [1]. En los ltimos aos se
han identificado RNAs
reguladores que desempean un importante papel en el control de la
expresin de genes eucariotas [2]. Wrap53 es un cis-NAT (Natural
Antisense Transcript) que se localiza en el cromosoma 17p13 y solapa
con la regin 5UTR de p53. Se ha descrito que los transcritos de Wrap53
controlan la expresin de p53 al interferir con la regin solapante [3]. Es
ms, se han identificado varios SNP (single nucleotide polymorphisms) en
Wrap53, especficos de humanos, que afectan a la capacidad de regulacin
de los niveles de p53.
En el presente trabajo nos propusimos estudiar la posible funcin de
Wrap53 en la regulacin de la expresin de p53 en neuronas, tras el insulto
isqumico. Adems, hemos analizado la asociacin entre SNP de Wrap53
(rs2287498 y rs2287497) y el estado funcional de pacientes de ictus
isqumico. La expresin de Wrap53 y p53 se analiz mediante RT q-PCR
en neuronas corticales y clulas de neuroblastoma humano (SH-SY5Y)
diferenciadas sometidas a 90 minutos isquemia experimental (deprivacin
de oxgeno y glucosa). Los estudios clnicos se realizaron en una cohorte
de pacientes de ictus isqumico procedente del Hospital Universitario de
Salamanca. El estado funcional de los pacientes se evalu a los 3 meses del
ictus, mediante la escala de Rankin modificada.
Nuestros resultados mostraron que los niveles de Wrap53 y p53 no
variaron significativamente durante la isquemia. Sin embargo, observamos
un incremento significativo en los niveles de Wrap53 a las 24 horas tras
el insulto isqumico. El anlisis genmico revel que el SNP rs2287498
modula el estado funcional de pacientes de ictus, de manera que pacientes
que portan el alelo T presentan peor pronstico funcional a los 3 meses del
infarto cerebral.
En conclusin, nuestros resultados sugieren que Wrap53, NAT de p53,
podra estar modulando la susceptibilidad de las neuronas a la isquemia. Es
ms, el estado funcional de pacientes de ictus isqumico est condicionado
por el SNP rs2287498 de Wrap53, de manera que hemos identificado un
nuevo biomarcador gentico de pronstico en ictus.
El trabajo ha sido financiado por el Instituto de Salud Carlos III
(PI12/0685;RD12/0014/0007), la Junta de Castilla y Len y el FSE (ISM).
119
Psters
Bibliografa
[1] Hong LZ, et al. (2010) Neurosci Bull 26:232-240.
[2] Watanabe T, et al. (2008) Nature 453:539-543.
[3] Mahmoudi et al. (2009) Molcel 33, 462-471.
P13-8
La expresin gnica de receptores de adenosina

y metabotrpicos de glutamato en cultivos
primarios de neuronas se modula por glutamato y
un derivado hidrosoluble de C60 fulereno
Davide Giust1, Tatiana Da Ros2, Mairena Martn3, Jos Luis
Albasanz3
1
Institute of Inflammation and Repair, University of Manchester,
Manchester, UK, 2Dipartimento di Scienze Chimiche e
Farmaceutiche, Universit degli Studi di Trieste, Trieste,
IT,3Departamento de Qumica Inorgnica, Orgnica y Bioqumica,
Facultad de Ciencias y Tecnologas Qumicas / Facultad de
Medicina de Ciudad Real, Centro Regional de Investigaciones
Biomdicas, Universidad de Castilla-La Mancha, Ciudad Real, ES
A concentraciones fisiolgicas el glutamato, como principal neurotransmisor
excitador, participa en procesos de aprendizaje y memoria a travs de receptores
ionotrpicos y metabotrpicos. Sin embargo, a elevadas concentraciones acta
como una neurotoxina, principalmente a travs de receptores ionotrpicos,
produciendo degeneracin y muerte celular propia de muchas enfermedades
neurodegenerativas. Los niveles de glutamato se regulan, entre otros, por la
accin del nuclesido adenosina sobre sus receptores, inhibiendo su liberacin,
a travs de receptores A1, o estimulando la misma, a travs de receptores
A2A. En el presente trabajo se estudi el efecto del L-Glutamato (L-Glu) en
la arquitectura celular y en la expresin de los receptores de adenosina y
metabotrpicos de glutamato; as como el posible papel neuroprotector del
t3ss, un derivado hidrosoluble del C60 fulereno. El estudio se llev a cabo
usando cultivos primarios de neuronas que fueron tratadas con L-Glu y t3ss. Se
determin la viabilidad celular por el mtodo de MTT, se valor la expresin
de las protenas MAP-2 y NEFH por inmunohistoqumica y se cuantificaron
los genes que codifican los mGluR y AdoR por PCR cuantitativa en tiempo
real. Los resultados mostraron que el L-Glu causaba muerte neuronal que
reverta en presencia de t3ss. Este derivado hidrosoluble de fulereno modulaba
los receptores mGlu y Ado en las mismas condiciones en las que reverta el
efecto txico del L-Glu. Por tanto, los resultados sugieren que el fulereno
protege las neuronas de la excitotoxicidad en un proceso que parece estar
asociado a la modulacin de la expresin gnica de los receptores de adenosina
y metabotrpicos de glutamato.

region remain unclear. It has been hypothesized that point mutations and/
or post-translational modifications on a-syn alter the stability of the protein
leading to an increase of its steady-state levels and eventually to neuronal
death. We have set up a microscope-based methodology to longitudinally
track individual neurons and determine the risk of neuronal death induced
by wild-type (wt) and mutant versions of a-syn. Furthermore, to determine
the stability of wt and mutant versions of a-syn in living neurons we
have applied a novel optical pulse-chase methodology based on the
photoswitchable protein Dendra2 and longitudinal analysis to measure
its half-life on neurons in situ. Among the pathological a-syn mutations,
the E46K mutation increases significantly the risk of neuronal death on
primary cortical neurons. Post-translational modifications modulate a-syn
dependent neuronal death risk. We are currently analyzing whether these
effects are associated with a change on the stability of the protein.
Supported by: FP7-Marie Curie IRG (Project ID:224849), MINECO
(BFU2012-39737), Ramn y Cajal Program MICINN (RYC2008-0325)
and UTE project CIMA.
P13-10
Anlisis de la expresin de SERCA2 en cerebros

humanos afectados por la enfermedad de
Alzheimer
M. Rosario Seplveda1, Julio Navascus1, Ana M. Mata2
1
Depto. Biologa Celular, Facultad de Ciencias, Universidad de
Granada, Granada, ES, 2Depto. Bioqumica y Biologa Molecular
y Gentica, Facultad de Ciencias, Universidad de Extremadura,
Badajoz, ES
P13-9 (R13-4)
La enfermedad de Alzheimer es una enfermedad neurodegenerativa que

se caracteriza por una prdida progresiva de memoria y una decadencia
cognitiva. Esta neurodegeneracin est asociada a la presencia de placas
seniles de pptido -amiloide y ovillos neurofibrilares de tau, as como a
una alteracin en la homeostasis del Ca2+ intracelular. Entre los mecanismos
reguladores del Ca2+ neuronal se encuentra la Ca2+-ATPasa de retculo
sarco(endo)plsmico (SERCA), que transporta Ca2+ del citoplasma al interior
del retculo a expensas de la hidrlisis de ATP. En este trabajo se ha analizado
la expresin de SERCA2 mediante inmunohistoqumica en secciones de
cerebros humanos sanos y afectados por la enfermedad de Alzheimer. Los
resultados mostraron localizacin de esta protena en el soma de neuronas
en ambas muestras, y una elevada expresin en otro tipo celular no neural
principalmente en los cerebros afectados. Se realizaron ensayos con
marcadores especficos de astrocitos y microgla, clulas que cada vez son
consideradas ms relevantes en la patognesis de la enfermedad. Nuestros
resultados sugieren una relacin entre la sobreexpresin de SERCA y la
participacin de la gla en la enfermedad de Alzheimer.
Effect of alpha-synuclein pathological mutations

and post-translational modifications on stability
and survival on neurons in situ
Financiacin: Ministerio de Economa y Competitividad (BFU201123313), Junta de Extremadura y FEDER (A.M.M); BFU2010-19981
(J.N.); CEI BioTic Granada mP_BS_35 (M.R.S.).
Ignacio Iigo1, Iker Zuriguel2, Laura Larrea3, Mara Esther Luquin3,

Rafael Aldabe3, Montserrat Arrasate3
1
Center for Applied Medical Research-CIMA, Graduate Program
on Neuroscience and Cognition, School of Medicine, University of
Navarra, Pamplona, ES, 2School of Sciences, University of Navarra,
Pamplona, ES, 3Center for Applied Medical Research-CIMA,
University of Navarra, Pamplona, ES
Alpha-synuclein (a-syn) is a presynaptic protein with a key role on
neurodegenerative disorders like Parkinsons disease (PD) and dementia
with Lewy Bodies (DLB). Although 90% of PD cases are sporadic,
mutations on the gene encoding a-syn (SNCA) cause autosomal
dominant PD. Indeed, duplications and triplications of SNCA gene cause
parkinsonism with prominent dementia linking a-syn levels with neuronal
death. Yet, the mechanisms that regulate a-syn steady-state levels on
sporadic and familial PD cases with point mutations on the SNCA coding
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P13-11
CSP-alpha is essential to maintain

the quiescence of radial-glia like stem cells
in postnatal neurogenesis
Jose Luis Nieto-Gonzalez, Leonardo Gmez-Snchez, Fabiola
Mavillard, Pedro Linares-Clemente, Jose Antonio Martinez-Lopez,
Ricardo Pardal, Rafael Fernandez Chacon
Instituto de Biomedicina de Sevilla (IBiS) HUVR/CSIC/Universidad
de Sevilla, Dpto. de Fisiologa Mdica y Biofsica and CIBERNED,
Sevilla, ES
Cysteine String Protein- (CSP-) is a synaptic co-chaperone that prevents
activity-dependent degeneration of nerve terminals. Mutations in the
Granada 2014
human CSP- gene cause neuronal ceroid lipofuscinosis characterized by
progressive dementia and seizures. Synapses formed onto granule cells
by parvalbumin (PV)-expressing basket cells progressively degenerate in
CSP- KO mice. Using electrophysiology in acute hippocampal slices,
we have found that the properties of GABA release from basket cells are
dysfunctional in CSP- KO mice. In addition, at the dentate gyrus of CSP-
KO mice, the number of calretinin-expressing neurons is significantly
increased. We have systematically used BrdU injections and specific
markers to uncover a severe deregulation of adult neurogenesis. We have
observed that the pool of radial-glia like stem cells becomes progressively
depleted due to hyper-proliferation, likely caused by a loss of stem cell
quiescence. Surprisingly, part of these alterations occurs before basket cell
synaptic dysfunction is established, suggesting that proliferation increase
is partly due to a cell autonomous mechanism. Indeed, in the absence
of CSP-, although the number of neurospheres formed is much higher
during the first passages, this number progressively becomes much lower
than in controls. Our data are compatible with two types of alterations
in adult neurogenesis: circuit-independent and circuit (PV neurons)dependent mechanisms. Remarkably, our study uncovers an unanticipated
requirement of CSP- to maintain the quiescence of radial-glia like cells in
postnatal neurogenesis.
P13-12
POMGnT1, a protein involved in muscle-eyebrain disease, is located in the Golgi complex

in the mouse retina and 661W photoreceptors
Mary Luz Uribe1, Carmen Haro1, Mara Paz Ventero1, Laura
Campello1, Jess Cruces2, Jos Martn Nieto1
1
Departamento de Fisiologa, Gentica y Microbiologa, Facultad
de Ciencias, Universidad de Alicante, Alicante, ES, 2Departamento
de Bioqumica, Facultad de Medicina, Universidad Autnoma
de Madrid, Instituto de Investigaciones Biomdicas UAM-CSIC,
IdiPAZ, Madrid, ES
The POMGNT1 gene, encoding protein O-linked mannose 1,2-Nacetylglucosaminyltransferase 1, is associated with muscle-eye-brain
disease (MEB) and other dystroglycanopathies. Its lack of function or
expression causes hypoglycosylation of alpha-dystroglycan (-DG) in
muscle and CNS (including brain and retina), where it catalyzes the
transfer of N-acetyl-glucosamine to O-mannosylated -DG. MEB patients
ocular symptoms include retinal dysplasia and detachment, glaucoma
and abnormal electroretinogram. In this context, -DG O-glycosylation
is known to be relevant for the establishment of functional synapses
between photoreceptors and their postsynaptic, bipolar cells in the retina.
Nonetheless, the POMGnT1 expression pattern in the normal mammalian
retina remains to be addressed.
In this work we have demonstrated by means of RT-PCR and
immunoblotting analyses that the POMGNT1 gene is expressed at the
mRNA and protein levels in all mammalian species studied, from rodents
to humans. Immunohistochemical studies have shown that it concentrates
in the myoid portion of mouse photoreceptor inner segments, where it
colocalizes with GM130, a Golgi complex protein marker. Its presence
in the Golgi has also been revealed in 661W cells, a mouse cone
photoreceptor immortalized cell line. However, and in contrast to retinal
tissue, POMGnT1 additionally accumulates in the nucleus in 661W
photoreceptors, where it colocalizes with POMT1 and POMT2, two
proteins known to form a heterodimer active in the O-linked addition
of mannose to (unglycosylated) -DG. These results are suggestive that
POMGnT1 not only participates in the O-glycosylation of -DG in the
Golgi complex, but also of other yet-to-be-identified proteins in the
nucleus of mouse photoreceptors.
Funding: Instituto de Salud Carlos III grant PI12/0157.
Psters
P13-13
Alterations in age-dependent spatial segregation

of secretory vesicles upon treatments associated
with Parkinsons disease
Mar Martnez Salvador, Elena Fernndez, Sabine Hilfiker
Instituto de biomedicina Lpez Neyra, CSIC, Armilla, ES
Neurons and neuroendocrine cells are packed with secretory vesicles,
only a few of which seem to be releasable upon appropriate stimuli.
Previous studies have shown that vesicles display functional and spatial
segregation according to age, whereby newly synthesized vesicles seem
to be preferentially released. Here, using various fluorescent cargo
proteins which change colour with time fused to either neuropeptides
or select neurotransmitter transporters, we evaluated effects of toxins
associated with Parkinsons disease on secretory vesicle age and
distribution in dopaminergic cells in culture. We found alterations in the
percentage of old versus newly synthesized vesicles largely consistent
with previously observed toxin-induced effects on secretion and/or axonal
transport. Various reagents which modulate either proteasome function
or macroautophagy indicate that preferentially older secretory vesicles
are subject to degradation by autophagy (crinophagy). Similarly, older
vesicles seem to be preferentially excluded from neurites, indicating the
existence of a cellular mechanism able to distinguish vesicles according to
their age for their intracellular transport as well as degradation. Additional
studies underway are attempting to address whether proteins associated
with familial Parkinsons disease also cause alterations in vesicle age with
implications for dopaminergic transmission.
P13-14
Increased intracellular iron load is associated

with lysosomal alterations and modulated by TPC
and TRPML2 channel regulation
Beln Fernndez, Sabine Hilfiker
(IPBLN-CSIC), Parque Tecnolgico de Ciencias de la Salud,
Granada, ES
Iron dyshomeostasis has been shown associated with various
neurodegenerative diseases including Parkinsons disease (PD), but the
underlying cellular mechanisms leading to eventual cellular demise remain
unclear. Here, we show increased iron content in postmortem samples from
PD patients as compared to age-matched controls. Artificially increasing
iron content in cultured dopaminergic neuronal as well as non-neuronal cells
causes increased apoptosis in a time- and dose-dependent manner, which
is most pronounced in dopaminergic cells. Under non-toxic conditions,
increasing iron content is associated with a pronounced proliferation of late
endosomal/lysosomal compartments, where iron seems to be preferentially
stored. Furthermore, increased intracellular iron load is associated
with increased oxidative stress and autophagy induction. Interestingly,
application of NAADP, an agonist of lysosomal TPC and TRPML
channels, potentiates cell death associated with increased cellular iron
content, whilst its antagonist Ned-19 inhibits the effects. Overexpression
of TPC1 and TPC2 potentiates cell death induced upon intracellular iron
increase, whilst the dominant-negative channel mutants are without effect.
NAADP triggers a large increase in cytosolic iron in TPC2-expressing
cells, indicating that iron is released from lysosomal stores in an NAADPmediated manner. Similar effects are observed with TRPML2, indicating
that both channels regulate iron release from lysosomal compartments.
Together, our data indicate that increased cellular iron load is associated
with an increase in lysosomal proliferation which may be a consequence
of enhanced iron storage in this organelle, and that iron release from acidic
stores involves TPC2 and TRPML2 channels in a manner regulatable by
NAADP and Ned-19. These data indicate that modulating lysosomal iron
storage and release capacities may be a beneficial therapeutic strategy for a
variety of disorders associated with iron dyshomeostasis.
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P13-15
Migration was studied using wound-healing assays, Time-Lapse live-cell

Imaging and Immunocytochemistry.
Results: Our results show that CPPs are internalized and reduced the rate
of cell growth. Interestingly, the effects of CPPs are stronger in GSCs
compared to other type of cells. TAT sequence did not significantly change
the rate of growth.
Although several studies show that Cx43 increases cell migration, our
results indicate that the region of Cx43 that interacts with c-Src does not
exert this effect. In fact, the CPPs used in this study reduced the rate of
GSC migration and locomotion.
Conclusions: Our results indicate that c-Src plays an essential role in the
effects of Cx43 in GSCs proliferation and migration and suggest these
CPPs as promising therapies .
El dficit en C-RAF incrementa la

neuroinflamacin y la apoptosis en el receptor
auditivo tras exposicin a estrs por ruido
Roco de Iriarte Rodrguez1, Marta Magarios Snchez2, Isabel
Varela-Nieto1, Ulf R. Rapp3
1
Instituto de Investigaciones Biomdicas Alberto Sols, CSICUAM - CIBERER, Unit 761, Instituto de Salud Carlos III , Madrid,
ES, 2Instituto de Investigaciones Biomdicas Alberto Sols,
CSIC-UAM - CIBERER, Unit 761, Instituto de Salud Carlos III
- Departamento de Biologa, Universidad Autnoma de Madrid ,
Madrid, ES, 3Department of Molecular Biology, Max-Planck-Institute
of Biochemistry, Madrid, ES
Las protenas RAF son serina treonina quinasas pertenecientes a la
ruta RAS-RAF-MERK-ERK. Son esenciales para la proliferacin,
supervivencia y diferenciacin celular durante el desarrollo, y en la
homeostasis de los tejidos adultos. El dficit a homocigosis de CRAF es
una enfermedad rara sindrmica que cursa sordera neurosensorial en el
hombre (ORPHA648), observndose el mismo fenotipo en el ratn nulo
C-Raf-/-. En contraste, los animales heterocigotos C-Raf+/- no muestran
alteraciones funcionales ni anatmicas.
Nuestro objetivo ha sido estudiar s el dficit parcial de C-Raf predispone
al dao auditivo. Para ello se ha utilizado como agente estresor el ruido y
ratones C-Raf +/+ y C-Raf +/- y se ha realizado el estudio comparado de
la neurofisiologa (potenciales auditivos del tronco cerebral), la morfologa
y citoarquitectura (histologa, inmunofluorescencia, TUNEL) y niveles de
marcadores de tipo celular, proliferacin, supervivencia e inflamacin (RTqPCR y Western blotting).
Los ratones de ambos genotipos presentaron un incremento de 50 dB SPL en
el umbral auditivo inmediatamente tras la exposicin al ruido. Los ratones
C-Raf +/+ recuperaron parcialmente la funcin auditiva 35 das despus,
por el contrario, los ratones C-Raf+/- presentaron una prdida auditiva
irreversible. As mismo, mostraron un mayor dao celular y un aumento del
90% en el nmero de clulas apoptticas. Las ccleas de los ratones C-Raf
+/- presentaron mayores niveles basales de activacin de JNK que las de
C-Raf +/+. Ambos genotipos expuestos a ruido incrementaron la actividad
de ERK y de protenas implicadas en la respuesta inflamatoria (p-JNK) en
la cclea, as como en marcadores de apoptosis (PARP-1 fragmentado).
En resumen, nuestros resultados indican que el receptor auditivo mantiene
su funcin aun cuando los niveles de C-RAF estn disminuidos, pero que
se reduce su capacidad de recuperacin tras la exposicin a estrs.
Agradecimientos: RdI tiene un contrato asociado al proyecto SAF201124391. El trabajo ha sido financiado con la ayuda de este proyecto y con
el FP7-AFHELO.
P13r-16
Specificity of cell-penetrating peptides based on

connexin43 and c-Src in glioma stem cells
Myriam Jaraz Rodrguez, Marta Domnguez-Prieto, Jos M. Medina,
Arantxa Tabernero
Instituto de Neurociencias de Castilla y Len-INCYL, Salamanca, ES
Glioma stem cells (GSCs) are thought to be responsible for tumor
initiation, relapse, and therapeutic resistance in gliomas, the most common
brain tumors. Connexin43 (Cx43), the main gap junction channelforming protein in astrocytes, is down-regulated in GSCs. Interestingly,
restoring Cx43 reverses GSC phenotype and consequently reduces their
tumorigenicity. Our previous work shows that the interaction of Cx43 with
c-Src is responsible for this antitumorigenic effect.
Aims: On this basis, we have designed several cell-penetrating peptides
(CPPs) containing different regions of Cx43 involved in c-Src interaction
and we have investigated their role on GSC proliferation and migration.
Methods: Human G166 GSCs, C6 rat glioma cells, neurons and astrocytes
cultures were treated with CPPs. Cell growth was analyzed by MTT.
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P13-17
Effect of Parkinsons disease-associated LRRK2

mutations and kinase inhibition on microtubule
interactions
Marian Blanca Ramrez1, Elena Fdez2, Sabine Hilfiker2
Granada, ES, 2Instituto de Parasitologa y Biomedicina LpezNeyra, CSIC, (IPBLN-CSIC), Parque Tecnolgico de Ciencias de la
Salud, Granada, ES
Mutations in LRRK2 (leucine-rich repeat kinase 2) are associated

with both sporadic and familial Parkinsons disease (PD). LRRK2 is
a large protein containing both GTPase and kinase activities. Various
studies indicate that LRRK2 interacts with microtubules and modulates
cytoskeletal functions by regulating microtubule dynamics. We find that
PD-related mutations in the GTPase domain, but not the kinase domain,
cause templating of LRRK2 onto stable, acetylated microtubules. A
similar templating is observed when inhibiting the kinase activity of
LRRK2 by various distinct, specific kinase inhibitors. Such interaction
can be partially disrupted by nocodazole, and fully disrupted upon coldinduced microtubule depolymerization. Microtubule regrowth assays
indicate that LRRK2 filaments form with a delay after microtubules
are reformed. Modulating the acetylation status of microtubules causes
alterations in filament formation consistent with the idea that pathogenic
LRRK2 interacts preferentially with stable microtubules. An artificial
mutation in LRRK2 which enhances GTP hydrolysis decreases filament
formation, whilst a mutation which abolishes an autophosphorylation
site in LRRK2 is without effect. Together, the data indicate that LRRK2
filament formation is dependent on its GTPase activity, modulated by
kinase inhibition, and occurs by preferential association with stable
microtubules. Further studies are under way to determine the impact
of such interactions on modulating posttranslational microtubule
modifications.
P13r-18
Targeted modulation of the glial inflammatory

response in Retinitis Pigmentosa attenuates
photoreceptor cell death
Mara Platn Corchado1, Catalina Hernndez-Snchez1, Alberto M.
Hernndez-Pinto1, Miguel Marchena1, Noem lvarez-Lindo1, Ana
I. Arroba1, Sean Jmaeff2, Pablo F. Barcelona2, H. Uri Saragovi2,
Enrique J. de la Rosa1
1
Centro de Investigaciones Biolgicas-CIB-CSIC, Madrid, ES,
2
McGill University, Lady Davis Institute-Jewish General Hospital,
Montreal, CA
Purpose: Retinitis Pigmentosa (RP) is a heterogeneous group of genetic
retinal dystrophies. In most forms of RP, photoreceptor cell death is
associated with disease progression. Reactive Mller cell gliosis and
microglial activation are also found, often prior to photoreceptor death.
Granada 2014
Here, we studied inflammatory pathways, arising from Mller cell and
microglia that regulate neuronal cell death.
Methods: We analyzed the expression of pro-inflammatory cytokines and
of markers of reactive Mller cell gliosis and microglial activation by qRTPCR and immunohistochemistry in retinas from wild type and RP mouse
models. Growth factors and pharmacological agents were tested in retinal
explants ex vivo and by intravitreal injection in vivo.
The agents were selected to modulate the glial inflammatory response.
The treatments included clodronate-liposomes (to cause depletion of
microglia), IGF-I (to polarize microglia response), and antagonists of the
p75NTR receptor (a receptor present in Mller and glial cells and whose
activity stimulates TNF production).
Results: A marked increase in GFAP and TNF transcription preceded
photoreceptor cell death in RP retinas. Reduced photoreceptor cell death,
better preservation of the outer nuclear layer, and decreased gliosis were
observed upon treatment.
Conclusions: Our results suggest the possible existence in the RP retinas
of a pro-inflammatory loop mediated by the activation of p75NTR in
Mller glial cells, which causes TNF production. Modulation of the
p75NTR inflammatory response is a possible target to attenuate RP
progression.
P13-19
LRRK2 delays degradative receptor trafficking

by impeding late endosomal budding through
decreasing Rab7 activity
Pilar Rivero-Ros1, Patricia Gmez-Suaga1, Elena Fdez1, Marian
Blanca Ramrez1, Isidro Ferrer2, Ana Aiastui3, Adolfo Lpez de
Munain3, Sabine Hilfiker1
1
Institute of Parasitology and Biomedicine Lpez-Neyra, Consejo
Superior de Investigaciones Cientficas-CSIC, Granada, ES,
2
Institute of Neuropathology, IDIBELL-University Hospital Bellvitge,
University of Barcelona, Hospitalet de Llobregat, ES, 3Neuroscience
Area, Biodonostia Institute, San Sebastin, ES
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause lateonset autosomal dominant Parkinsons disease (PD), and sequence
variations at the LRRK2 locus are associated with increased risk
for sporadic PD. LRRK2 contains both GTPase and kinase domains
flanked by protein interaction motifs, and mutations associated with
familial PD have been described for both catalytic domains. LRRK2
has been implicated in diverse cellular processes, and recent evidence
pinpoints to an important role for LRRK2 in modulating a variety of
intracellular membrane trafficking pathways. However, the underlying
mechanisms are poorly understood. Here, by studying the classical,
well-understood, degradative trafficking pathway of the epidermal
growth factor receptor (EGFR), we show that LRRK2 regulates
endocytic membrane trafficking in a Rab7-dependent manner. Mutant
LRRK2 expression causes a slight delay in early-to-late endosomal
trafficking, and a pronounced delay in trafficking out of late endosomes,
which become aberrantly elongated into tubules. This is accompanied
by a delay in EGFR degradation. The LRRK2-mediated deficits in
EGFR trafficking and degradation can be reverted upon coexpression of
active Rab7 and of a series of proteins involved in bridging the EGFR
to Rab7 on late endosomes. Moreover, expression of TBC1D15, the
Rab7 GAP, mimicks the effects of mutant LRRK2 on EGFR trafficking.
Effector pull-down assays indicate that pathogenic LRRK2 decreases
Rab7 activity both in cells overexpressing LRRK2, as well as in
fibroblasts from pathogenic mutant LRRK2 PD patients as compared
to healthy controls. Together, these findings provide novel insights into
a previously unknown regulation of Rab7 activity by mutant LRRK2
which impairs membrane trafficking at very late stages of the endocytic
pathway. Current studies are underway to determine whether LRRK2mediated changes in intraluminal endolysosomal calcium may account
for altered Rab7 activity.
Psters
P13-20
Pathogenic mutations in LRRK2 cause

alterations in cell cycle progression
and premature centrosome splitting
Jess Madero Prez1, Elena Fdez1, Patricia Gmez-Suaga1, Angus
C. Nairn2, Ana Aiastui3, Adolfo Lpez de Munan3, Sabine Hilfiker1
1
Instituto de Parasitologa y Biomedicina Lpez-Neyra-CSIC,
Armilla, ES, 2Dept. of Psychiatry, Yale University School of
Medicine, New Haven, US, 3Neuroscience Area, Biodonostia
Institute, San Sebastin, ES
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common
cause of familial and sporadic Parkinsons disease (PD). The effects
of pathogenic mutations in LRRK2 have generally been studied in
differentiated, non-dividing neuronal cells. However, recent studies indicate
that pathogenic LRRK2 may play a role in dividing cells as well, as indicated
by alterations in adult neurogenesis or the increased cancer risk in LRRK2
PD patients. Here, we report that three pathogenic LRRK2 mutants cause
alterations in cell cycle progression, nuclear shape alterations and premature
centrosome splitting. Premature centrosome splitting is associated with
partial displacement of the intercentrosomal linker protein rootletin, and can
be partially or fully rescued when expressing rootletin, dominant-negative
Nek2a or active PP1a, indicating a LRRK2-mediated deregulation of the
balance of the Nek2a/PP1 pathway controlling centrosome cohesion via
intercentrosomal linkers. Alterations in centrosome cohesion and nuclear
shape changes are reverted upon pharmacological LRRK2 kinase inhibition.
Centrosomal and nuclear shape alterations are also observed in human
dermal fibroblasts from G2019S LRRK2 mutant PD patients compared to
healthy controls. Together, our data indicate a mechanism for pathogenic
LRRK2 function in dividing cells.
P13-21 (R13-5)
Chronic hypoxia aggravates Alzheimers disease

pathology by causing microglial dysfunction
Rosana March-Diaz1, Antonio Heras-Garvn1, Adrian Viehweger1,
Sebastian Jimenez1, Alberto Serrano-Pozo1, Victoria Navarro1,
Almudena Gerpe2, Marisa Vizuete1, Antonia Gutierrez3, Jos LpezBarneo1, Edurne Berra2, Javier Vitorica1, Alberto Pascual Bravo1
1
Instituto de Biomedicina de Sevilla-IBiS, Hospital Universitario
Virgen del Roco/CSIC/Universidad de Sevilla, Sevilla, ES, 2Centro
de Investigacin Cooperativa en Biociencias, CIC bioGUNE - Parque
Tecnolgico de Bizkaia, Derio, ES, 3Dept. Biologa Celular, Gentica
y Fisiologa - Facultad de Ciencias. Instituto de Investigacin
Biomdica de Mlaga-IBIMA - Universidad de Mlaga, Sevilla, ES
Alzheimers disease (AD) is the most prevalent neurodegenerative disorder
and the most common form of dementia. In many cases AD patients present
concomitant vascular pathology. Low oxygen levels are also frequently
found in the brain of AD patients. The most accepted hypothesis to explain
the correlation between hypoxia and AD is the deposition of amyloid
(A) occurring in the microvasculature (amyloid angiopathy) and the
affectation by the disease of the locus coeruleus, a brain region involved
in the control of brain blood flow. However, few data has been collected to
understand the relation between hypoxia and AD progression.
We show here the accumulation of the hypoxic marker HIF1 (Hypoxiainducible-factor 1), the major transcription factor for the adaptation to
hypoxic conditions, in the brain of AD patients by western blot. We have
also characterized the consequences of chronic exposition to hypoxia in the
progression of the disease using a widely accepted AD mice model.
AD mice were exposed to physiologic hypoxia (8.5% oxygen, 21 days) at
initial and advances stages of the pathology. Brains from hypoxic animals
showed no differences in the A content and number of plaques, but they
showed a clear reduction in the total number of microglial cells that was
even more evident around the A plaques. In vitro analyses suggest that
hypoxia slows down proliferation and chemotaxis towards polymeric A
in both cell line and primary microglial cultures.
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Psters
Interestingly, the brain cortex from the hypoxic animals showed a high
increase in the number of dystrophic neurites surrounding the microgliafree A plaques. We observed also a decrease in the mRNA levels of
two markers of interneurons, Somatostatin and Neuropeptide-Y, in the
hippocampus of hypoxic mice. These data suggest that hypoxia accelerates
the progression of AD pathology.
The pathway underlying microglial affectation by hypoxia has an enormous
potential in neurodegenerative disorders where microglia function is
correlated with the progression of the disease.
P13-22
GPCR homo- and heteroreceptor complexes

in the brain
Dasiel Oscar Borroto Escuela1, Manuel Narvez2, Michael Di
Palma1, Wilber Romero-Fernndez1, Ismel Brito3, Michael Bader4,
Malgorzata Filip5, Luigi F. Agnati1, Pierre Trifilieff6, Jonathan
Javitch7, Kjell Fuxe1
1
Department of Neuroscience, Karolinska Institutet, Stockholm,
SE, 2Department of Physiology, School of Medicine, University of
Malaga, Mlaga, ES, 3IIIA-CSIC, Artificial Intelligence Research
Institute, Spanish National Research Council, Barcelona, ES, 4MaxDelbrck-Center for Molecular Medicine, Berln, DE, 5Laboratory
of Drug Addiction Pharmacology and Department of Pharmacology,
Institute of Pharmacology, Polish Academy of Sciences, Krakw,
PL, 6University Bordeaux 2 INRA, Bordeaux, FR, 7Department of
Neuroscience, Columbia University, New York, US
G proteincoupled receptors (GPCRs) play critical roles in cellular processes
and signaling and have been shown to form homo and heteroreceptor
complexes with diverge biochemical and/or pharmacological activities.
However, despite extensive experimental results supporting the formation
of GPCR homo and heteroreceptor complexes in heterologous systems, the
existence of such receptor-receptor complexes in their native environment
remains largely unknown, mostly because of the lack of appropriate
methodology. For instance, until recently years the methods that have
been developed to study receptor-receptor interactions require that genetic
constructs be expressed in the cells to enable detection of the receptor
interactions, thus excluding the use of tissue samples. In order to demonstrate
in native tissue the existence of GPCR homo and heteroreceptor complexes,
especially in a manner that can be generally applicable to different receptor
pairs, a well-characterized in situ proximity ligation assay (in situ PLA) has
been adapted to confirm the existence of GPCR homo- and heteroreceptor
complexes in brain slices ex vivo. We also describe the in situ PLA procedure
as a high selectivity and sensitivity assay to image GPCR homo- and
heteroreceptor complexes in brain sections by confocal microscopy and how
the assay is performed. We point out as well the method advantages and
disadvantages and compare it to other available techniques.
P13-23 (R13-6)
The neuronal-specific serum and glucocorticoidregulated kinase 1.1 protects against seizureinduced neuronal death
Natalia Armas-Capote1, Adoracin Martnez-Palacin2, Diego lvarez
de la Rosa1, Teresa Girldez3
1
Departamento de Ciencias Biomdicas, Facultad de Medicina y Centro
de Investigaciones Biomdicas de Canarias-CIBICAN, Universidad
de La Laguna, La Laguna, ES, 2Dep. of Biochemistry and Molecular
Biology II, School of Pharmacy, Complutense University of Madrid and
Instituto de Investigacin Sanitaria del Hospital Clnico San CarlosIdISSC, Madrid, ES,3Departamento de Ciencias Biomdicas, Facultad
de Medicina y Centro de Investigaciones Biomedicas de CanariasCIBICAN, Universidad de La Laguna, La Laguna, ES
The M-current formed by tetramerization of Kv7.2 and Kv7.3 subunits is
a neuronal voltage gated K+ conductance that controls resting membrane
124

potential and cellular excitability. Our group has previously shown that
the neuronal isoform of the serum and glucocorticoid-regulated kinase
(SGK1.1), with distinct subcellular localization and modulation, upregulates the Kv7.2/3 current in neurons. Moreover, transgenic mice
(Tg.sgk) expressing a constitutively active form of SGK1.1 (S515D)
were resistent to seizures induced by kainic acid, unveiling a novel role
for SGK1.1 as a physiological regulator of the M-current and neuronal
excitability (Miranda et al, J Neurosci 2013 33:2684). The ubiquous
isoform of this kinase, SGK1, has been involved in protection from
apoptosis (Mikosz et al JBC 2001 276:20) and neurogenesis (Anacker
et al PNAS 2013 110:8708). Both mechanisms have been associated to
seizure-induced neuronal death. Therefore, to explore whether SGK1.1
also protects against seizure cell death, we compared the levels of neuronal
death in Tg.sgk vs wild-type mice, using the Fluoro-Jade staining approach
in different brain regions in mice with comparable seizure development.
Our results show a significant decrease in neuronal death in transgenic
mice after kainate treatment, even under status epilepticus. Experiments are
underway in our laboratory to test signalling pathways involved in SGK1.1
protective role. This result is clinically relevant, since both seizure-induced
cognitive impairments and epileptic seizure severity are influenced by the
extent of damage caused by seizures.
P13-24
Dopamine D1 and D2 receptor immunoreactivities

in the arcuate median eminence complex
Wilber Romero Fernndez1, Dasiel Borroto-Escuela2, Vctor VargasBarroso3, Manuel Narvez Narvez4, Michael Di Palma5, Luigi F
Agnati6, Jorge Larriva Sahd3, Kjell Fuxe2
1
Faculty of Health Sciences, Technical University of Ambato,
Ambato, EC, 2Department of Neuroscience, Karolinska Institute,
Stockholm, SE, 3Institute of Neurobiology, National Autonomous
University of Mexico, Campus Juriquilla, Quertaro, MX,
4
Department of Physiology, School of Medicine, University of
Mlaga, Mlaga, ES, 5Department of Earth, Life and Environmental
Sciences, Section of Physiology, Carlo Bo University of Urbino,
Urbino, IT, 6IRCCS Lido Venice, Venice, IT
Dopamine D1 and D2 receptor (D1DR and D2DR) immunohistochemistry
was used to determine the distribution of the D1DR and D2DR
immunoreactivities (IR) in the rat arcuate-median eminence complex.
Punctate D1DR and D2DR immunoreactivities were enriched in the
lateral palisade zone, while the densities were low to modest in the medial
palisade zone. D1DR but not D2DR IR nerve cell bodies were found
in the ventromedial part of the arcuate nucleus and in the lateral part of
the internal layer of the median eminence. A large number of tyrosine
hydrolase (TH) IR nerve cell bodies in dorsomedial part showed punctate
D1DR IR but only a few presented D2DR IR which were mainly found
in a substantial number of TH cell bodies of the ventral periventricular
hypothalamic nucleus. This immunohistochemical work could explain the
role of D1DR and D2DR in this region.
P13-25 (R13-2)
Super-resolution imaging of cargo transport

Melike Lakadamyali
ICFO, Instituto de Ciencias Fotnicas, Castelldefels, Barcelona, ES
Intracellular transport is essential for maintaining cell function and
morphology. Transport is carried out by molecular motors that convert the
energy of ATP hydrolysis to mechanical motion. Several single molecule
in vitro studies produced a wealth of information on the biophysical
properties and function of these motors. However, the cellular environment
imposes further challenges on motors. Multiple, opposing polarity motors
must coordinate or compete to overcome road-blocks in the crowded
cellular environment to effectively deliver their cargo in the right place at
the right time. We are combining super-resolution microscopy with single
Granada 2014
Psters
particle tracking to understand how motors tackle these challenges. Our

studies have shown that, even the dense microtubule network can present
obstacles to motor-mediated cargo transport, but motors are well-adapted
to circumvent these obstacles.
P13-26
Ultrastructural localization of the core-machinery

for vesicle secretion
Israel Salcedo Gonzlez, Heidi de Wit, Jan van Weering
Department of Functional Genomics, Center for Neurogenomics and
Cognitive Research, Neuroscience Campus Amsterdam, Amsterdam, NL
Neurons communicate with each other through synapses. The secretion
of neurotransmitters is highly regulated by voltage-sensitive channels that
allow Ca2+ influx in the cell triggering the fusion of the vesicles, where the
neurotransmitters are stored, with the active zone in the cell membrane. All
this process is controlled by a set of proteins, including SNARE proteins.
The SNARE proteins are a large family of proteins connected to the cells
membrane system, some, to the vesicles (v-SNAREs) and some others
to the cell membrane (t-SNAREs), while other proteins help this core to
operate correctly. Four proteins are essential for neurotransmitter release:
Syntaxin, Munc18, SNAP25 and Synaptotagmin. This process starts with
the assembly of Syntaxin and SNAP25 in the membrane, helped by Munc18.
After, Synaptotagmin and Synaptobrevin bind to them and form the SNARE
complex, docking the vesicles close to the membrane. When the SNARE
complex is zippered, Complexin inhibits fusion until Synaptotagmin detects
Ca2+ influx, promoting membranes fusion and neurotransmitter release.
It is known that, after fusion, the SNARE complex is dissolved by the
enzyme NSF, but somehow the proteins should be prevented to bind again.
Also, the axon button constitutes a very independent system as it is far
away from the cell soma, making necessary to recycle the vesicles to gain
functionality again.
The aim of this research is to clarify the redistribution and recycling of the
proteins involved in neurotransmitter release after vesicle fusion. To address
this question, in our lab we use primary cultures of mouse chromaffin cells
as a suitable model for neuron vesicle cycle. The cells are stimulated with a
highly concentrated K+ solution to trigger vesicle secretion. They are fixed at
several times after stimulation. To visualize the proteins, immunofluorescent
stainings are used for confocal microscopy, analyzing the redistribution of
the proteins through the cell with the program Image J. To follow the exact
organelle where the proteins are while recycling, electron-microscopy is
used, labeling the proteins with immunogold particles. The data obtained
from these techniques will allow insight for the first time into the cycle of the
proteins responsible of vesicle fusion.
P14. Parasitologa molecular
on-a-chip incorporates for the first time two compartments with different flow
velocities (according to the physiological flow division) and two physical
barriers representing the reticular mesh and the interendothelial slits (IES)
where cells first slow down increasing the hematocrit and then traverse the
IES in a unidirectional matter. To validate the use of this platform, several
experiments were carried out with different types of red blood cells (RBCs). As
a proof of concept, we described that old RBCs showed less deformability than
freshly drawn RBCs when traversing the microconstrictions. Posterior analysis
allowed studying the passage and deformability of infected RBCs through the
IES using peripheral blood of BALB/c mice experimentally infected with
the P. yoelii 17X-GFP strain. Results showed that infected reticulocytes are
significantly more deformable than non-infected reticulocytes, in agreement
with the higher deformability of reticulocytes parasitized by P. vivax, a human
malarial parasite with reticulocyte tropism. These results suggest that the device
is able to reproduce physiological conditions and to distinguish different types
of RBCs by means of deformation/mechanical properties. Presently, we are
determining if there is hemolysis during the pass of blood through the device,
if there is pitting in the absence of macrophages and the rheological properties
of malarial infected RBCs.
P14-2
Cambios inducidos por choque trmico en el

transcriptoma de Leishmania major
Alberto Rastrojo, Begoa Aguado, Jos M. Requena
Centro de Biologa Molecular Severo Ochoa-CSIC-UAM, Madrid, ES
Los parsitos del gnero Leishmania, causantes de un grupo de patologas
conocidas como leishmaniasis, son transmitidos a humanos y otros mamferos
a travs de insectos hematfagos. Durante la transmisin, entre otros cambios,
el parsito debe enfrentarse a un aumento significativo en la temperatura
ambiente, siendo considerado un factor disparador del proceso de diferenciacin.
Para comprender mejor la relacin entre el aumento de temperatura y la
diferenciacin en Leishmania, en este estudio, hemos analizado mediante
RNASeq los cambios en el transcriptoma de la forma promastigote (propia
del insecto vector) asociados a la temperatura de crecimiento propia de sus
huspedes (26 o 37C). La cuantificacin de los niveles de expresin relativa
de los 11.523 transcritos anotados para L. major y su anlisis con diferentes
programas (Cuffdiff, DESeq y DESeq2), han puesto de manifiesto importantes
cambios en el transcriptoma inducidos por choque trmico. Considerando los
resultados comunes a los 3 programas, observamos la expresin diferencial de
111 transcritos, de los que 92 mostraron una acumulacin y 19 disminuyeron
sus niveles. Los transcritos codificantes de amastinas y otras protenas
especficas del estadio amastigote (forma del parsito en el husped mamfero)
aumentaron su expresin, as como transcritos codificantes de quinasas, ciclinas
y protenas de unin a RNA. Tambin observamos la acumulacin durante
el choque trmico de 12 transcritos correspondientes a RNA estructurales
(snoRNA y rRNA). Adems, hemos observado que el choque trmico tiene
efectos importantes sobre mecanismos clave de la regulacin de la expresin
gnica en Leishmania como son el trans-splicing, la poliadenilacin y la
terminacin transcripcional.
P14-1 (R14-2)
Functional microengineered model of the human

splenon-on-a-chip
1
Aleix Elizalde-Torent , Lus Rigat-Brugarolas , Mara Bernabu ,

Mariana De Niz1, Lorena Martn-Jaular1, Carmen FernndezBecerra1, Antoni Homs-Corbera2, Josep Samitier2, Hernando A. Del
Portillo1
1
CRESIB-Barcelona Center for International Health Research,
Barcelona, ES, 2Nanobioengineering group, Institute for
Bioengineering of Catalonia-IBEC, Barcelona, ES
We have developed a newfangled microfluidic device that mimics the
hydrodynamic behavior and filtering functions of the splenon, the minimal
functional unit of the red pulp of the spleen able to maintain its filtering
functions. Unlike any other previous microfluidic devices, the human splenon-
P14-3
Adenylate cyclase toxin promotes bacterial

internalization into non phagocytic cells
Asier Etxaniz Iriondo1, Csar Martn1, Kepa B. Uribe1, Aitor
Etxebarria1, David Gonzlez Bulln1, Jon Arluzea2, Felix M. Goi1,
Juan Archaga2, Helena Ostolaza1
1
Bilbao, ES, 2Departamento de Biologa Celular, Facultad de
Medicina, Universidad del Pas Vasco, Bilbao, ES
Bordetella pertussis causes whooping cough, a respiratory infectious disease
that is the fifth largest cause of vaccine-preventable death in infants. Though
historically considered an extracellular pathogen, this bacterium has been
125
Psters
detected both in vitro and in vivo inside phagocytic and non-phagocytic cells.
However the precise mechanism used by B. pertussis for cell entry, or the
putative bacterial factors involved remain largely unknown. Here we find that
adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is
sufficient to promote bacterial internalization into nonphagocytic cells. After
exhaustive characterization of the entry route we show that uptake of toxincoated bacteria proceeds via a clathrin-independent, caveolaedependent
entry pathway, allowing the internalized bacteria to survive within the cells.
Intracellular bacteria were found inside non-acidic phagosomes enriched
in sphingomyelin and cholesterol, or free in the cytosol of the invaded
cells, suggesting that the ACT-induced bacterial uptake does not follow the
classical phagolysosomal pathway. Activation of Tyr kinases and toxininduced Ca2+-influx are essential for the entry process. We hypothesize that
B. pertussismight use ACT to activate the endocytic machinery of nonphagocytic cells and gain entry into these cells, which represents a possible
mechanism by which it might evade the host immune system.
P14r-4
Molecular epidemiology and analysis of the

risk of infection by Anisakis spp. (Nematoda:
Anisakidae) in sardines (Sardina pilchardus) from
Iberian waters
Dolores Molina Fernndez, David Malagn, Gema Merino Espinosa,
Roco Bentez, F. Javier Adroher, Joaquina Martn Snchez
Departamento de Parasitologa, Facultad de Farmacia, Universidad
de Granada, Granada, ES
Sardine (Sardina pilchardus) is a fish commonly consumed and appreciated
in Mediterranean countries. A molecular epidemiological survey with 190
sardines from 5 fishing areas of Mediterranean (Mlaga, southern Spain)
and Atlantic Spanish coasts (southern: Cdiz and Isla Cristina; northern: A
Corua and Ondarroa) were carried out to analyse the presence of Anisakis
spp. larvae. All found larvae in fish were in third larval stage. The higher
prevalence of these larvae was observed in fish from A Corua (28.3%),
followed from Ondarroa (5%) and Cdiz (2.5%). No Anisakis larvae were
found in fish from Mlaga and Isla Cristina. Three Anisakis genotypes were
identified by polymerase chain reaction followed by restriction fragment
length polymorphism of the ribosomal fragment ITS1-5.8S-ITS2: A. simplex
sensu stricto, A. pegreffii and a hybrid genotype between these two species. A.
pegreffii was the more prevalent species in A Corua (71.0 % of larvae). Only
three Anisakis larvae (9.1% collected larvae) were located in the musculature
of sardines: two of them were identified as A. pegreffii, and the remaining
one was a hybrid genotype. Sardine infection was associated with fishing
area and fish length/weight. Risk factor multivariate analysis shows that the
risk of infection increases 1.6 times per cm in the length of the sardines in the
same fishing area. Also, in equal length, sardines from A Corua have a risk
of parasitization 11.5 times higher than those from other fishing areas. In any
case, the low number of larvae found in the muscle supposes a low human
anisakiasis risk. This risk is less, however, if people eat smaller sardines and/
or sardines from fishing areas with low prevalence of Anisakis infection,
which reduces significantly the presence of Anisakis in this fish.
P14-5
Caracterizacin multignica de los vectores

transmisores de fascioliasis humana y animal en
Chile, basada en la utilidad de marcadores del
ADN ribosomal y mitocondrial
Vernica H Agramunt, Maria Dolores Bargues, Santiago Mas-Coma
Departamento de Parasitologa, Facultad de Farmacia, Universidad
de Valencia, Burjassot, Valencia / Departamento de Ciencias
Biomdicas, Universidad CEU-Cardenal Herrera, Castelln, ES
Las implicaciones de los lymnaeidos en la transmisin de la fascioliasis,
su epidemiologa y control, insta a desarrollar nuevas herramientas para
126

facilitar la clasificacin de muestras, caracterizacin gentica de las
poblaciones naturales y cepas de laboratorio, y para dilucidar la sistemtica
y taxonoma de la famlia Lymnaeidae. Los marcadores de ADN ribosomal
(ADNr) y de ADN mitocondrial (ADNmt) han demostrado ser tiles en esta
tarea en los invertebrados en general. Su aplicacin tambin ha demostrado
ser til en caracoles lymnaeidos a niveles especficos, genricos y de
taxones supragenricos. Los espaciadores transcritos internos del ADNr,
ITS-2 e ITS-1, son las secuencias ms tiles para clasificar especies de
lymnaeidos. Un fragmento del gen de la subunidad I de la citocromo c
oxidasa (cox1) del ADNmt tambin result til, aunque los marcadores
del ADNmt deben ser empleados con precaucin. Cada uno de los tres
marcadores de ADN se amplificaron mediante PCR independientemente
para cada especmen de lymnaeido, y cada producto de PCR se secuenci
para una caracterizacin de haplotipos. Los datos de las secuencias de
nucletidos de los ITS-2 e ITS-1 del ADNr nuclear y de cox1 del ADNmt
de depositaron en el GenBank. Los resultados obtenidos cambian el
escenario de los lymnaeidos conocido hasta ahora en Chile. Se demostr
la presencia de Galba truncatula en Chile, especie que ha sido siempre
confundida con Lymnaea viator. Esta ltima podra ser la principal
responsable de la infeccin animal, mientras que G. truncatula lo sera
de la mayora de casos humanos. Especmenes de Pectinidens diaphana
mostraron una gran variabilidad intraespecfica en su ADN, con un nuevo
haplotipo en ITS-1 y tres nuevos en cox1. Lymnaea patagonica demostr
ser un sinnimo de P. diaphana. Las escasas diferencias nucleotdicas a
nivel de ITS-2 e ITS-1 mantienen a L. patagonica como una subespecie
de P. diaphana. Tampoco Lymnaea lebruni present diferencias suficientes
para considerarlo un taxn distinto respecto a P. diaphana y L. patagonica.
Todo ello proporciona una nueva lnea de base para llevar a cabo futuros
estudios apropiados sobre la transmisin, epidemiologa y control de la
Fascioliasis humana y animal en Chile.
Financiacin: SAF2010-20805, Ministerio de Economa y Competitividad;
RD06/0021/0017, Red de Investigacin Cooperativa en Enfermedades
Tropicales-RICET, RETICS-FEDER, Ministerio de Sanidad, Madrid; y
PROMETEO 2012/042, Programa I+D de Grupos de Investigacin de
Excelencia, Generalitat Valenciana, Valencia.
P14-6 (R14-1)
Carbohydrate-binding agents act as potent

trypanocidals that elicit modifications in VSG
glycosylation
Vctor M. Castillo-Acosta1, Antonio E. Vidal1, Luis Miguel RuizPrez1, Els J.M. Van Damme2, Yasuhiro Igarashi3, Jan Balzarini4,
Dolores Gonzlez-Pacanowska1
1
Instituto de Parasitologa y Biomedicina Lpez - Neyra,
CSIC, Armilla (Granada), ES, 2Laboratory of Biochemistry and
Glycobiology, Department of Molecular Biotechnology, Ghent
University, Ghent, BE, 3Biotechnology Research Center, Toyama
Prefectural University, Toyama, JP, 4Rega Institute for Medical
Research, Leuven, BE
The protozoan parasite Trypanosoma brucei is the etiologic agent of
African trypanosomiasis. The chemotherapy relies on a few drugs
which have adverse side-effects. The cell surface of bloodstream forms
dwelling in the mammalian host is covered by a densely packed coat
of glycosylphosphatidylinositol-anchored glycoproteins. The major
component is the variant surface glycoprotein (VSG) which is glycosylated
by both paucimannose and oligomannose N-glycans for building up the
protective barrier against the immune system. Surface glycans are poorly
accessible and killing mediated by peptide lectin-VSG complexes is
hindered by active endocytosis. In contrast to previous belief, here we
show that high affinity carbohydrate binding agents (CBAs) bind to
surface glycoproteins and abrogate growth of T. brucei bloodstream
forms. Specifically, binding of the mannose-specific Hippeastrum hybrid
agglutinin (HHA) triggered intense perturbations in endocytosis and
further parasite lysis. Prolonged exposure to HHA induced parasites
Granada 2014
resistance based on their diminished capacity to bind the lectin. These
parasites exhibited a loss of triantennary oligomannose at the expense of
paucimannose structures in surface glycoproteins as a result of genetic
rearrangements that abolished expression of the oligosaccharyltransferase
TbSTT3B gene together with the appearance of novel chimeric enzymes.
In addition, we have evaluated the trypanocidal activity of a series of
prokaryote non-peptidic CBAs that rendered parasitological cure in acute
models of African trypanosomiasis. Thus specific glycosylation patterns
are important for CBA cytotoxicity and parasite fitness in vivo and agents
binding efficiently to surface glycoproteins emerge as a new strategy for
therapy for African trypanosomiasis.
P14-7
Funcin, estructura y regulacin de la expresin

de la tirosina aminotransferasa de Leishmania
infantum
Miguel ngel Moreno Izquierdo1, Ana Alonso Ayala1, Pedro Jos
Alcolea Alcolea1, Peter John Myler2, Antonio Jimnez3, Vicente
Larraga Rodrguez1
1
2
Seattle Biomedical Research Institute, Seattle, Washington, US,
3
Universidad de Alcal de Henares, Alcal de Henares, ES
Leishmania infantum es el agente responsable de la leishmaniasis visceral
en la cuenca mediterrnea. En su ciclo biolgico se alternan dos estados:
promastigote (extracelular en el insecto vector, donde desarrolla un proceso
por el que adquiere mayor infectividad conocido como metaciclognesis)
y amastigote (intracelular en el macrfago del hospedador mamfero). La
falta de nutrientes esenciales hace que el parsito utilice rutas alternativas
para obtener energa, como la degradacin de aminocidos aromticos
mediante transaminacin, relacionada con la patogenia y la infectividad por
la toxicidad para el hospedador de los productos finales de degradacin. En
L. infantum esta transaminacin la lleva a cabo la tirosina aminotransferasa
(LiTAT). LiTAT es una protena citoplsmtica, que se expresa en
amastigotes y en promastigotes logartmicos y metacclicos en cultivo
axnico. Por otro lado se sobreexpresa en cepas resistentes a xido ntrico
de L. chagasi. Dicha sobreexpresin podra explicarse por su interaccin
con la malato descarboxilasa, responsable de la sntesis de piruvato a partir
de malato produciendo NADPH, requerido en la proteccin frente a estrs
oxidativo. LiTAT, en ausencia de hierro, sufre una degradacin mediada por
proteasoma, la cual puede ser eludida por la delecin del dominio N-terminal.
Por ello, LiTAT es un buen candidato para un diseo racional de inhibidores
y su estructura unida al cofactor fue resuelta por cristalografa de rayos X
a una resolucin de 2.3 (PDB: 4ix8). Mediante acoplamiento molecular
in silico, los residuos responsables de la diferente especificidad de sustrato
entre LiTAT y el ortlogo en mamferos fueron identificados, permitiendo
generar un grupo farmacforo para la identificacin de nuevos inhibidores
frente a la leishmaniasis.
P14r-8
Identificacin por cribado de una librera de

sobreexpresin de genes implicados en el
mecanismo de resistencia al suero humano de
Trypanosoma brucei gambiense
Jean-Mathieu Bart1, Carlos Cordon-Obras2, Fabian Lorenzo3, Basilio
Valladares3, Agustin Benito1, Miguel Navarro2
1
Centro Nacional de Medicina Tropical Instituto de Salud Carlos
III, Armilla, ES, 2Instituto de Parasitologa y Biomedicina Lpez Neyra, CSIC, Armilla, ES, 3Instituto Universitario de Enfermedades
Tropicales y Salud Pblica de Canarias, Santa Cruz de Tenerife, ES
Trypanosoma es un protozoo que vive en el torrente sanguino de los
mamferos. Desarroll a lo largo de la evolucin una serie de mecanismos para
eludir las respuestas inmunes especficas e innatas de sus huspedes. De las
Psters
tres subespecies de Trypanosoma brucei, dos son infectivas para el humano,
T. b. rhodesiense y T. b. gambiense, mientras T. b. brucei es sensible al accin
del suero humano (SH). Se ha descrito la Apolipoproteina L1 (ApoL1) como
el componente del suero humano responsable de la lisis de los tripanosomas,
formando poros al nivel del lisosoma del parasito. En T. b. rhodesiense, la
expresin de un nico gen, el SRA (Serum Resistante Associated gene) es
suficiente para conferir resistencia al SH, impidiendo la accin de ApoL1. En
T. b. gambiense, el parasito responsable de 95% de los casos de enfermedad de
sueo, el mecanismo de resistencia no est todava bien entendido.
El objetivo de nuestro trabajo era descubrir genes implicados en el
mecanismo desarrollado por T. b. gambiense para resistir al SH. Nuestra
estrategia consisti en complementar la cepa sensible al SH de T. b. brucei
por una librera de expresin construida a partir del ADN genmico de T.
b. gambiense. La seleccin de los clones se hizo por adicin de 1% de SH.
Dos genes han sido identificados: HRF-1 y HRF-2 (Human Resistant
Factor), cuya sobreexpresin permite a T. b. brucei proliferar en presencia
de 10% de SH. HRF-1 se expresa en el nuclolo de la clula, y su papel
regulador esta estudiado va secuenciacin masiva de ARN. HRF-2 es una
protena glicosilada perteneciendo a la familia de la lectinas. El anlisis
de su localizacin subcelular por inmunofluorescencia nos permiti
colocalizar HRF-2 con protenas implicadas en exocytosis. El KO de
ambos genes HRF-1 y 2 en T. b. gambiense est en proceso y nos permitir
evaluar si su falta de expresin impide al parasito de resistir el SH.
A pesar de la ausencia de la protena TgsGP publicada recientemente como
implicada en el mecanismo de resistencia al SH de T. b. gambiense, hemos
caracterizados dos genes HRF-1 y HRF-2 cuya sobreexpresin confiere
resistencia parcial.
P14-9
ABCI3, un nuevo transportador mitocondrial de

Leishmania major implicado en la susceptibilidad
a antimoniales
Talia Arcari, Jos Ignacio Manzano, Santiago Castanys, Francisco
Gamarro
Granada, ES
Los parsitos del genero Leishmania son responsables de un grupo
de enfermedades devastadoras con diversas manifestaciones clnicas.
La ausencia de vacunas y la frecuente resistencia a los frmacos, hace
necesario identificar nuevas dianas teraputicas. Los transportadores
ABC (ATP-binding cassette) son una familia de protenas implicadas en
la resistencia a frmacos en diversos organismos. En el presente trabajo
describimos la caracterizacin funcional del transportador ABCI3 de
Leishmania major, que pertenece a un grupo de transportadores nicos
de tripanosomtidos. Utilizando parsitos que sobreexpresan ABCI3
fusionada a GFP (GFP-ABCI3) y ensayos de permeabilizacin con
digitonina, hemos determinado que ABCI3 se localiza en la membrana
externa de la mitocondria del parsito. Hemos obtenido parsitos con un
alelo delecionado para ABCI3 (LmABCI3+/-), y hemos realizado ensayos
de viabilidad celular en presencia de frmacos leishmanicidas. Los
promastigotes LmABCI3+/- son cinco veces ms resistentes a Sb(III) y
As(III); al exponer los parsitos a concentraciones subletales de Sb(III),
estos acumulan menos Sb(III), presentan unos niveles de ATP mayores, una
menor despolarizacin de la membrana mitocondrial, y menores niveles de
ROS que los parsitos control. Adems, los parsitos que sobreexpresan
una versin mutada inactiva del transportador (LmABCI3K1088M) son
tambin ms resistentes a Sb(III). Actualmente estamos realizando ensayos
de complementacin de LmABCI3+/- con una copia episomal del gen y
posteriormente trataremos de obtener parsitos mutantes nulos.
Financiado por el Ministerio de Economa y Competitividad, Plan
Nacional de I+D+i Proyectos SAF2012-34267 (F.G.), SAF2011-28102
(S.C.) y por el Plan Andaluz de Investigacin (Proyecto de Excelencia
CTS-7282).
127
Psters
P14r-10
several bioinformatic tools in T. cruzi and L. major. We also demonstrated

that Jean3 is differentially expressed during the stages of growth and
proliferation. In addition, the infective forms of these parasites exhibit high
gene expression levels of this protein kinase.
We are now performing several experiments in order to study the
localization of Jean3 and its implication in different processes such as
treatment resistance, cell cycle and viability, cell death and apoptosis, and
parasites virulence phenomena.
Identificacin y caracterizacin de nuevos frmacos

de reposicin activos frente a Trypanosoma brucei
Marta Martnez-Garca1, Luis Carvalho1, Rubn Cebrin Castillo2,
Jenny Campos-Salinas1, Ignacio Prez-Victoria3, J. Ignacio Manzano
Gonzlez1, Santiago Castanys1, Francisco Gamarro1, Vanessa Yardley4,
Elena Gonzlez1, Mercedes Maqueda Abreu2, Jos M Prez-Victoria1
1
IPBLN-CSIC, Granada, ES, 2Universidad de Granada, Granada, ES,
3
Fundacin MEDINA, Granada, ES, 4LSHTM, Londres, UK
El protozoo parsito Trypanosoma brucei es responsable de la enfermedad del
sueo, una de las enfermedades consideradas como olvidadas por la OMS
que cuenta con un peor control farmacolgico. Los pocos medicamentos
disponibles son muy txicos y difciles de administrar, por lo que se
necesitan nuevos frmacos capaces de cruzar la barrera hematoenceflica
sin producir neurotoxicidad, a ser posible de administracin oral y de un
precio asumible por los pacientes. La reposicin de frmacos (utilizacin
de medicamentos en uso para tratar otras enfermedades) es una estrategia
interesante para identificar este tipo de compuestos, ya que permite reducir el
tiempo transcurrido desde la identificacin del compuesto hasta su entrada en
fase clnica de estudio. En este trabajo describiremos la actividad tripanocida
y el mecanismo de accin de varios de estos prometedores compuestos frente
a T. brucei. Un ejemplo lo constituye la tafenoquina (TFQ), un frmaco oral
en fase clnica avanzada (IIb/III) para tratar y prevenir la malaria y que
ha demostrado ser eficaz in vivo frente a Leishmania, aunque no frente a
Trypanosoma cruzi. Mostramos que la TFQ es activa a concentraciones
submicromolares frente a distintas subespecies de T. brucei, incluyendo
T. b. rhodesiense. La TFQ provoca una despolarizacin mitocondrial en el
parsito, aumento de Ca2+ intracelular y produccin de especies reactivas del
oxgeno, que acaban produciendo alteraciones celulares a nivel del bolsillo
flagelar, el ncleo y la mitocondria. Tambin mostraremos el efecto de otros
frmacos de reposicin muy activos frente a T. brucei (IC50%= 3 nM) cuyo
mecanismo de accin estamos caracterizando.
Financiacin: Ministerio de Economa y Competitividad SAF2011-28215
(JMPV),SAF2012-34267 (FG), Junta de Andaluca BIO1786(JMPV) y
Fondos FEDER de la UE (SC, FG y JMPV).
P14r-11
Analysis of the Trypanosomatid serine/threonine

protein kinase Jean3, a novel potential
therapeutic target
Andrs Vacas-Oleas1, Celia Fernndez-Rubio1, Carmen Sanmartin2,
Socorro Espuelas3, Paul Nguewa1
1
Instituto de Salud Tropical, University of Navarra/Departamento
de Microbiologa y Parasitologa, University of Navarra, Pamplona,
ES, 2Instituto de Salud Tropical/Departamento de Qumica Orgnica
y Farmacutica, University of Navarra, Pamplona, ES, 3Instituto
de Salud Tropical/Department of Pharmacy and Pharmaceutical
Technology, School of Pharmacy, University of Navarra, Pamplona, ES
Leishmaniasis, Sleeping sickness and Chagas disease are neglected diseases
caused by intra-cellular parasites that belong to the Trypanosomatidae
family. These pathologies affect several million people around the world.
The transmission of the parasites between invertebrate vectors and
mammalian hosts, involves morphological and physiological changes
needed to acquire virulence and to adapt to the environment. This
mechanism of differentiation is called metacyclogenesis. We identified
a number of genes apparently involved during this process. One of our
objectives is the validation of these genes as therapeutic targets.
After screening the Leishmania and Trypanosoma genomes, we found
plausible candidates involved in the parasites differentiation and adaptation
phenomena. We mainly focus on protein kinases as promising druggable
molecules described against different pathologies. After identifying
Jean3 as a therapeutic target candidate in several trypanosomatids, the
analysis of its structure and the prediction of its function were assessed by
128
P14r-12
Identificacin funcional de posibles secuencias

promotoras de la polimerasa de ARN tipo II en
tripanosomas africanos
Carlos Cordon-Obras1, Andreu Saura1, Diana Lpez Farfn1, Fabin
Lorenzo2, Nick Dickens3, Basilio Valladares2, Miguel Navarro1
1
Granada, ES, 2Instituto Universitario de Enfermedades Tropicales
y Salud Pblica de Canarias, Santa Cruz de Tenerife, ES,3The
Wellcome Trust Centre for Molecular Parasitology, Glasgow, UK
Los tripanosomtidos son protozoos flagelados pertenecientes al orden
Kinetoplastida, organismos ancestrales que se separaron muy pronto del
resto de la rama eucariota. Su particular evolucin hace que presenten una
combinacin de rasgos procariotas y eucariotas nica, fundamentalmente a
nivel de transcripcin y organizacin genmica. Con la salvedad del Spliced
Leader (SL), un ARN que se aade al 5 de todos los ARN mensajeros, no
se ha identificado ningn otro promotor de ARN polimerasa de tipo II (pol
II), e incluso ste, es bastante particular, puesto que inhibidores especficos
de pol II y pol III no reducen su actividad en la medida esperada. La
transcripcin, mediada en la mayora de los marcos abiertos de lectura
por la pol II, est organizada en policistrones, y se ha sugerido que los
sitios donde cambia el sentido de la transcripcin (SSR), son secuencias de
iniciacin de la transcripcin. Sin embargo, no se ha identificado ninguna
secuencia especfica que pueda ser considerada como promotor.
Para realizar este trabajo primero se gener un antisuero purificado por
afinidad frente a la subunidad mayor de la pol II, lo que nos permiti analizar
su expresin por Western blot, y su localizacin en el ncleo del Kinetoplastida
modelo (Trypanosoma brucei) mediante inmunofluorescencia. Empleando este
antisuero para inmunoprecipitacin de cromatina y posterior secuenciacin
masiva identificamos posibles promotores de la pol II. Esta tcnica revel ms
de 200 picos significativos de acumulacin de la pol II, la mayora de ellos
asociados con los SSR divergentes del genoma de T. brucei. La alta resolucin
de esta tcnica nos permiti acotar con gran precisin regiones candidatas para
actuar como promotores. El potencial de estas regiones como promotores se
est evaluando mediante el anlisis de actividad de un gen reportero insertado
de forma estable en el genoma. Resultados preliminares muestran que algunas
de estas regiones permiten la transcripcin del gen reportero, al contrario de lo
que ocurre con la misma construccin en ausencia de promotor. Estos datos, de
confirmarse, supondran la modificacin del paradigma que dicta la ausencia
de promotores de ARN pol II en los Kinetoplastida.
P14-13
Caracterizacin de un nuevo transportador

ABC esencial en Trypanosoma brucei, agente
etiolgico de la enfermedad del sueo
Lina Orrego, Viviana Snchez-Martns, Marta Martnez-Garca, Mara
Cabello-Donaire, Sophie Malagerie-Cazenave, Jenny CamposSalinas, Antonio Estevez, Santiago Castanys, Francisco Gamarro,
Jos M. Prez-Victoria
Instituto de Parasitologa y Biomedicina Lpez-Neyra-CSIC, Granada, ES
La enfermedad del sueo es una enfermedad tropical causada por el
protozoo parsito Trypanosoma brucei. La transmite la mosca TseTse,
que inyecta las formas procclicas del parsito al hospedador mamfero,
Granada 2014
donde se transforman en formas sanguneas que causan la enfermedad,
mortal en ausencia de tratamiento. Como los medicamentos tripanocidas
no son adecuados debido a su toxicidad y a la aparicin de resistencias,
es prioritario encontrar nuevas dianas teraputicas para el desarrollo de
frmacos. En este trabajo presentamos la caracterizacin de TbABCG6,
un nuevo transportador ABC de T. brucei. La protena ortloga en el
parsito Leishmania, se encuentra localizada en membrana plasmtica
y confiere resistencia a medicamentos leishmanicidas. En primer
lugar, ensayos de northern y western blot indicaron que TbABCG6 se
expresa mayoritariamente en las formas sanguneas del parsito, estado
sobre el que se realiz el resto de la caracterizacin. Los ensayos de
inmunofluorescencia usando anticuerpos generados frente a esta protena
mostraron que se localiza en vesculas cercanas al bolsillo flagelar del
parsito, donde se encuentra concentrada la maquinaria endoctica y
exoctica. La sobreexpresin de TbABCG6 no confiri resistencia a
frmacos tripanocidas. Por el contrario, increment la sensibilidad a
suramina, un frmaco que entra por endocitosis mediada por receptor,
aunque esta mayor sensibilidad no se debe a diferencias en la acumulacin
de suramina. Por otra parte, la interferencia parcial de la expresin
de TbABCG6 mediante RNAi caus una dramtica disminucin del
crecimiento del parsito asociada con una anormal morfologa celular
(ensanchamiento del bolsillo flagelar y presencia de mltiples flagelos).
La importancia de dicha protena en la viabilidad del parsito sugiere
un papel esencial de TbABCG6 en T. brucei que estamos tratando de
elucidar.
(JMPV), SAF2012-34267 (FG), Junta de Andaluca BIO1786 (JMPV) y
Fondos FEDER de la UE (SC, FG y JMPV).
P14r-14
El transportador ABCG2 de Leishmania est

implicado en la resistencia a antimoniales
Ana Perea, Jos Ignacio Manzano, Kirsten Verachtert, Santiago
Castanys, Francisco Gamarro
Granada, ES
La quimioterapia es actualmente la nica herramienta de lucha disponible
frente a la leishmaniasis, aunque el reducido arsenal de frmacos
disponibles y el incremento del fallo teraputico limitan el control de esta
enfermedad parasitaria. La familia de transportadores ABC (ATP-binding
cassette) en Leishmania incluye 42 genes distribuidos en 8 subfamilias (AH), llevando a cabo funciones biolgicas relevantes. Se han caracterizado
varios miembros de la subfamilia ABCG, los cuales son half-transporters,
implicados en la resistencia a frmacos, translocacin de fosfolpidos a la
membrana plasmtica e infectividad. Nuestro grupo ha descrito que lneas
dominantes negativas para el transportador ABCG2, presentan una menor
infectividad en modelo murino. Hemos comprobado que la sobreexpresin
de ABCG2 confiere una resistencia significativa a Sb(III) as como al in
metlico As(III), debido a una reduccin en su acumulacin por incremento
de su eflujo. Igualmente, hemos observado que formas amastigotas
de Leishmania que sobreexpresan el transportador ABCG2, son ms
resistentes a antimonio que la lnea salvaje. Asimismo, comprobamos que
la resistencia a metales revierte tras la reduccin de los niveles de tioles
mediante el pretratamiento con butionina-sulfoximida. Los estudios de
localizacin subcelular en parsitos que sobreexpresan ABCG2-GFP,
sugieren que el transportador se localiza en vesculas intracelulares del
parsito.
Financiado por el Ministerio de Economa y Competitividad, Plan
Nacional de I+D+i Proyectos SAF2012-34267 (F.G.), SAF2011-28102
(S.C.) y por el Plan Andaluz de Investigacin (Proyecto de Excelencia
CTS-7282).
Psters
P14m-15
Desarrollo y validacin de un sistema HTS

en un modelo experimental de LV para
el descubrimiento de nuevos frmacos
leishmanicidas
Estefana Calvo lvarez, Jos Miguel Escudero Martnez, Rosa
Reguera Torres, Rafael Balaa Fouce, Estefana Calvo lvarez
Dpto. Ciencias Biomdicas, Facultad de Veterinaria, Universidad de
Len, Len, ES
El descubrimiento de nuevos frmacos para tratar la leishmaniosis visceral
(LV), la forma ms severa de los distintos tipos de leishmaniosis, es cada vez
ms urgente y necesario, debido a la ineficacia de los tratamientos actuales,
su toxicidad y a la aparicin de resistencias. El desarrollo de sistemas de
alto rendimiento o HTS (High-Throughput Screening), permitira testar
miles de compuestos de manera rpida, efectiva y reproducible. Debido a
la influencia crtica de la respuesta inmune del hospedador en el tratamiento
de la infeccin, el sistema empleado incluye la totalidad de las poblaciones
celulares involucradas en la interaccin parsito-hospedador y la forma
intracelular del parsito. Nuestro grupo ha validado un sistema HTS en
placas de 384 pocillos, mediante el empleo de un modelo ex vivo de bazos
infectados con una cepa de L. infantum que sobreexpresa de forma estable la
protena infrarroja iRFP. La regulacin de la expresin gnica en Leishmania
involucra las zonas 3 no traducidas (3UTR), las cuales contienen seales
necesarias para el procesamiento adecuado de los ARNm. La mxima
expresin de la protena iRFP en la fase de amastigote fue analizada mediante
el clonaje de distintas regiones 3UTR de genes regulados diferencialmente
en la fase intracelular del ciclo (A2, AMASTIN), y del gen HSP70II de L.
infantum expresado de forma constitutiva, corriente abajo del reportero
infrarrojo. La caracterizacin de la fluorescencia emitida fue realizada en
promastigotes, amastigotes aislados de infecciones in vitro en la lnea humana
THP-1, y amastigotes purificados de bazos de ratones BALB/c infectados.
Las infecciones experimentales en ratones BALB/c para la obtencin de los
cultivos ex vivo se llevaron a cabo con la cepa iRFP-3HSP70+ L. infantum.
La validacin del sistema se realiz mediante el testado de una coleccin
de 320 compuestos en placas de 384 pocillos, obteniendo valores Z>0.5.
La capacidad para identificar compuestos mediante sistemas HTS es vital
para el descubrimiento de nuevos frmacos que puedan ser empleados en un
futuro en la terapia contra la leishmaniosis.
Agradecimientos: trabajo financiado por los proyectos AGL2010 16078/
GAN y CYTED 214RT0482, del Ministerio de Economa y Competitividad
(MINECO).
P14-16
Fosforilacin de la histona H2AX en Leishmania

major como respuesta al dao producido en el
ADN por los derivados camptotecnicos
Raquel lvarez-Velilla, Rafael Balaa-Fouce, Rosa M RegueraTorres, Yolanda Prez-Pertejo
Len, Len, ES
La camptotecina y sus derivados estabilizan el complejo de escisin que se
genera entre la Topoisomerasa IB (TopIB) y la molcula de ADN durante el
proceso de replicacin. Esta estabilizacin a menudo implica la formacin
de roturas de la doble cadena debido a la colisin entre este complejo y
la horquilla de replicacin, lo que puede desencadenar tanto en un arresto
del ciclo celular como en la muerte de la clula. Como consecuencia del
dao producido se activan los procesos de reparacin del ADN a travs
de modificaciones o variantes de las histonas. Uno de los marcadores mas
tempranos del dao en el material gentico en eucarioras es la H2AX, una
variante de la histona H2A con un serina fosforilada. En tripanosomatidos,
esta fosforilacin se produce en la treonina 130 situada en el extremo
C terminal de la histona. Por lo tanto, este marcador nos es til para una
129
Psters
correcta evaluacin del dao sufrido en el ADN provocado por la accin
de los inhibidores camptotecnicos. Diseamos un pptido para la posterior
inmunizacin de un conejo y as obtener anticuerpos especficos frente a
la histona fosforilada. Estudiamos el dao producido por la camptotecina
y tres de sus derivados: gimatecan, topotecan y el metabolito activo del
irinotecan, denominado SN38, en promastigotes de Leishmania major.
Estos compuestos fueron evaluados a una concentracin fija y distintos
tiempos, observndose en todos los casos una seal positiva para la histona
fosoforilada que va aumentando hasta un mximo de 2 horas. Adems se
observa un arresto de ciclo celular en distintas fases como consecuencia de la
rotura de doble cadena durante las primeras horas de exposicin a la droga.
Financiacin: PI12/00104 del Instituto de Salud Carlos III y CYTED
214RT0482 del Ministerio de Economa y Competitividad (MINECO).
P14-17
Respuesta inmune de dendrmeros carbosilanos en

ganglio de ratn infectado con Leishmania major
Ana Tejera-Bengoechea, Jos Miguel Escudero-Martnez, Yolanda
Prez-Pertejo, Rafael Balaa Fouce, Rosa M Reguera
Len, Len, ES
Se ha estudiado la respuesta inmune ex vivo de 10 nanopartculas
dendrimricas derivadas de carbosilano con diferente carga, sobre
gangliocitos murinos infectados con Leishmania major, el agente responsable
de la leishmaniosis cutnea en el Viejo Mundo. Ratones BALB/c infectados
en la almohadilla plantar con promastigotes metacclicos de L. major
modificados genticamente con el gen que codifica la protena fluorescente
mCherry, se sacrificaron la 16 semana postinfeccin y se reseccion el
ganglio poplteo que drenaba la lesin infectada. Los explantes de ganglio
infectados se cultivaron en presencia de una concentracin de cada
dendrmero toxicolgicamente no comprometida. Tras 72h de cultivo, se
recogieron los sobrenadantes para valorar las citoquinas: IL-4, IL-12, TNF
e INF. El dendrmero BDEF 031 y los cinco derivados de ste, mostraron
una disminucin significativa de la IL-4 y un aumento tanto en la seal de
TNF como en la del INF, ambas correspondientes a una respuesta Th1,
cabe destacar, que ninguno de los dendrmeros mostr cambios en IL-12 con
respecto al control. Los otros 4 dendrmeros, de generaciones superiores a los
anteriores, fueron capaces de disminuir la respuesta Th2 mediada por la IL4, pero no fue significativa, mientras que en el caso de la respuesta Th1, no
se encontraron seales de un aumento del TNF y del INF. Para completar
estos resultados de la respuesta inmune se realiz la cuantificacin de las
actividades iNOS y arginasa de las clulas hospedadoras, obteniendo unos
niveles altos de NO en el sobrenadante de los cultivo y una baja actividad
arginasa atribuible a un ratio Th1/Th2 elevado.
Trabajo financiado por los proyectos PI12/00104 del Instituto de
Salud Carlos III y CYTED 214RT0482, del Ministerio de Economa y
Competitividad (MINECO).
P14-18 (R14-4)
Amino alcohols as an appealing and versatile

scaffold for Leishmania chemotherapy.
Unraveling their mechanism of action
M. ngeles Abengozar Infantes1, Raquel Garca-Hernndez2, Pilar
Fernndez de Palencia1, Luis Antonio Bustos3, Ricardo Escarcena3,
Santiago Castanys2, Esther Del Olmo3, Francisco Gamarro2, Arturo
San Feliciano3, Luis Rivas1
1
Centro de Investigaciones Biolgicas (CSIC), Unidad
Metabolmica, Interacciones y Bioanlisis (UMIB), Madrid, ES,
2
Armilla, ES, 3Facultad de Farmacia, Departamento de Qumica
Farmacutica (USAL), Salamanca, ES
130

The quest for new leads against the human infections caused by the
protozoans of the genus Leishmania, known collectively as leishmaniasis,
is urgently demanding, as chemotherapy, based on a paucity of drugs,
is threatened by rising resistance. Aminoalcohols are a versatile
pharmacological scaffold successfully assayed for anti-inflammatory,
antitumoral, antibiotic and antiparasitic activities.
In this work, a set of 15 alkyl-substituted aminoalcohols were assayed
for leishmanicidal activity. All were active at micromolar range, being
compound 5 the best candidate on intracellular L. donovani parasites (IC50=
0.7M, and selectivity index = 71.4).
The most active compounds induced depletion of intracellular levels
of ATP; however, they displayed a non-homogenous mechanism lethal
pattern, depending on subtle structural differences, as assessed by
biochemical and microscopical techniques. Two polarized modes of
action were unveiled; whereas for compound 5 is based on mitochondrial
dysfunction, with inhibition of complex II of the respiratory chain,
compound 8 behaved as a membrane-active agent inducing an irreversible
loss of internal homeostasis with entrance of vital dyes. Aside from these
two extreme prototypes, others, as compound 9, fell in an intermediate,
gray area, between them with alteration of membrane traffic with
intracellular vesiculation and accumulation of material at the flagellar
pocket.
Altogether aminoalcohols resulted as appealing new candidates for
Leishmania chemotherapy.
Supported by grants VI PN de I+D+I 2008-2011 ISCIII -Subdireccin
General de Redes y Centros de Investigacin Cooperativa RICET
(RD12/0018/0007, RD12/0018/0017, RD12/0018/0002), PI12-02706 (LR),
SAF2012-34267 (F.G.), Proyecto de Excelencia CTS-7282 (F.G.) and JCyL
221U13.
P14-19
Clonaje y expresin de la enzima E1, activadora

de ubiquitina, en Leishmania infantum
Jaime Larraga, Pedro Jos Alcolea, Ana Alonso, Vicente Larraga
Centro de Investigaciones Biolgicas - CSIC, Madrid, ES
La leishmaniosis es una enfermedad producida por protozoos del gnero
Leishmania que afecta a dos millones de personas en el mundo. Es
una zoonosis transmitida a travs de la picadura de dpteros del gnero
Phlebotomus. Las principales formas clnicas son cutnea, visceral y
mucocutnea. En Europa el agente causante de la enfermedad visceral es
L. infantum.
La enzima activadora de ubiquitina (E1) cataliza el primer paso en la
reaccin de ubiquitinacin que marca las protenas para su degradacin
va proteasoma. Al inicio de la cascada de la ubiquitinacin, la enzima
E1 une ATP-Mg2+ y la ubiquitina. En el siguiente paso, una cistena
cataltica del enzima E1, ataca el complejo ubiquitina-AMP formado a
travs de una sustitucin aclica, creando simultneamente una unin
tioster, liberndose AMP. Finalmente el complejo E1-ubiquitina
transfiere la ubiquitina a la enzima cataltica E2 a travs de una reaccin
de transesterificacin.
Se ha realizado el clonaje del gen activador de la ubiquitina E1
(LinJ.07.0010) en el vector pQE-30 y se llev a cabo la expresin en la
cepa M15 de E. coli. Las condiciones ptimas de expresin resultaron ser
2h a 30 C, siendo parcialmente soluble. Se purific usando cromatografa
de afinidad y se est procediendo a su caracterizacin.
Mediante el uso de los programas ClustalW y BlastP se ha realizado el
alineamiento, siendo el grado de homologa obtenido aproximadamente
del 90% con L. major y L. braziliensis, reducindose este valor al 41%
en el caso de Trypanosoma cruzi, siendo todava menor en el caso de
los mamferos. Se ha llevado a cabo el modelado de la protena con el
programa PyMol.
Granada 2014
P14-20
Efecto de la sobreexpresin de las protenas

fosfatasas 1 (PP1) en Leishmania infantum
Mara Degayn, Ana Alonso, Pedro J. Alcolea, Miguel Angel Moreno,
Vicente Larraga
La leishmaniasis visceral es una enfermedad zoontica causada por
el protozoo Leishmania infantum en la Cuenca Mediterrnea, donde
los perros son el reservorio principal. La transmisin se produce
por la picadura del vector Phlebotomus perniciosus. A lo largo del
ciclo biolgico digentico, el parsito se desarrolla desde su forma
extracelular y mvil en el hospedador invertebrado, promastigote;
a su estado intracelular y no mvil, en el hospedador mamfero,
amastigote. Las PP1 desempean un papel esencial en la regulacin
de una gran variedad de procesos en los organismos eucariotas. Sin
embargo, las rutas de sealizacin celular que desencadenan estos
cambios morfognicos en L. infantum apenas han sido caracterizadas,
lo que promueve este estudio. El alineamiento comparativo de las
PP1 codificadas por el cromosoma 34 en L. infantum revela un
ncleo cataltico altamente conservado que difiere nicamente en el
extremo N-terminal. Los perfiles de expresin gnica analizados por
qRT-PCR a lo largo del ciclo biolgico muestran que diversas PP1 se
encuentran sobreexpresadas en promastigotes infectivos diferenciados
en el flebtomo respecto a promastigotes de cultivo axnico. La
sobreexpresin de las PP1 LinJ.15.0240 y LinJ.34.0840 en trasfectantes
estables de L. infantum mediante la integracin del plsmido pIR-mcs,
altera la cintica de crecimiento en promastigotes de cultivo axnico.
Asimismo, los estudios por Western blot reflejan un drstico descenso
en la sobreexpresin de las mencionadas PP1 tras 48 h de incubacin.
Estas observaciones sugieren que las PP1 podran desempear una
funcin esencial en los procesos de diferenciacin celular.
P14-21
Reticulocyte-prone malaria parasites

predominantly invade CD71hi immature cells:
implications for the development of an in vitro
culture for Plasmodium vivax
Lorena Martn Jaular1, Aleix Elizalde-Torrent1, Richard ThomsonLuque1, Mireia Ferrer1, Jose Carlos Segovia2, Esperanza HerrerosAviles3, Carmen Fernandez-Becerra1, Hernando A del Portillo4
1
CRESIB (Barcelona Center for International Health Research),
Barcelona, ES, 2CIEMAT, madrid, ES, 3Tres Cantos Medicines
Development Campus, GlaxoSmithKline, madrid, ES, 4ICREA CRESIB (Barcelona Center for International Health Research),
barcelona, ES
The lack of an in vitro culture for malaria parasites with a unique
tropism for reticulocytes, such as Plasmodium vivax or the P. yoelii
17X strain hinders research in these organisms. In order to advance
in the characterization of infected cells during P.yoelii 17X-Balb/c
experimental infections we have assessed CD71 expression in erythroid
cells (TER119+) as a correlate of maturation. Parasites were found
mainly in immature cells from blood after day 5 post infection (p.i.).
Immature reticulocytes (CD71hi) were present in low levels at the
beginning of the infeccion but its number increases as the infection
progresses. The increase of CD71hi cells in blood concurred with
changes in erythropoietic organs. CD71hi cells appeared in spleen at
day 10p.i and increased in bone marrow from day 15 p.i. being the
cells mainly infected. Interestingly, mean fluorescence intensity (MFI)
of CD71 in infected cells was higher in both, bone marrow and spleen
than in the blood at day 15 p.i. This fact strongly suggests that the
parasite would begin the infection in erythropoietic organs; thereafter,
immature cells would be released to blood. The parasite preference
for immature cells that are rare in normal blood could have important
Psters
implications for the development of an in vitro culture. As a proof of
concept, we attempted in vitro culture of P.yoelli adding RBCs from
mice with a pyruvate kinase deficiency (PKD). PKD mice presented
high percentages of CD71hi cells in blood comparing with normal
animals. In these conditions, we were able to see merozoite reinvasion
and viable parasites after 120 h of culture.
P14r-22
Trfico de hemo en Leishmania, un protozoo

parsito auxtrofo para este metabolito esencial
Mara Cabello Donaire1, Alfonso Calvo2, Francisco J. Glvez3, Noem
Vergara-Segura1, Alba Rodrguez-Martnez1, Lina Orrego1, Marta
Martnez-Garca1, Sophie Malagarie-Cazenave1, M. Carmen RuizCantero1, Antonio M. Estvez1, Jos M Prez-Victoria1
1
Institute of Parasitology and Biomedicine Lpez-Neyra, Consejo
Superior de Investigaciones Cientficas-CSIC, Armilla, ES, 2Centro
de Investigacin Mdica Aplicada-CIMA/Departamento de Histologa
y Patologa, Universidad de Navarra, Pamplona, ES, 3Estacin
Experimental del Zaidin, CSIC (EEZ-CSIC), Granada, ES
La leishmaniasis es una devastadora enfermedad tropical causada por el
protozoo parsito Leishmania. Su control se basa en la quimioterapia,
pero los frmacos leishmanicidas en uso clnico no son adecuados,
por lo que es prioritario encontrar nuevos tratamientos. Una estrategia
atractiva para la identificacin de nuevas dianas farmacolgicas consiste
en aprovechar diferencias bioqumicas entre el parsito y el hospedador
humano. El metabolismo del grupo hemo, un metabolito esencial en
todos los organismos aerbicos, constituye una de estas diferencias,
ya que como el resto de tripanosomtidos patgenos, Leishmania es
auxtrofo para esta porfirina y debe rescatarlo de la persona infectada.
En este trabajo caracterizamos el trfico de porfirinas a travs de la
membrana plasmtica de L. major. Mostramos que la entrada de
porfirinas es dependiente de protena y que se afecta por variaciones
de pH y temperatura as como por la presencia iones y de inhibidores
de la produccin de energa. A su vez, existe un eflujo de porfirinas que
tambin caracterizamos. Por otra parte mostramos la caracterizacin
de una nueva familia de transportadores de membrana pertenecientes
a la superfamilia MFS. Algunos miembros estn implicados en trfico
de porfirinas en el parsito y otros confieren resistencia a frmacos
leishmanicidas. Finalmente, estamos analizando la implicacin de
transportadores ABCB en el trfico de porfirinas en la mitocondria, el
destino principal del hemo.
(JMPV), Junta de Andaluca BIO1786 (JMPV) y Fondos FEDER de la UE
(JMPV).
P14-23
Identification of a novel chromatin factor (CPF)

associated with VSG expression in African
Trypanosomes
Andreu Saura, Diana Lpez Farfn, Isabel Vidal-Cobo, Miguel
Navarro
Granada, ES
Antigenic variation in Trypanosoma brucei involves a sophisticated
mechanism to elude the host immune response by changes in the
expression of different Variant Surface Glycoprotein (VSG). The VSG
is expressed in a mono-allelic manner out of 1000 VSG genes, from one
telomeric site known as VSG-expression site (VSG-ES). The monoallelic expressed VSG gene is recruited in a single extra-nucleolar
nuclear body (ESB) that seems to be controlled at several levels. In
131
Psters
this study, we have identified a novel chromatin factor that function
by enabling and disabling the accessibility of transcriptional factors.
We first developed a monoclonal antibody (11C10E4 mAb) against the
recombinant protein and designed a trypanosome cell line that express
an HA-tagged version of CPF. Western blot analysis using the mAb
11C10E4 showed a single band corresponding to the endogenous CPF
(105 KDa), also detected by epitope-tagging. Subcellular localization
of CPF by Immunofluorescence microscopy using the mAb localized
the protein in the nucleus, as expected. Functional analysis by RNA
interference confirmed first the depletion of CPF by Western blot
analysis. To deepen the phenotype we carried out a reverse transcriptase
quantitative PCR (RT-qPCR), after CPF depletion which showed a
decreased expression of VSG221 mRNA, as well as a slightly increase
of Procyclin mRNA, the surface protein expressed in the Procyclic
insect stage. However, no changes in the ribosomal RNA level were
detected although is also transcribed by the same RNA polymerase.
Next, we investigate the occupancy of CPF along the VSG-ES locus
by Chromatin Immunoprecipitation (ChIP) experiments using the mAb
anti-CPF. ChIP-qPCR data analysis demonstrated that CPF is present
in VSG221 coding region and the pseudo-VSG, genes located within
the active VSG-ES. Future aims are directed to delineate the specific
function of CPF in VSG-ES expression.
P14-24 (R14-5)
Caracterizacin funcional de las condensinas

en la variacin antignica de Trypanosoma brucei
Domingo I. Rojas-Barros1, Jean-Mathieu Bart2, Diana Lpez-Farfn1,
Miguel Navarro1
1
Granada, ES, 2Instituto de Salud Carlos III, Madrid, ES
Los tripanosomas africanos eluden la respuesta inmunolgica del
hospedador expresando consecutivamente distintos tipos de la
Glicoprotena Variable de Superficie (VSG) lo que permite al parsito
una infeccin persistente. Trypanosoma brucei dispone de un complejo
sistema gentico dedicado a proporcionar todo un repertorio de miles de
genes que codifican diferentes VSG. Sin embargo, para una variacin
antignica eficaz, el parsito debe expresar en la membrana un solo tipo
de VSG en un momento dado, lo que requiere la expresin monoallica
de la familia multignica de VSG. Recientemente hemos descrito que
el complejo Cohesina es importante para la regulacin de la VSG, ya
que la interferencia de RNA de las cohesinas conduce a la separacin
prematura de las cromtidas hermanas del VSG-ES y a un incremento
en la frecuencia de aparicin de nuevas variantes antignicas (Landeira
et al., JCB. 2009).
Las condensinas, junto con las cohesinas, son complejos cuya funcin
es esencial para mantener la topologa de los cromosomas a lo largo del
ciclo celular. Recientemente se ha sido descrito que las condensinas estn
involucradas en la regulacin de la expresin gnica (Hirano et al. G&D,
2012). Para estudiar la funcin de las condensinas en T. brucei hemos
caracterizado funcionalmente las subunidades TbSMC4 y TbCND2 del
complejo. Para ello, hemos generado un anticuerpo monoclonal y un
antisuero de conejo frente a TbSMC4. Anlisis por imunoflorescencia nos
ha permitido determinar la localizacin nuclear de TbSMC4. Experimentos
de interferencia de RNA (RNAi) de TbSMC4 muestran un incremento
significativo del porcentaje de clulas en fase G2, demostrando la funcin
esencial de las condensinas en el control del ciclo celular en este protozoo
parsito. Adems, RNAi de TbSMC4 y TbCND2 provoc un incremento
en la frecuencia de variacin antignica, detectndose la expresin de
otras VSG. Estos resultados sugieren que el complejo Condensina juega
un papel importante en el establecimiento y/o mantenimiento del estado
transcripcional del VSG-ES.
132

P14-25
Identificacin de polimorfismo F200Y del

gen de -tubulina en una poblacin de
Haemonchus contortus aislada en la Facultad
de Estudios Superiores Cuautitln, UNAM,
Estado de Mxico
Rosa Isabel Higuera Piedrahita1, Jorge Alfredo Cullar Ordaz1, Maria
Eugenia Lpez Arellano2, F. Alba Hurtado1, Gabriel Ramrez Vargas2,
Csar Cuenca Verde1
1
Maestra en Ciencias de la Produccin y Salud Animal. Facultad de
Estudios Superiores Cuautitln. UNAM, Cuautitln, MX, 2Instituto
Nacional de Investigaciones Forestales, Agrcolas y Pecuarias.
Centro Nacional de Investigacin Disciplinaria en Parasitologa
Veterinaria, Jiutepec, MX
Haemonchus contortus es uno de los parsitos gastrointestinales
con mayor impacto en los sistemas productivos mexicanos. La
genotipificacin del polimorfismo F200Y del gen de -tubulina permite
monitorear la resistencia en fincas y puede ayudar a correlacionar con
factores de riesgo y prcticas de manejo en campo. El aislamiento
de la cepa de Haemonchus contortus en la FES Cuautitln muestra
susceptibilidad del 80- 90% a bencimidazoles en ensayos in vitro
(Moreno y col., 2013), sin embargo se requiere confirmar como cepa
susceptible por mtodos moleculares. El objetivo de ste estudio fue
determinar el polimorfismo F200Y del gen de -tubulina de esta cepa
provenientes de ovinos. El estudio se realiz en la Unidad de Helmintos
del CENID-PAVET, INIFAP, Jiutepec, Morelos. Se realiz la extraccin
y digestin de DNA de larvas infectantes (L3) a 56 C por 12 horas, se
realiz la cuantificacion DNA en un nanodrop, obtenindose 2,8 ug/
uL, con una correlacin de 260/280 de 2,04. Paso seguido, se realiz la
tcnica de Alelo Especfico - PCR (AS-PCR) para la identificacin del
polimorfismos, se utilizaron 10uL de Gotaq que contiene 1X de buffer,
0.25uM de primer, 0,2 mM de dNTP, 1.5 mM de MgCl2 y 1 U de Taq
polimerasa en 22uL y 40 ciclos de amplificacin. En la primera reaccin
se utiliz 1uL de PN1: (5-GGCAAATATGTCCCACGTGC-3) y 1uL
de PN2 (5-GAAGCGCGATACGCTTGAGC- 3) y 8uL de DNA de
larvas tres. El segundo PCR se realiz utilizando 8uL de DNA del primer
PCR, se utiliz 1uL de PH1 (5-GGAACGATGGACTCCTTTCG-3);
1uL de PH2 (5- GATCAGCATTCAGCTGTCCA-3) y 1ul de PH3
(5- CTGGTAGAGAACACCGATGAAACATA-3) para resistencia
y 1ul de PH4 (5-ATACAGAGCTTCGTTGTCAATACGA-3) para
susceptibilidad (Mo y col., 2012). Los resultados muestran que la cepa
de Haemonchus contortus de la FES Cuautitln present un patrn de
bandas de 250 (fragmento resistente) y 550 (fragmento susceptible), lo
cual indica que es una cepa heterocigota, resultados que contrastan con
lo notificado por Moreno y col. (2013) e Higuera y col. (2014) donde
haba sido susceptible en estudios in vitro e in vivo. stos resultados
nos indican que la presin que se realiza el aislamiento para mantenerla
debe ser mnima manteniendo individuos susceptibles en su mayora,
adems de que las pruebas moleculares son mucho ms sensibles para
realizar el diagnstico de resistencia a bencimidazoles.
P14-26
Estudio de nuevos compuestos con actividad

leishmanicida, inhibidores de la expresin de
genes implicados en la resistencia y virulencia
Celia Fernndez-Rubio1, Daphne Campbell1, Elena Ibaez2, Esther
Moreno3, Andrs Vacas-Oleas1, Juana Schwartz3, Juan Antonio
Palop4, Alfonso Calvo5, Socorro Espuelas3, Carmen Sanmartin4,
Paul Nguewa1
1
Instituto de Salud Tropical, University of Navarra/Departamento
de Microbiologa y Parasitologa, University of Navarra, Pamplona,
ES, 2Centro de Investigacin Mdica Aplicada-CIMA, Universidad
de Navarra, Pamplona, ES, 3Instituto de Salud Tropical/Department
Granada 2014
of Pharmacy and Pharmaceutical Technology, School of Pharmacy,
University of Navarra, Pamplona, ES,4Instituto de Salud Tropical/
Departamento de Qumica Orgnica y Farmacutica, University of
Navarra, Pamplona, ES, 5Centro de Investigacin Mdica AplicadaCIMA/Departamento de Histologa y Patologa, Universidad de
Navarra, Pamplona, ES
La OMS clasifica a la leishmaniasis como una de las enfermedades
tropicales desatendidas y de inters mundial. Entre las prioridades
actuales en el abordaje de estas patologas, est la bsqueda de nuevos
tratamientos. A partir de un banco de compuestos, cuatro fueron
seleccionados segn los criterios empricos de un buen frmaco. Dos
de ellos (2b y 4b) presentaron en ensayos de dosis-respuesta frente a
promastigotes de L. major bajas IC50s (< 3 M) y altos ndices de
selectividad debido a su escasa toxicidad en macrfagos. Se midi el
potencial teraputico de los dos posibles antiparasitarios, observndose
una reduccin de la carga parasitaria cercana al 80% en macrfagos
murinos infectados a concentraciones subletales de ambos. El
frmaco leishmanicida de referencia, la paromomicina, present una
disminucin menor, cercana al 60%. Adems, 2b y 4b inducen por s
mismos la liberacin en los macrfagos de xido ntrico cuya actividad
leishmanicida ha sido ampliamente descrita. El estudio de la actividad
de estos compuestos demostr alteracin del ciclo celular con arresto
en la fase G1, siendo muy significativo en el caso del compuesto 2b.
Se analiz la expresin de genes relacionados con la proliferacin
(PCNA), resistencia a frmacos (ABC-transporter y -tubulina) y
virulencia (QDPR). Se observa una disminucin de los niveles de
PCNA, -tubulina y QDPR tras el tratamiento con ambos compuestos.
Sin embargo, no vara la expresin de ABC-transporter, que se ha
descrito como una de las principales familias de protenas implicadas
en la multiresistencia a frmacos. Por los resultados mencionados,
consideramos estos compuestos con actividad leishmanicida como
buenos candidatos para futuros estudios en un modelo animal de
leishmaniasis cutnea.
P14-27 (R14-3)
Papel de la protena de unin a RNA RBP33

en el silenciamiento de la expresin gnica en
Trypanosoma brucei
Sandra Fernndez Moya1, Mark Carrington2, Antonio M. Estvez3
1
LMU-Mnchen, Departamento de Anatoma III-Biologa Celular,
Mnich, DE, 2University of Cambridge Department of Biochemistry ,
Cambridge, UK, 3Instituto de Parasitologa y Biomedicina LpezNeyra, IPBLN-CSIC, Armilla, ES
En esta comunicacin presentamos la caracterizacin inicial de la
protena de unin RNA RBP33 de Trypanosoma brucei. RBP33 es
una protena nuclear y esencial para la viabilidad del parsito. Hemos
identificado el conjunto de los RNA asociados a RBP33 mediante
inmunoprecipitacin de los complejos RNA-protena y posterior
secuenciacin masiva (RNAseq). La mayora de los transcritos unidos
a RBP33 estn descritos como no codificantes, siendo un tercio de stos
producidos por genes localizados al final de las unidades de transcripcin
policistrnicas o localizados en orientacin antisentido dentro de una
unidad transcripcional. La deplecin mediante RNAi de RBP33 conduce
a un aumento en los niveles de RNA provenientes de regiones que
estn normalmente silenciadas, tales como zonas de cambio de hebra,
retrotransposones o secuencias repetidas, siendo este fenmeno ms
acusado en la forma sangunea del parsito. Este trabajo supone el primer
ejemplo de una protena de unin a RNA implicada en la regulacin del
silenciamiento gnico en tripanosomas.
Psters
P15. Radicales libres y estrs oxidativo

P15-1
Cambios en la homeostasis del hierro (Fe) y

expresin de xido ntrico sintasa inducible
(iNOS) en el preacondicionamiento (P) heptico
con hierro frente a isquemia reperfusin (IR)
Virginia Fernandez, Angela Novoa Arce, Arlette Romn Salinas,
Romina Vargas Villagrn, Luis A. Videla Cabrera, Pamela Cornejo
Zamorano
Universidad de Chile, Facultad de Medicina, ICBM, Programa de
Farmacologa, Santiago, CL
La isquemia-reperfusin (IR) asociada a cirugas hepticas bajo oclusin
vascular, desencadena inflamacin en el hgado, asociado a la activacin
temprana de las clulas de Kpffer que liberan especies reactivas del
oxgeno, gatillando un estrs oxidativo drstico. El uso del protocolo de
proteccin contra la IR consistente en 6 dosis de 50 mg/kg de Fe en das
alternos (P con Fe; PFe), genera una ventana de estrs oxidativo transiente
que recupera la actividad del NF-B, factor de transcripcin redox sensible.
Considerando que NF-B regula la expresin de la iNOS, y los efectos
hepatoprotectores de esta enzima, en este estudio se evalu la expresin
(RT-PCR) y actividad (niveles de nitritos y nitratos sricos) de iNOS
en ratas sometidas al protocolo de PFe o dosis equivalentes de salino
(controles), seguido de 1 h de isquemia y 20 h de reperfusin o ciruga
sham (laparatoma sin isquemia). Adems, se evalu la expresin de
protenas que regulan la homeostasis del Fe, hepcidina (qPCR) que
degrada a la ferroportina (exportador celular de Fe), ferroportina y ferritina
(almacenador celular de Fe) (RT-PCR) en hgado de rata.
En relacin a controles-sham, la IR per se increment la expresin de iNOS,
efecto exacerbado 5,2 veces por el PFe, el cual aument 1,3 veces (p<0,05)
los niveles de nitritos/nitratos sricos. En estas condiciones, el PFe frente a
la IR indujo la expresin de hepcidina (7 veces) y la de ferritina (2 veces),
sin cambios en la expresin de ferroportina.
Se concluye que el PFe se asocia a la induccin de iNOS, enzima de
accin antioxidante ya que NO neutraliza radicales superxido. La
induccin de hepcidina limita los niveles de ferroportina, favoreciendo el
almacenamiento del Fe en la ferritina, evitando sus efectos deletreos.
Financiado por FONDECYT 1110006
P15-2
Protein kinase CK2 as a potential therapeutic

target in glioblastoma: preliminary preclinical
studies with apigenin based inhibition
Laura Ferrer-Font1, Estefania Alcaraz1, Maria Plana1, Ana Paula
Candiota2, Emilio Itarte1, Carles Ars1
1
Departament de Bioqumica i Biologia Molecular, Universitat
Autnoma de Barcelona, Cerdanyola del Valls, ES, 2Centro de
Investigacin Biomdica en Red en Bioingeniera, Biomateriales y
Nanomedicina (CIBER-BBN), Cerdanyola del Valls, ES
Glioblastoma (GBM) is the most aggressive human glial tumour, with
a median survival of 14-15 months. Temozolamide (TMZ) is a standard
chemotherapeutic for GBM treatment. Cancer stem cells (CSC) mediate
chemoresistence, indicating their persistence in these tumours after
treatment. Protein kinase CK2 contributes to tumour development,
proliferation, and suppression of apoptosis in cancer, and it is overexpressed
in human GBM. Targeting CK2 in GBM treatment could benefit patients.
We have studied the CK2 expression level, using Western Blot analysis,
normalized to constitutive tubuline content in preclinical GBM. Orthotopic
GBM growing in C57Bl/6 mice (n=3) were generated by injecting 105
GL621 cells. Tumour volumes were assessed by T2W MRI during 15
days and mice sacrificed (final volume 77.3+10.9mm3). Both contralateral
133
Psters
non-affected brain parenchyma and wild type mice brains (n=3) were
investigated. We also evaluated the effect of TMZ and apigenin (API),
a CK2 inhibitor, in cultured GL261 cells. For this, incubation of GL261
cells in 6-well plates with 20, 40 and 100M drug was performed and cell
viability was assessed by the Trypan Blue method. The expression level
of CK2 catalytic subunit (CK2) was found higher in tumour (4-fold)
and in contralateral brain parenchyma (2-fold) than in wild type tissue
(p <0.05). In contrast, no significant changes were detected in CK2
regulatory subunit (CK2) expression, suggesting a predominance of free
CK2 in tumours. In in vitro experiments, API decreased cell viability
with respect to control as compared to TMZ (28.8+5% and 64.3+1,5%,
respectively) at equal drug concentration (100M). Our data suggest
that CK2 targeted inhibitors could offer an alternative treatment for
chemoresistance to TMZ in preclinical GBM studies.

Cortical neurons in primary culture were stimulated with glutamate (100 M)
and mitochondrial function and generation of radical oxygen species were
measured by flow cytometry. Here we described that glutamate promoted
oxidative stress and mitochondrial membrane potential depolarization by
a mechanism involving cyclin B1 accumulation. Moreover, expression
of either cyclin B1 or hEmi1, a well-known APC/C inhibitor, induced
oxidative stress, mitochondrial dysfunction and neurodegeneration.
Our results suggest that NMDAR stimulation increased Cdk5 kinase
activity leading to Cdh1 phosphorylation, APC/C inactivation and cyclin
B1 stabilization. As a consecuence, cyclin B1 promoted oxidative stress,
mitochondrial dysfunction and neuronal apoptotic death. These results
reveal Cdh1 as a novel Cdk5 substrate that mediates cyclin B1 neuronal
accumulation in excitotoxicity.
Supported by: SAF 2011-23870, SGR191-2014.
Funded by ISCIII (PS09/0366, RD06/0026/1008) MICINN (SAF201020008), and Junta de Castilla y Len.
P15-3 (R15-4)
P15r-5
Activacin por hipoxia del nicho neurognico

del cuerpo carotdeo
NMDA receptor activity in astrocytes sustains

neuronal survival through a p35/Cdk5-Nrf2
pathway
Aida Platero-Luengo1, Susana Gonzlez-Granero2, Blanca DazCastro1, Jos Ignacio Piruat1, Jos Manuel Garca-Verdugo2, Ricardo
Pardal1, Jos Lpez-Barneo1
1
Instituto de Biomedicina de Sevilla, Universidad de Sevilla,
Sevilla, ES, 2Instituto Cavanilles de Biodiversidad y Biologa
Evolutiva, Universidad de Valencia, Valencia, ES
El mecanismo por el cual los estmulos ambientales y la demanda
fisiolgica son capaces de modular los nichos neurognicos en los
organismos adultos es una cuestin clave para entender la funcin de los
procesos de formacin de nuevas neuronas en la etapa postembrionaria.
Nuestros estudios con clulas madre neurales del cuerpo carotideo, un
rgano quimiosensor responsable de la respuesta ventilatoria arterial, han
revelado cmo el estmulo hipxico es capaz de activar la proliferacin del
nicho neurognico del cuerpo carotdeo a travs del establecimiento de una
estructura tipo sinapsis entre la clula glmica neuronal madura y la clula
progenitora de donde proviene. De este modo, se establecen mecanismos
de control donde los elementos maduros pueden influir sobre los elementos
indiferenciados para conseguir un equilibrio en el desarrollo de la funcin
del rgano. El modelo establecido para el cuerpo carotdeo demuestra
cmo los distintos elementos de un nicho neurognico se coordinan para
permitir al tejido adaptarse a situaciones fisiolgicas cambiantes.
P15-4 (R15-3)
Cyclin B1: mediator of mitochondrial dysfunction

in excitotoxicity
Carolina Maestre1, Miguel Veas-Prez de Tudela2, Juan P. Bolaos2,
ngeles Almeida2
1
Centro Nacional de Investigaciones Oncolgicas, Madrid, ES,
2
Instituto de Investigacin Biomdica de Salamanca, Hospital
Universitario de Salamanca - Instituto de Biologa Funcional y
Genmica, Universidad de Salamanca-CSIC, Salamanca, ES
Anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase
that destabilizes cell cycle proteins, is activated by Cdh1 in post-mitotic
neurons, where it regulates axonal growth, synaptic plasticity and survival.
The APC/C-Cdh1 substrate, cyclin B1, has been found to accumulate in
degenerating brain areas in Alzheimers disease and stroke. Recently, we
have reported that stimulation of N-methyl-D-aspartate receptors (NMDAR)
that occurs in neurodegenerative diseases promoted the phosphorylation of
Cdh1 by the Cdk5/p25 complex, a condition necessary and sufficient for its
translocation to the cytosol and APC/C-Cdh1 inactivation. This led to the
stabilization of cyclin B1 in cortical neurons and cell death. These results
highlight the importance of elucidating the role of cyclin B1 in neurons
under excitotoxic conditions relevant to neurological disease.
134
Daniel Jimenez Blasco1, Patricia Santofimia-Castao2, Antonio

Gonzalez2, Angeles Almeida1, Juan P. Bolaos1
1
Institute of Functional Biology and Genomics (IBFG), University
of Salamanca-CSIC, Salamanca, ES, 2Department of Physiology,
Faculty of Veterinary, University of Extremadura, Caceres, ES
Glutamatergic neurotransmission unavoidably enhances reactive oxygen
species (ROS) that in excess can trigger oxidative damage eventually leading
to neurodegeneration. Neurons are equipped with discrete antioxidant
defense that is insufficient to fully provide self-protection under certain
stressing conditions. Their neighbor astrocytes complement the antioxidant
defense and survival of neurons by releasing glutathione precursors,
which neurons take up for de novo glutathione biosynthesis. However, the
molecular mechanism by which neurons communicate astrocytes the need
for glutathione precursors to support survival during neurotransmission
is unknown. Here, we show that N-methyl-D-aspartate (NMDA), a
glutamate-receptor subtype agonist, at a dose sustaining neurotransmission
activates NMDA receptors in astrocytes triggering a signaling cascade
involving protein kinase Cd-dependent p35/cyclin-dependent kinase-5
(Cdk5) activation. Active p35/Cdk5 poly-phosphorylates nuclear factorerythroid 2-related factor 2 (Nrf2), which translocate to the nucleus to
promote the expression of antioxidant genes. Furthermore, by releasing
glutathione precursors, NMDA receptor-mediated Nrf2 activation in
astrocytes sustains the antioxidant defense and survival of closely spaced
neurons. Thus, during neurotransmission, glutamate-receptor activation
in astrocytes decisively contributes to boost the antioxidant defense and
survival of neurons.
P15-6 (R15-2)
Micro RNAs involved in redox responses:

some examples related to organ fibrosis
Santiago Lamas Pelez
Consejo Superior de Investigaciones Cientificas - Centro de Biologia
Molecular Severo Ochoa, Madrid, ES
Recent evidences point to the existence of a set of microRNAs that
participate actively in redox homeostasis and hormesis, and the term
redoximirs has been coined to define them. Reciprocally, proteins
involved in the basic machinery of microRNA processing can be
influenced by the cellular redox state. MicroRNAs may regulate central
pathways of the antioxidant response such as that of Nrf2 or directly
regulate the generation of reactive oxygen species by targeting mRNAs
that code for proteins directly involved in this process such as NOXs
Granada 2014
or components of mitochondrial complexes. We have focused on two
different potential targets: a) miRNAs regulating the levels of the
most relevant endogenous antioxidant, glutathione (GSH) b) miRNAs
targeting ROS generating proteins involved in fibrogenesis. We
identified mir-433 as a redoximir capable of targeting both the catalytic
and modulatory subunits of Glutathione-cysteine ligase (GCL) and of
reducing GSH levels. In addition we could demonstrate the participation
of mir-433 in fibrotic processes by using models of fibrosis in the liver
(bile duct ligation) and kidney (unilateral ureteral obstruction). We have
also identified a novel microRNA that participates in the regulation of
fibrogenesis by targeting both NOX-4 and the TGF-b pathway in cellular
and animal models of fibrosis as well as in patients with idiopathic
pulmonary fibrosis. We believe redoximirs constitute an important
potential pathway to understand the involvement of redox responses in
pathophysiological processes leading to organ fibrosis.
P15-7
Influencia del estrs oxidativo en la va

de activacin de la protena quinasa activada
por AMP (AMPK) heptica en ratas tratadas
con hormona tiroidea (T3)
Pamela Cornejo Zamorano1, Romina Vargas Villagran2, Virginia
Fernndez Arancibia2, Luis A. Videla Cabrera2
1
Universidad Diego Portales, Facultad de Salud y Odontologa,
Escuela de Tecnologa Mdica, uoa, CL, 2Universidad de
Chile, Facultad de Medicina, ICBM, Programa de Farmacologa,
Independencia, Santiago, CL
La administracin de T3 conduce a estrs oxidativo (EOx) heptico,
mediado por el efecto calorignico de la hormona. AMPK es un
sensor del estado energtico celular, regulada por alosterismo (AMP)
y por fosforilacin reversible de la subunidad cataltica por distintas
protenas quinasas tales como TAK1, CaMKK y LKB1. El objetivo de
este estudio es determinar el efecto del EOx en la expresin de AMPK
en hgado de rata por la administracin de T3 y determinar su correlacin
con la expresin de TAK1 y CaMKK. Para esto los animales fueron
pretratados 0,5 horas antes de la administracin con T3 (0,1 mg/Kg de
peso) con el antioxidante N-acetilcisteina (NAC) 0,5 g/Kg. Se midieron
los niveles de mRNA con qPCR, niveles proteicos mediante Western
Blot y 8-isoprostanos hepticos y plasmticos como parmetro de EOx
con un kit comercial. La administracin de T3 produjo un incremento
estadsticamente significativo en los niveles de mRNA de AMPK en
comparacin con los controles (p<0,05), efecto suprimido parcialmente
con el pretratamiento con NAC. Por otra parte, se observ aumento de
6,4 veces y 291 veces en los niveles de mRNA de CaMKK y TAK1
respectivamente luego del tratamiento con la hormona en comparacin
con los controles, efecto abolido por la administracin de NAC. Se
determin el efecto sobre la relacin TAK1 fosforilada/TAK1 no
fosforilada, observndose un incremento estadsticamente significativo
despus de tratamiento con T3 comparado con los valores controles
(190%), efecto parcialmente suprimido en los animales que fueron
pretratados con NAC (p<0,05). Los niveles de CaMKK aumentaron
65% en relacin a los controles, efecto anulado con el pretratamiento
con NAC. Los niveles de 8-isoprostanos plasmticos y hepticos de
ratas tratadas con T3 aumentaron en relacin a controles, efecto abolido
en los animales pretratados con NAC. Se concluye que el incremento
en la expresin de AMPK heptica inducida por T3 est mediado por un
mecanismo redox al cual podra contribuir el aumento en la expresin y/o
activacin de TAK1 y CaMKK.
Financiado por FONDECYT 1120034.
Psters
P15r-8
Anlisis de las micorredoxinas de

Corynebacterium glutamicum y su papel
en el control del estrs oxidativo
Almudena F. Villadangos, Jose A. Gil, Luis M. Mateos
rea de microbiologa, departamento de biologa molecular,
facultad de ciencias biolgicas y ambientales, Universidad de Len,
Len, ES
Las clulas sufren oxidaciones moleculares indeseables derivadas del propio
metabolismo aerobio y de agentes exgenos txicos. Los mecanismos de
respuesta celular combaten esas oxidaciones manteniendo un ambiente
reductor en el citoplasma mediante el uso de sistemas de buffer Redox.
Un sistema Redox exclusivo de actinomicetos, el cual depende de redoxinas
especficas (micorredoxinas; Mrx) y de compuestos de bajo peso molecular
(micotiol; MSH), conforma el par Redox Mrx/MSH.
Representantes del grupo de actinomicetos estn ampliamente distribuidos
en la naturaleza y presentan gran inters desde el punto de vista industrial
(p.e. Streptomyces es el principal productor de antibiticos) y mdico.
Con respecto a este ltimo aspecto, el par Mrx/MSH podra ejercer
un papel relevante en los procesos de supervivencia de los miembros
de actinomicetos patgenos ms representativos (Mycobacterium
tuberculosis, Rhodococcus equi o Corynebacterium diphtheriae) frente al
desafo con agentes oxidantes del hospedador.
El conocimiento detallado de las micorredoxinas a nivel molecular
y funcional en representantes de actinomicetos facilitara la tarea
indicada. Nuestro grupo de trabajo ha identificado varias micorredoxinas
en Corynebacterium glutamicum, M. tuberculosis y R. equi, siendo
denominadas Mrx1 y Mrx3. Las micorredoxinas son proteinas pequeas
con un centro activo conservado CxxC y prximo al extremo N-terminal;
estas Mrx son especficas de MSH y no se acoplan con otros compuestos
de bajo peso molecular (glutation o bacilitiol) de otros sistemas Redox. En
C. glutamicum se han obtenido mutantes knockout simples y dobles para
los genes mrx1 y mrx3, siendo estos mutantes sometidos a anlisis de estrs
oxidativoin vivo por presencia de metales pesados (cadmio, manganeso
o arsenico) o agentes qimicos como perxido de hidrgeno y diamida.
Los resultados indican que Mrx1 y Mrx3 tienen un papel importante en
el restablecimiento de la viabilidad celular despus de ser somentidos a
estrs oxidativo. La proteina Mrx1 de C. glutamicum [1] y M. tuberculosis
[1,2] ha sido recientemente caracterizada in vitro. Los ensayos in vitro
realizados con Mrx1 para determinar la cascada multienzimtica mostraron
que la enzima es reducida mediante MSH [1,2]. La protena Mrx3 de C.
glutamicum presenta igualmente el motivo tpico CxxC del centro activo,
as como una cistena adicional atpica (C65), aunque hasta el momento se
desconoce su cascada enzimtica in vitro.
Bibliografa
[1] E. Ordez, K. Van Belle, G. Roos, S. De Galan, M. et al. J Biol Chem
2009; 284:15107-15116.
[2] K. Van Laer, L. Buts, N. Foloppe, D. Vertommen, et al. Mol Microbiol.
P15-9
DHA diet supplementation modulates the

activation of neutrophils in post-exercise
conditions
Miquel Martorell, Xavier Cap, Antoni Sureda, Joan Miquel Batle,
Isabel Llompart, Josep A Tur, Antoni Pons
Research Group on Community Nutrition and Oxidative Stress,
IUNICS, University of the Balearic Islands - CIBER: CB12/03/30038
Fisiopatologa de la Obesidad y la Nutricin, CIBERobn, Instituto de
Salud Carlos III-ISCIII, Palma de Mallorca, ES
Background and objectives: Docosahexaenoic acid (DHA, C22:6n3)
increases phagocytic and fungical activity of neutrophils. We aimed to
135
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study the effects of training and DHA diet supplementation on the reactive
oxygen and nitrogen species production by neutrophil in football players.
Methods: Subjects were randomly included into two groups: placebo and
experimental groups. The placebo group took one liter five times a week
of a placebo drink and the experimental group consumed the experimental
drink enriched with DHA. Blood samples, used to purify neutrophils and
serum, were collected at the beginning in basal conditions and after 8 weeks
of training and diet intervention in basal and in post-exercise conditions.
ROS production by neutrophils after the activation with zymosan and with
phorbol myristate acetate (PMA) were determined by chemiluminescence
techniques. Nitrate levels were determined both in serum and in neutrophil
using a nitric oxide analyzer (NOA) 280i (Sievers).
Results: Neutrophils counts were not influenced by the training season or
the DHA diet supplementation, but the acute exercise realized at 8 weeks
significantly increased neutrophil counts. No effects were observed in the
chemiluminescence produced by neutrophils after their activation with
zymosan or PMA in the basal condition, however, the exercise significantly
increased neutrophil ROS production in response to immune stimulus
without effects of DHA diet supplementation. Moreover, the exercise
significantly decreased the time at which the maximal ROS production was
attained, and this decrease was higher in the experimental group. Exercise
significantly decreased nitrate levels in neutrophil but their increased in
serum, in both placebo and experimental groups, but this effect was higher
in the experimental group.
Conclusion: The consumption of an enriched DHA beverage for eight
weeks accelerates the postexercise neutrophil production of ROS after
stimulation with zymosan and increases the neutrophil secretion of nitrate
in post-exercise conditions.
Keywords: Docosahexaenoic acid; DHA; neutrophil; exercise; zymosan;
PMA; nitrate.
Acknowledgments: Programme of Promotion of Biomedical Research
and Health Sciences, Projects 11/01791, Red Predimed-RETIC
RD06/0045/1004, and CIBERobn CB12/03/30038.
P15r-10 (R15-5)
The effects of NQO1 deletion in Nrf2/Akt

pathways and growth deregulation of mouse
embryonic fibroblasts
Julia Ariza Gmez1, Laura Jdar Montilla1, Evi Mercken2, Michel
Bernier2, Rafael de Cabo Moreno2, Jos Manuel Villalba Montoro1
1
Department of Cell Biology, Physiology and Immunology, University
of Crdoba, Campus de Excelencia Internacional Agroalimentario,
CeiA3, Crdoba, ES, 2Experimental Gerontology Section,
Translational Gerontology Branch, National Institute on Aging,
National Institutes of Health, Baltimore, US
NAD(P)H-quinone oxidoreductase 1 (NQO1) is a 31kDa ubiquitously
expressed flavoenzyme that plays an important role in antioxidant defense,
being one of the best characterized detoxifying enzymes whose expression is
regulated by Nrf2. NQO1 catalyzes the reduction of several quinone substrates
using both NADH and NADPH. Besides its role as antioxidant, NQO1 has
been involved in the regulation of cell growth and development. Although
cells from many solid tumors contain increased levels of NQO1, low levels of
NQO1 are also associated with tumor development and carcinogenesis. Our
results show that the lack of NQO1 results in higher proliferation rates and
extended lifespan of immortalized MEFs, which is consistent with a role for
decreased NQO1 expression in tumor development. We have also observed
increased nuclear translocation of Nrf2 in NQO1KO MEFs, as well as in liver
of NQO1KO mice when compared to their Wt counterparts. An enhancement
of Nrf2-mediated antioxidant response might constitute a hormetic response
to the lack of NQO1 which, together with the activation of the Akt pathway
and higher levels of the glucose transporter GLUT1, could result in increased
growth and lifespan of NQO1KO cells. In fact, teratomas are generated when
NQO1KO cells are subcutaneously injected into immunodeficient mice.
However, NQO1KO MEFs also exhibited higher levels of apoptosis when
136

compared to their Wt counterparts. This could be driven by the presence
of dysfunctional mitochondria in NQO1KO cells, which causes outer
mitochondrial membrane permeabilization and the release of apoptotic factors
that trigger the apoptosis process. Deregulation of mitogenic pathways could
surpass this increase of apoptotic signaling resulting in the overall stimulation
of growth rate and extended long-term survival of MEFs lacking NQO1.
P15-11
Hyperbaric oxygen therapy in the management

of chronic wound induces an antioxidant response
and modulates plasma VEGF and endothelin-1
Antoni Sureda1, Juan M. Batle1, Xavier Cap1, Miquel Martorell1,
Silvia Tejada2, Josep A. Tur1, Antoni Pons1
1
Research Group on Community Nutrition and Oxidative Stress,
IUNICS, University of the Balearic Islands, Palma de Mallorca,
Spain and CIBER: CB12/03/30038 Fisiopatologa de la Obesidad
y la Nutricin, Instituto de Salud Carlos III (ISCIII), Palma de
Mallorca, ES, 2Experimental Laboratory, Research Unit, Son Lltzer
Hospital, IUNICS and University of the Balearic Islands , Palma de
Mallorca, ES
Hyperbaric oxygen (HBO) treatment is the medical use of oxygen at higher
than 1 atmosphere of pressure. The aim of the study was to evaluate the
effects of HBO clinical treatment on the antioxidant response and the levels
of vascular endothelial growth factor (VEGF) and endothelin-1 in patients
with chronic wounds. Ten patients with a chronic wounded situation (diabetic
wounds, osteomyelitis, enteritis radica and osteoarthritis) were volunteered
to participate in the study. The average time wounds without healing before
starting the HBO treatment were 20.5 10.1 months. The patients performed
20 HBO sessions and blood samples were taken before and 2 hours after
the sessions 1, 5 and 20. No significant differences were reported in the
hematological parameters. Serum CK activity was progressively decreasing
with the sessions performed. No significant differences were reported in any
enzymatic activity of erythrocytes after HBO treatments. Plasma catalase
activity responded to the HBO therapy with a significant increase after the
first and fifth sessions. Plasma MPO activity was significantly reduced after
all analysed sessions. VEGF levels significantly increased after the sessions
respect to the pre-session values. Endothelin-1 levels were progressively
decreasing during the HBO therapy, with significant differences at the
session 20. No significant differences were evidenced in plasma nitrite
levels. The levels of MDA adducts in plasma were significantly lower
at the last analysed session. In conclusion, HBO therapy increases the
plasma antioxidant defenses and regulates angiogenesis and vascular tone
by increasing VEGF and decreasing endothelin-1, which may be important
factors in promoting enhanced wound healing.
Keywords: Antioxidants, Chronic wound, Hyperbaric chamber, Oxidative
damage, VEGF.
Acknowledgments: Programme of Promotion of Biomedical Research and
Health Sciences, Projects 11/01791, Red Predimed-RETIC RD06/0045/1004,
and CIBERobn CB12/03/30038.
P15-12
Assessment of environmental pollution applying

oxidative stress biomarkers in the mussel Mytilus
galloprovincialis
Silvia Tejada1, Antoni Sureda2, Ainhoa Zuazu3, Miguela Mndez2,
Antonino Natalotto4, Salvatore Fasulo4, Salud Deudero3
1
University of the Balearic Islands / Experimental Laboratory, Research
Unit, Son Lltzer Hospital, IUNICS, Palma de Mallorca, ES, 2Research
Group on Community Nutrition and Oxidative Stress, IUNICS,
University of the Balearic Islands, Palma de Mallorca, Spain and
CIBER: CB12/03/30038 Fisiopatologa de la Obesidad y la Nutricin,
Granada 2014
Instituto de Salud Carlos III (ISCIII), Palma de Mallorca, ES, 3Spanish
Institute of Oceanography, Palma de Mallorca, BI, ES, 4Dep. of Animal
Biology and Marine Ecology, University of Messina, IT
In polluted environments, and especially in coastal waters, living organisms
are most often exposed to complex mixtures of chemical contaminants.
Mussels are desirable as bioindicators since they are likely to reflect
changes in the environment pollution status. Transplanting mussels from a
reference site to different locations allows biomonitoring the alterations due
to environmental pollutants. The aim of the present study was to analyse the
biochemical response of the mussels (Mytilus galloprovincialis) exposed to
petrol refinery. Mussels purchased from a mussel farm in Messina (Italy)
were transplanted for two months in a control clean site (Brucoli) and a
polluted site (Priolo refinery). A third group was maintained in the same
conditions in the farm for two months. Gills from each specimen (n=8 for
each station) were dissected and used for biochemical analysis. Mussels
from the three studied areas presented the same length and width. Mussels
from the polluted site presented higher levels of malondialdehyde respect
to the clean site. The activities of catalase and glutathione-S transferase
(GST) were significantly higher in the polluted site, whereas the activity
of acetylcholinesterase was significantly lower respect non-polluted sites.
A significant increase in the gene expression of metallothionein (MT)-10,
MT-20 and cytochrome P450 were reported in the polluted site respect
to the other areas, whereas the increase in catalase and GST was not
statistically significant. In conclusion, pollutants induce oxidative stress
and increase the antioxidant and detoxifying system in mussels. The use
of biomarkers in gills of the mussel M. galloprovincialis is a good tool to
categorize differences between polluted and non-polluted areas.
Keywords: Antioxidants, Biomarkers, Oxidative damage, Pollution.
Acknowledgments: National Parks Program 024/2010 and CIBERobn
CB12/03/30038.
P15-13
Drug screening in Drosophila models of FRDA

and PD
Pablo Calap Quintana1, Francisco Jos Sanz Lpez1, Luca Benito
Jardn1, Vernica Muoz Soriano2, Sandra Lpez Domnech2, Mara
Dolores Molt Ruiz3, Nuria Paricio Ortiz2, Mara Jos Martnez
Sebastin1
1
Departamento de Gentica, Universidad de Valencia, Burjassot,
ES, 2Departamento de Gentica, Universidad de Valencia - ERI
de Biotecnologa y Biomedicina, Burjassot, ES,3Departamento
de Gentica, Universidad de Valencia - Red de Salud Mental,
CIBERSAM - Fundacin Investigacin Clnico de Valencia, INCLIVA,
Burjassot, ES
Friedreich ataxia (FRDA) is the most common inherited ataxia in
the western population and is caused by a reduction in the level of the
mitochondrial protein frataxin. Parkinsons disease (PD) is the second
most common neurodegenerative disease in the world and is caused
by the selective loss of dopaminergic neurons in the substantia nigra
pars compacta and the decrease of the striatal dopamine levels. In both
neurodegenerative diseases oxidative stress and mitochondrial alterations
seem to have an important role in the pathogenesis of both diseases.
Many groups are trying to identify possible therapeutic molecules that may
alleviate the symptoms of both disorders. For FRDA, the drug candidates
under more intense study are antioxidants, iron chelators and compounds
that increase the synthesis of frataxin. Current therapy for PD, based on a
dopamine replacement strategy, provides effective control in the first stages
of the disease, but fails in retard, stop or reverse the neurodegeneration.
We have developed a model of FRDA in D. melanogaster that employs an
iRNA expressed in the whole organism by means of the GAL4-UAS system
with the purpose of reducing the expression of the Drosophila fh gene
(which codifies Drosophila frataxin). Mutations in DJ-1 are associated to
autosomal recessive forms of PD. As a D. melanogaster model of PD, we
Psters
have used a Drosophila line carrying loss of function mutations in DJ-1
(ortholog of DJ-1).
The individuals of both disease models show a decrease of their motor
performance. Using this phenotype, we started a screening of molecules
that could recover the motor ability of these flies. So far, we have
identified some possible candidates that belong to different functional
categories. Among these compounds, there are antioxidants, chelators
of different metals and metabolism modulators. Interestingly, some of
the compounds are able to improve motor performance in flies of both
disease models, while others seem to have a specific effect over either
FRDA flies or PD flies.
P15-14
La genistena afecta a la eficacia de los

tratamientos contra el cncer de mama
modulando la produccin de especies radicales
de oxgeno
Daniel Gabriel Pons Miro, Daniel Palou Gramn, Adamo Valle
Gmez, Jordi Oliver Oliver, Pilar Roca
Algunos tratamientos del cncer de mama tienen como efecto la generacin
de especies radicales de oxgeno (ROS), lo que favorece la muerte celular.
La genistena (GEN), un fitoestrgeno presente en la soja, podra modular la
produccin de ROS gracias a su interaccin con los dos tipos de receptores
estrognicos (ER), ER y ER, siendo ms afn por ER.
Nuestro objetivo fue determinar los efectos de la GEN sobre la eficacia del
cisplatino (CDDP), el paclitaxel (PTX) y el tamoxifeno (TAM) en lneas
celulares de cncer de mama con diferente ratio ER /ER. Se estudiaron
las lneas MCF-7 (elevada ratio ER/ER) y T47D (baja ratio ER/ER)
tratadas con 1M GEN, 10M CDDP, 10nM PTX y 10M TAM, as como
la combinacin de los citotxicos con la GEN, durante 48h. Se determin
la viabilidad celular (Cristal Violeta), la produccin de ROS (Amplex Red)
y la autofagia (monodansilcadaverina, indicador de apoptosis).
En MCF-7 la GEN promovi una mayor viabilidad celular y una
disminucin de la autofagia provocada por los tratamientos excepto con
CDDP, sin cambios significativos en la produccin de ROS. En T47D la
GEN provoc una disminucin en la produccin de ROS en los tratamientos
de CDDP y TAM con la GEN respecto a los tratamientos solos, sin cambios
significativos en la viabilidad celular ni en la autofagia.
Los resultados parecen indicar que el tratamiento con GEN en clulas con
una ratio ER/ER alta podra favorecer la proliferacin celular e inhibir
la apoptosis debido a su actividad estrognica. En cambio, en clulas con
una ratio ER/ER baja la presencia de GEN favorecera una reduccin de
la produccin de ROS, pudiendo disminuir la eficacia de los tratamientos
anticancergenos.
Financiacin: CAIB-FSE, ISCIII (PI12/01827) y FEDER-Unin Europea
P15-15
Efecto del silenciamiento o inhibicin de la

UCP2 sobre el estrs oxidativo en la lnea de
cncer de mama MCF-7
Mercedes Nadal-Serrano, Daniel G. Pons, Adamo Valle, Pilar Roca,
Jordi Oliver Oliver
El estrs oxidativo juega un papel dual en el desarrollo del cncer; por
una parte una elevada produccin de radicales libres de oxgeno (ROS)
137
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promueve la muerte celular, pero a niveles menores los ROS pueden
actuar como segundos mensajeros promoviendo la proliferacin celular.
La mitocondria es la principal fuente de produccin de ROS en la clula,
por lo que la UCP2 juega un papel importante en el control del estrs
oxidativo, desacoplando la cadena respiratoria y evitando de esta manera
la produccin de ROS.
El objetivo que nos propusimos fue determinar el efecto del silenciamiento
con siRNA o la inhibicin con genipin de la UCP2 sobre la proliferacin y
el estrs oxidativo en la lnea de cncer de mama MCF-7. Concretamente
se ha determinado la proliferacin (cristal violeta), la produccin de ROS
(Amplex Red) y el dao oxidativo en lpidos y protenas (4-HNE y grupos
carbonilo, respectivamente).
Mediante siRNA se consigui una importante disminucin de los
niveles de ARN mensajero de la UCP2, que se acompa de una cada
de los niveles de protena. El silenciamiento y la inhibicin de la UCP2
provocaron una disminucin de la viabilidad de las clulas cancerosas, en
el caso del genipin dependiente de la dosis. Adems se observ un aumento
importante en la produccin de ROS, que se acompa de un aumento de
los daos oxidativos, tanto en lpidos como protenas.
De los resultados obtenidos se puede desprender que la inhibicin de
la UCP2 podra utilizarse como una nueva estrategia en el tratamiento
coadyuvante del cncer.
Financiacin: CAIB-FSE, ISCIII (PI12/01827) y FEDER-Unin Europea
P15r-16 (R15-6)
Calorie restriction abolishes age-related changes

of coenzyme Q levels in liver and skeletal muscle
from mice fed a polyunsaturated fatty acidsenriched diet
Luca Fernndez del Ro1, Guillermo Lpez-Lluch2, Plcido Navas2,
Jon J. Ramsey3, Mara Isabel Burn1, Jos Manuel Villalba1
1
Departamento de Biologa Celular, Fisiologa e Inmunologa,
Universidad de Crdoba, Campus de Excelencia Internacional
Agroalimentario, ceiA3, Crdoba, ES, 2Centro Andaluz de Biologia
del Desarrollo, Universidad Pablo de Olavide-CSIC, CIBERER,
Instituto de Salud Carlos III, Sevilla, ES, 3VM Molecular
Biosciences. UCDavis, Davis (California), US
Coenzyme Q (Q) is a lipid-soluble electron carrier and a powerful
antioxidant. Dietary n-6 polyunsaturated fatty acids (n-6 PUFA) increase
hepatic Q levels with aging. To investigate if these effects were altered
by calorie restriction (CR) we established two groups of mice fed lifelong
diets containing soybean oil (high in n-6 PUFA) as the main fat source,
either ad libitum or under CR. We also studied two additional CR groups
fed diets containing lard (high in saturated and n-9 monounsaturated
fatty acids) or fish oil (high in n-3 PUFA). Liver and skeletal muscle
samples were taken after 6 or 18 months of dietary intervention starting
at an age of 3 months. Hepatic Q9 and Q10 levels increased and Q9/Q10
ratio decreased with age in the control but not in the CR group. When
comparing the three CR diets it was found that Q9 and Q10 levels were
maximal in CR-fish, and Q9/Q10 ratio was the lowest in CR-soy and CRfish. In skeletal muscle, levels of coenzyme Q9 did not change either by
CR or age but Q10 levels increased significantly with age in the control
although not in CR group. Q9 levels were higher in CR-soy compared
with CR-lard, whereas CR-fish displayed the lowest Q9/Q10 ratio. We
demonstrate that: i) CR abolishes age-induced changes of Q levels in
liver and skeletal muscle from mice fed PUFA-enriched diets, and ii)
liver and skeletal muscle tissues adapt to a PUFA-enriched CR diet by
increasing Q levels and decreasing Q9/Q10 ratio.
138

P15r-17
Effects of exogenous lipid source on mitochondrial

physiology and mTOR pathway in an in vitro model
Elena Gutirrez Casado1, Luca Fernandez del Ro1, Jos Antonio
Gonzlez Reyes1, Mara Isabel Burn Romero1, Chary Lpez
Pedrera2, Jos Manuel Villalba Montoro1
1
Departamento de Biologa Celular, Fisiologa e Inmunologa,
Universidad de Crdoba, Campus de Excelencia Internacional
Agroalimentario, ceiA3, Crdoba, ES, 2Instituto Maimnides de
Investigacin Biomdica de Crdoba (IMIBIC), Universidad de
Crdoba, Crdoba, ES
Over the last few decades many scientific research groups have focused
their interest on the knowledge of those processes that are involved
in aging, as well as on their possible manipulation in order to stop or
retard it. Nowadays, the unsaturation degree of membrane phospholipids
(Membrane Theory of Aging) (Hulbert, 2003) and the endogenous
generation of reactive oxygen species (ROS) (Mitochondrial/Free Radicals
Theory of Aging) (Harman, 1956) are two of the most widely accepted
factors that affect aging.
Unsaturation degree of cellular membranes is inversely proportional to
longevity, although unsaturated fatty acids are also crucial to maintain the
fluidity of the lipid membranes. Fatty acid content of the diet can modify the
composition of membrane phospholipids (Ochoa-Herrera et al., 2001), and
can also affect the function of the electron transport chain (ETC) located
in the inner mitochondrial membrane (Aoun et al., 2012). The ETC is not
only the most important cellular energetic machinery for ATP generation,
but also one of the main sources of ROS in cells. ROS can interact with
lipids and proteins altering the permeability of cellular membranes and the
functional conformation of proteins.
The Ser/Thr-kinase mTOR is a crucial kinase in sensing the availability of
nutrients and growth factors in cells, and promoting the activation/inhibition
of different molecules involved in processes such as autophagy. Optimal
nutrient sensing through the mTOR pathway is impaired with aging. On
the other hand, inhibition of mTOR pathway by dietary manipulations as
calorie restriction increases lifespan (Lpez-Otn et al., 2013).
The aim of our work was to determine how the mitochondrial physiology,
cellular oxidative damage, and the mTOR signaling pathway could be
modified by the supplementation with exogenous lipids using an in
vitro model. A mouse hepatocarcinoma cell line (Hepa 1.6) was grown
in culture medium containing either low (1g/l) or high glucose (4,5g/l),
and supplemented or not with two different lipid emulsions (at 7g/ml):
Lipofundin, based on -6 fatty acids (thus mimicking a diet enriched
in soybean oil) or Lipoplus which also contains -3 fatty acids (thus
mimicking a diet containing fish oil). After 48 hours of treatment with the
corresponding emulsion, different parameters related with mitochondrial
function (including mitochondrial potential and activities of ETC
complexes) and oxidative stress (including superoxide and peroxide levels
and oxidative damage), as well as the activation state of components of
the mTOR pathway were assessed. Our results indicate that exogenous fat,
targets mitochondrial physiology, oxidative stress and mTOR pathway in a
manner that is dependent on fatty acid composition.
P15-18 (R15-1)
Papel de p38alfa y del estrs oxidativo en el

control de la citoquinesis
Ana M. Tormos1, Raquel Talns-Visconti2, Mara Jorques1, Salvador
Prez-Garrido1, Ana Bonora-Centells1, ngel R Nebreda3, Juan Sastre1
1
Departamento de Fisiologa, Facultad de Farmacia, Universidad
de Valencia, Burjasot, ES, 2Departamento de Farmacia y Tecnologa
Farmacutica, Facultad de Farmacia, Universidad de Valencia,
Burjasot, ES, 3Instituci Catalana de Recerca i Estudis Avanats
(ICREA), Barcelona, ES
La citoquinesis es la ltima etapa de la mitosis e implica una reorganizacin
del citoesqueleto de actina. Un fallo de la citoquinesis es uno de los
Granada 2014
principales mecanismos de formacin de clulas poliploides en mamferos,
que pueden resultar genticamente inestables. Nuestros resultados indican
que la citoquinesis est regulada por la MAP quinasa p38alfa sensible al
estado redox y puede verse afectada por el estrs oxidativo. La activacin
de MNK-1 es dependiente de p38alfa y MK2 y se requiere para la escisin
del puente intercelular. Los ratones viejos con deficiencia especfica de
p38alfa en hgado tienen disminuida la fosforilacin de MNK-1. La va
de la pequea GTPasa Rho a travs de fosfo-PRK-2, fosfocofilina,
y p27- tambin est deteriorada en los ratones knock-out cuando son
viejos. Asimismo, la ausencia de p38alfa condujo a una disminucin de la
fosforilacin de HSP27, que se requiere para la polimerizacin de la actina.
Por ello, se observa un grave deterioro de los filamentos de F-actina en el
hgado de ratones viejos con deficiencia de p38alfa. No obstante, no se
produce estrs oxidativo en estos animales. En consecuencia, la deficiencia
de p38alfa a largo plazo afecta el ensamblaje de la actina, sin producir
estrs oxidativo, dando lugar a un fallo de la citoquinesis y binucleacin
de los hepatocitos con el envejecimiento. Por otro lado, el aislamiento de
hepatocitos produce marcada deplecin de glutatin, incremento en la
generacin de especies reactivas del oxgeno y activacin de la entrada
en ciclo celular con bloqueo de la citoquinesis y binucleacin. La adicin
de N-acetil cistena previno la deplecin de glutatin y fren la entrada
en el ciclo celular, evitando el fallo de la citoquinesis y la formacin de
hepatocitos binucleados durante el aislamiento. En consecuencia, nuestros
resultados muestran que los hepatocitos entran en fase S pero no completan
la division celular cuando existe deficiencia de p38alfa o durante su
aislamiento, dando lugar en ambos casos a un fallo de la citoquinesis y
binucleacin.
P15r-19
N-based ligands with intracellular oxidative

activity as potential anti-tumor agents
Marta Gonzlez-Brtulos1, Olaf Cuss1, Miquel Costas1, Xavi Ribas1,
Anna Massaguer2
1
Bioinorganic and Supramolecular Chemistry group (QBIS),
Department of Chemistry, University of Girona, Girona, ES,
2
Biochemistry and Molecular Biology Unit, Department of Biology,
University of Girona, Girona, ES
Tumor cells have persistently higher levels of Reactive Oxygen Species
(ROS) than normal cells which is a consequence of their abnormal
metabolism together with genetic alterations [1,2]. The constitutive
oxidative stress of cancer cells provides a specific target for the design
of novel redox-based anticancer therapies, as cancer cells are very
vulnerable to pro-oxidant agents such as transition metal based-complexes
[3]. When accumulated inside the cells, metals such as iron, manganese
or copper, undergo cycling redox reactions that generate high levels of
ROS, generating additional ROS, which likely increase the oxidative stress
above the cytotoxic threshold, leading to a cell death [4]. Therefore, we
evaluated the antitumoral properties of five polyamine Fe-Complexes
and their corresponding uncomplexed ligands. Only the ligands displayed
antiproliferative activity against breast adenocarcinoma MCF-7 and
pancreas adenocarcinoma CAPAN-1 cancer cell lines. The most active
compounds were selected to further analyze their cytotoxicity against
a broad panel of tumor and non-malignant cell lines and their ability to
inhibit the clonogenicity of cancer cells. In addition, the ability of the
compounds to generate ROS inside the cells and the influence of the
intracellular iron in their cytotoxicity was evaluated. Our results reveal that
three ligands display an interesting anticancer activity, with higher IC50
values in non-malignant cells than in tumor cells and exhibit inhibitory
effect on the clonogenicity of tumor cells. Moreover, the ligands are not
hemolytic against human red blood cells. We determine that the ligands are
inducers of oxidative stress and that ROS induction is associated to their
strong capactity to bind intracellular iron and generate the highly oxidant
iron coordination complexes inside the cells, inducing the cell death. These
results indicate that these ligands may be a potential pro-oxidant anticancer
agents.
Psters
References
[1] Cairns RA et al. Nat Rev Cancer 2011.
[2] Luo J et al. Cell 2009.
[3] Montero AJ et al. Drugs 2011.
[4] Dixon S et al. Natu Chem Bio 2014.
P15-20
Presence of antioxidant enzymes and

peroxisomes in olive fruits (Olea europaea L.)
Eduardo Lpez-Huertas, Luis A del Ro
Group of Antioxidants, Free Radicals and Nitric Oxide in
Biotechnology, Food and Agriculture - Estacin Experimental del
Zaidn, Consejo Superior de Investigaciones Cientficas (CSIC),
Granada, ES
The occurrence in olive (Olea europaea L.) fruit tissue of peroxisomes and
different antioxidant enzymes is reported for the first time. Ultrastructural
analysis showed that olive cells were characterized by the presence of large
vacuoles and lipid drops. Plastids, mitochondria and peroxisomes were
placed near the cell wall, showing some type of association with it. Olive
fruit peroxisomes were purified by sucrose density-gradient centrifugation,
and catalase, glutathione reductase and ascorbate peroxidase were found in
peroxisomes. In olive fruit tissue the presence of a battery of antioxidant
enzymes was demonstrated, including catalase, four superoxide dismutase
(SOD) isozymes (mainly an Fe-SOD plus 2 Cu,Zn-SOD, and a Mn-SOD),
all the enzymes of the ascorbate-glutathione cycle, reduced and oxidized
glutathione, ascorbate, and four NADPH-recycling dehydrogenases. The
knowledge of the full composition of antioxidants (enzymatic and nonenzymatic) in olive fruits is crucial to be able to understand the processes
regulating the antioxidant composition of olive oil.
Supported by the ERDF-cofinanced grant AGL2011-24428 from the
Ministry of Economy and Competitiveness, Spain.
P15-21
NADPH oxidasas como dianas teraputicas

en la leucemia mieloide crnica
Beatriz Snchez-Snchez1, Sara Gutirrez-Herrero2, Guillermo
Lpez Ruano1, Rodrigo Prieto Bermejo1, Marta Romo-Gonzlez1,
Marcial Llanillo1, Atanasio Pandiella2, Carmen Guerrero2, Jess F.
San Miguel3, Fermn Snchez-Guijo3, Consuelo del Caizo3, Angel
Hernandez Hernandez1
1
Salamanca - Instituto de Investigacin Biomdica de SalamancaIBSAL, Salamanca, ES, 2CIC-Centro de Investigacin del Cncer,
CSIC, Salamanca, ES, 3Instituto de Investigacin Biomdica
de Salamanca-IBSAL, Hospital Universitario de Salamanca,
Salamanca, ES
Las clulas cancerosas muestran una excesiva produccin de especies
reactivas del oxgeno (ROS), lo que parece estar relacionado con el inicio y
la progresin del cncer. Las NADPH oxidasas son uno de los principales
sistemas productores de ROS en la clula. La leucemia mieloide crnica
(LMC) est provocada por la expresin de la quinasa BCR-ABL, cuya
actividad conduce a un aumento de la produccin de ROS, en parte a travs
de NADPH oxidasas. La terapia de la LMC basada en el uso de inhibidores
de BCR-ABL es muy efectiva, sin embargo la existencia o aparicin de
resistencias a este tratamiento es an un serio problema.
En este estudio se evala la familia de las NADPH oxidasas como dianas
teraputicas en la LMC. Se ha analizado el efecto de diferentes inhibidores
de NADPH oxidasas, ya sea solos o en combinacin con inhibidores de
BCR-ABL, en clulas de LMC y en dos modelos animales diferentes. Una
de las conclusiones a las que llega el estudio es que la inhibicin de las
NADPH oxidasas deteriora drsticamente la proliferacin y viabilidad de
las clulas de LMC e inhibe el crecimiento de tumores in vivo. Adems, la
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Psters
combinacin de inhibidores de las NADPH oxidasas con inhibidores de
BCR-ABL era altamente sinrgica. Los investigadores sugieren que esta
estrategia podra utilizarse no solo en LMC sino tambin en otros tipos de
tumores.
Por tanto, este estudio demuestra que las NADPH oxidasas son una
interesante diana teraputica para la LMC, que podra usarse como
alternativa al uso de inhibidores de BCR-ABL, o para potenciar el efecto
de dichos inhibidores. Ser interesante en el futuro profundizar en la
posibilidad de trasladar estos resultados a la prctica clnica.
P15-22
Role of the NADPH oxidase NOX4 in liver

tumorigenesis
Eva Crosas-Molist1, Esther Bertran1, Joan Fernando1, Isabel
Fabregat2
1
Bellvitge Biomedical Research Institute-IDIBELL, LHospitalet de
Llobregat, ES, 2Bellvitge Biomedical Research Institute-IDIBELL Departament de Cincies Fisiolgiques II, University of Barcelona,
Barcelona, ES
NOX4 has been implicated in a variety of physiological processes, including
cellular senescence, apoptosis, survival, migration, endoplasmatic reticulum
stress and differentiation. We have previously described that in liver cells,
NOX4 upregulation is necessary for TGF-beta-induced suppressor effects,
in particular, for cell death (Carmona-Cuenca et al., J Hepatol. 49:965,
2008). NOX4 is also required for TGF-beta-induced stellate cell activation
during liver fibrosis (Sancho et al. PLoS One, 7(9):e45285, 2012) and
pre-clinical assays have suggested NOX4 inhibitors as useful tools to
ameliorate this process (Jiang et al., Free Radic Biol Med., 53:289, 2012).
However, the potential consequences of sustained treatment of liver cells
with NOX4 inhibitors are unknown yet.
Here we show that stable knockdown of NOX4 expression in liver tumour
cells increase their proliferative capacity. In vivo analysis in mice revealed
that NOX4 expression is down regulated during diethyl-nitrosamine (DEN)induced hepatocarcinogenesis. Furthermore, xenograft experiments in athymic
mice indicated that NOX4 silencing confers advantage to human hepatocellular
carcinoma (HCC) cells to form earlier and bigger tumours in vivo. Furthermore,
silencing NOX4 in HCC cells induces a decrease in cell-to-cell and cell-tomatrix adhesion and an increase in cell migration. NOX4 knockdown resulted
in decreased expression of E-cadherin and ZO-1, components of adherent and
tight junctions respectively, lower adhesion capacity and a decrease in focal
adhesions. Moreover, a change in the integrin pattern in the plasma membrane
was observed when NOX4 was silenced. All together, these results point to
a role for NOX4 in maintaining epithelial structures, increasing cell-to-cell
and cell-to-matrix adhesions, and preventing cell migration. In summary,
NOX4 may play a relevant tumor suppressor role during early stages of liver
tumorigenesis. In support of this idea, immunochemical analyses of NOX4
expression in human liver tumor cell lines and tissues revealed decreased
NOX4 protein levels in liver tumorigenesis.
P15-23
Amitriptyline, used alone or in combination

therapy, modifies expression of several
antioxidants in breast cancer cell lines
Eduardo Daz-Parrado, Jssica Nez-Vasco, Matilde Illanes,
Ana Fernndez-Rodrguez, Ana Mara Moreno-Fernndez, Manuel
De Miguel
Dpto. Citologa e Histologa Normal y Patolgica, Facultad de
Medicina, Universidad de Sevilla, Sevilla, ES
Introduction: Oxidative therapy is a promising anticancer strategy based
on the production of high levels of ROS and/or the depletion of antioxidants
of tumor cells. We have already demonstrated that Amitriptyline (Amit), a
tricyclic antidepressant, induces an increase of ROS production, leading to
140

apoptosis through the caspase-dependent pathway. In this work, in order
to continue studying the potential antitumor activity of amitriptyline, we
have analyzed the effects of amitriptyline on the expression of several
antioxidants in human breast cancer cell lines, and in combination with the
chemotherapeutic camptothecin (CPT).
Materials and Method: MCF-10A (non-tumoral line cell) and MCF-7
and MDA-MB-231 cells (breast cancer cell lines with different hormonal
characteristics) were treated 24h with Amit (20M), 10M CPT and a
combination of both. Expression of several antioxidants (MnSOD, catalase,
GSR, HO-1) was analyzed by RT-qPCR.
Results: MCF-10A: Antioxidant expression was decreased in the different
treatment conditions, except for HO-1 that was significantly increased.
MCF-7: With Amit, an increase of GSR and a decrease of HO-1 were
observed. With the combination therapy, expression of the four antioxidants
was decreased.
MDA-MB-231: With Amit, all antioxidants showed a decreased expression
except for HO-1, which was higher than in control. With the combination
therapy, similar results to MCF-7 were obtained, with the exception of the
expression of HO-1, which showed a significant increase.
Conclusions: In general, Amit induces a reduction in the antioxidant
system, especially dramatic for catalase in the combination therapy.
However, HO-1 showed a significant increase in the non-tumoral cells and
in the combination therapy of MDA-MB-231 tumor cells. HO-1 could be
an important antioxidant against oxidative stress in breast cells since two of
the cell lines tested reacted increasing notably its expression after oxidative
treatment. After inducing oxidative stress, cells increase the expression of
some antioxidants, different ones depending on cell type.
P15-24
Caracterizacin de la enzima ascorbato

peroxidasa en extractos crudos de Dunaliella
salina
Antonio Len Vaz, Rocio Rengel Domnguez, Maria del Carmen
Romero Cruz, Javier Vigara Fernndez
Dpto. Qumica y Ciencia de los Materiales, Facultad de Ciencias
Experimentales, Universidad de Huelva, CEIMAR, Huelva, ES
Las especies reactivas de oxgeno (ROS), tales como el perxido de
hidrgeno, el radical superxido, el radical hidroxilo y el oxgeno singlete,
son continuamente producidas en plantas y microalgas en distintas rutas
metablicas y con distinta localizacin subcelular. La produccin de estas
especies se incrementa en situaciones de estrs ambiental, excediendo
la capacidad detoxificadora del organismo y causando daos oxidativos
a protenas, ADN, lpidos y otras macromolculas. Para evitar estos
daos oxidativos los distintos organismos han desarrollado eficientes
sistemas enzimticos. La ascorbato peroxidasa (APX; EC 1.1.1.11) es una
hemoprotena que juega un importante papel en la eliminacin de H2O2
(la forma ms estable entre las distintas ROS) utilizando ascorbato como
donador de electrones. En este trabajo se presenta la caracterizacin del
ensayo de actividad de la enzima de la APX en extractos crudos de la
microalga eucaritica halfila Dunaliella salina. La actividad se muestra
dependiente de sus sustratos (ascorbato y agua oxigenada) y se estabiliza
con la presencia de EDTA en el medio de reaccin, el ensayo tambin
requiere un medio tamponado, siendo su pH ptimo 6,0, en tampn
MES 100 mM. Se ha determinado una temperatura ptima de 34 C y
una energa de activacin de 13,31 kJ/mol para el ensayo de actividad.
La APX de Dunaliella salina muestra una cintica de Michaelis-Menten
para el perxido de hidrgeno, mostrando un valor de Km de 47,52 M
y una Vmx de 449,33 U/ml; presentando una cintica sigmoidal para el
ascorbato, con valores de S0,5 de 3,05 M y un factor de Hill de n=1,06.
La actividad APX de Dunaliella salina se inhibe drsticamente por azida
y cinuro, inhibidores caractersticos de hemoprotenas, mostrndose una
inhibicin parcial por Mn+2 y por DTT. Por otro lado, la carencia de fuente
nitrogenada o la presencia de metales pesados (Cd+2) en el medio de cultivo
de Dunaliella salina, gener estrs oxidativo en la microalga, provocando
el aumento de los niveles de actividad de la ascorbato peroxidasa.
Granada 2014
P16. Regulacin de la expresin gnica

y dinmica del genoma
P16-1 (R16-4)
Temporal and spatial regulation of the Yen1

Holliday junction resolvase prevents genome
instability
Miguel Gonzlez Blanco1, Joao Matos2, Stephen C. West1
1
Clare Hall Laboratories, London Research Institute, Cancer
Research UK, Potters Bar, UK, 2Institute of Biochemistry,
Department of Biology, ETH Zrich, Otto-Stern-Weg 3, Zrich, CH
The careful orchestration of cellular events such as DNA replication,
repair, and segregation is essential for equal distribution of the duplicated
genome into two daughter cells. To ensure that persistent recombination
intermediates are resolved prior to cell division, the Yen1 Holliday junction
resolvase is activated at anaphase. Here, we show that the master cell-cycle
regulators, cyclin-dependent kinase (Cdk) and Cdc14 phosphatase, control
the actions of Yen1. During S phase, Cdk-mediated phosphorylation of
Yen1 promotes its nuclear exclusion and inhibits catalytic activity by
reducing the efficiency of DNA binding. Later in the cell cycle, at anaphase,
Cdc14 drives Yen1 dephosphorylation, leading to its nuclear relocalization
and enzymatic activation. Using a constitutively activated form of Yen1,
we show that uncontrolled Yen1 activity is detrimental to the cell: spatial
and temporal restriction of Yen1 protects against genotoxic stress and, by
avoiding competition with the noncrossover-promoting repair pathways,
prevents loss of heterozygosity.
P16-2 (R16-3)
Protein neddylation controls the timing of DNA

end resection and DNA double strand break
break repair
Pablo Huertas1, Sonia Jimeno1, Mara Jess Fernndez vila2,
Cristina Cepeda Garca2, Daniel Gmez Cabello2, Andrs Cruz Garca1
1
CABIMER/Universidad de Sevilla, Sevilla, ES, 2CABIMER, Sevilla, ES
DNA double strand breaks are the most cytotoxic lesions that can occur
on the DNA. They can be repaired by different mechanisms and optimal
survival requires a tight control between them. Here we show that
protein deneddylation as a major controller of repair pathway choice.
Neddylation inhibition changes the normal repair profile towards an
increase on homologous recombination. Indeed, after DNA damage fast
and local RNF111-mediated neddylation acts as an inhibitor of BRCA1
and CtIP-mediated DNA end resection, a key process in repair pathway
choice. A secondary wave of deneddylation by the COP9-signalosome
allows resection to take place at later times. By controlling DNA resection,
protein neddylation not only affect the choice between NHEJ and
homologous recombination, but also controls the balance between different
recombination subpathways. Thus, protein neddylation act as a molecular
clock, controlling the timing of DNA end resection and repair events.
Psters
the genome is not well understood. Here we report that in breast
cancer cells the unliganded progesterone receptor binds genomic sites
and targets a repressive complex containing HP1g, LSD1, HDAC1/2,
CoREST, JARID1B and the RNA SRA (Steroid Receptor Activator) to
20% of hormone-inducible genes, keeping these genes silenced prior to
hormone treatment. The complex is anchored via binding of HP1g to
H3K9me3. SRA interacts with PR, HP1g and LSD1, and its depletion
compromises the loading of the repressive complex to target chromatin
promoting aberrant gene de-repression. Upon hormonal treatment the
HP1g-LSD1 complex is displaced from these constitutively poorly
expressed genes as a result of rapid phosphorylation of histone H3 at
serine 10 mediated by MSK1, which is recruited to the target sites by
the activated PR. On the other hand, the HP1g-LSD1 complex is also
recruited to the hormone-repressed genes BCAS1 and KRT23 promoting
a close chromatin conformation. These results highlight the importance
of the HP1g-LSD1 complex as a key factor involved in hormonal gene
regulation of breast cancer cells.
P16-4
La cromatina-quinasa VRK1 regula la histona

H2AX y la formacin de los focos de reparacin
de ADN inducidos por radiacin ionizante
Marcella Salzano, Marta Sanz-Garca, Diana M. Monsalve, David S.
Moura, Pedro A. Lazo
Instituto de Biologa Molecular y Celular del Cncer de Salamanca,
(CSIC). Instituto de Investigacin Biomdica de Salamanca-IBSAL,
Hospital Universitario de Salamanca, Salamanca, ES
El dao local al ADN altera la estructura de la cromatina y el detectar y
el responder a esta alteracin es probablemente el inicio de la Respuesta
al Dao del ADN (RDA). En este contexto, las cromatina quinasas son
unos buenos candidatos por participar en la deteccin de la alteracin
local de la cromatina por dao al ADN y en sus respuestas celulares.
VRK1 es una Ser-Thr quinasa nucleosomal identificada en el nuestro
laboratorio como un nuevo sensor de la RDA. Est descrito que VRK1
regula muchos procesos celulares como por ejemplo el ciclo celular,
condensacin de la cromatina y proteccin de las clulas al dao gnico
por regulacin positiva de p53. Nuestros resultados demuestran que su
deplecin por knock-down causa una prdida global de la acetilacin
de las histonas independientemente de ATM. Tambin hemos demostrado
que VRK1 interacciona directamente con H2AX fosforilndola en
Ser139 inmediatamente tras el dao al ADN inducido por radiacin
ionizante. El efecto sobre la fosforilacin de H2AX y la formacin de los
focos es prevenido por el knock-out de VRK1 y rescatado por la quinasa
activa, pero no con la inactiva, demostrando que es necesaria la actividad
de VRK1 para la respuesta al dao del ADN. Adems, el pretratamiento
con los inhibidores de ATM demostr una cooperacin de ATM y VRK1
sobre la formacin de los focos de reparacin. Concluimos que VRK1 es
un nuevo componente de la cromatina, que reacciona a sus alteraciones
y que participa a la respuesta muy temprana al dao gnico, funcionando
ella sola o en colaboracin de ATM.
P16r-5
P16-3 (R16-2)
Role of chromatin dynamics on hormonedependent gene regulation in breast cancer cells
Papel de SF3B en la reparacin de los cortes

de doble cadena en el DNA
Guillermo Pablo Vicent Martnez

Centre de Regulaci Genmica (CRG), Barcelona, ES
M Rosario Prados Carvajal1, Cristina Cepeda Garca1, Pablo

Huertas2
1
CABIMER, Sevilla, ES, 2CABIMER/Universidad de Sevilla, Sevilla, ES
A close chromatin conformation prevents gene expression in eukaryotic

cells. Genes activated by external cues have to overcome this repressive
state by locally changing chromatin structure to a more open state.
Although much is known about hormonal gene activation, how basal
repression of regulated genes is targeted to the correct sites throughout
De los muchos tipos de daos que puede sufrir el DNA, los cortes de
doble cadena (DSB) son de los ms citotxicos, ya que causan mutaciones,
reordenamientos cromosmicos e inestabilidad genmica. As, las clulas
han desarrollado diferentes mecanismos para la deteccin, sealizacin y
reparacin de estas roturas. La reparacin de dicho dao puede realizarse
141
Psters
por recombinacin homloga (Homologous Recombination; HR) o
por unin de extremos no homlogos (Non-Homologous End Joining;
NHEJ). La decisin entre ambos mecanismos de reparacin depende de
la activacin o no del proceso denominado reseccin de los extremos de
DNA. Dicho mecanismo es, por tanto, un proceso muy regulado y crtico
en la supervivencia de las clulas.
Una de las protenas ms importantes implicada en la reseccin del DNA es
la protena CtIP, que controla si el proceso de reseccin ha de activarse o no.
Esta decisin depende de la integracin de mltiples seales celulares por
parte de CtIP, garantizando as que las DSB sean sealizadas y reparadas
de la mejor manera posible. Para ello, CtIP sufre mltiples modificaciones
postraduccionales e interacciona con diversas protenas.
Como parte de un estudio para entender como se regula la reseccin
a nivel celular, hemos aislado nuevas protenas que interaccionan
fsicamente con CtIP. Entre ellas, hemos encontrado diversas subunidades
del complejo SF3B. Como la deplecin de varios componentes de dicho
complejo disminuyen la eficiencia de la recombinacin homloga, estamos
estudiando su papel en reseccin con ms detalles. Aqu, presentamos
evidencias de que la deplecin de distintas subunidades de SF3B reduce
la reseccin de cortes de doble cadena inducidos en el DNA de manera
artificial. Como se ha descrito que la subunidad SF3B2 del complejo es un
sustrato de ATM, la quinasa principal del checkpoint de dao en el DNA,
estamos analizando el papel de esas fosoforilaciones en la funcin del
complejo SF3B en reseccin.
P16r-6
Regulacin del mtodo de reparacin de roturas

de doble cadena en el DNA por HDAC1, HDAC2
y PARP3
Cintia Checa Rodrguez, Pablo Huertas Snchez, Daniel Gmez
Cabello
Centro Andaluz de Biologa Molecular y Medicina Regenerativa
(CABIMER), Sevilla, ES
Los cortes de doble cadena (DSB) son el tipo de dao ms citotxico
que puede sufrir el DNA, pudiendo causar mutaciones e inestabilidad
genmica. Existen dos mecanismos para reparar estos cortes, la unin
de extremos no homlogos (Non-Homologous End Joining; NHEJ) y la
recombinacin homloga (Homologous Recombination; HR). No se conoce
bien el mecanismo por el que los DSB son reparados por una u otra va.
Sin embargo, se considera que el procesamiento de los extremos del corte,
o reseccin, es un paso clave en esta decisin conduciendo a la reparacin
por HR e impidiendo la va de NHEJ. La deteccin y sealizacin del dao
(checkpoint) influye tambin en el mtodo de reparacin.
El dao en el DNA conduce a una cascada de eventos coordinada, conocida
como respuesta al dao en el DNA (DNA Damage Response, DDR)
donde el reclutamiento de protenas a la lesin est regulado en parte por
modificaciones postraduccionales incluyendo la fosforilacin y la poli
(ADP-ribosilacin). Recientemente, se ha demostrado que la poli (ADPribosa) polimerasa PARP3 es activada especficamente por los DSB in vitro
y se ha propuesto que pueda tener un papel en HR.
Por otra parte, la estructura de la cromatina influye en el reconocimiento,
sealizacin y reparacin de daos en el DNA. Las modificaciones de
histonas pueden alterar sus propiedades e influir en la respuesta al dao.
Se ha visto que las deacetilasas de histonas humanas HDAC1 e HDAC2
responden al dao del DNA y promueven la sealizacin y reparacin de
los DSB. Ambas protenas parecen implicadas tanto en NHEJ como en HR.
En este trabajo hemos analizado el efecto de la deplecin de HDAC1,
HDAC2 y PARP3 sobre el balance entre NHEJ y HR. Para entender su
funcin, hemos estudiado su posible papel en el proceso de reseccin y
checkpoint.
142

P16m-7
Identification of novel SND1 target genes

involved in glycerolipid metabolism in human
hepatoma cells under inflammatory conditions
Enara Arretxe, Sandra Armengol, Sarai Mula, Leire Enzunza,
Yolanda Chico, Begoa Ochoa, Mara Jos Martnez
Universidad del Pas Vasco, Leioa, ES
SND1 (Staphylococcal nuclease and Tudor domain-containing 1) is a
transcriptional corregulator involved in adipogenesis, stress response and
cancer progression. Current evidence indicates that SND1 upregulation
is a hallmark of several types of cancers. In particular in human
hepatocarcinoma, SND1 overexpression is correlated with malignancy
and a crosstalk between SND1, miRNA221, NF-B and inflammation
signaling pathways may be envisaged. Work in our lab demonstrated
that SND1 gene activity in human hepatoblastoma cells is upregulated
by a transcriptional network involving NF-B, Sp1 and NF-Y binding
in response to the inflammatory cytokine TNFa. Very recently, we have
performed the first genome-wide search for endogenous SND1 binding
sites by Chromatin Immunoprecipitation (ChIP)-chip assays on HepG2
human hepatoma cells under basal and TNF-induced inflammatory
conditions. Here we validated by ChIP-qPCR the binding of SND1 to the
promoter of four target genes, CHPT1, LPGAT1, LPIN1 and PTDSS1,
involved in regulation of glycerophospholipid metabolism. When SND1
protein was augmented in the nucleus by TNF treatment, the transcript
expression of CHPT1, LPGAT1 and LPIN1 was increased whereas that of
PTDSS1 was decreased. These results were compatible with the increment
in the cellular phosphatidylcholine content detected in the TNF treated
cells. SiRNA-mediated SND1 silencing abolished the TNF-induced
changes on the mRNA level of the target genes as well as the increment in
cellular PC content. However, under non-inflammatory conditions, SND1
silencing did not affect the expression of LPIN1 and PTDSS1 but reduced
the expression of CHPT1 and LPGAT1. Our findings indicate that SND1
binding is essential for the TNF action on the expression of these SND1
target genes involved in regulating cellular phosphatidylcholine content.
Supported by Gobierno Vasco (IT336/19, AE-2011-1-34, Saiotek calls) and
UPV/EHU (UFI11/20).
P16r-8 (R16-5)
VRK1 y AurKB interaccionan en mitosis

regulando la fosforilacin especfica de la histona
3 y la correcta progresin del ciclo celular
David da Silva Moura, Marta Vzquez-Cedeira, Pedro A. Lazo
Instituto de Biologa Molecular y Celular del Cncer, CSIC,
Universidad de Salamanca; e Instituto de Investigacin Biomdica
de Salamanca (IBSAL), Hospital Universitario de Salamanca,
Salamanca, ES
El ciclo celular es un proceso altamente regulado en el que participan
diversas protenas quinasa, como la Aurora B y la VRK1. Estas dos
quinasas controlan varios procesos para una correcta progresin del ciclo
celular, entre los cuales se destaca la condensacin de la cromatina. Durante
la mitosis, las dos serina-treonina quinasas contribuyen significativamente
en la condensacin de la cromatina en varios mecanismos, entre los que
se destaca la fosforilacin de las histonas, ms precisamente de la histona
3. VRK1 fosforila especficamente la treonina 3 (T3) de la histona 3 y la
Aurora B fosforila especficamente la histona 3 en la serina 10 (S10).
En este trabajo, hemos demostrado que VRK1 interacciona con Aurora B
y que esta interaccin se produce durante la mitosis, concretamente en las
fases ms tardas. Adems, analizamos el efecto de la interaccin VRK1Aurora B en la capacidad de fosforilar a la histona 3. As, confirmamos
que cantidades crecientes de Aurora B, afectaban a la fosforilacin de la
histona 3, en el residuo T3, por VRK1 y que este efecto era independiente
de la actividad quinasa de la Aurora B. De la misma manera, cantidades
Granada 2014
crecientes de VRK1, activa o inactiva, afectaban a la fosforilacin de la
histona 3, en el residuo S10, por la Aurora B. El mismo efecto sobre la
histona 3 fue verificado en ensayos quinasa con (32P[ATP]). Posteriormente,
hemos analizado durante el ciclo celular los niveles de fosforilacin de
la histona 3, en los dos residuos T3 y S10, y hemos demostrado que la
disminucin de la fosforilacin de la histona 3 en los dos residuos coincide
con la interaccin VRK1-Aurora B.
Con estos resultados concluimos que la interaccin VRK1-Aurora B afecta
negativamente la fosforilacin de la histona 3 por las dos quinasas, y que
este puede ser un mecanismo regulatorio para una correcta salida de la
mitosis.
P16-9
GtrS and GltR form a two-component system:

The central role of 2-ketogluconate in the
expression of exotoxin A and glucose catabolic
enzymes in Pseudomonas aeruginosa
Abdelali Daddaoua1, Carlos Molina-Santiago2, Jess de la Torre2,
Tino Krell2, Juan-Luis Ramos3
1
Estacin Experimental de Zaidn, CSIC, Otura, Granada, ES, 2EEZ CSIC, Granada, ES, 3ABENGOA RESEARCH, Sevilla, ES
In the human pathogen Pseudomonas aeruginosa the GltR regulator is
required for glucose transport whereas GtrS is a sensor kinase that plays a
key role in mediating bacteria-host interaction and pathogen dissemination
in the host. We show that GtrS and GltR form a two-component system and
this novel TCS was found to regulate the expression from the promoters
Pedd/gap-1, PoprB and Pglk, which control the expression of genes involved in
glucose metabolism and transport. In addition, the GtrS/GltR pair regulates
the expression of toxA that encodes exotoxin A, the primary virulence factor.
Microcalorimetry-based ligand screening of the recombinant GtrS ligand
binding domain revealed specific binding of 2-ketogluconate (2-KG) (KD=
5 M) and 6-phosphogluconate (KD= 98 M). These effectors accelerate
GtrS autophosphorylation, with concomitant transphosphorylation of
GltR leading to a 3-fold increase in transcription. Surprisingly, in vivo a
similar increase in expression from the above promoters was observed
for the mutant deficient in GltR regardless of the presence of effectors.
Using footprinting assays, the GltR operator site was found to contain the
consensus sequence 5-tgGTTTTTc-3. We propose that 2-ketogluconate is
a key metabolite in the stringent transcriptional control of genes involved
in virulence and glucose metabolism. Surprisingly, we found that the GltR
response regulator is a repressor that is released from its target operators of
the toxA and glucose metabolism genes upon phosphorylation. This system
corresponds to another mode that guarantees a concerted regulation of
carbohydrate metabolism and exotoxin A expression.
P16-10
La inhibicin de la actividad Aurora quinasa

B mediada por VHC regula la respuesta
antiinflamatoria en las fases iniciales
de la infeccin
Irene Francisco Recuero1, Antonio Madejn2, Julie Sheldon3, Celia
Perales3, Ana Isabel Gil2, Jordi Muntan4, Esteban Domingo3, Javier
Garca-Samaniego Rey2, Aurora Snchez Pacheco1
1
Bioqumica, Universidad Autnoma de Madrid, Madrid, ES,
2
Departamento de Digestivo, Ciberhed. Hospital Carlos III, Madrid,
ES, 3Consejo Superior de Investigaciones Cientficas (CSIC),
Madrid, ES, 4Instituto de Biomedicina de Sevilla (IBIS) - Hospital
UniversitarioVirgen Del Rocio de Sevilla, Sevilla, ES
Introduccin: Los cambios epigenticos, entre los que se encuentran las
modificaciones covalentes de histonas, son cruciales en la induccin de
inestabilidad gentica asociada a enfermedades en humanos, incluyendo
el desarrollo de fibrosis heptica y carcinoma hepatocelular. Sin embargo,
Psters
se desconoce el papel que este tipo de modificaciones pueda tener en la
evolucin de la enfermedad heptica producida por el virus de la hepatitis
C (VHC).
Objetivos: Estudiar las modificaciones covalentes de histonas inducidas
por la infeccin por VHC y determinar los mecanismos moleculares
implicados.
Mtodos: Para determinar el papel del VHC en la induccin de cambios
epigenticos se utiliz un sistema de infeccin in vitro de VHC (VHCcc)
sobre la lnea celular Huh7.5. Alternativamente la regin codificante de
la protena del core viral (genotipos 1a, 1b y 2a) se clon en un vector
de expresin eucariota con el que se realizaron ensayos de transfeccin
transitoria sobre la lnea celular Huh7.5 y en cultivos primarios de
hepatocitos humanos. Las modificaciones de histonas se analizaron por
Western Blot, utilizando anticuerpos especficos. Para determinar las
actividades enzimticas implicadas se ensay el efecto de inhibidores
especficos como el ZM443979 y ensayos de siRNA. La expresin de
genes implicados en el control de la actividad inflamatoria, como NF-B y
COX-2, se determin por RT-PCR cuantitativa.
Resultados: Hemos demostrado que el HCV a travs de la protena
core inhibe la fosforilacin del residuode Serina 10 de la histona H3
(H3Ser10ph), un marcador epigentico asociado a la regulacin de la
actividad transcripcional y la mitosis celular. Los ensayos con inhibidores
de actividades enzimticas implicadas en la fosforilacin de histonas
demostraron que el efecto sobre la H3Ser10 inducida por la protena del
core viral se inhiba en presencia de ZM443979, un inhibidor de la actividad
Aurora quinasa B (AKB). Mediante ensayos de coinmunoprecipitacin
observamos una interaccin directa entre la protena del core viral y la
AKB. La interaccin con la protena del core indujo una inhibicin de
la actividad quinasa de la AKB. Asociado a la inhibicin de la actividad
AKB se observ una inhibicin de transcripcin de NF-B y COX-2, genes
implicados en el control de la respuesta inflamatoria. La sobreexpresin de
AKB revirti este efecto y disminuy la infectividad extracelular del virus,
de manera opuesta la inhibicin de la actividad de Aurora B aument la
infectividad viral.
Conclusiones: La protena del core del VHC interacciona con AKB
inhibiendo su actividad quinasa y disminuyendo los niveles de fosforilacin
de la H3Ser10ph as como la expresin de NF-B y COX-2, dos genes
implicados en la regulacin de la respuesta inflamatoria. Este mecanismo
podra ser una nueva estrategia del VHC para asegurar su infectividad y
persistencia en la clula husped.
P16-11
Vitamin D has wide regulatory effects on histone

modifying enzymes in human colon cancer cells
Antonio Barbchano Becerril, Fbio Pereira, Asuncin FernndezBarral, Gemma Ferrer-Mayorga, Alberto Muoz, Mara Jess Larriba
Instituto de Investigaciones Biomdicas Alberto Sols, CSIC-UAM,
Madrid, ES
Vitamin D3 is obtained from the diet or mainly synthesized in the skin and
is converted into the active metabolite 1alpha, 25-dihydroxyvitamin D3
(1,25(OH)2D3). Epidemiological data suggest a protective role of vitamin D
against several neoplasias, particularly colorectal cancer. 1,25(OH)2D3 is a
major regulator of gene expression. Histones are subject to posttranslational
modifications including methylation, acetylation and others. Misregulation
of histone modifications alters gene expression and cell phenotype, which
may contribute to cancer initiation, progression and/or metastasis. We
found that 1,25(OH)2D3has a wide regulatory action on the expression
of genes coding for histone demethylases of the Jumonji C domain and
lysine-specific demethylase families (Pereira et al., 2011; 2012). Notably,
1,25(OH)2D3induces the expression of the histone demethylase JMJD3/
KDM6B that specifically demethylates the lysine 27 of histone H3
(H3K27) and has tumour suppressor activity. We found that the induction
of JMJD3 is necessary for the adequate gene regulatory, antiproliferative,
and prodifferentiation actions of 1,25(OH)2D3 in human colon cancer cells.
Conversely, 1,25(OH)2D3 inhibits the expression of several Polycomb
143
Psters
group proteins that participate in the Polycomb repressive complexes

(PRC) 1 and 2, which are responsible for H3K27 methylation and promote
tumorigenesis. The inhibition of PRC2 activity by DZNep potentiates the
gene regulatory action of 1,25(OH)2D3 and also its effects on colon cancer
cell proliferation and differentiation. Thus, 1,25(OH)2D3 exerts an ample
regulatory effect on the expression of histone modifying enzymes involved
in epigenetic regulation that, in turn, mediate 1,25(OH)2D3 actions on gene
expression and cell phenotype in colon cancer.
directly regulates the expression of virulence functions in response

to a host signal. In this work we aimed at understanding the molecular
mechanism that leads to the activation of P. aeruginosa ECF sigma factors.
We have identified two proteases, Prc and RseP, involved in the processing
of several anti-sigma factors in response to their cognate inducing signal.
Moreover, we have also observed that some anti-sigma factors are already
processed in an N- and a C-domain prior to the perception of the signal.
The biological significance of this observation is discussed.
P16-12
P16-14
New strategies in relationship between lncRNAs

and the SWI/SNF complex
Genetic analysis of the role of the Mlp1 export

factor in genetic stability
Antonio Herrera Merchn, Carmen Callejas Lechuga, Purificacin

Fernndez Espinosa, Pedro Pablo Medina Vico
Francisco Garca Bentez, Hlne Gaillard, Andrs Aguilera Lpez

CABIMER/Universidad de Sevilla, Sevilla, ES
Long non-coding RNAs (lncRNAs) are a new class of non-coding gene

regulators. But unlike their smaller counterparts, microRNAs, relatively
less is known about the roles and functions of lncRNAs. Current evidence
suggests that lncRNAs may play important roles in a wide range of
biological processes in human cancers.
Many of the LncRNAs have been functionally associated with chromatinremodeling complexes.
SWI/SNF is chromatin-remodeling complex, which alters the interactions
between DNA and histones and modifies the availability of the DNA for
transcription. The latest deep-sequencing of tumor genomes has reinforced
the important and ubiquitous tumor suppressor role of the SWI/SNF
complex in cancer. However, although SWI/SNF complex play a key role in
gene expression, the regulation of this complex itself is poorly understood.
We hypothesize that LncRNAs could be also functionally related SWI/SNF
playing a role in its function in carcinogenesis. Supporting this hypothesis,
recently, an LncRNA was found functionally associated with the SWI/SNF
complex, in prostate cancer.
We are gathering preliminary results using new technologies of sequencing
and immunoprecipitation of RNA (RNA-IP) to identify new LncRNAs
associated with SWI/SNF in different tumor cell lines for subsequently
develop functional assays to infer its biological role in tumor development.
Transcription of a DNA sequence increases its recombination frequency, a

phenomenon referred to as transcription-associated recombination (TAR).
During transcription, negative supercoils are accumulated behind the
advancing RNA polymerase, facilitating the unwinding of the DNA helix.
This transient DNA opening favours the annealing of the nascent mRNA to
the transcribed strand (TS) forming a DNA:RNA hybrid (R-loop). R-loops
can be formed naturally as intermediates in specific cellular processes, such
as mitochondrial DNA replication or immunoglobulin class switching and
have been suggested to play a role in transcription termination. A number of
observations in E. coli, yeast and humans indicate that the non-transcribed
strand (NTS) is more susceptible to damage than the TS. The formation of
R-loops, in which the NTS remains single stranded, renders the NTS more
vulnerable to DNA damage and contributes to TAR.
We are interested in understanding the different mechanisms associated with
the optimal biogenesis of mRNA and the control of R-loops formation. We
have found mlpD in a screening for deletions of non essential nuclear genes
that show hyper recombination when we overexpress a human deaminase
that preferentially targets ssDNA in Saccharomyces cerevisiae. The MLP1
gene encodes a nuclear pore basket protein that has an important role in
unspliced mRNA retention, SUMO regulation and telomere organization.
We investigate the role of Mlp1, its paralog Mlp2 and their human homolog
TPR in preventing genomic instability. Current results of Mlp1 will be
presented and discussed.
P16r-13
Unravelling the molecular mechanism activating

ECF sigma factor in Pseudomonas aeruginosa
1
P16-15
Novel roles for the protein kinase DYRK1A as a

chromatin-associated transcriptional regulator
Joaqun R. Otero-Asman , Cristina Civantos , Karlijn Bastiaansen ,

Marian Llamas1
1
Dept. of Environmental Protection, Estacin Experimental del
Zaidn-CSIC, Granada, ES, 2Department of Molecular Microbiology
Vrije Universiteit Amsterdam Faculty of Earth and Life Sciences,
Amsterdam, NL
Chiara Di Vona1, Daniela Bezdan1, Abul B.M.M.K. Islam2, Nuria

Lopez-Bigas2, Stephan Ossowski1, Susana de la Luna1
1
Centro de Regulacin Genmica-CRG, Barcelona, ES,
2
Departament de Cincies Experimentals i de la Salut, Universitat
Pompeu Fabra, Barcelona, ES
Pseudomonas aeruginosa is an opportunistic pathogen that lives in a

wide range of environments. There are many interactions between the
environment and the bacterium that permit this niche variability. Many of
these interactions are possible because of the action of a regulatory system
known as cell surface signaling (CSS). CSS systems recognize signals
from the environment and transmit them into the cytosol, affecting gene
expression.The system is constituted by an outer membrane receptor, a
cytoplasmic membrane located anti-sigma factor and an extracytoplasmic
function (ECF) sigma factor in the cytosol. In absence of the inducing
signal, the anti-sigma factor binds to and keeps inactive the sigma factor.
The regulatory cascade starts with the recognition of the signal by the
receptor, which promotes the proteolytic degradation of the anti-sigma
factor and the activation of the ECF sigma factor. The ECF sigma factor
can then interact with the RNA polymerase and induce expression of target
genes. Most P. aeruginosa CSS systems have a role in the regulation of iron
uptake, which is an important process for the pathogen during infection.
However, this bacterium has a CSS system, called PUMA3 or Vre, that
Our perception of how kinases regulate gene expression has recently

been expanded to consider not only their influence on transcription
factors and co-regulators but also that on histones, chromatin remodelers
or components of the basal transcription machinery, all of which may be
directly modified by kinases at specific genomic loci. DYRK1A (dualspecificity tyrosine-regulated kinase) belongs to a highly conserved family
of kinases represented in all eukaryotes and it is known to fulfil key roles
during brain development. While DYRK1A is present both in the nucleus
and cytoplasm of mammalian cells, very little is known about the nuclear
activities of DYRK1A. We show here that nuclear DYRK1A is an active
kinase that participates in high molecular weight complexes, interacting
with several components of the basal transcriptional machinery, including
RNA polymerase II. We mapped the genome-wide profile of DYRK1A
interactions with chromatin and found that the kinase is recruited to RNA
polymerase II-dependent promoters, where it activates transcription in a
kinase dependent manner. DYRK1A binds chromatin regions displaying a
highly conserved palindromic sequence that lies close to the transcription
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Granada 2014
start site of target genes, a sequence that is apparently necessary for
DYRK1A-mediated transcriptional activation. Growth-dependent
expression of a subset of DYRK1A target genes depends on DYRK1A
protein levels and/or activity, and indeed, downregulation of DYRK1A
diminishes cell growth. Thus, we propose a novel role for DYRK1A as a
chromatin-associated transcriptional regulator and as part of the machinery
controlling cell growth.
P16-16
SMARCA4 expression regulation by miRNAs

in lung carcinogenesis
IF Coira1, EE. Rufino-Palomares1, P. Peinado1, C. Metheetrairut2, L.
Boyero1, OA Romero3, J Carretero4, E. Farez-Vidal5, M. Cuadros5, FJ
Reyes-Zurita5, JA Lupiez5, M Snchez-Cespedes3, FJ Slack2, PP
Medina1
1
University of Granada, Department of Biochemistry and Molecular
Biology. Centre for Genomics and Oncological Research (GENYO),
Granada, ES, 2Yale University, New Haven, US,3IDIBELL,
Barcelona, ES, 4University of Valencia, Department of Physiology,
Valencia, ES, 5University of Granada. Department of Biochemistry
and Molecular Biology, Granada, ES
SWI/SNF complex is chromatin-remodeling complex able to alter the
interactions between DNA and histones and modify the availability
of the DNA information to the cell machinery, using the energy of the
ATP hydrolysis. Last deep-sequencing efforts on tumoral genomes have
underlined the important and ubiquitous role of the SWI/SNF complex
and cancer. It has been known that the expression of SMARCA4, the
helicase/ATPase catalytic subunit of the SWI/SNF complex, is frequently
lost in NSCLC cell lines, mainly by mutations. In primary tumours, the
loss of expression of SMARCA4 is also frequent, however it lost cannot
be explained by mutations nor other gene silencing mechanisms such us
promoter hyper-methylation. So, the regulation of SMARCA4 is partially
unknown. In this assay, we study if the microRNAs contributes to the
loss of expression of SMARCA4 in lung tumors. For this purpose we
have determined experimentally the 3UTR, and analysed functionally
the microRNA binding sites previously predicted by bioinformatics tools.
Results obtained indicated that the loss of expression of SMARCA4
observed in primary lung tumors can be due to microRNA activity.
P16-17 (R16-1)
RNAPII dephosphorylation is facilitated

by the Rpb4/7 heterodimer
Olga Calvo
Instituto de Biologa Funcional y Genmica. CSIC/Universidad de
Salamanca, Salamanca, ES
The eukaryotic RNAPII enzyme, whose activity is essential for
transcription of protein-coding genes, is a complex of 12 subunits, Rpb1
to Rpb12. The Rpb4 and Rpb7 subunits bind to each other forming a
complex in archaebacteria and in eukaryotic cells, and display some unique
features that distinguish them from the remaining subunits. In the case of
S. cerevisiae, the Rpb4/7 heterodimer can dissociate from the rest of the
holoenzyme, and has been shown to be involved in several gene expression
processes. Thus, they are important for transcription initiation, elongation
and, in particular, Rpb4 contributes to contranscriptional recruitment
of 3-end processing factors. Moreover, recent evidences suggest that
they are also involved in DNA repair, mRNA export and decay, as well
as in translation. Here, I present additional data showing that the Rpb4/7
heterodimer is important to maintain proper RNAPII phosphorylation levels
in S. cerevisiae, regulating several kinases and phosphatases. In fact, in the
absence of a functional Rpb4/7 heterodimer, RNAPII phosphorylation is
dramatically enhanced all along the transcription cycle due to altered CTD
phosphatases recruitment.
Psters
P16-18
Roturas de ADN y topoisomerasas: el peligro de

jugar con cuchillos
Felipe Cortes Ledesma
CABIMER, Sevilla, ES
Las topoisomerasas de ADN son enzimas nucleares muy conservadas en la
evolucin que pueden considerarse un arma de doble filo. Por un lado, su
accin es necesaria para regular los cambios topolgicos inherentes a todos
los procesos fundamentales que implican a la molcula de ADN, incluyendo
tanto la transcripcin, replicacin y reparacin como la condensacin y
segregacin cromosmica. Sin embargo, su ciclo cataltico emplea un
mecanismo de rotura transitoria y religacin que, si se ve interrumpido, puede
dar lugar a roturas de ADN persistentes, con las evidentes consecuencias
que stas pueden tener para la supervivencia celular y la integridad del
genoma. Adems, esta peculiaridad del mecanismo de accin de las
topoisomerasas es la base de la eficacia antitumoral de los llamados venenos
de topoisomerasas, un grupo qumicamente heterogneo de compuestos
que inhiben especficamente el paso de religacin catalizado por la enzima,
induciendo as la formacin de roturas de ADN que, por su alto ndice de
proliferacin, afectan preferentemente a las clulas tumorales. Adems de
esta interesante relacin con la terapia del cncer, se ha demostrado que una
deficiente reparacin de roturas generadas por topoisomerasas puede ser la
base de diversas patologas neurolgicas humanas. Por lo tanto, comprender
en su totalidad los mecanismos y la regulacin que rigen la reparacin de
las roturas de ADN inducidas por topoisomerasas adquiere una importancia
fundamental para la salud, con posibles implicaciones en el desarrollo de
futuras herramientas, tanto de diagnstico y pronstico como teraputicas.
P16-19
Pitx2 differently controls the expression of host

genes and gene-embedded microRNAs in cardiac
and skeletal muscle cells
Francisco Hernandez-Torres, Amelia E Aranega, Diego Franco Jaime
Departamento de Biologa Experimental, Universidad de Jan,
Jan, ES
The Pitx2 gene is a member of the Bicoid-like homeobox family that
plays an importantrole in embryonic left-right signalling and subsequently
during cardiac and skeletal muscle organogenesis. Pitx2 misregulation
has been as well associated with skeletal and cardiac muscle diseases.
Although three Pitx2 isoforms are expressed in mice, Pitx2a, Pitx2b
and Pitx2c, only Pitx2c plays a determinant role in leftright signalling
and organogenesis. Over the last years we have gained insights into the
transcriptional regulatory mechanisms driven by Pitx2 in both cardiac
and skeletal muscle. Interestingly, besides playing a role in regulating a
large number of protein-coding genes, microRNAs are also regulated by
Pitx2. Whereas the functional role of distinct microRNAs in cardiac and
skeletal muscle development and homeostasis is progressively emerging,
their transcriptional control and the genetic networks in which they are
involved remains largely unexplored. Among 34 differentially regulated
microRNAs by Pitx2 in either skeletal or cardiac muscle cells, 10 (~30%)
display putative Pitx2 binding sites in the -3000 bp proximal promoter
sequence, among with other muscle-enriched transcription factor binding
sites. Curiously, only two of these microRNAs were intergenic, supporting
thus their own transcriptional regulatory mechanisms, while the majority
of them were embedded into distinct host genes. Since transcriptional
regulation of gene-embedded microRNAs seemed to be modulated in
accordance with transcriptional regulation of the host gene,we evaluated
the host gene/microRNA expression levels in Pitx2 gain-of-function
experiments using Sol8 skeletal muscle and HL-1 atrial cardiomyocytes,
respectively. Surprisingly, our data demonstrate that most of these
microRNAs are independently regulated from its host gene, and moreover
such transcriptional regulation is cell-type specific. We our now working
to establish the mechanisms by with Pitx2 can exert this microRNAs
transcriptional regulation.
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Psters
P16-20
datos obtenidos resultaron ser ms claros al analizarlos por esta tcnica que
por western. En la cepa salvaje, se obtienen niveles altos de GFP expresado
con la 3-UTR de CYC1 y bajos con la de KlCYC1. Hemos comprobado
por tanto, que se puede modular la expresin de GFP con estas UTR, y que
el efecto del mutante nup84 vara en funcin de la 3-UTR contigua a la
regin codificadora.
Adult exposure to Bisphenol A decrease

the expression of 5alpha-reductase type I
in the prefrontal cortex of female rat. A novel
mechanism underlying Bisphenol A-associated
psychopathologies?
Esperanza Ortega Snchez1, Beatriz Castro Bohorquez2, Pilar
Snchez Medina2, Jess M. Torres de Pinedo2
1
Universidad de Granada, Granada, ES, 2Dpto. de Bioqumica,
Biologa Molecular 3 e Inmunologa. Facultad de Medicina.
Most people in developed countries are exposed almost continuously to
Bisphenol A (BPA), an endocrine-disrupting chemical presents in food
packaging and dental sealants. BPA is able to interfere with cognitive
functions and behavior, but the mechanisms underlying these effects remain
unknown. Allopregnanolone (AlloP) is a neuroactive steroid involved in
modulating behavioral functions, stress, and neuroendocrine axes and its
deregulation is implicated in the development of several psychopathologies.
In these processes a key role is held by 5-reductase (5-R), the rate-limiting
enzyme of AlloP synthesis. Given 5-R type 1 is the isozyme mainly
implicated in the biosynthesis of AlloP, we examined the effects of BPA on
5-R1 expression in the prefrontal cortex (PFC) of female rats. We focused
on PFC since it is an important area for cognitive control and complex
behaviors. Adult female Wistar rats were subcutaneously injected during
4 days with 50 g/kg/day of BPA, the current Environmental Protection
Agency (EPA) reference dose. Rats were euthanized at 30 min after the
final administration. Quantitative RT-PCR and Western blot analysis were
performed to quantify mRNA and protein levels of 5-R1 respectively. Under
the conditions assayed, BPA decreased the expression of 5-R1. This finding
is very interesting because reduced brain levels of 5-R1 and, consequently
AlloP, may contribute to increased the vulnerability for mental and emotional
pathology in females. Thus, mood changes during the menstrual cycle,
postpartum, major depression and epilepsy are pathologies associated with
low AlloP levels. We present here a possible novel mechanism underlying
BPA-associated psychopathologies but also raises questions about the safety
of adult exposure to BPA.
P16r-21
Anlisis de la expresin de GFP con diferentes

3-UTR en un mutante nup84 del NPC
Brbara Mara Varela-Rodrguez, Tania Alvarez-Felgar, Ana Mara
Rodrguez-Torres, Maria Angeles Freire Picos
rea de Bioqumica y Biologa Molecular, Facultad de Ciencias,
Universidad de la Corua, A Corua, ES
Uno de los aspectos clave de regulacin de la expresin gnica es
el procesamiento del extremo 3de los RNA transcritos por la RNA
polimerasa II. En las regiones 3-UTR (3-Untranslated) de los mensajeros
se encuentran seales que determinan, entre otras, su estabilidad y en
general todos los procesos post-transcripcionales.
Menos conocido es el proceso que determina el emplazamiento de un
mRNA en el citosol por su paso a travs del Complejo del Poro Nuclear
(NPC). Datos de nuestro grupo [1] indican que la mutacin del factor
Nup84 (que forma parte del complejo Y del NPC) de la levadura
Saccharomyces cerevisiae afecta a la abundancia de transcritos detectables
en los genes con poliadenilacin alternativa. Esto nos llev a buscar un
sistema para identificar el producto final de la expresin, la protena.
Para ello expresamos GFP con variantes de regiones 3-UTR de genes de
levaduras: por un lado la 3-UTR del gen CYC1 y por otro las variantes de
la UTR de un gen con poliadenilacin alternativa, KlCYC1, expresado en S.
cerevisiae [2]. El anlisis de la expresin de GFP se llev a cabo tanto por
microscopa de fluorescencia como por fluorimetra y por western blot. Los
resultados de fluorimetra permitieron apreciar diferencias importantesen la
expresin de GFP en el mutante nup84 expresando la UTR de KlCYC1. Los
146
Bibliografa
[1] T. Alvarez-Felgar, B.M Varela-Rodrguez y MA Freire-Picos.
Variaciones en la expresin de genes con procesamiento alternativo en
mutantes del poro nuclear. XXXVI Congreso de la SEBBM 2013.
[2] B.M. Varela-Rodrguez. Expresin de formas recombinantes de GFP
en levaduras: efecto de la cepa y componentes del poro nuclear. Tesis de
licenciatura. Facultad de ciencias, UDC 2013.
P16-22
DbdR, nuevo miembro de la familia de

LysR, regulador transcripcional de la ruta de
degradacin anaerobia de 3,5-dihidroxibenzoato
en Thauera aromatica AR-1
Molina-Fuentes, A., Pacheco, D., Marn, P., Daz-Romero, A.,
Marqus, S.
Departamento de Proteccin Ambiental, Estacin Experimental del
Zaidn, CSIC, Granada, ES
T. aromatica AR-1 es una bacteria desnitrificante capaz de crecer en 3,5
dihidroxibenzoato (3,5-DHB) como nica fuente de carbono y energa
mediante una ruta oxidativa independiente de oxgeno utilizando nitrato
como aceptor final de electrones [1]. La agrupacin gnica que codifica para
las enzimas de la ruta y protenas transportadoras consta de 20 genes en una
regin cromosmica de 29 Kb [2]. Un anlisis de RT-PCR ha permitido definir
una organizacin transcripcional en cinco operones, cuatro de los cuales son
inducibles por sustrato. A ambos extremos de la agrupacin se encuentran
sendos reguladores de la familia de LysR. Mediante mutagnesis dirigida
hemos determinado que el nico gen regulador esencial de la ruta es dbdR.
Un anlisis de expresin en un fondo mutante dbdR revela que los operones
principales de la ruta son dependiente de este regulador. Mediante extensin
a partir de cebador hemos definido los promotores de estos operones, que
muestran una notable conservacin en estructura, y en los que se predicen los
posibles sitios de unin para reguladores de la familia de LysR. Estos sitios
de unin se estn confirmando mediante ensayos de retardo en gel (EMSA)
y de footprinting con DNAsa utilizando protena DbdR sopreexpresada y
purificada. El gen regulador forma un opern con qorA, una de las enzimas
responsable del segundo paso de la ruta. Este opern presenta niveles basales
de expresin, inducibles en presencia de sustrato. Su actividad est regulada
negativamente por la presencia de DbdR en ausencia del sustrato. Se han
construido fusiones de cuatro promotores de la ruta (Porf18, Porf20, Pdctp y Pdbdr)
al gen reportero lacZ para analizar su actividad en un husped heterlogo
en presencia y ausencia del regulador. Los resultados confirman el papel del
regulador en la expresin de estos promotores, aunque la actividad basal y la
fuerza de los distintos promotores varan. Esto siguiere distintos mecanismos
de activacin para los distintos promotores. Estas construcciones se han
utilizado para analizar la expresin en T. aromatica AR-1. Los resultados
confirman que DbdR regula la expresin desde al menos Porf18, Porf20 y Pdctp.
Por otra parte se confirma la represin catablica inicialmente descrita como
la represin de la utilizacin de 3,5-DHB en presencia de benzoato(3):
observamos que la presencia simultnea de succinato y 3,5-DHB produce
una cintica tpica de crecimiento diuxico, donde el 3,5-DHB slo se
utiliza una vez consumido el succinato. Esta represin se ejerce a nivel de
la expresin desde al menos los promotores Porf18 y Porf20. Finalmente, el
anlisis de la expresin de los promotores regulados en fondos mutantes para
los distintos pasos de la ruta nos ha permitido determinar que la molcula
efectora es el sustrato 3,5-DHB y no un producto de su metabolismo. Este
extremo se analizar bioqumicamente mediante microcalorimetra isoterma
de titulacin (ITC) con la protena reguladora purificada.
Granada 2014
Psters
Proyecto financiado por fondos FEDER, proyecto del MICINN BIO201123615.
del gen. Tras la activacin del gen existe un desplazamiento de N-2 y

una desestructuracin parcial o total en los nucleosomas N-1 y N+1,
dejando accesible los elementos en cis del gen. Tambin se estudia el
posicionamiento de Egr1 en condiciones de silenciamiento para investigar
la implicacin de la protena EGR1 en el desplazamiento del nucleosoma
N-2. Los resultados reiteran la importancia del estudio de los mecanismos
de regulacin de la cromatina a nivel mononucleosomal.
P16r-23
P16-25
Electrophoretic mobility of catenated, knotted

and supercoiled DNA molecules
BCL7As role in haematological malignancies
Bibliografa
[1] Gallus, C., and Schink, B. (1998) Arch Microbiol. 169, 333-338.
[2] Molina-Fuentes, A. (2012) Tesis doctoral.
[3] Philipp B & Schink B. (2000) Arch Microbiol. 173, 91.
Jorge Cebrin , Alicia Castn , Maridian J. Kadomatsu-Hermosa ,

Vctor Martnez2, Cristina Parra2, Mara Jos Fernndez-Nestosa2,
Christian Schaerer2, Pablo Hernndez1, Jorge B. Schvartzman1,
Dora B. Krimer1
1
Department of Cellular and Molecular Biology. Centro de
Investigaciones Biolgicas (CSIC), Madrid, ES, 2Scientific and Applied
Computing Laboratory. Polytechnic School. National University
of Asuncin. P.O. Box 2111 SL. San Lorenzo, Asuncin, PY
Two-Dimensional (2D) agarose gel electrophoresis is the method of
choice to separate the stereoisomers for any given circular DNA molecule.
Supercoiled, catenated and knotted families are clearly identified as they
exhibit different electrophoretic mobility. Here we used classical genetics,
treatments with Norfloxacine and 2D agarose gel electrophoresis run at
different conditions (voltage and agarose concentration) to analyze the
mobility of four families of stereoisomers of the same mass: monomeric
CatAs (where both duplexes are nicked), monomeric CatBs (where
one duplex is nicked and the other is covalently closed), covalently
closed dimers and knotted dimers. The results obtained indicate that the
contribution of catenane, knot and supercoil nodes to electrophoretic
mobility varies significantly depending on the electrophoretic conditions
employed. Whereas increasing voltage has little effect on the catenanes
mobility, it changes significantly the mobility of supercoiled and knotted
dimers. This observation was confirmed for CatBs. Moreover, for partially
replicated molecules displaying two regions: one already replicated and
the other not, the electrophoretic mobility is determined by the size of the
region and the distribution of supercoiled and pre-catenane nodes.
Carlos Balias Gavira1, Pedro P. Medina2

1
Centre for Genomics and Oncological Research (GENYO), Granada,
ES, 2University of Granada, Departement of Biochemistry and
Molecular Biology I. Centre for Genomics and Oncological Research
(GENYO), Granada, ES
Gene expression regulation increases the functional versatility and
adaptability of the cell by allowing it to express certain proteins when
needed. It is, therefore, one of the most important and complex processes
of biology. Changes in the gene expression patterns are key in cancer cell
transformation, through an increase in expression of genes that promote
carcinogenesis (oncogenes) and/or a decrease in expression of genes that
prevent it (tumour suppressor genes).
SWI/SNF chromatin-remodeling complex alters the interactions between
DNA and histones and modifies the availability of the DNA for transcription.
The latest deep-sequencing of tumor genomes has reinforced the important
and ubiquitous tumor suppressor role of the SWI/SNF complex in cancer.
However, although SWI/SNF complex play a key role in gene expression,
the regulation of this complex itself is poorly understood.
BCL7A was recently identified as a new member of the human SWI/SNF
complex. Recently, some reports found BCL7A expression inactivated
in hematological malignancies. After screening for BCL7A genetic
alterations over a battery of hematological cell lines, we found two cell
lines homozygously mutated for BCL7A. We are currently using these
cell lines to analyze tumor phenotype changes after BCL7A expression
restoration. Upon the completion of this task, we will go through functional
analysis to determine BCL7A role in tumorigenesis.
P16-26
P16-24
Mecanismos de regulacin de la cromatina:

posicionamiento mononucleosomal en el gen Egr1
Angela L. Riffo-Campos, Josefa Castillo, M. Isabel Rodrigo, Gerardo
Lpez-Rodas, Luis Franco
Laboratorio de Epigentica y cromatina. Departamento de
Bioqumica y Biologa Molecular, Universidad de Valencia e
INCLIVA, Valencia, ES
La cromatina es una estructura compleja formada por DNA, RNA y
protenas, que permite el empaquetamiento ordenado del genoma en las
clulas eucariticas. Representa un nivel adicional de regulacin de todos
los procesos metablicos del DNA, como la replicacin, reparacin,
recombinacin y transcripcin. El gen Egr1 (Early growth response 1)
pertenece al tipo denominados genes inmediato tempranos (IEGs), dado
que se activa rpidamente en respuesta a seales mitogenicas que activan
la proliferacin celular fundamentalmente mediante la va de sealizacin
de las MAP Quinasas.
En la presente investigacin se estudia el posicionamiento nucleosomal
del gen Egr1 en los modelos biolgicos in vivo (hepatectoma parcial en
ratn) e in vitro (lnea celular MLP29 de precursoras de hepatocitos de
ratn). El posicionamiento se determina mediante ensayos de digestin con
la enzima MNasa y posterior anlisis por PCR en tiempo real utilizando
amplicones solapantes que cubren la regin del promotor y el inicio de
la transcripcin del gen. Los resultados demuestran que la posicin de
los nucleosomas se mantiene en ambos modelos antes de la activacin
El tirosol regula el patrn global de expresin de

ARN mensajeros implicados en la reproduccin,
desarrollo y crecimiento de Caenorhabditis elegans
Ana Cauelo Navarro1, Francisco J. Esteban Ruiz2, Juan Peragn
Snchez1
1
rea de Bioqumica y Biologa Molecular, Departamento de
Biologa Experimental, Universidad de Jan, Jan, ES, 2rea
de Biologa Celular, Departamento de Biologa Experimental,
Universidad de Jan, Jan, ES
El tirosol (Tir, 2-(-4-hidroxifenil)etanol) es uno de los compuestos
fenlicos presentes en el fruto y hoja de olivo beneficiosos para la salud. La
incorporacin de 250 M de Tir en el medio de cultivo de Caenorhabditis
elegans incrementa la longevidad, la termotolerancia y la resistencia
al estrs oxidativo. En estudios previos hemos identificado, mediante
el anlisis de los cambios inducidos en el proteoma de esta especie, las
protenas dianas celulares sobre las que termina influyendo. En este trabajo
hemos determinado los cambios que se producen en el patrn global de
expresin de ARN mensajeros mediante un anlisis de microarrays de
ARN utilizando la metodologa Affymetrix.
Utilizando C.elegans GeneChipR Genome Array se han medido los
cambios que se producen en la expresin gnica de 22625 transcritos. En
respuesta a 250 M de Tir se producen cambios en los niveles de ARNm de
208 genes; en 206 el nivel de expresin estaba aumentado y en 2 de ellos
disminuido. Un posterior anlisis de enriquecimiento funcional puso de
manifiesto que estos genes estn implicados en procesos biolgicos tales
147
Psters
como la reproduccin, el metabolismo lipdico, el desarrollo embrionario
y larvario, la morfognesis y la regulacin de la velocidad de crecimiento,
transcripcin, transporte, as como en otras funciones. Destaca la induccin
de 19 genes que codifican para la Protena Mayor del Esperma (msp).
Estos resultados permiten relacionar el incremento en la longevidad con la
reproduccin, desarrollo y crecimiento en C. elegans mediado por tirosol.
Este trabajo ha sido financiado por el Plan de Apoyo a la Investigacin,
Desarrollo Tecnolgico e Innovacin de la Universidad de Jan
(R1/13/2010/02).
P16-27 (R16-6)
Mip6p, a putative mRNA binding protein

Manuel Martn Expsito, Susana Rodrguez Navarro
Las protenas de unin a ARN (RBP) juegan un papel clave en el control
post-transcripcional de diferentes ARN, que, junto a la regulacin
transcripcional, es una de las vas para regular los niveles de expresin
de genes durante el desarrollo. El control postranscripcional puede ocurrir
a diferentes niveles en el metabolismo de ARN, incluyendo splicing,
poliadenilacin, estabilidad, localizacin y transporte del ARN mensajero
del ncleo al citoplasma.
La protena Mip6 (MEX67-interacting protein 6) es una protena con
dominios de unin a ARN, que interacciona fsicamente con las protenas
Mex67 y Sus 1. Mip6p posee tres dominios de unin a ARN en su extremo
N-terminal.
A travs de distintas aproximaciones se ha tratado de identificar las dianas
(RNA unidos) a estos RBP o identificar RBP que se unen a secuencias
regulatorias conocidas. En general, los anlisis genticos indican que los
RBP deben regular numerosos RNA ya que la prdida de un RBP presenta
mayores efectos que cepas desreguladas de ARNm conocidos.
En nuestro laboratorio estamos realizando la caracterizacin funcional
de Mip6. Hemos identificado que la localizacin de Mip6 vara en base a
cambios de temperatura. Por otro lado, el dominio C-terminal es necesario
para la interaccin con Mex67 adems de ser imprescindible para el paso
de Mip6 entre el ncleo y el citoplasma. Adems, el mutante mip6 presenta
resistencia a estrs severo por temperatura.
Por ltimo, estamos estudiando cules son los ARN diana de Mip6 bajo
condiciones de estrs como choque trmico. Se presentarn los ltimos
resultados ms relevantes obtenidos en la caracterizacin de Mip6.
P16r-28
RNAi-mediated silencing of plekstrin in murine

erytrholeukemia cell differentiation
Vanessa Fernndez Calleja1, Pablo Hernndez2, Jorge B.
Schvartzman2, Dora B. Krimer2
1
2
Department of Cellular and Molecular Biology, Centro de
Investigaciones Biolgicas-CSIC, Madrid, ES
Murine erythroleukemias are experimental models that have been
successful to comprehend tumour leukemogenesis mechanisms and
normal erythropoiesis development. Friend murine erythroleukemia
cells (MEL) derived from transformed erythroblasts immortalized by the
Friend complex virus were used in our lab to study the cell differentiation
blockade that characterize these cell lines. We established resistant cell
lines (MEL-R) that unlike their parental line, are refractive to inducers
of differentiation (Fernndez et al., Leukemia Research (2008) 32,
121; SpringerPlus (2013) 2, 392). Recent results using next generation
sequencing recognized 487 genes that increased their expression
greater than two fold between MEL undifferentiated and MEL-R cells.
Among these genes we identified pleckstrin (plek), a protein implicated
in signaling and cytoskeletal function. Cytoskeletal proteins undergo
reorganization during terminal erythropoiesis and are involved in erythroid
148

cell enucleation. After validation of the pleck results by real-time PCR and
Western, we generated shRNAs encoded by mammalian vectors that were
used to transfect MEL cells and down-regulate plek in transient or stable
transfectants. We analyzed also the effect of plek in early and late stages
of cell differentiation.
P16-29
Accurate chromatin structure is required for

proper Top2 and condensin activities during
chromosome decatenation and centromere
biorientation
Marina Murillo-Pineda1, Marta Clemente-Ruiz1, Fernando Monje
Casas2, Flix Prado Velasco1
1
CABIMER-CSIC, Sevilla, ES, 2CABIMER-USE, Sevilla, ES
The structural organization of chromosomes is essential for their correct
function and dynamics during the cell cycle. The assembly of DNA into
chromatin provides the substrate for topoisomerases and condensin, which
introduce the different levels of superhelical torsion required for DNA
metabolism. In particular, Top2 and condensin are directly involved in
both the resolution of precatenanes that form during replication and the
formation of the intramolecular loop that detect tension at the centromeric
chromatin during chromosome biorientation. Here we show that histone
depletion activates the spindle assembly checkpoint (SAC) and impairs
sister chromatid decatenation, leading to chromosome missegregation and
lethality in the absence of the SAC. We demonstrate that histone depletion
impairs chromosome biorientation and activates the Aurora-dependent
pathway, which detects tension problems at the kinetochore. Interestingly,
SAC activation is suppressed by the absence of Top2 and Smc2, an
essential component of condensin. Indeed, smc2-8 suppresses catenanes
accumulation, mitotic arrest and growth defects induced by histone
depletion at semi-permissive temperature. Therefore, our results reveal the
importance of chromatin structure for the function of Top2 and condensin
during precatenanes resolution and centromere biorientation, two essential
processes for chromosome segregation.
P16-30
Cambio de accesibilidad de la subunidad

ribosomal 40S al IRES del virus de la hepatitis C
tras la unin del miR-122
Ascensin Ariza-Mateos, Jordi Gmez
Instituto de Parasitologa y Biomedicina Lpez - Neyra, CSIC
- CIBER de enfermedades hepticas y digestivas (CIBERehd),
Armilla, Granada, ES
El virus de la hepatitis C (HCV) es un virus de RNA de cadena sencilla
y polaridad positiva perteneciente a la familia Flaviviridae. El genoma
comprende una regin 5 no traducible que funciona como un sitio de
entrada interna del ribosoma (IRES), incluyendo el codn de inicio de la
traduccin AUG, esencial para la sntesis de protenas independiente de la
estructura 5-cap.
Se ha demostrado que la enzima RNasa P, que procesa los precursores de
los tRNA del hospedador, puede cortar in vitro el genoma del RNA de
HCV en el IRES cerca del triplete iniciador AUG, indicando la presencia
de una estructura tipo-tRNA en el IRES. Otros grupos y nosotros hemos
demostrado previamente que el IRES de HCV reside en la regin genmica
1-570 en una conformacin cerrada que es capaz de cambiar a una
conformacin abierta por la unin de molculas del microRNA-122 en su
flanco 5.
En este estudio hemos probado cuantitativamente la capacidad del miR122 para interaccionar con el RNA-HCV tanto en la forma cerrada como
en la abierta, determinando dos nuevos sitios de unin en tandem para el
miR-122 en el flanco 3 del IRES.
Granada 2014
Los efectos de unin del miR-122 en el flanco 3 del IRES fueron
determinados mediante RNasa especficas de simple y doble cadena (RNasa
A, T1 y V1) y agentes qumicos (DMS). Sin embargo, la accesibilidad a
la estructura tipo-tRNA por las RNasa que reconocen especficamente
este regiones tipo-tRNA (RNasas P y Z) fue afectada, al igual que la
accesibilidad por la RNasa H, indicando que la unin del miR-122 en el
flanco 3 del IRES provoca un cambio conformacional local en torno al
codn de inicio de traduccin, que podra afectar directamente a la unin
de las subunidades ribosomales. Mediante ensayo de unin de la subunidad
ribosomal 40S en presencia de miR-122, se pudo concluir que el miR-122
se une al RNA-HCV en ambos flancos del IRES-HCV y que esto podra
ser un proceso sincrnico que induce un cambio conformacional global de
la estructura del IRES afectando directamente a la unin de la subunidad
ribosomal 40S.
P16-31
The inhibitory state of the T-cell receptor alpha

enhancer in T lymphocytes is not reverted upon
T cell helper differentiation of CD4+ lymphocytes
rsula Angulo Urrecho, Beatriz del Blanco, Jennifer Lpez Ros,
Cristina Hernndez-Munain
Armilla, ES
The T-cell receptor (Tcra) enhancer (E) is essential for Tcra locus
germline transcription and primary V-to-J recombination during
thymocyte development. We found that E is inhibited late during
thymocyte differentiation and in peripheral T cells, indicating that it is not
required to drive transcription of rearranged Tcra genes. E inactivation in
peripheral T lymphocytes resulted in the lost of the enhancer-dependent
chromatin modifications and in the disruption of functional long-range
enhancer-promoter interactions as a consequence of the downregulation of
E2A and GATA-3 in mature T cells. Since GATA-3 is essential for T helper
(Th) 2 differentiation and has a role in regulatory T cell (Treg) maintenance
and function, we hypothesized that E function might be re-activated
during CD4+ cell differentiation. We have differentiated T cells in vitro to
different Th subsets: Th1, Th2, Th17 and Treg to evaluate whether there is a
direct correlation between E activity and Gata3 and Tcfe2a transcription.
Our results indicate that the high induction of GATA-3 transcription
observed during Th2 differentiation is not sufficient to activate E activity
in peripheral CD4+ T cells and that other factors are clearly required for
enhancer function in these cells. Further experiments are currently being
performed to extend these analyses during CD8+ T lymphocyte activation.
P16-32
Regulacin epigentica del promotor PRDM1 en

mieloma mltiple
Raquel Romero Garca, Laura Gmez Jaramillo, Francisco Mora
Lpez, Antonio Campos Caro
Hospital Universitario Puerta del Mar, Cdiz, ES
Objetivo: El gen PRDM1 codifica para un factor de transcripcin clave
en el proceso de diferenciacin de linfocitos B a clulas plasmticas (CP).
Existen dos isoformas de PRDM1, denominadas PRDM1 y PRDM1,
que se originan a partir de dos promotores diferentes. La protena PRDM1
acta como represor de la transcripcin mientras que se desconoce la
funcin de la isoforma truncada PRDM1 aunque se ha observado que, en
las lneas celulares de mieloma estudiadas, su expresin est aumentada
con respecto a la de la protena normal. Por ello, hemos estudiado si el
estado de metilacin de su promotor est relacionado con la expresin en
mielomas.
Metodologa: Se han utilizado lneas celulares de mieloma que expresan
la isoforma PRDM1 (U266, NCI-H929) y otras lneas de linfocitos que
Psters
no la expresan (Daudi, Raji) as como poblaciones celulares de linfocitos
B y de CP normales y patolgicas derivadas de pacientes con mieloma.
Se modific el DNA genmico con bisulfito y se analiz el grado de
metilacin de las diferentes CpG por secuenciacin. Tambin se trataron
las clulas con 5-Aza-2-deoxycytidine, un inhibidor de la metilacin del
DNA, para analizar, mediante PCR a tiempo real, si haba cambios en los
niveles de transcripcin de PRDM1.
Resultados: Hemos analizado hasta 12 posiciones CpG presentes en el
promotor de PRDM1. Lo ms destacado es que la lnea celular U266
prcticamente no presenta metilacin en ninguna de las CpG, solo en las
posiciones CpG 7 y 8 en menos del 25% de los clones analizados. Sin
embargo en lneas celulares como Daudi y Raji, as como en linfocitos B y
CP normales, se observa un patrn diferente donde las posiciones 4, 5, 6, 9 y
12 estn preferentemente metiladas. Para evaluar si la expresin de PRDM1
tiene relacin directa con el estado de metilacin del promotor, el tratamiento
con 5-Aza-2-deoxycytidine mostr un aumento en los niveles de transcritos
de PRDM1 en cultivos de clulas Namalwa pero no con U266.
Conclusin: Estos datos indican que la metilacin de las CpG en el
promotor es, al menos, uno de los mecanismos por el cual se regula la
expresin de PRDM1.
P17. Regulacin metablica

P17-1
Profiling of promoter occupancy by SND1

coactivator in human hepatoma cells via
ChIP-chip analysis
Enara Arretxe, Begoa Ochoa, Sandra Armengol, Sarai Mula,
Yolanda Chico, Mara Jos Martnez
Department of Physiology, University of the Basque Country Medical
School, UPV/EHU, Leioa, ES
Staphylococcal nuclease domain-containing protein 1 (SND1), also
known as p100 coactivator or Tudor-SN, is an evolutionarily conserved
multifunctional protein implicated in a variety of cellular processes,
including adipogenesis, stress response, gene transcription, RNA
metabolism and cancer progression. Originally identified as a transcriptional
coactivator of EBNA-2, we now know that SND1 can modify the
expression of several genes by interacting with transcription factors
STAT5, STAT6, c-Myb and PPAR. However, the relevance of SND1 as
a transcriptional coactivator remains unresolved and no comprehensive
picture of potential SND1 target genes has yet been reported. Interestingly,
we found overexpression of SND1 in human hepatoma cells exposed to
TNF through NF-B binding to gene promoter, supporting a role for
SND1 in inflammation signalling pathways. To gain insight into the role of
SND1 in gene transcription regulation, we performed genome-wide search
for endogenous SND1 binding sites by Chromatin Immunoprecipitation
(ChIP)-chip analysis on HepG2 human hepatoma cells left untreated and
upon TNF stimulation. We found a set of 645 target genes in basal cells
and 822 in cytokine-treated cells, of which 281 genes were exclusively
bound in the treated group. Transcription factor binding site analysis of
target genes at CEAS server revealed enrichment of motifs for established
partners and novel transcription factors involved in stress response (i.e.
HSF and ATF), viral infection (i.e. STAT1 and STAT3), cell proliferation
(i.e. MEIS1/AHOXA9, E2F, E2F1 and PAX) and metabolic regulation
(i.e. p300 and CREB). Gene Ontology classification of SND1 target genes
by DAVID showed enriched ontologies such as regulation of information
molecules, metabolism, organ morphogenesis and central nervous system
development. Major differences of TNF-treated vs untreated human
hepatoma cells were a few ontologies involving phospholipid metabolism,
heat shock protein binding and the TGF- signaling pathway.
Supported by Basque Government grants IT-336/10 and S-PE13UN139.
149
Psters
P17r-2
P17m-4
La sensibilidad del metabolismo de poliaminas a

la privacin de glucosa es mayor en clulas
de neuroblastoma con amplificacin de n-myc
Es la resistencia a la insulina dependiente

de la remodelacin del tejido adiposo?
M Victoria Ruiz Prez, Jos Luis Urdiales, Francisca Snchez

Jimnez, Miguel ngel Medina
Departamento de Biologa Molecular y Bioqumica, Universidad de
Mlaga, Mlaga, ES
El oncogn n-myc es capaz de potenciar la sntesis de poliaminas, molculas
esenciales para la proliferacin celular cuyos niveles estn elevados en muchos
tumores. El neuroblastoma, el tumor slido extracraneal ms frecuente
en nios, muestra amplificacin de n-myc en el 25% de los casos, y dicha
amplificacin est asociada a mala prognosis y escaso xito del tratamiento.
En este trabajo, hemos evaluado la relevancia de la amplificacin de n-myc
sobre varias caractersticas metablicas de clulas de neuroblastoma humano,
y hemos analizado los efectos de la inhibicin de la gluclisis sobre el
metabolismo de poliaminas en dichas clulas. Los resultados de este trabajo
muestran una relacin previamente desconocida entre la gluclisis y los
niveles de poliaminas: la inhibicin de la gluclisis desencadena eventos de
sealizacin que llevan a una disminucin de los niveles proteicos de N-Myc
y a un descenso de la expresin de la enzima ornitina descarboxilasa y de los
niveles de poliaminas, todo ello acompaado por un bloqueo del ciclo celular.
Esta relacin entre la privacin de glucosa y la deficiencia de la sntesis de
poliaminas, y su aparente relacin con la amplificacin de n-myc, podra ser
explotada para el desarrollo de nuevas terapias antitumorales contra tumores
que presenten amplificado este oncogn.
P17m-3 (R17-5)
Modulacin del metabolismo de los cuerpos

lipdicos hepticos de ratn en la endotoxemia
Lino Arisqueta Herranz, Hiart Navarro-Imaz, Yuri Rueda Estvez,
Olatz Fresnedo Aranguren
Departamento de Fisiologa, UPV/EHU, Leioa, ES
Durante la respuesta de fase aguda frente a patgenos se producen
cambios especficos en el metabolismo de los lpidos que conducen a la
denominada lipemia de la sepsis. Esta adaptacin se considera parte de
la respuesta inmune innata ya que favorece la eliminacin del torrente
sanguneo de endotoxinas como el lipopolisacrido (LPS). La lipemia se
origina, entre otros, como consecuencia de adaptaciones hepticas que
afectan al ensamblaje de VLDL, un proceso complejo en el que intervienen
los cuerpos lipdicos (CL). Este estudio se propuso con el fin de analizar
en qu medida el metabolismo de los CL hepticos est implicado en la
respuesta inmune innata. Para ello se administr LPS a ratones salvajes y
ratones deficientes en leptina (ob/ob), cepa que presenta esteatosis heptica
(acumulacin de CL) y mayor sensibilidad a la endotoxemia. Uno de los
efectos ms destacables del tratamiento con LPS es una menor acumulacin
de CL hepticos. El segundo es la reduccin en el contenido de colesterol
esterificado (CE) de estos CL. La cuantificacin de la actividad de
enzimas implicados en el metabolismo de lpidos neutros as como de la
expresin de protenas relacionadas condujo a proponer dos mecanismos
para explicar estas adaptaciones: uno dependiente de la modulacin de
actividades enzimticas y otro dependiente de la movilizacin de lpidos.
Estudios con cultivos primarios de hepatocitos confirman que las citoquinas
sobresecretadas tras el tratamiento con LPS participan en la regulacin
diferencial de las actividades triglicrido hidrolasa y diglicrido hidrolasa
hepticas, lo que probablemente conduce a una canalizacin de sustratos
hacia la biognesis de VLDL. Este mecanismo conducira a la menor
acumulacin de CL en el hgado. Por otro lado, el menor contenido de CE
de los CL se debe probablemente a una mayor secrecin de colesterol al
sinusoide mediada por sobreactivacin del receptor nuclear LXR. En los
ratones ob/ob estos mecanismos se encuentran atenuados, lo que podra
condicionar una respuesta innata deficiente.
Subvencionado por Gobierno Vasco (IT-336-10) y UPV/EHU (UFI11/20).
150
Martn Alcal Daz-Mor1, Julio Sevillano Fernndez1, Jimena Pita

Santibaez1, Isabel Snchez-Vera Gmez-Trelles2, Mara del Pilar
Ramos lvarez1, Marta Viana Arribas1
1
Departamento de Qumica y Bioqumica, Facultad de Farmacia,
Universidad San Pablo CEU, Madrid, ES, 2Departamento de
Ciencias Mdicas Bsicas, Facultad de Medicina, Universidad San
Pablo CEU, Madrid, ES
Introduccin: La capacidad de expansin del tejido adiposo ante la
acumulacin de grasa en la obesidad es clave en el desarrollo de patologas
asociadas. Sin embargo, se desconoce la cronologa en la aparicin de los
cambios estructurales del tejido adiposo, as como la implicacin de cada
uno en la prdida de funcionalidad.
Objetivos: Determinar el papel de la remodelacin de la matriz extracelular
y la infiltracin de macrfagos en el estroma vascular sobre la resistencia
a la insulina (RI) en el tejido adiposo visceral (TAV) de ratones obesos.
Mtodos: Ratones CBL57/6J de 3 semanas de edad fueron divididos
en 2 grupos: un control (C), alimentado con dieta estndar y un grupo
alimentado con dieta rica en grasa (O), durante 14 y 28 semanas. Se
analizaron parmetros metablicos, protenas de la cascada de sealizacin
de la insulina en TAV y se analiz su estructura por inmunohistoqumica.
Resultados: Se observ RI en el grupo O desde la semana 14. A pesar de
que ya exista una infiltracin de macrfagos inducida por HIF-1, todava
no se vieron cambios ni en el acmulo de colgeno ni en la sealizacin de
la insulina en el TAV. Sin embargo, en la semana 28, la RI en los animales O
se caracteriz por la disminucin en el receptor de insulina y el aumento en la
fosforilacin de p38 en el TAV. A nivel estructural, adems de la infiltracin de
macrfagos, se observ un marcado acmulo de colgeno total, tipo I y tipo III.
Conclusiones: Durante el desarrollo de obesidad, la llegada de macrfagos
parece ser uno de los primeros cambios en el TAV, aunque no es
suficiente para bloquear all la sealizacin de la insulina. Esta prdida
de funcionalidad del tejido se ve aumentada por los cambios estructurales
posteriores, principalmente mediados por el acmulo de colgeno.
P17-5
The lack of GlnB and YejB compensates

the detrimental effect of spoT deletion in E. coli
cells expressing wild type relA
Manuel Montero1, Goizeder Almagro1, Alejandro Viale1, Angel Sevilla2,
Manuel Cnovas2, Cristina Bernal2, Ana Beln Lozano2, Francisco
Jos Muoz1, Edurne Baroja-Fernndez1, Javier Pozueta-Romero1
1
Mutilva, Navarra, ES, 2Departamento de Bioqumica y Biologa
Molecular e Inmunologa, Facultad de Qumica, Campus de
Excelencia Internacional Regional Campus Mare Nostrum,
Universidad de Murcia, Murcia, ES
In Escherichia coli, intracellular ppGpp content in response to nutritional
deficiency is controlled by the balanced action of RelA (synthetase I) and
the dual-function (synthetase/hydrolase) SpoT. Generally ascribed to the
detrimental effects of high (p)ppGpp levels, the co-existence of spoT null
(DspoT) with relA proficient alleles has been considered synthetically lethal.
However, in a recent work we have reported the construction of DspoT mutants
in a relA+background accumulating nearly wild type (WT) ppGpp levels when
cells were cultured on a rich complex medium. Sequencing of the genomes
from various selected DspoT clones identified in all of them suppressor
mutations located in relA. In two of them, additional inactivating mutations
were found in glnB and yejB, two genes that have not been characterized as
genetically linked to relA. To know whether mutations resulting in the total
abolishment of the glnB and yejB genes could compensate the detrimental
effects of spoT deletions in E. coli in this work we obtained multiple independent
DspoTDglnB and DspoTDyejB clones. Importantly, these clones expressed
WT RelA, displayed a nearly WT growth phenotype, and accumulated nearly
Granada 2014
WT ppGpp and glycogen contents when cultured in a rich complex medium.
None of these mutants, however, could grow on glucose minimal medium. The
overall data (a) show that different mutations other than in relA can be selected
in E. coli cells compensating the detrimental effects of spoT deletion, and (b)
point to the occurrence in E. coli of mechanism(s), other than SpoT-mediated
ppGpp hydrolytic breakdown, that prevent ppGpp over-accumulation. To our
knowledge this is the first report describing the production of spoT null mutants
of E. coli expressing WT RelA.
P17-6 (R17-6)
The development of insulin resistance can be

regulated by the levels of G-protein coupled
Receptor kinase 2 in myeloid cells
Roco Vila-Bedmar1, Elisa Lucas1, Marta Cruces-Sande1, Hanneke
Willemen2, Annemieke Kavelaars3, Cobi Heijnen3, Cristina Murga1,
Federico Mayor Jr.1
1
Centro de Biologa Molecular Severo Ochoa (UAM-CSIC),
Universidad Autnoma de Madrid - Instituto de investigacin
Sanitaria La princesa, Madrid, ES, 2Laboratory of Neuroimmunology
and Developmental Origins of Disease (NIDOD), University Medical
Center Utrecht, Utrecht, NL, 3Department of Symptom Research,
Division of Internal Medicine, The University of Texas MD Anderson
Cancer Center, Houston, Texas, US
Introduction: G protein-coupled receptor kinase 2 (GRK2) is a kinase
classically involved in the modulation of the signalling mediated by
many G protein-coupled receptors (GPCR), but it has also been recently
described to contribute to the development of insulin resistance (IR) and
fat mass accretion in vivo. Accordingly, our previously published studies
have described that lowering GRK2 levels can confer protection against
the development of IR in different mice models of this condition, and also
that this kinase plays an important role in the regulation of obesity and
energy expenditure. In this regard, obesity, which is currently considered
as a chronic low-level inflammatory disease, is a risk factor for the
development of IR. Particularly, macrophages that infiltrate adipose tissue
during obesity have been recognized to have a key contribution to this
metabolic disorder. In this work, we evaluate the potential effect of changes
in the levels of GRK2 specifically in macrophages to the insulin resistant
and obese phenotype observed after a high fat diet (HFD) in mice.
Materials and Methods: The specific contribution of GRK2 in
macrophages/myeloid cells in the context of IR and obesity has been
assessed in a mice model with a specific partial deletion of GRK2 in
myeloid cells, including microglia/macrophages/granulocytes (LysMGRK2+/-). These mice were fed a HFD and insulin and glucose tolerance
as well as insulin signalling in different tissues were explored.
Results: Our study showed that LysM-GRK2+/- mice are partially protected
against diet induced obesity (DIO), with no differences in food intake, and
display lower fasting plasma glucose levels after high fat feeding. Moreover,
HFD-fed LysM-GRK2+/- mice show improved glucose tolerance and a more
potent activation of insulin signals in different insulin target tissues, and are
protected against the development of non alcoholic fatty liver disease (NAFLD).
Conclusion: Our data establish that changes in GRK2 levels specifically in
myeloid cells can regulate the development of diet-induced IR.
P17r-7 (R17-8)
Hacking the master transcriptional program of

prostate cancer metabolism
Lorea Valcrcel1, Veronica Torrano1, Ana Rosa Cortazar1, Sonia
Fernndez Ruiz1, Patricia Zuiga Garca1, Mar Lorente2, Alfredo Caro
Maldonado1, Natalia Martn Martn1, Amaia Zabala1, Pere Puigserver3,
Brett Carver4, Paolo Pinton5, Guillermo Velasco2, Ana Maria Aransay6,
Arkaitz Carracedo7
1
CIC bioGUNE, Derio, ES, 2Biochemistry and Molecular Biology
Department, School of Biology, Complutense University, Madrid,
Psters
ES, 3Department of Cancer Biology, Dana-Farber Cancer Institute,
Harvard Medical School, Boston, MA, US, 4Surgery, Memorial
Sloan-Kettering Cancer Center, New York, US, 5Department of
Morphology, Surgery and Experimental Medicine Section of General
Pathology, University of Ferrara, Ferrara, IT, 6CIC bioGUNE, Centro
de Investigacin Biomdica en Red de Enfermedades Hepticas
y Digestivas (Ciberehd), Derio, ES, 7CIC bioGUNE, Ikerbasque,
Basque foundation for science; Biochemistry and Molecular Biology
Department, University of the Basque Country (UPV/EHU), Derio, ES
Prostate cancer (PCa) is the third most common tumor found and the
third leading cause of cancer death in men in Europe. The main driver of
PCa is the PI3K pathway, which regulates cell proliferation, survival and
importantly, metabolism. The availability of a mouse model that is faithful
to the human disease is an invaluable tool for the study of this type of
cancer. While much is known about the signaling requirements of PCa, the
metabolic needs of this tumor remain vastly obscure.
We have previously demonstrated that in order to meet their energetic
demands, cancer cells reprogram their metabolism through alterations in
the transcriptional landscape.
We have approached the study of cancer metabolism starting from a
systematic analysis of transcriptional regulators that potentially contribute
to the metabolic switch. To define these factors, we have applied metaanalysis constrains that ensure the selection of relevant candidates,
approach that has been shown to be valid to identify novel metabolic cues.
We have focused on metabolic transcriptional regulators that i) are altered
in a significant proportion of publicly available databases, and ii) that show
association with disease-free survival and metastatic disease.
This approach allowed us to unveil the tumor suppressive potential of
the transcriptional co-activator PGC1A. Comparative analysis of PGC1A
expression in melanoma, where it has been shown to be up-regulated, has
led us to define the appropriate expression level for gene re-expression in
prostate cancer. PGC1A re-expression leads to a tumor-suppressive reverse
Warburg effect in vitro and in vivo, reflected as an increase in mitochondrial
oxidative metabolism and decrease in lactate production as a consequence
of the transcriptional regulation. Our data suggest that PGC1A is at the
top of prostate cancer metabolic reprogramming and that this event is of
importance for the development of metabolic therapeutic strategies.
P17-8
La osteopontina provoca cambios en el

metabolismo lipdico y biliar en hgado de ratn
Maitane Nez, Beatriz Gmez-Santos, Olatz Fresnedo, Patricia
Aspichueta
University of the Basque Country (UPV/EHU), Leioa, ES
La osteopontina (OPN) participa en numerosos procesos fisiolgicos y
patolgicos. Diferentes estudios han relacionado el aumento de OPN con
la aparicin de hepatocarcinoma pero se desconoce si este aumento juega
algn papel importante en el metabolismo heptico y en la reprogramacin
metablica, caracterstica de este tipo de enfermedades. El objetivo
principal de este trabajo fue estudiar si el aumento de OPN provoca cambios
en el metabolismo lipdico y biliar en ratn. Para ello, se administr OPN
recombinante (rOPN) va intravenosa en dos das alternos. Se determinaron
parmetros corporales, niveles de lpidos y expresin de genes relevantes.
Los resultados muestran que la administracin de rOPN provoca un
aumento de la masa heptica sin cambios de la corporal. Estos cambios estn
asociados a una remodelacin lipdica, con un aumento en el contenido en
colesterol libre (CL) y sin modificaciones en el colesterol esterificado (CE),
diglicrido (DG) y triglicrido (TG). Paralelamente ocurre un descenso
en la expresin de genes implicados en la sntesis de CE, el transporte
de CL y en el metabolismo de lipoprotenas. La administracin de rOPN
induce cambios en el metabolismo de glicerofosfolpidos, aumentando el
contenido heptico de fosfatidilcolina (PC) y fosfatidiletanolamina (PE) y
disminuyendo la expresin de genes relacionados con las vas de sntesis
de PC. Estas modificacines del metabolismo de CL y PC pueden estar
directamente relacionadas con un descenso en la expresin de genes
151
Psters
implicados en el transporte de sales biliares y en la regulacin de la
sntesis de cidos biliares. En conclusin, la exposicin a OPN produce
una remodelacin del metabolismo lipdico y biliar en el hgado de ratn.
Subvencionado por GV (IT-336-10) y UPV/EHU (UFI11/20).
P17-9
Adaptation of T lymphocyte effector function to

metabolic stress
Sonia Tejedor Vaquero, Cristina Lpez-Rodrguez, Jos Aramburu
Beltrn
Departament de Cincies Experimentals i de la Salut, Universitat
Pompeu Fabra, Barcelona, ES
In the last years it has been reported that nutrients and metabolism play
essential roles in T lymphocyte activation and differentiation. T lymphocytes
exhibit specific metabolic features depending on their activation and
polarization stage, being this crucial for their role in adaptive immune
responses. Glucose is necessary for T lymphocyte expansion and polarization
towards effector Th cells upon antigen stimulation, since it is both a main
fuel for ATP production and a source of building blocks for macromolecule
biosynthesis. T lymphocytes can encounter substantial variations in glucose
concentration at inflammation sites and tumors, which raises the question of
how glucose fluctuations affect their capacity to maintain ATP and appropriate
responses to cytokines. We have explored the response of T lymphocytes to
glucose deprivation during cytokine-driven polarization. Our results show
that T lymphocytes can resist substantial glucose restriction and maintain
ATP levels by arresting their proliferation. Results indicate that expression
of different cytokines is differentially sensitive to glucose deprivation, and
we are currently analyzing the contribution of various energy-regulatory
pathways to cytokine expression. Our results suggest that local nutrient
conditions during T lymphocyte activation can have a significant impact in
their function over polarizing pressure from cytokines.
Funded by the Spanish Government (SAF2009-08066, SAF2012-36535
to CL-R; SAF2011-24268 to JA), Fundaci la Marat TV3 (080730,
122530, CL-R and JA) and Generalitat de Catalunya (2009 SGR601,
2014 SGR1153). STV is supported by a predoctoral fellowship BES-2013062670.
P17m-10
Alterations of cellular metabolism of podocytes in

a lipotoxic context
Adriana Izquierdo Lahuerta1, Cristina Martnez-Garca1, T-K Yeo2, Yurena
Vivas1, Patricia Corrales1, Sheldon Chen2, Gema Medina-Gmez1
1
Universidad Rey Juan Carlos, Madrid, ES, 2Division of Nephrology/
Hypertension, Northwestern University, Chicago, US
In last decades there has been a rapid change in life style that has
led to an alarming increase in the prevalence of obesity and obesityassociated complications. Obese patients are at increased risk of
developing hypertension, heart disease, insulin resistance, dyslipidemia,
type 2 diabetes and renal disease. The excess of calories are stored as
triglycerides in adipose tissue, and also accumulate ectopically in other
organs, including kidney, which contributes to the damage through a toxic
process named lipotoxicity. Recently, evidences suggest that renal lipidic
accumulation leads to glomerular damage and more specifically, whether
this accumulation produces podocyte dysfunction. The aim of this study
was to analyze the mechanisms underlying the process of lipotoxicity in
podocytes, key cells in glomerular filtration barrier maintenance. We have
used conditional immortalized cultured mouse podocytes treated with
different doses (100, 500 and 750 M) of palmitic acid (PA) and oleic acid
(OA), for 24h. PA treatment produced an intracellular accumulation of lipid
droplets and abnormal glucose and lipid metabolism. Such accumulation
led to an inflammation associated to an increased phosphorylation at Ser
152

307 of the IRS-1. Also, a lack of response to Akt phosphorylation via
mTOR in the presence of insulin was observed. Furthermore, this fatty
acid produced oxidative stress and endoplasmic reticulum stress as well as
rearrangements of the actin cytoskeleton. These effects were not observed
with treatment OA. It suggests that saturated fatty acids promote insulin
resistance and disturb the podocyte metabolism, playing an important role
in the renal dysfunction in the context of lipotoxicity.
Acknowledgements: Fundacin de la SEEN, MINECO (BFU2012- 33594),
CAM (S2010/BMD-2423) y Ayudas a la Movilidad 2012 URJC.
P17r-11
Mitochondrial ATP governs neuronal response to

NMDA, and is maintained by calcium activation
of ATP-Mg/Pi carrier, SCaMC-3
Carlos Rueda, Javier Traba, Ignacio Amigo, Irene Llorente-Folch,
Beatriz Pardo, Araceli del Arco, Jorgina Satrustegui
Departamento de Biologa Molecular, Centro de Biologa Molecular
Severo Ochoa, Consejo Superior de Investigaciones Cientficas-CSICUAM, Universidad Autnoma de Madrid - Centro de Investigacin
Biomdica en Red de Enfermedades RarasCIBERER - Instituto de
Investigacin Sanitaria Fundacin Jimnez Daz-IIS-FJD, Madrid, ES
Glutamate excitotoxicity is caused by sustained activation of neuronal
NMDA receptors causing elevations in cytosolic Ca2+ and Na+, activation
of PARP-1, fall in cytosolic NAD+ and ATP, and delayed Ca2+deregulation
(DCD) followed by neuronal death. Mitochondria undergo early changes in
membrane potential during excitotoxicity but their relation with these events
is still controversial. SCaMC-3/Slc25a23 is a mitochondrial ATP-Mg/Pi
carrier which transports ATP or ADP in a strictly Ca2+-dependent way and
is a candidate to play a role in the initial mitochondrial response to NMDA.
We have studied the early responses to NMDA in cortical neurons including
a rapid increase in oxygen consumption rate (OCR) which was found to be
due to Ca2+-dependent upregulation of respiration. NMDA exposure resulted
in a rapid fall in mitochondrial ATP ([ATP]mit) in SCaMC-3 KO neurons, but
not in control neurons, in which Ca2+-dependent adenine nucleotide uptake
through the carrier maintained [ATP]mit. The fall in [ATP]mit in the KO neurons
was associated with a blunted increase in respiration and a further decrease
in cytosolic ATP levels, indicating that maintenance of [ATP]mit was required
to upregulate OXPHOS upon NMDA exposure. The rapid fall in [ATP]
and blunted respiratory response were prevented by PARP-1 inhibitors,
mit
indicating a rapid NMDA-induced PARP-1 activation as cause. A second
consequence of the lack of SCaMC-3 was an early appearance of DCD. This
was associated with reduced Ca2+ retention capacity (CRC) and increased Ca2+dependent swelling in mitochondria from SCaMC-3 KO mice incubated with
physiological millimolar concentrations of ADP or Mg-ATP, suggesting that
failure to maintain matrix AdN is responsible for both the impaired CRC in
mitochondria and earlier neuronal DCD. SCaMC-3 KO neurons were more
vulnerable to glutamate excitotoxicity in vitro and SCaMC-3 KO mice were
more susceptible to kainate-induced seizures, showing that SCaMC-3 is an
early player in excitotoxic cascade both in vitro and in vivo.
P17r-12
G protein-coupled receptor kinase 2 regulates:

The development of non-alcoholic fatty liver disease
M Cruces Sande1, R Vila Bedmar1, E Lucas1, HL Willemen2,
CJ Heijnen3, A Kavelaars3, Gonzlez-Rodrguez4, M Valverde4,
F Mayor Jr1, C Murga1
1
Centro de Biologa Molecular Severo Ochoa UAM-CSIC,
Universidad Autnoma de Madrid - Instituto de Investigacin
Sanitaria La Princesa, Madrid, ES, 2Laboratory of Neuroimmunology
and Developmental Origins of Disease (NIDOD), University Medical
Center Utrecht, Utrecht, NL, 3Department of Symptom Research,
Division of Internal Medicine, The University of Texas MD Anderson
Granada 2014
Cancer Center, Houston, Texas, US, 4Centro de Investigacin
Biomdica en Red de Diabetes y Enfermedades Metablicas
Asociadas (CIBERDEM), ISCIII, Barcelona - Instituto de
Investigaciones Biomdicas `Alberto Sols CSIC-UAM, Madrid, ES
Background: Insulin resistance (IR) and obesity are major health problems
and important risk factors for the development of non-alcoholic fatty liver
disease (NAFLD), a disease spectrum that includes hepatic steatosis,
non-alcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. G proteincoupled receptor kinase 2 (GRK2), first identified as a G protein-coupled
receptor regulator, has been recently described to play a relevant role in
IR and obesity in vivo. GRK2 hemizygous (GRK2+/-) mice are protected
against high fat diet (HFD)-induced systemic and hepatic insulin resistance
and obesity. However, the effect of GRK2 in the development of HFDinduced NAFLD has not been studied.
Materials and methods: Given the lack of a potent and specific GRK2
inhibitor, we used a global tamoxifen (Tx)-inducible murine model
in which GRK2 levels are decreased once HFD-induced IR has been
established. Also, a long term chronic state of IR and obesity was triggered
by feeding wild type (WT) and GRK2+/- mice a 32 week-long HFD. To
discriminate the effects of the differential body weight gain, we fed mice
with a methionine-choline deficient diet (MCD), which induces NAFLD
independently of fat mass accretion.
Results: Systemic insulin sensitivity was preserved after decreasing GRK2
levels in the middle of a high fat feeding using tamoxifen, and accordingly
enhanced insulin signaling was observed in the liver of HFD-fed Tx-induced
GRK2-/- mice. Moreover, hepatic steatosis, a hallmark of NAFLD, was
decreased in these mice. Also, different signs of inflammation present in WT
mice were absent in Tx-induced GRK2-/- animals. Nevertheless, after a long
term HFD we found no differences in steatosis between WT and GRK2+/mice, despite the decrease in body weight gain and liver weight. Finally, we
found an increase in GRK2 protein levels in the liver of mice fed a MCD,
a well established model of NASH, similar to what is detected during HFD
feeding in WT animals, and we are further characterizing this model.
Conclusion: Taken together, these results suggest a role for GRK2 in the
establishment and development of NAFLD.
P17r-13
Obesity related cardiac hypertrophy is modulated

by G protein-coupled receptor kinase 2
Elisa Lucas1, Mara Jurado_Pueyo1, Roco Vila-Bedmar1, Juan J.
Lazcano2, Javier Gmez-Ambrosi3, Gema Frhbeck3, Javier Dez4,
Cristina Murga1, Federico Mayor Jr.1
1
Departamento de Biologa Molecular y Centro de Biologa Molecular
Severo Ochoa (UAM-CSIC) - Instituto de Investigacin Sanitaria
La Princesa, Madrid, ES, 2Centro Nacional de Investigaciones
Cardiovasculares (CNIC), Madrid, ES, 3Metabolic Research
Laboratory, Universidad de Navarra, CIBERobn, Pamplona, ES,
4
Division of Cardiovascular Sciences, Centre for Applied Medical
Research (CIMA) and Department of Cardiology and Cardiovascular
Surgery, University Clinic, University of Navarra, Pamplona, ES
The heart is a constitutive energy-demanding organ. Although
mitochondrial lipid oxidation is the principal energy source in the healthy
heart, the maintenance of glucose utilization is necessary for normal
cardiac function. Nevertheless, alterations in cardiac energy metabolism
downstream neurohormonal stimulation play a crucial role in the
pathogenesis of heart failure (HF). One biomarker molecule upregulated
in human and several animal models of HF is G protein-coupled receptor
kinase 2 (GRK2), a kinase originally discovered to regulate G proteincoupled receptor desensitization. Previous studies have described the
potential role of GRK2 as a direct modulator of insulin signaling in
several tissues in addition to its classical role in cardiac AR signaling
regulation which can also have an indirect effect on insulin signaling.
Since we have observed that GRK2 levels are increased in the hearts
of high fat diet (HFD)-fed animals and also in ob/ob mice, we aimed
to explore the effect of lowering GRK2 levels during HFD feeding in
Psters
order to study whether GRK2 could be a good target to ameliorate the
detrimental consequences of HFD in the heart. Given the lack of selective
GRK2 pharmacological inhibitors, we have used GRK2 hemizygous
mice (GRK2+/-) as a model to assess the effects of a sustained systemic
inhibition of GRK2 on cardiac tissue. We find that, after 12 weeks of
HFD, insulin-dependent cardiac responses are impaired in young
wild type (WT) while preserved in GRK2+/- mice. Besides, adult 10
month-old WT mice fed a HFD for 32 weeks presented cardiomyocyte
hypertrophy and pathological cardiomegaly, while in HFD-fed GRK2+/animals heart size was indistinguishable from that of chow-fed mice.
In addition, we detected that cardiac tissue of GRK2+/- adult mice fed
with standard diet shows enhanced cardiac insulin sensitivity compared
with aged-matched WT hearts, and displays features of non-pathological
mild cardiac hypertrophy. These results highlight the importance of
maintaining GRK2 levels under a certain physiological level to preserve
insulin-mediated cardioprotection.
P17-14
Increased expression of carnitine

palmitoyltransferase 1A in rat ventromedial
hypothalamus produces hyperphagia, overweight
and alters the hyphothalamic lipidomic profile
Joan Francesc Mir1, Paula Mera1, Gemma Fabrias2, Fina Casas2, Sofia
Costa2, Minia Weber1, Raquel Fucho1, Mara Caldern-Domnguez1,
Harald Petri3, Nria Casals4, Laura Herrero1, Dolors Serra1
1
Department of Biochemistry, Facultat de Farmcia, Universitat de
Barcelona, Institut de Biomedicina de la Universitat de Barcelona
(IBUB) and CIBER Fisiopatologa de la Obesidad y la Nutricin
(CIBEROBN), Instituto Carlos III, Barcelona, ES, 2Department
of Biomedicinal Chemistry, Institute of Advanced Chemistry of
Catalonia (IQAC)/CSIC, Barcelona, ES, 3UniQure, Amsterdam, NL,
4
Universitat Internacional de Catalunya, Sant Cugat del Valls, ES
Lipid metabolism in hypothalamic neurons is a key pathway in food intake
modulation and glucose homeostasis. Carnitine palmitoyltransferase
(CPT) 1A is the rate-limiting enzyme in mitochondrial fatty acid oxidation
and it has recently been proposed as a crucial mediator of fasting and
ghrelin orexigenic signaling. However, the relationship between changes in
CPT1A activity and the intracellular downstream effectors that contribute
to appetite modulation in hypothalamus is not fully understood.
The aim of our study is to analyze the peripheral and central effect of
an increased CPT1A expression in the ventromedial nucleus of the
hypothalamus (VMH).
We used adeno-associated virus (AAV) to express a permanently active
form of CPT1A (malonyl-CoA-insensitive CPT1A, here CPT1AM) in rat
VMH. We have observed that permanent activation of CPT1A in VMH led
to hyperphagia, overweight, hyperglycemia and the development of insulin
resistance. These effects seem to be triggered by changes in the expression
of neurotransmitter transporters and hypothalamic changes in structural
and bioactive complex lipids such as ceramides and phospholipids.These
data highlight the role of ventromedial CPT1A in the modulation of food
intake and consequently glucose homeostasis.
P17-15
Modulacin de la composicin lipdica

de cuerpos lipdicos en hgado de ratn. Efecto
de una dieta rica en grasa
Hiart Navarro Imaz, Ibone Labiano Ciriza, Yuri Rueda Estvez, Lino
Arisqueta Herranz, Olatz Fresnedo Aranguren
Departamento de Fisiologa Universidad del pais vasco/ Euskal
Herriko Unibersitatea (UPV/EHU), Leioa, ES
Los cuerpos lipdicos (CL) citoplasmticos son estructuras especializadas
en el almacenaje y movilizacin de lpidos que se acumulan en una
153
Psters
amplia diversidad de situaciones fisiolgicas y patolgicas. La dieta y los
condicionantes metablicos afectan a los niveles hepticos de CL. Se han
realizado diversos estudios protemicos con la finalidad de establecer en
qu modo se regula la composicin de este orgnulo pero se desconocen
las adaptaciones cuantitativas en el lipidoma. En este estudio describimos
cmo afecta una dieta rica en grasa a la composicin de los CL hepticos
en ratones salvajes y deficientes en leptina (ob/ob), que presentan esteatosis
heptica por hiperfagia. Es destacable la similitud en la composicin lipdica
(triglicridos, diglicridos, colesterol libre y esterificado, fosfatidilcolina
y fosfatidiletanolamina) entre las dos cepas analizadas alimentadas con
una dieta control. La caracterstica ms remarcable de los CL aislados
de hgado de animales tratados con dieta rica en grasa es la disminucin
en el contenido de colesterol esterificado (CE). Esta disminucin es
muy marcada en los ratones ob/ob y va acompaada de modificaciones
relevantes en los parmetros bioqumicos plasmticos e ndice heptico. El
conjunto de resultados indica que la mejora de la funcin heptica de los
ratones obesos alimentados con la dieta rica en grasa podra estar vinculada
a una mejora en la gestin del colesterol acumulado en los CL hepticos.
El incremento en el transporte reverso de colesterol podra jugar un papel
central en esta adaptacin.
Subvencionado por Gobierno Vasco (IT-336-10) y UPV/EHU (UFI11/20).
P17m-16 (R17-7)
Alteraciones en la va tumoral Wnt/-catenina

en linfocitos circulantes inducidas por
metabolitos relacionados con la diabetes
Valle Montalvo-Romeral1, Mara Gutirrez-Salmern1, Ana ChocarroCalvo2, Jos Manuel Garca-Martnez1, Antonio De La Vieja3,
Custodia Garca-Jimnez1
1
Universidad Rey Juan Carlos, Alcorcn, ES, 2Ludwig Institute for
Cancer Research - University of Oxford, Oxford, UK, 3Instituto de
Salud Carlos III, Majadahonda (Madrid), ES
Introduccin: La poblacin diabtica presenta un riesgo incrementado de
desarrollar ciertos cnceres tejido-especficos. Los cnceres asociados a
diabetes suelen presentar alteraciones en la va Wnt/-catenina. Nuestro
grupo ha demostrado que la acumulacin nuclear de -catenina, efector de la
sealizacin tumoral por Wnt, slo se produce en presencia de alta glucosa
(hiperglucemia). Hipotetizamos que otros metabolitos caractersticos
de la diabetes pueden inducir alteraciones en protenas relacionadas con
la va Wnt/-catenina cooperando con la alta glucosa para favorecer la
aparicin del fenotipo tumoral. Los linfocitos circulantes expresan los
componentes de la va Wnt y en pacientes diabticos estn expuestos a
dichos metabolitos, por tanto planteamos la posibilidad de encontrar en
ellos, marcadores tempranos de riesgo de cncer asociado a diabetes.
Objetivo: Identificar alteraciones moleculares en la va Wnt/-catenina en
linfocitos circulantes humanos producidas por el ambiente metablico de
la diabetes.
Metodologa: Se usan linfocitos humanos procedentes de voluntarios
sanos y la lnea tumoral inmortalizada de linfocitos Jurkat. Se compara la
respuesta de los linfocitos sanos y los tumorales a estmulos metablicos
caractersticos de la diabetes. Los resultados se analizan por western-blot,
qPCR y citometra.
Resultados: Se observan cambios en los componentes de la sealizacin
tumoral Wnt/ -catenina a nivel de expresin gnica (p300), niveles
protecos (-catenina) y funcionalidad (exhibicin de receptor LRP6)
en linfocitos humanos sanos que difieren de la respuesta observada en
linfocitos tumorales (Jurkat).
Conclusiones: Es posible detectar cambios a distintos niveles de la va
Wnt/-catenina en linfocitos humanos expuestos al ambiente metablico
de la diabetes. Nuestros resultados sugieren la posibilidad de usar linfocitos
circulantes de pacientes diabticos para la deteccin precoz de marcadores
predictivos de tumores asociados a diabetes.
154

P17-17
Activacin de blancos metablicos ro abajo

de la protena quinasa activada por AMP (AMPK)
heptica por la hormona tirodea (T3)
Luis Videla Cabrera1, Romina Vargas Villagrn1, Pamela Cornejo
Zamorano2, Virginia Fernndez Arancibia1
1
Facultad de Medicina Universidad de Chile, Santiago, CL, 2Faculad
de Medicina Universidad Diego Portales, Santiago, CL
La administracin de T3 activa a la AMPK heptica como respuesta
adaptativa frente a condiciones de alta demanda energtica. El hgado
de ratas macho Sprague-Dawley tratadas con 0,1 mg T3/kg present un
aumento (P<0,05) en los niveles de pAMPK (ELISA) y de sus blancos
acetil-CoA carboxilasa (pACC) y protena de unin al elemento de
respuesta a AMP-cclico (pCREB) (Western blot), con mayor expresin
de los ARNm de ACC, CREB y del receptor activado por proliferadores
peroxisomales- coactivador-1 (PGC-1)(qPCR). Conjuntamente,
T3 aument (P<0,05) la expresin proteica y de ARNm de la carnitina
palmitoil transferasa-1 (CPT-1), acil-CoA oxidasa 1 (ACOX1) y acilCoA tioesterasa 2 (ACOT2) hepticas, con mayores niveles sricos de
-hidroxibutirato. Se concluye que la fosforilacin de AMPK heptica por
T3 se asocia a la mayor activacin de sus blancos directos ACC, CREB y
PGC-1 conduciendo a un incremento en la capacidad de oxidacin de
cidos grasos, asociado al aumento en las enzimas relacionadas CPT-1,
ACOX1 y ACOT2, y evidenciado por la respuesta cetognica consecuente.
La activacin de la AMPK constituira un mecanismo de sealizacin
clave para la regulacin de la dinmica energtica heptica en apoyo de
los mecanismos de preacondicionamiento, proteccin y/o reparacin
otorgados por T3.
Financiado por FONDECYT 1120034.
P17r-18
Mathematical modelling of the amyloidogenic

pathway during Alzheimers disease in the
context of the lipid membrane
Guido Rosales Santos1, Mario Daz2, Nstor V. Torres1
Grupo de Biologa de Sistemas y Modelizacin Matemtica.
Departamento de Bioqumica, Microbiologa, Biologa Celular y
Gentica. Universidad de La Laguna, La Laguna, ES, 2Laboratorio
de Fisiologa y Biofsica de Membranas, Departamento de Biologa
Animal y Edafologa y Geologa, La Laguna, ES
Alzheimers disease (AD) is the most common cause of dementia. There is

over 30 million people affected worldwide, and this number is increasing
every year. There is not therapy for AD. It is caused by the neuronal death
produced mainly by the toxic effect of amyloid- peptides accumulation,
after the precursor protein (APP) is cleaved by a specific beta secretase
(BACE). APP and BACE proteins are located in the cellular membrane
of neurons. Specifically, both APP and BACE reside within detergent
resistant domains called lipid rafts. It is known that alterations in the lipid
composition of lipid rafts play an important role in the disease, being this
issue a very important aspect for finding a cure to AD.
Systemic approach to the study of AD let us to quantify the importance
of each component of the causal pathway to produce amyloid- and its
relationship with the lipid composition of the membrane. The objective
of this work is to use mathematical modeling to understand the role of the
membrane composition on AD and try to find an effective therapy for this
disease.
This work was funded by research Grants from Spanish MINECO
(BIO2011-29233-C02-02 and SAF2010-22114-C02-0) and from Agencia
Canaria de Investigacin, Innovacin y Sociedad de la Informacin (Ref.
Project PIL2070901 and PIL2071001). GS is recipient of a fellowship from
Fundacin Cajacanarias para posgraduados.
Granada 2014
Psters
P17r-19 (R17-1)
P17-21
Adaptation of T lymphocyte effector function

to metabolic stress
Regulation of hypoxia response by NO

in pluripotent stem cells
Sonia Tejedor Vaquero, Cristina Lpez Rodrguez, Jos Aramburu

Beltrn
Departamento de Ciencias Experimentales y de la Salud,
Universidad Pompeu Fabra, Barcelona, ES
Estefana Caballano Infantes1, Ana B. Hitos2, Irene Daz2, Gladys M.

Cahuana2, Abdelkrim Hmadcha3, Fran Martn4, Bernat Soria5, Juan
R. Tejedo4, Francisco J. Bedoya4
1
(CABIMER) y Universidad Pablo de Olavide, Sevilla, ES, 2CABIMER,
Universidad Pablo de Olavide, y Centro de Investigacin Biomdica
en Red de Diabetes y Enfermedades Metablicas Asociadas
(CIBERDEM), Sevilla, ES, 3CABIMER, Fundacin Progreso y
Salud, y Red de Terapia Celular (Tercel), Sevilla, ES,4CABIMER,
Universidad Pablo de Olavide; Red de Terapia Celular (Tercel), y
CIBERDEM, Sevilla, ES, 5CABIMER, Fundacin Progreso y Salud;
Red de Terapia Celular (Tercel), y CIBERDEM, Sevilla, ES
In the last years it has been reported that nutrients and metabolism
play essential roles in T lymphocyte activation and differentiation. T
lymphocytes exhibit specific metabolic features depending on their
activation and polarization stage, being this crucial for their role in adaptive
immune responses. Glucose is necessary for T lymphocyte expansion and
polarization towards effector Th cells upon antigen stimulation, since it
is both a main fuel for ATP production and a source of building blocks
for macromolecule biosynthesis. T lymphocytes can encounter substantial
variations in glucose concentration at inflammation sites and tumors, which
raises the question of how glucose fluctuations affect their capacity to
maintain ATP and appropriate responses to cytokines. We have explored the
response of T lymphocytes to glucose deprivation during cytokine-driven
polarization. Our results show that T lymphocytes can resist substantial
glucose restriction and maintain ATP levels by arresting their proliferation.
Results indicate that expression of different cytokines is differentially
sensitive to glucose deprivation, and we are currently analyzing the
contribution of various energy-regulatory pathways to cytokine expression.
Our results suggest that local nutrient conditions during T lymphocyte
activation can have a significant impact in their function over polarizing
pressure from cytokines.
Funded by the Spanish Government (SAF2009-08066, SAF2012-36535
to CL-R; SAF2011-24268 to JA), Fundaci la Marat TV3 (080730,
122530, CL-R and JA) and Generalitat de Catalunya (2009 SGR601,
2014 SGR1153). STV is supported by a predoctoral fellowship BES-2013062670.
P17-20
Implicacin de FAT/CD36 en la modulacin

del metabolismo lipdico heptico
Larraitz Fernndez-Ares, Daniela Mestre, Juan Luis GarcaRodrguez, Igor Aurrekoetxea, Xabier Buqu, Patricia Aspichueta
Universidad del Pas Vasco (UPV/EHU), Bilbao, ES
CD36 es una glicoprotena que en la enfermedad del hgado graso no
alcohlica se sobreexpresa en la membrana plasmtica del hepatocito
donde juega un papel clave en la entrada de AG, siendo su destino
metablico desconocido. El objetivo fue investigar la implicacin de
CD36 en la modulacin del metabolismo lipdico heptico e investigar
el mecanismo.
Se infectaron hepatocitos de ratn C57/6J con adenovirus sentido para
CD36 y LacZ. Se analiz la incorporacin de sustratos radiactivos en
los principales lpidos hepticos, y en los metabolitos solubles cidos
secretados. Se investig la expresin de protenas implicadas en estrs
de retculo endoplsmico, actividad mTORC1 y se analiz su relacin
con el proceso de autofagia en presencia de inhibidores de la funcin
lisosomal.
La sobreexpresin de CD36 aument la incorporacin de oleato en
triglicridos y en los metabolitos solubles cidos secretados y de acetato
en fosfolpidos. Adems, provoc el incremento en p-S6 y la inhibicin del
flujo de autofagia, compatible con el incremento en la actividad mTORC1.
La inhibicin de la funcin lisosomal disminuy los niveles de p-S6 y
revirti la secrecin de solubles cidos y la esterificacin lipdica.
Como conclusin, la sobreexpresin de CD36 en el hepatocito induce una
remodelacin metablica ligada a la activacin de mTORC1. El incremento
en la sntesis de novo de fosfolpidos y esterificacin de triglicridos sugiere
una mayor actividad de fosfolipasas lo que podra inducir mTORC1.
The expansion of pluripotent cells (ESCs and iPSCs) under conditions that
maintain their pluripotency is necessary to implement a cell therapy program.
Previously, we have described that low nitric oxide donor diethylenetriamine/
nitric oxide adduct (DETA-NO) added to the culture medium, promote the
expansion of these cell types. The mechanisms involved in this response
are not yet known. We present evidences that when ESC and iPSCs are
grown under normoxia in presence of Nitric Oxide (NO) triggers a similar
response to hypoxia, thus maintaining the pluripotency. We have studied
the stability of protein HIF-1 (Hypoxia Inducible Factor) in presence of
low NO. Because of the close relationship between hypoxia, metabolism,
mitochondrial function and pluripotency we have analyzed by qRT-PCR
the expression of genes involved in glucose metabolism and the glycolytic
pathway such as: HK2, LDHA and PDK1; besides other gene targets of
HIF. We further analyzed the expression of genes involved in mitochondrial
biogenesis such as PGC1, TFAM and NRF1 and we have observed that
low NO maintains the same expression that in hypoxia. The study of the
mitochondrial membrane potential using MitoTracker dye, showed a
slight decrease in the membrane potential in cells with NO in normoxia ,
this indicated that NO might decrease the mitochondrial function. We will
analyze other metabolic parameters, to determinate if this molecule regulates
mitochondrial function and mimics Hypoxia Response. The knowledge of
the role of NO in the Response to Hypoxia and the mechanisms that help
to maintain self renewal in pluripotent cells grown under normoxia, can
help to the design of culture media where NO could be optimal for stem cell
expansion in the performance of future cell therapies.
Subvencionado por:
- Ministerio de Economa y Competitividad-Secretara de Estado de
Investigacin Desarrollo e Innovacin (IPT-2011-1615-900000)
- Junta de Andaluca (CTS- 7127/2011)
P17-22
Papel de los factores de transcripcin E2F1

y E2F2 en la esteatosis heptica
Daniela Mestre Congregado1, Igor Aurrekoetxea1, Larraitz
Fernndez-Ares1, Xabier Buqu1, Ainhoa Iglesias2, Ana Zubiaga2,
Patricia Aspichueta1
1
Departamento de Fisiologa, Facultad de Medicina y Odontologa,
Universidad del Pas Vasco UPV/EHU, Leioa, ES, 2Departamento de
Gentica, Antropologa Fsica y Fisiologa Animal, Facultad de Ciencia
y Tecnologa, Universidad del Pas Vasco UPV/EHU, Leioa, ES
La enfermedad del hgado graso no alcohlica (EHGNA) se desarrolla en
un 80% de pacientes obesos, lo que se considera un factor de riesgo para el
desarrollo de carcinoma hepatocelular (CHC). En el desarrollo de EHGNA
y de CHC se producen importantes adaptaciones metablicas. E2F1,
regulador del metabolismo oxidativo y cuya ausencia confiere resistencia
a obesidad, es junto con otros factores de transcripcin E2F importante
regulador del ciclo celular. El objetivo fue definir el papel de los factores de
transcripcin E2F1 y E2F2 en la instauracin de hepatoesteatosis.
155
Psters
Se utilizaron ratones macho de 3 meses E2F1-/-, E2F2-/- y sus controles.
Se indujo esteatosis administrando una dieta rica en grasa (HFD) durante
10 semanas. Se analiz la secrecin heptica de triglicridos (TG), el
contenido heptico en TG y su modulacin en condiciones pro-esteatticas
como la inhibicin de la secrecin de VLDL y el ayuno de 24 horas.
La HFD indujo esteatosis en ratones control. En ratones E2F2-/- el contenido
heptico en TG era el 40% del de los ratones control y permaneci invariable
tras la administracin de la HFD. Sin embargo, el almacn de TG inducido
por el ayuno de 24 horas era superior en los ratones E2F2-/- que en los E2F1/y sus controles. En animales E2F1-/-, la HFD provoc el incremento de TG,
aunque no lleg a los valores de sus controles, lo que es atribuible al aumento
en la secrecin de TG como lo demuestra su inhibicin farmacolgica.
En conclusin, los factores de transcripcin E2F1 y E2F2 estn implicados
en la instauracin de la hepatoesteatosis por HFD. E2F1 ejerce un efecto
sobre la secrecin heptica de TG y E2F2 podra estar implicado en los
procesos de sntesis de novo de lpidos.
Gobierno Vasco IT-336-10.
P17-23
Papel de la osteopontina en la modulacin del

metabolismo heptico durante el envejecimiento
Beatriz Gmez-Santos, Maitane Nez-Garca, Olatz Fresnedo,
Patricia Aspichueta
Departamento de Fisiologa Universidad del pais vasco/ Euskal
Herriko Unibersitatea (UPV/EHU), Leioa, ES
El envejecimiento est caracterizado por una prdida progresiva de
la integridad fisiolgica. La osteopontina (OPN) es una glicoprotena
multifuncional que muestra expresin incrementada en patologas derivadas
del sndrome metablico y en varios tipos de cncer, siendo la prevalencia de
estas patologas mayor con la edad. Nuestro objetivo fue determinar si OPN
modula el metabolismo lipdico heptico durante el envejecimiento y definir el
mecanismo implicado. Se emplearon ratones deficientes en OPN (OPN-KO) y
sus controles, de 3 y 10 meses de edad. Se estudi el contenido lipdico heptico
y actividades enzimticas involucradas en el metabolismo de triglicridos y
colesterol. Se analizaron parmetros sricos y rutas de sealizacin ligadas
a procesos de sntesis y oxidacin de lpidos. Los resultados mostraron que
en animales control los niveles sricos de OPN aumentaban con la edad. La
deficiencia en OPN durante el envejecimiento result en un aumento del
ndice heptico, del contenido heptico en colesterol esterificado y triglicrido
as como en glicerofosfolpidos. Los cambios en las actividades enzimticas
analizadas no parecan ser causantes del incremento lipdico observado en
los ratones OPN-KO de mayor edad, en los que la actividad mTORC1 estaba
disminuida. Se observ mayor expresin proteica de la acetil-CoA carboxilasa
y menor porcentaje de su forma fosforilada compatible con su mayor actividad.
Como conclusin, OPN modula el metabolismo lipdico heptico durante el
envejecimiento. Su falta resulta en el incremento del almacn lipdico, debido,
al menos en parte, al incremento de la sntesis de novo.
Subvencionado por GV (IT-336-10) y UPV/EHU (UFI11/20).
P17-24
Dimorfismo sexual en el efecto de la rosiglitazona

sobre la lipotoxicidad del msculo cardaco
asociada a una dieta hiperlipdica
Miquel Sbert-Roig, Marco Bauz-Thorbrgge, Bel M. GalmsPascual, Francisco J. Garca-Palmer, Isabel Llad, Ana M. Proenza,
Magdalena Gianotti
Grup de Metabolisme Energtic i Nutrici, Dept. Biologia Fonamental
i Cincies de la Salut y IUNICS, Universitat de les Illes Balears.
IdISPa, Palma de Mallorca. Centro de Investigacin Biomdica
en Red Fisiopatologa de la Obesidad y la Nutricin (CIBERobn,
CB06/03/0043), Instituto de Salud Carlos III, Palma de Mallorca, ES
156

La ingesta de dietas hiperlipdicas (HL) provoca una hiperlipidemia que
conlleva una mayor captacin de cidos grasos por parte del msculo
cardaco. La situacin de lipotoxicidad que se genera se acompaa de
resistencia a la insulina, estrs oxidativo, disfuncin mitocondrial y
disminucin de la utilizacin de glucosa, que puede conducir a una prdida
de la capacidad contrctil y, por ende, a un fallo cardaco. La rosiglitazona
(Rsg) es una tiazolidinediona que aumenta la sensibilidad a la insulina y
disminuye la lipotoxicidad perifrica, a travs de la mejora de la funcin
mitocondrial. En estudios previos hemos demostrado la existencia de un
dimorfismo sexual en la funcin y biognesis mitocondriales, lo cual nos
lleva a plantearnos si existen diferencias entre sexos en el efecto de la Rsg
sobre la lipotoxicidad cardaca asociada a la administracin de una dieta
HL durante 15 semanas en ratas. La Rsg (100mg/kg dieta) se administr a
la mitad de los animales durante las dos ltimas semanas del tratamiento.
En respuesta a la Rsg, las hembras responden de manera ms acusada
a la disminucin de triglicridos en el miocardio provocada por la HL,
lo que se acompaa de una mayor activacin de la va de sealizacin a
la insulina y el consiguiente incremento en la utilizacin de la glucosa.
Paralelamente, las ratas hembra tratadas con Rsg muestran un mayor
metabolismo mitocondrial y un menor estrs oxidativo en el miocardio.
En conclusin, la Rsg promueve una mayor movilizacin lipdica en el
msculo cardaco de las hembras, mejorando del fenotipo lipotxico tpico
que es caracterstico de la cardiomiopata diabtica.
Financiado por MECD (SAF2010-21792, una beca FPU), CAIB
(PCTIB-31/2011, una beca FPI), FSE y FEDER-Unin Europea Una
manera de hacer Europa.
P17r-25 (R17-3)
Protein Kinase A-dependent phosphorylation

of the ATPase Inhibitory Factor 1 (IF1)
determines the metabolic status of the cell
Javier Garca-Bermdez1, Mara Snchez-Arag2, Jos M. Cuezva2
Centro de Biologa Molecular, Madrid, ES, 2Centro de Biologa
Molecular Severo Ochoa (CSIC-UAM), Madrid, ES
1
Mitochondria play key roles in cellular metabolism and bioenergetics

and are the master regulators in the execution of cell-death mediating
intracellular signaling by calcium and reactive oxygen species (ROS).
During the last years, the interest in mitochondria has experienced
a boost because of its involvement in a growing number of human
diseases displaying very different phenotypes. In mitochondria, the H+ATP synthase is the rotatory engine of the inner membrane that utilizes
the proton gradient generated by respiration for the synthesis of most
cellular ATP in normal aerobic differentiated cells. The expression level
of the nuclear encoded inhibitor peptide called ATPase Inhibitory Factor
1 (IF1) regulates the synthase activity of the H+-ATP synthase, being a
central player in controlling the flux of ATP being produced by oxidative
phosphorylation (OXPHOS). IF1 is a very short half-life protein that
is highly overexpressed in most prevalent human carcinomas. The
overexpression of IF1 in cancer promotes an increase in the flux of aerobic
glycolysis and a ROS-mediated signal that results in retrograde signaling to
the nucleus to activate programs of cell survival. However, the mechanisms
that regulate the expression and activity of IF1 remain elusive. Herein,
we demonstrate in human cancer cell lines that IF1 is regulated by posttranslational mechanisms. We show that the phosphorylation of IF1 on a
serine residue by cyclic AMP-dependent protein kinase A (PKA) prevents
its binding to the H+-ATPsynthase complex. Moreover, de-phosphorylated
IF1 is strongly bound to the complex and exerts an enhanced inhibition on
its enzymatic activity.
Supported by SAF2013-41945-R.
Granada 2014
P17-26
Dimorfismo sexual en la respuesta de la

paraoxonasa a la suplementacin de una dieta
hiperlipdica con rosiglitazona
Bel M. Galms-Pascual, Marco Bauz-Thorbrgge, Miquel SbertRoig, Magdalena Gianotti, Ana M. Proenza, Francisco J. GarcaPalmer, Isabel Llad
i Cincies de la Salut y IUNICS, Universitat de les Illes Balears.
IdISPa, Palma de Mallorca. Centro de Investigacin Biomdica
en Red Fisiopatologa de la Obesidad y la Nutricin (CIBERobn,
CB06/03/0043), Instituto de Salud Carlos III, Palma de Mallorca, ES
La ingesta de dietas hipercalricas induce inflamacin y estrs oxidativo
y aumenta el riesgo cardiovascular. Estudios previos han puesto de
manifiesto que las ratas hembra presentan un menor dao oxidativo tisular
como resultado de una mayor capacidad antioxidante y funcionalidad
mitocondrial. El enzima paraoxonasa 1 (PON1) es el principal responsable
de la funcin antiaterognica de las HDL, vindose alterada su actividad
por la dieta. La rosiglitazona (Rsg), un frmaco que mejora el perfil de
sensibilidad insulnica, estimula la funcin mitocondrial y reduce el estado
de inflamacin.
El objetivo fue determinar las diferencias entre sexos en la respuesta a
la Rsg de marcadores de estrs oxidativo y, en particular, de la actividad
y los niveles sricos de la PON1. Para ello se utilizaron ratas macho y
hembra Wistar de 22 semanas, alimentadas durante 16 semanas con una
dieta hiperlipdica (22,5% de materia grasa). Durante las ltimas dos
semanas de tratamiento, la mitad de los animales recibieron la misma dieta
suplementada con Rsg (100mg/kg dieta).
La Rsg mejor el perfil de sensibilidad a la insulina, aument la capacidad
antioxidante total y disminuy la peroxidacin lipdica srica de manera
ms acusada en las ratas macho que en las hembras. La suplementacin
de la dieta hiperlipdica con Rsg aument la expresin heptica de PON1
en las ratas macho, mientras que en las hembras produjo un aumento de la
actividad del enzima. Estos resultados ponen de manifiesto un dimorfismo
sexual en los efectos de la Rsg a nivel de proteccin antioxidante que
podran condicionar el riesgo cardiovascular en funcin del sexo.
Estudio financiado por MECD (SAF2010-21792, una beca FPU), CAIB
P17-27 (R17-4)
Dimorfismo sexual en la funcin y biognesis

mitocondrial del tejido adiposo retroperitoneal en
respuesta a la suplementacin con rosiglitazona
en ratas alimentadas con dieta hiperlipdica
Marco Bauz-Thorbrgge, Miquel Sbert-Roig, Bel M.
Galms-Pascual, Magdalena Gianotti, Francisco J. Garca-Palmer,
Isabel Llad, Ana M. Proenza
Grup de Metabolisme Energtic i Nutrici, Dept. Biologia
Fonamental i Cincies de la Salut, IUNICS, Universitat de les
Illes Balears. IdISPa, Palma de Mallorca. Centro de Investigacin
Biomdica en Red Fisiopatologa de la Obesidad y la Nutricin
(CIBERobn, CB06/03/0043), Instituto de Salud Carlos III, Palma
de Mallorca, ES
La poblacin mitocondrial del tejido adiposo blanco retroperitoneal (TABr)
de ratas hembra es menos diferenciada y ms abundante que la de los
machos. Este dimorfismo sexual se atena en respuesta a la alimentacin
con una dieta hiperlipdica (HL).
El objetivo de este estudio fue determinar si la rosiglitazona (Rsg), un
frmaco agonista del PPAR que mejora la capacidad oxidativa y aumenta
el contenido mitocondrial en los adipocitos, ejerce efectos dependientes
del sexo en el TABr.
Psters
Se utilizaron ratas Wistar de ambos sexos alimentadas durante 16 semanas
con dieta HL que, a dos semanas del sacrificio y para la mitad de los
animales, se suplement con Rsg (100mg/kg dieta). En muestras de TABr
se determinaron parmetros marcadores de funcin, biognesis, dinmica
y capacidad oxidativa mitocondriales, as como mecanismos antioxidantes.
La suplementacin con Rsg provoc un aumento de la masa y la dinmica
mitocondriales en ambos sexos, aunque mucho ms acentuado en los
machos. Sin embargo, la capacidad oxidativa nicamente se increment
en las hembras. La respuesta antioxidante, en cambio, se estimul en igual
medida en ambos sexos.
En conclusin, el TABr parece responder de manera diferencial segn
el sexo a los efectos inductores de la biognesis de la Rsg, aumentando
la proliferacin mitocondrial en las ratas macho y la diferenciacin
mitocondrial en las hembras. Esta respuesta heterognea podra deberse
a la accin de las hormonas sexuales, de acuerdo con resultados previos
obtenidos in vitro que muestran un efecto opuesto del estradiol y de la
testosterona sobre la biognesis mitocondrial.
P17-28
La suplementacin de una dieta hipercalrica

con rosiglitazona mejora la sensibilidad heptica
a la insulina en las ratas macho pero no en las
hembras
Bel M. Galms-Pascual, Miquel Sbert-Roig, Marco BauzThorbrgge, Ana M. Proenza, Magdalena Gianotti, Isabel Llad,
Francisco J. Garca-Palmer
i Cincies de la Salut y IUNICS, Universitat de les Illes Balears,
IdISPa - Centro de Investigacin Biomdica en Red Fisiopatologa de
la Obesidad y la Nutricin (CIBERobn, CB06/03/0043), Instituto de
Salud Carlos III, Palma de Mallorca, ES
La ingesta de dietas hipercalricas conduce a resistencia a la insulina y a
acumulacin de grasa en el hgado. La rosiglitazona (Rsg) es un agonista de
PPAR que mejora la sensibilidad a la insulina de forma directa en el tejido
adiposo blanco y, gracias a la secrecin de adiponectina por los adipocitos,
en el msculo y en el hgado.
El objetivo fue determinar el efecto de la suplementacin con Rsg
de una dieta hipercalrica sobre la sensibilidad heptica a la insulina y
a la adiponectina en ratas macho y hembra. Se utilizaron ratas Wistar
alimentadas durante 16 semanas con una dieta hiperlipdica rica en
sacarosa (HLS, 22,5% materia grasa). Durante las dos ltimas semanas
de tratamiento, la mitad de los animales se alimentaron con la misma
dieta HLS suplementada con Rsg (100mg/kg). Se recogieron el suero, los
depsitos grasos y el hgado. En el hgado se determin la actividad de la
fosfoenolpiruvato carboxiquinasa y se analiz la va de sealizacin de la
insulina mediante la activacin de Akt, y la de la adiponectina a travs de
AdipoR2 y la activacin de AMPk. En el suero se determinaron los niveles
de adiponectina.
En comparacin con las ratas hembra, la adiposidad y los niveles de
triacilglicridos hepticos fueron ms elevados en los machos HLS y
disminuyeron de forma ms acusada por efecto de la Rsg. Adems, en
las ratas macho, la Rsg mejor la tolerancia a la glucosa aumentando
los niveles sricos de adiponectina y los hepticos de AdipoR2 e
incrementando la activacin de Akt. La disminucin de la actividad
fosfoenolpiruvato carboxiquinasa indica una menor produccin heptica
de glucosa. Por el contrario, en las ratas hembra los efectos de la dieta HLS
y de la suplementacin con Rsg fueron ms moderados.
En conclusin, la mayor acumulacin ectpica de grasa en el hgado de
los machos por la alimentacin con la dieta HLS altera su sensibilidad a
157
Psters
la insulina de forma ms acusada que en las hembras, alteraciones que se
revierten, al menos en parte, por la Rsg.
P17-29 (R17-2)
Mitochondrial glutamate transporter (Aralar):

influence over myelin formation and dopamine
metabolism
Beatriz Pardo1, Irene Llorente-Folch1, Sara Laine-Menndez1,
Carlos B. Rueda1, Paloma Gonzlez-Snchez1, Ins Juaristi1, Paula
Martnez-Valero1, Araceli del Arco2, Jorgina Satrstegui1
1
Dept. de Biologa Molecular, Centro de Biologa Molecular Severo
Ochoa UAM-CSIC, CIBER de Enfermedades Raras, Universidad
Autnoma de Madrid, Madrid, ES, 2rea de Bioqumica, Centro
Regional de Investigacin Biomdica (CRIB), Facultad de Ciencias
Ambientales y Bioqumica, Universidad de Castilla - La Mancha,
Toledo, ES
The mitochondrial transporter of aspartate-glutamate Aralar/AGC1 is a
regulatory component of the malate-aspartate shuttle, mainly expressed
in neurons. Aralar-deficiency in mouse and human causes a shutdown
of brain shuttle activity and global cerebral hypomyelination (OMIM
ID # 612949) associated with a drastic drop in brain aspartate and
N-acetylaspartate (NAA) levels. Aralar-knockout (Aralar-KO) mice
show deficits in motor coordination, ataxia, hyperactivity, anxiety-like
behaviour and hyperreactivity (a phenotype very similar to what is
observed in the human patients). In Aralar-KO mice, the striatum is the
brain region most affected in terms of size, amino acid and monoamine
content. We find a fall in DA and in vesicular monoamine transporter-2
(VMAT2) levels which causes an increase both in non-vesicular dopamine
and dopamine turnover (DOPAC/dopamine ratio) through MAO activity.
Our results suggest that Aralar-deficiency results in a failure to produce
mitochondrial NADH and to an increase of ROS in the cytosol causing
a fall in VMAT2 and GSH/GSSH ratio in striatum. Aralar-hemizygous
(Aralar-HT) mice, with half-a-dose of Aralar and decreased NAA, have
also a heightened DOPAC/DA ratio with oxidative stress via MAO and
increased sensitivity to amphetamine; as well as a delayed postnatal
myelination.
NAA is one of the most concentrated metabolites in human and mice brain,
but the physiological functions served by NAA remain controversial.
NAA is proposed to be involved in providing acetyl CoA needed for
oligodendrocyte maturation and postnatal myelin synthesis, neuronal
osmoregulation and energy metabolism. These data make further the point
about the putative relevance of NAA in higher brain functions and control
of behavior.The correlation between reduced NAA, hypomyelination
and DA dysfuntion is relevant because its involvement in neurological
and psychiatric human diseases and needs to be further investigated and
discussed.
P17-30
Deteccin temprana del dao oxidativo

en pacientes hipercolesterolmicos: efectos
del tratamiento con estatinas
Lidia Lpez Lpez, Sandra Tavrez Alonso, Ana Martn Vega, Eulalia
Alonso Iglesias
Departamento de Bioqumica y Biologa Molecular, Facultad de
Medicina, Universidad de Valencia, Valencia, ES
Numerosas evidencias clnicas y experimentales indican que las
alteraciones en el balance oxidativo estn implicadas en la instauracin
y progresin de las patologas de RCV como la hipercolesterolemia, y
158

que parte de los efectos pleiotrpicos de las estatinas en esta patologa
se relacionan con sus beneficios sobre el estrs oxidativo. Entre los
marcadores ms recientemente introducidos de peroxidacin lipdica
se encuentran los 8-isoprostanos, compuestos estables resultantes de
la oxidacin del cido araquidnico por radicales libres. Evaluando
los niveles de 8-isoprostanos (ELISA) en plasma y orina de individuos
control, hipercolesterolmicos e hipercolesterolmicos tratados
con estatinas en este trabajo demostramos, tanto que los niveles de
8-isoprostanos pueden ser un marcador temprano de oxidacin en
humanos hipercolesterolmicos, como que el tratamiento con estatinas
se asocia a prevencin del dao oxidativo. Los niveles de 8-isoprostanos
se elevan significativamente en plasma del grupo hipercolesterolmico
(52,084,01 vs 39,063,17 pg/mL; p=0,016), normalizndose
prcticamente por el tratamiento con estatinas (52,084,01 vs
41,342,5 pg/mL; p=0,04). Los resultados en orina corroboran estas
observaciones, sin alcanzar las diferencias significacin estadstica.
De las estatinas evaluadas, la atorvastatina es la que muestra un mejor
perfil en la normalizacin de los niveles de 8-isoprostanos, as como en
la reduccin del colesterol total. De la comparacin de estos resultados
con los de otros indicadores de dao oxidativo a lpidos y protenas,
proponemos considerar a los 8-isoprostanos como el indicador ms
temprano de dao oxidativo en la deteccin y posterior evaluacin del
tratamiento con estatinas.
Financiado con la ayuda CONSOLIDER-INGENIO CSD2007-00063.
P17-31
Integrando el papel de la resistina en un modelo

de riesgo cardiovascular: estudio en ratones ob/ob
Ana Martn Vega1, Sandra Tavrez Alonso1, Lidia Lpez Lpez1, Pilar
Codoer Franch2, Eulalia Alonso Iglesias1
1
Departamento de Bioqumica y Biologa Molecular, Facultad de
Medicina, Universidad de Valencia, Valencia, ES, 2Departamento de
Pediatra , Facultad de Medicina, Universidad de Valencia; Servicio
de Pediatra, HU Dr Peset, Valencia, ES
La obesidad es uno de los factores principales de RCV. De origen
multifactorial, la acumulacin excesiva de grasa en el tejido adiposo
promueve la liberacin por este rgano endocrino de molculas
inflamatorias y adipoquinas implicadas, por diferentes vas, en las
alteraciones metablicas de la obesidad y en su asociacin con el RCV.
Para evaluar el papel de la adipoquina resistina en este contexto, hemos
cuantificado sus niveles circulantes (Multiplex) y su relacin con el estado
metablico (perfil lipdico y resistencia insulnica), el dao oxidativo
(8-isoprostanos), la inflamacin (IL-6) y el metabolismo de la arginina
(xido ntrico (NO) y poliaminas) en ratones ob/ob, un modelo gentico
de RCV. Los resultados sobre los niveles de resistina en este modelo de
obesidad son contradictorios. Segn nuestros resultados, los niveles de
resistina en ratones ob/ob son significativamente ms altos que en los
controles lean (3269297 vs 135274 pg/mL; p<0,0001), y se correlacionan
positivamente con aumentos significativos del ndice HOMA (p=0,002),
colesterol (p=0,018), TGs (p=0,015), IL-6 (p<0,0001), 8-isoprostanos
(p=0,003), espermina (p=0,005) y peso de los paquetes grasos (p<0,0001).
Los niveles de resistina tambin se correlacionan, aunque de forma
inversa, con el descenso en la produccin de NO (p=0,003) y el cociente
espermidina/espermina (p<0,0001). Estos resultados apoyan el papel de la
resistina en la RI en este modelo de obesidad, as como los mecanismos
propuestos para su actuacin que incluiran activacin de la respuesta
inflamatoria, del estrs oxidativo y descenso en la produccin de NO. Los
posibles efectos de la resistina sobre el metabolismo de las poliaminas
merecen ser analizados.
Financiado con la ayuda CONSOLIDER-INGENIO CSD2007-00063.
Granada 2014
P18. Sealizacin celular

P18-1 (R18-3)
Glutaminolisis y mTOR: interaccin entre el

metabolismo y la sealizacin celular en cncer
Ral V. Durn
Group of Metabolism and Cell Signaling, Institut Europen de
Chimie et Biologie VINCO Unit, U916 INSERM, Bordeaux, FR
Tradicionalmente se ha contemplado la transformacin metablica de
las clulas cancergenas como un fenmeno secundario encaminado a
abastecer a la clula de la energa necesaria para permitir una rpida
proliferacin. Sin embargo, en los ltimos aos esta visin ha cambiado
con el descubrimiento de que los genes que codifican para determinados
enzimas metablicos pueden ser considerados como oncogenes o genes
supresores de tumor. De esta manera, la transformacin metablica puede
localizarse en el origen del cncer. Esta conclusin lleva a la pregunta
de cmo consiguen los cambios metablicos celulares alterar las rutas
de sealizacin para permitir el crecimiento descontrolado de la clula
tumoral. En este sentido, nuestra investigacin se centra en el estudio de
los mecanismos por los que los nutrientes, en especial los aminocidos,
activan la va de sealizacin mTOR (mammalian target of rapamycin), un
regulador principal del crecimiento celular. Nuestros resultados muestran
que la glutamina, un aminocido crucial para la bioenergtica de las clulas
cancergenas, activa la ruta mTORC1 (mTOR complex 1) por medio de
su propia degradacin a travs de la glutaminolisis. La glutaminolisis es
la doble desaminacin de la glutamina para producir -cetoglutarato. El
-cetoglutarato intracelular activa a la familia de protenas PHD (prolyl
hydroxylases), necesarias para la activacin de mTORC1. Por lo tanto,
la ruta glutamina/PHD/mTORC1 constituye una cascada de sealizacin
principal en la respuesta celular a la disponibilidad de nutrientes. Tanto
la glutaminolisis como la ruta mTORC1 aparecen activados en una gran
variedad de tumores. De esta manera, nuestros resultados suponen un claro
ejemplo de cmo el metabolismo alterado de la clula tumoral afecta a
procesos de sealizacin celular, fundamentales para el crecimiento rpido
del tumor. La existencia de una relacin mecanstica entre glutaminolisis
y mTORC1 abre las puertas a futuras investigaciones para determinar si
ambos procesos tienen un efecto sinergstico como dianas teraputicas
contra el cncer.
P18r-2
Non-invasive spatio-temporal activation of

engineered receptor tyrosine kinases with light
lvaro Ingls Prieto1, Eva Reichhart1, Karin Schelch2, Robert
Riedler1, Michael Grusch2, Harald Janovjak1
1
Institute of Science and Technology Austria, Viena, AT, 2Institute of
Cancer Research, Department of Medicine I, Comprehensive Cancer
Center Vienna, Medical University of Vienna, Viena, AT
Receptor tyrosine kinases (RTKs) are a large family of cell surface
receptors that sense growth factors and hormones and regulate a variety
of cell behaviours in health and disease. Contactless activation of RTKs
with spatial and temporal precision is currently not feasible. Here, we
generated RTKs that are insensitive to endogenous ligands but can be
selectively activated by low intensity blue light. We screened lightoxygen-voltage (LOV)-sensing domains for their ability to activate
RTKs by light-activated dimerization. Incorporation of LOV domains
found in aureochrome photoreceptors of stramenopiles resulted in robust
activation of the fibroblast growth factor receptor 1 (FGFR1), epidermal
growth factor receptor (EGFR) and RET receptor. In human cancer and
endothelial cells, light induced cellular signalling with spatial and temporal
precision. Furthermore, light faithfully mimicked complex mitogenic
and morphogenic cell behaviour induced by growth factors. RTKs under
optical control (Opto-RTKs) provide a powerful optogenetic approach to
actuate cellular signals and manipulate cell behaviour.
Psters
P18-3
Critical role of p38MAPK in the alternative

activation of macrophages
Sonsoles Hortelano Blanco, Lidia Jimenez, Sandra Herranz, Alfonso
Luque
Unidad de Terapias Farmacolgicas. Instituto de Investigacin
de Enfermedades Raras (IIER), Instituto de Salud Carlos III,
Majadahonda, ES
Alternative activation of macrophages plays an important role in a range
of physiological and pathological processes, including inflammation,
wound repair and tissue remodeling as well as parasite clearance and tumor
progression. This alternative phenotype, also known as M2 macrophages, is
induced by type 2 cytokines such as IL-4. The binding of IL-4 to its receptor
leads to activation of two major signaling pathways: STAT-6 and PI3K.
However, recent studies have described that p38MAPK might play a role in
IL-4-dependent signaling in some types of cells. Since IL-4 is one of the most
relevant cytokines involved in the alternative activation of macrophages,
we investigated whether p38MAPK plays a role in the polarization of
macrophages. Our results reveal that IL-4 induces phosphorylation of
p38MAPK in peritoneal macrophages, in addition to STAT-6 and PI3K
activation. Furthermore, p38MAPK inactivation, by gene silencing or
pharmacological inhibition, suppressed IL-4-induced typical M2 markers,
indicating the involvement of p38MAPK in the signaling of IL-4 leading
to M2-macrophage polarization. Moreover, p38MAPK inhibition blocked
phosphorylation of STAT-6 and Akt, suggesting that p38MAPK is upstream
of these signaling pathways. Taken together, our results establish a new role
for p38MAPK during IL-4-induced alternative activation of macrophages.
P18r-4
Insights into novel DDR functions of GRK2 and

repercussions on cell cycle arrest
Adolfo Javier Molejn Garca, Vernica Rivas Guerrero, Federico
Mayor Menndez, Petronila Penela Mrquez
Centro de Biologa Molecular Severo Ochoa (CSIC-UAM),
Universidad Autnoma de Madrid - Instituto de investigacin
Sanitaria La princesa, Madrid, ES
DNA Damage Response (DDR) is a complicated and conserved signalling
mechanism utilised by cells to repair errors in DNA in order to avoid genomic
lesions that may arise from non-pathological origins (DNA replication,
oxidative metabolism) or genotoxic insults (radiation, genotoxins). As such,
DDR mechanisms exist as an anti-tumourigenic generated from a variety
of genomic lesions from point mutations to chromosomal aberrations.
Furthermore, defects in DDR signalling are associated to a variety of
tumourigenic predispositions such as loss of p53, BRCA or ATM. In order to
efficiently orchestrate a DDR response, cells require sensors (MRN, 53BP1),
signal transducers (ATM/ATR, Chk1/Chk2, p53) and ultimately effectors
(p21, Puma, Noxa). Depending on the type of signal in a genomically intact
cell, the outcome will be either DNA repair and re-entry into cell cycle, or cell
cycle arrest via senescence or apoptosis. GRK2, a GPCR Receptor Kinase, has
emerging functions outside of its traditional role of phosphorylating GPCR
receptors for desensitisation and subsequent internalisation for degradation
or receptor recycling. We have recently uncovered that GRK2 is degraded
by default during G2/M transition, which allows cell cycle progression.
However, upon G2-checkpoint activation GRK2 is stabilized and cell cycle
is arrested. In this cellular context, GRK2 helps cells to cope with damage
by attenuating the p53 response and the expression of pro-apoptotic factors.
We have unveiled evidence of GRK2 further modulating the DDR cascade
upstream of p53 in response to acute and chronic genotoxic insults. As such,
we suggest a complex intertwinement of GRK2 with ATM wherein GRK2
could be a target of ATM based on structural features and in vitro and in situ
phosphorylation assays, while downstream functionality of ATM would be
attenuated by GRK2 in a kinase dependent manner. Furthermore, we note an
inverse correlation between GRK2 protein levels and degree of senescence
in cellular models and physiological settings. In this light, we propose GRK2
159
Psters
as a potential therapeutic target owing to novel modulatory functions as well
as putative substrate in DDR response.
P18-5
Funcin de 2-quimerina como supresor de

tumores en cncer de mama Her2+
Victoria Casado Medrano1, Mara Jos Caloca Roldn2
1
Instituto de Biologa y Gentica Molecular, Valladolid, ES, 2CSIC
(IBGM), Valladolid, ES
Las quimerinas son una familia de protenas GAP (GTPase activating
proteins) que esta formada por cuatro miembros: 1-, 2-, 1-, y 2quimerinas. Estas protenas tienen una estructura caracterstica con un
dominio C1 que une DAG y steres de forbol, un dominio GAP que inactiva
selectivamente a la GTPasa Rac, y un dominio SH2 N-terminal, solo
presente en las isoformas 2- y 2 de las quimerinas. Estas caractersticas
hacen nicas a las quimerinas dentro de las Rho-GAP y las situa como
reguladores clave que conectan la sealizacin por DAG con la activacin
de Rac. La hiperactivacin de GTPasas de la familia Rho, incluida Rac,
es comn en cncer, por lo que a las protenas inactivadoras de estas
GTPasas se las atribuye un posible papel como supresores de tumores. Para
comprobar este papel de 2-quimerina en cncer de mama, hemos generado
un modelo de ratn en el que hemos eliminado la expresin de esta protena
en ratones MMTV-ErbB2/Neu, que desarrollan cncer de mama por la
sobreexpresin del protooncogn ErB2/Her2/Neu en el epitelio mamario.
Hemos evaluado el efecto de la deficiencia de 2-quimerina sobre el tiempo
de latencia, la multiplicidad tumoral, la velocidad de crecimiento de los
tumores, el nmero de lesiones preneoplsicas y la metstasis pulmonar.
Nuestros resultados demuestran que la eliminacin de 2-quimerina
reduce significativamente la latencia en la aparicin de tumores de mama
y aumenta el nmero de lesiones preneoplsicas. Sin embargo, no hemos
observado diferencias estadsticamente significativas en la multiplicidad o
el crecimiento de los tumores en ausencia de 2-quimerina. Para estudiar
los mecanismos moleculares regulados por 2-quimerina implicados en
la transformacin maligna hemos estudiado los niveles de Rac activado
y las principales vas de sealizacin reguladas por el receptor ErbB/Neu
(activacin de Akt y ERK). En conjunto nuestros resultados indican que 2quimerina regula los pasos iniciales del desarrollo de los tumores de mama
mediados por activacin del receptor ErbB2 a travs de un mecanismo que
implica la activacin de la GTPasa Rac.
P18-6 (R18-4)
ERK5 binds and regulates the androgen receptor

in prostate cancer cells: molecular and functional
characterization
Tatiana Erazo Andrade, Pau Muoz Guardiola, Jos Ramn
Bayascas, Nstor Gmez, Jos Miguel Lizcano
Protein Kinases and Signal Transduction Lab, Departament de
Bioquimica, Institut de Neurocincies, Facultat de Medicina,
Universitat Autnoma de Barcelona, Bellaterra (Barcelona), ES
Prostate cancer is the second commonest cause of cancer related death in
men in the western world. Usually, prostate cancer is initially androgendependent so patients are usually treated with androgen deprivation
therapy. However, after remission the tumor frequently progresses into
a state of castration resistance, and available treatment options are only
palliative in most cases.
The MAP kinase ERK5 is activated in response to a wide range of growth
factors and cellular stresses and controls cell proliferation and progression
through the cell cycle, and the ERK5 pathway is associated with poor
prognosis of prostate cancer.
Here we show that ERK5 interacts with the AR protein in the cytosol
of resting cells, and that activation of AR with androgens induces the
translocation of both proteins to the nucleus, where they continue to interact
160

with each other. Conversely, specific activation of ERK5 by the upstream
kinase MEK5 also induces the nuclear translocation of both ERK5 and
AR. We also provide evidences showing a functional link between these
proteins. Over-expression of active ERK5 enhances the ligand-dependent
trancriptional activity of AR (PSA promoter activity, a specific clinical
biomarker for prostate cancer), whereas silencing ERK5 inhibits the AR
transcriptional activation in response to androgens. Finally, we tested in
prostate cancer cells the compound XMD8-92, a newly sinthetized ERK5
speficic inhibitor. XMD8-92 inhibits cell proliferation and induces cell
death in both LnCaP (functional AR) and C4-2 (castrate resistant) cells,
whereas inhibition of the ERK1/2 pathway using MEK1 inhibitors had not
significative effect. In conclusion, we propose ERK5 inhibiton as a new
treatment fort androgen-dependent and castrate-resistant prostate cancers.
P18r-7
ABTL0812, a novel dual mTORC1/2

and dihydrofolate reductase (DHFR) inhibitor
with a novel mechanism of action
Ingrid Tatiana Erazo Andrade1, Jos Alfon2, Mariana GomezFerreria2, Mara Salazar3, Mar Lorente3, Marc Cortal2, Guillermo
Velasco3, Carles Domenech2, Jos Miguel Lzcano1
1
Protein Kinases and Signal Transduction Lab, Departament de
Bioquimica, Institut de Neurocincies, Facultat de Medicina,
Universitat Autnoma de Barcelona , Bellaterra, ES, 2Ability
Pharmaceuticals SL, Edifici Eureka, Campus de la UAB Universitat
Autnoma de Barcelona , Bellaterra, ES, 3Departamento de
Bioqumica y Biologa Molecular, Facultad de Biologa, Universidad
Complutense de Madrid , Madrid, ES
ABTL0812 is a novel multitarget drug with high efficacy by oral route in
mice models of cancer. In vivo ABTL0812 induces the regression of tumor
volume in xenograft models derived from human lung (A549) and human
pancreatic cancer cells (MiaPaca-2), and very recently started a Phase
I First in Human Clinical Trial in patients with advanced solid tumors.
We have shown that ABTL0812 induces the inhibition of two clinically
validated targets: mTORC1 (mTOR complex-1) and dihydrofolate
reductase (DHFR). Despite of this, ABTL0812 does not bind any of these
proteins, thus its precise mechanism of action remains to be described.
Here we show that ABTL0812 blocks the proliferation of lung and pancreatic
cancer cell lines. ABTL0812 induces autophagic cell death, but no apoptosis,
and inhibition of fusion of autophagosomes with lysosomes prevents its
citotoxicity. We also show that ABTL0812 induces the expression of TRB3
(tribbles homologue 3), a known Akt protein inhibitor in both A549 and
MiaPaca-2 human cancer cells. Overexpressed TRB3 interacts with Akt and
prevents Akt phosphorylation (Ser473) by mTORC2. This, in turn, results in
low levels of phosphorylated TSC2 and inhibition of mTORC1 (ribosomal
S6 phosphorylation). Finally, ABTL0812-induced mTORC1 inhibition
would render the loss of expression of the Cyclin D1, of E2F1 transcription
factor (controlled by Cyclin D1) and DHFR (controlled by E2F1).
Since TRB3 is down-regulated in some tumor types, the induction of TRB3
expression by ABTL0812 might account for the anti-tumoral action of
this compound. We proposed TRB3 expression levels as a biomarker to
monitor the antitumor efficacy of ABTL0812 in humans.
P18r-8
Role of the APC/C complex mediated degradation

of GRK2 in mitosis
Clara Reglero, Vernica Rivas, Federico Mayor Jr., Petronila Penela
Centro de Biologa Molecular Severo Ochoa (CSIC-UAM),
Universidad Autnoma de Madrid - Instituto de Investigacin
Sanitaria La Princesa, Cantoblanco, ES
Proteasome-dependent degradation of key kinases represents a major
regulatory mechanism in the control of cell cycle. We have recently
Granada 2014
reported that timely degradation of GRK2, a serine/threonine G proteincoupled receptor kinase, is part of an intrinsic pathway that ensures cell
cycle progression. Besides being a key player in the desensitization of
manifold GPCR receptors, GRK2 emerge as a signaling node due to its
ability to regulate a variety of signaling proteins and cellular functions.
We have previously found that GRK2 protein levels progressively decay
during G2/M as a result of the sequential cooperation of the E3 ligases
Mdm2 and APC/C. Thus, during G2 the functional interaction of Pin1 with
the CDK2/cyclinA-dependent phosphorylated GRK2 triggers its Mdm2mediated ubiquitination. At mitosis onset, GRK2 decay is promoted by the
APC/C-Cdc20 complex that recognizes a D-Box in GRK2, in a spindle
checkpoint-independent manner.
It is expected that this tightly regulated degradation of GRK2 will have
significant physiological consequences in mitosis. In this regard, recent
data of our group have unveiled the functional interaction of GRK2 with
regulatory factors involved in cytokinesis. Thus, we have demonstrated
that GRK2 is a key modulator of microtubule dynamics through the
phosphorylation of the tubulin deacetylase HDAC6. In addition, we have
found that GRK2 influences the extent of PI3K activation by a direct
interaction with the regulatory subunit p85, which also acts as a scaffold to
regulate the Rho-family GTPase Cdc42 and Septins at the cleavage furrow.
Our data reveal the presence of GRK2 in the midbody, a structure wherein
HDAC6 and p85 are also found. Dynamic changes in the levels and
functionality of GRK2 might help to timely activate HDAC6 or/and PI3K
in order to orchestrate microtubule dynamics and to recruit/activate factors
involved in organizing the contractile ring and setting the symmetry of the
division plane. Our results suggest that defective or sustained degradation
of GRK2 during mitosis could impair cytokinesis and contribute to
polyploidy and chromosomal instability.
Psters
P18-10
The neurosteroid-regulated 1 receptor engages

NMDA receptor negative control for opioid
analgesia
Pilar Sanchez Blazquez1, Maria Rodriguez-Muoz1, Manuel Merlos2,
Jose Miguel Vela2, Javier Garzn1
1
Instituto Cajal, CSIC, Madrid, ES, 2Drug Discovery & Preclinical
Development, Esteve. Scientific Park of Barcelona, Barcelona, ES
The antagonists of the sigma 1 receptor (1R) are of therapeutic interest
because these drugs reduce neuropathic pain and enhance mu-opioid
receptor (MOR)-mediated analgesia. However, the precise molecular events
implicated in the positive effects of these antagonists remain unknown.
The present study has identified the 1R within the protein assembly,
supporting MOR-glutamate N-methyl-D-aspartate (NMDA) receptor
cross-regulation, in which this receptor couples the negative influence of
NMDARs to restrain opioid signalling in response to MOR activation. The
1R is a neurosteroid-regulated receptor that associates with NMDAR NR1
subunits to maintain NMDAR signalling within physiological limits. The
exogenous 1R ligands likely mimic this physiological regulation through
the use of agonists and antagonists to respectively enhance and reduce the
ability of morphine-activated MORs to recruit NMDAR activity. Thus, the
absence of 1Rs or 1R antagonists disconnects both systems, enhancing
morphine analgesia and diminishing the development of tolerance. At
the molecular level, 1R antagonists through the removal of 1Rs from
their binding to NR1 subunits promote the entrance of negative regulators
of NMDARs, likely Ca2+-CaM, which impair the negative feedback on
opioid analgesia and reduce the impact of glutamate in neuropathic pain.
Therefore, the negative effects of 1R antagonists on NMDAR function
alleviate neuropathic pain and also reduce the requirements of opioid
receptor occupancy as a suitable adjuvant of opioid analgesia. (Supported
by MSC, Plan Nacional Drogas 11-014 and MINECO, SAF 2012-034991).
P18-9
PAR-3 participates in Thy-1-induced astrocyte

migration without affecting polarization
Areli Crdenas, Milene Kong, Alvaro Alvarez, Alejandra Valdivia,
Andrew F.G. Quest, Lisette Leyton
Facultad de Medicina, Universidad de Chile, Independencia, CL
PAR-3 is an important adaptor protein that participates in polarization
of many cell types by forming a complex with PAR-6/aPKC; however,
PAR-3 is not part of this complex during astrocyte polarization. We have
shown that the neuronal protein Thy-1 induces astrocyte polarization and
migration; however, the signaling mechanisms involved and the role of
PAR-3 in these processes have not been completely elucidated. Here,
we evaluated whether PAR-3 participates in adhesion, polarization and
migration of astrocytes induced by Thy-1. To this end, DITNC1 astrocytes
were transfected with PAR-3-specific siRNA or siRNA control. Then, cells
were seeded and treated with Thy-1-Fc or control TRAIL-R2-Fc. Cell
polarity and focal adhesions were assessed by immunofluorescence staining
of cells with anti-giantin and anti-vinculin, respectively, and analyzed by
confocal microscopy. Cell migration was assessed in wound-healing assays
and the cell-free area was calculated after 24h of Thy-1 stimulation.
The polarization of DITNC1 cells upon Thy-1 stimulation was not affected
by silencing of PAR-3, whereas these cells exhibited longer focal adhesions
than control cells either non-stimulated or stimulated with the negative
control protein (TRAIL-R2-Fc). Accordingly, astrocytes lacking PAR-3
migrated less than the control cells in the presence of Thy-1.
Our results implicate PAR-3 in Thy-1-induced astrocyte migration, but not
polarization. Together, these observations suggest a novel role for PAR-3 in
cell signaling pathways activated by Thy-1 in astrocyte migration.
Acknowledgements: FONDECYT 3140471 (AC), FONDECYT 1110149
(LL), 1130250 (AFGQ); Iniciativas Cientficas Milenio BNI P09-015-F
(LL); Anillo ACT1111 (AFGQ).
P18-11
Cytoneme-mediated delivery of Hedgehog

regulates the expression of bone morphogenetic
proteins to maintain germline stem cells
in Drosophila
Patricia Rojas Ros1, Isabel Guerrero2, Acaimo Gonzlez Reyes3
1
Institute of Human Genetics (IGH) mRNA Regulation and
Development , Montpellier, FR, 2Centro de Biologia Molecular
Severo Ochoa, CSIC/Universidad Autonoma de Madrid, Madrid,
ES,3Centro Andaluz de Biologa del Desarrollo, CSIC/Universidad
Pablo de Olavide, Sevilla, ES
Stem cells are characterised by their undifferentiated state, their potential
for self-renewal and their capacity to produce one or multiple types of
differentiated cells. These properties sustain homeostasis and repair of adult
tissues and open a high expectative about the use of these cells in regenerative
medicine. Adult stem cells are found in specialised microenvironments
or niches that control their proliferation, self-renewal and differentiation
of the progeny. The niches are composed of different support cells and
signals like morphogens. The Drosophila ovary is an excellent system to
study the molecular mechanisms that operate in stem cell niches because
this organ contains a well-defined niche, the Germline Stem Cell (GSC)
niche. Our data demonstrate that Hedgehog (Hh) signalling pathway is
essential in the niche of the GSCs for their maintenance. The expression of
Hh is regulated by Engrailed in a type of support cells of the GSC niche,
the Cap Cells (CpCs). Hh is secreted from the CpCs and received by the
Escort Cells, another type of niche cell, where the Hh pathway induces
the expression of two essential GSC factors, Dpp and Gbb. Interestingly,
Hh protein decorates short projections emanating from CpCs that remind
to the filopodia described in different tissues of Drosophila larvae called
cytonemes. When the Hh signalling is impaired within the niche, wildtype
CpCs project longer Hh-coated cytonemes suggesting that the niche senses
161
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and responds to changes in Hh signalling. In addition, we have found that
the perturbation of the formation and/or dynamic of cytonemes in the niche
compromised the GSC self-renewal demonstrating that cytonemes are
essential for the maintenance of stem cells.

due to the induction of the indole-dependent efflux pump demonstrating that
indole acts as an interspecies signalling molecule in antibiotic resistance.
P18r-14
P18r-12
Papel de TCFL5 en el desarrollo tumoral

Javier Galn Martnez, Ins Snchez Gmez, Konstantinos
Stamatakis, Nria Girons Pujol, Manuel Fresno Escudero
Centro de Biologa Molecular Severo Ochoa (CSIC-UAM), Madrid,
ES
Las clulas madre tumorales, tambin denominadas Cancer Stem Cells
(CSC) son parte de la poblacin heterognea del tumor siendo las
responsables del mantenimiento de este, de la recurrencia y de la metstasis.
Comprender el papel que desempean este tipo de clulas durante el
desarrollo tumoral se ha convertido recientemente en un importante campo
de estudio para el desarrollo de nuevas terapias contra el cncer. Una va
para la obtencin de estas CSC es la formacin de esferoides multicelulares
tumorales (MCTS), un modelo de cncer in vitro generado a partir de la
agregacin de clulas en suspensin. Estudios recientes muestran niveles
elevados del factor de transcripcin TCFL5 despus de la formacin
de MCTS por la lnea celular de cncer de colon HT-29. El gen TCFL5
codifica para al menos cuatro isoformas de las cuales, de solo dos, se tienen
referencias a nivel experimental, denominadas TCFL5 y CHA. TCFL5
presenta la secuencia cannica compuesta por 6 exones mientras que CHA
carece del primer exn. Para determinar el papel de estas isoformas en la
formacin de MCTS y de CSC in vitro se sobreexpresaron las isoformas
TCFL5 y CHA en diferentes lneas de cncer de colon y se estudi el
fenotipo tumoral de estas clulas as como la expresin de marcadores
tpicos de CSC. Adems, se generaron tumores xenoinjertados a partir de
estas clulas para determinar el crecimiento tumoral in vivo. Los resultados
obtenidos muestran que, in vitro, las lneas celulares que sobreexpresan
CHA presentan un fenotipo ms tumoral y mayor expresin de marcadores
de CSC que las lneas sobreexpresantes de TCFL5 que presentan el fenotipo
opuesto. In vivo, sin embargo, las lneas que sobreexpresan CHA crecieron
ms lentamente que las TCFL5.
P18r-13
Regulation of TtgGHI efflux pump by indole

increases antibiotic resistance of Pseudomonas
putida DOT-T1E
Carlos Molina Santiago1, Abdelali Daddaoua1, Juan Luis Ramos2
1
Consejo Superior de Investigaciones Cientficas. Estacin
Experimental del Zaidn, Granada, ES, 2ABENGOA RESEARCH,
Sevilla, ES
In Gram-negative bacteria, multidrug efflux pumps are responsible for
the extrusion of chemicals. Some of these efflux pumps are induced by
endogenously produced effectors, while abiotic or biotic signals induce the
expression of other efflux pumps. In Pseudomonas putida, the TtgABC
efflux pump is the main antibiotic extrusion system and it is modulated
by TtgR regulator. The plasmid-encoded TtgGHI efflux pump in P. putida
DOT-T1E, regulated by TtgV regulator, plays a secondary role in antibiotic
resistance in the parental strain; however, its role is critical in isogenic
backgrounds deficient in TtgABC. TtgV recognizes indole as effector
which is not produced by Pseudomonas species, therefore, the indoledependent antibiotic resistance seems to be part of an antibiotic resistance
programme at the community level.
Transcriptomic analyses revealed that the indole specific response involves
the expression of the TtgGHI pump but also a set of genes involved in
iron homeostasis, as well as genes for amino acid catabolism. In a
ttgABC-deficient P. putida, background ampicillin and other bactericidal
compounds lead to cell death. Co-culture assays of E. coli and P. putida
ttgABC allowed growth of P. putida mutant in the presence of ampicillin
162
Crosstalk between HGF and BMP9 signaling

pathways in hepatic progenitor oval cells
Annalisa Addante1, Mara Garca-lvaro1, Nerea Deleyto1, Margarita
Fernndez1, Csar Roncero1, Isabel Fabregat2, Blanca Herrera1,
Arnzazu Snchez1
1
Instituto de Investigacin Sanitaria, Hospital Clnico San Carlos
(IdISSC). Dep Bioqumica y Biologa Molecular II, Facultad
de Farmacia, Universidad Complutense de Madrid, Madrid,
ES,2Laboratori dOncologia Molecular, Universitat de Barcelona,
Institut dInvestigaci Biomdica de Bellvitge, LHospitalet de
Llobregat, Madrid, ES
Oval cells constitute a bi-potential progenitor cell population from adult
liver. Under chronic liver disease (CLD), they become activated and start
to proliferate and differentiate into cholangiocytes and/or hepatocytes to
compensate for the cellular loss and maintain liver homeostasis. It is well
recognized the critical role of the TGF-b in CLD, but the role of other
members of the TGF-b superfamily, the BMPs, is only partly understood.
We focused our attention in BMP9, since previous results from our group
and others indicated that BMP9 has a pro-tumorigenic action in HCC cells.
To study BMP9 role in hepatic progenitor cells, we used an in vitro model
of oval cell lines harbouring a genetically inactivated met tyrosine kinase
(Met-/- cells) and its control (Metflx/flx cells) that also allow us to explore a
possible signaling interaction between HGF and BMP9.
Our data show that BMP9 induces canonical and non-canonical signaling
pathways in both Metflx/flx and Met-/- oval cells, although Met appears to
increase BMP9-induced Smad1/5/8 activation. Interestingly, BMP9
decreases oval cell number, effect that is amplified in absence of Met. To
elucidate the underlying mechanisms; we investigated a potential effect of
BMP9 on apoptotic oval cell death. Our results show that BMP9 elicits a
moderate but significant increase in apoptosis, which is more pronounced
in Met-/- cells. Furthermore, HGF is able to prevent BMP9 effects in oval
cells.
All these data suggest that BMP9 is a suppressor (pro-apoptotic) factor
in oval cells and they also evidence a novel functional crosstalk between
Met/HGF and BMP9 signaling pathways in oval cell survival, with a
mechanism that will be discussed.
P18r-15
The Calcineurin splicing isoform CnA1

is implicated in early embryonic stem cell
differentiation and reduces pathological cardiac
hypertrophy
Jess Mara Gmez-Salinero, Mara Villalba-Orero, Marina
Mercedes-Olaeta, Paula Ortiz-Sanchez, Enrique Lara-Pezzi
Centro Nacional de Investigaciones Cardiovasculares (CNIC),
Madrid, ES
Embryonic stem cells (ESCs) have the ability to proliferate indefinitely
in culture, and to differentiate into all embryonic lineages. Although
the transcriptional program that coordinates pluripotency has been
progressively unveiled during the past few years, the signalling pathways
that regulate early differentiation events are not completely understood.
We have recently described that CnA1, an alternative splicing variant of
the phosphatase calcineurin, enhances muscle regeneration and improves
cardiac function after myocardial infarction. CnA1 lacks the autoinhibitory
domain typical of calcineurin, and instead has a unique C-terminal domain
with no similarity to any known protein. Unlike other calcineurin isoforms,
CnA1 has no impact on NFAT-regulated genes and instead activates the
Akt pathway. Interestingly, we have currently determined that CnA1 is
Granada 2014
specifically located at the Golgy contrary to other calcineurin isoforms, and
that this localization is mediated by its unique C-terminal domain. CnA1
is strongly expressed in stem and progenitor cells, although its role in these
cells is unknown. We found that CnA1 downregulation had no effect on
ESC pluripotency. However, CnA1 depletion in mESCs during the first
48 hours of differentiation specifically affected differentiation towards
the cardiac mesoderm lineage promoting hematopoietic differentiation.
In contrast, CnA1 overexpression promoted differentiation towards
the cardiac mesoderm lineage. Our results suggest that CnA1 regulates
mESC differentiation through the control of the Akt/GSK3 signalling
pathway. Interestingly, CnA1 knockout mice show increased cardiac
hypertrophy in response to trans-aortic banding, whereas transgenic
mice overexpressing CnA1 have decreased hypertrophy and improved
cardiac function. Together, these results suggest that CnA1 is implicated
in early mESCs differentiation, and that it reduces pathological cardiac
hypertrophy.
P18-16
A proteomics strategy reveals novel roles for

the protein kinase DYRK1A within the nucleus
Julia Roewenstrunk1, Chiara Di Vona1, Eva Borras2, Eduard Sabido2,
Susana de la Luna1
1
Centro de Regulacin Genmica-CRG, Barcelona, ES, 2Proteomics
Unit, Centro de Regulacin Genmica-CRG y Universitat Pompeu
Fabra-UPF, Barcelona, ES
The protein kinase DYRK1A (dual-specificity tyrosine-regulated kinase)
is an evolutionary conserved kinase highly sensitive to gene dosage
alterations. Reduced levels of DYRK1A in individuals due to gene
truncation or microdeletions cause microcephaly, intrauterine growth
retardation and developmental delay. On the other hand, DYRK1A
overexpression is associated to some neuropathological traits in Down
syndrome. Evidence obtained in different model systems indicates that
DYRK1A regulates brain growth across evolution, by playing a role in
neuron survival and differentiation. These activities have been associated to
DYRK1A-mediated phosphorylation of mostly cytosolic targets. However,
DYRK1A is also localized in the nucleus, but information on functional
roles for nuclear DYRK1A is still pretty scarce. To search for the identity
of physiological targets of nuclear DYRK1A, we have implemented a
quantitative interaction proteomic approach based on DYRK1A affinity
purification from nuclear extracts followed by label-free quantification
mass spectrometry. By using statistical values, we have generated a rankedlist of putative interactors that includes already known DYRK1A partners
as the scaffold DCAF7 or members of the 14-3-3 family of proteins. To
gain insight into the biological role of nuclear DYRK1A, we have looked
for the enrichment of gene ontology (GO) terms for the proteins found
in the screen. A significant enrichment in GO terms associated with RNA
splicing, transcription and DNA repair was found. We have validated
the interaction of DYRK1A with several of its putative targets by direct
and reverse co-immunoprecipitation experiments. Moreover, we have
generated experimental evidence indicating that some of the DYRK1A
putative interactors are novel DYRK1A substrates. In summary, we have
defined a nuclear DYRK1A interactome that will be a powerful tool for
the characterization of the functional participation of DYRK1A in specific
nuclear processes.
P18-17 (R18-2)
Regulation of satellite cell functions in aging

Eusebio Perdiguero Santamara
Universidad Pompeu Fabra (UPF), Barcelona, ES
Among the principal properties that distinguish adult mammalian stem
cells from their more differentiated progeny is the capacity of stem cells
to remain in a quiescent state for prolonged periods of time. However, the
molecular pathways for the maintenance of stem-cell quiescence remain
Psters
largely unknown. Recent studies reported that, in skeletal muscle of old
mice, the muscle fiber expresses grow factors, driving satellite cells to
break quiescence by undergoing proliferation, and this would explain the
previously accepted skeletal muscle regenerative decline with aging. A
deep analysis of muscle stem cell aging has provided us with a novel vision
on why aged muscle stem cells lose their homeostatic quiescent state.
In very old, geriatric mice, satellite cells do not undergo a proliferative
pathway; instead, they switch to a pre-senescence stage. This switch
has dramatic consequences, since it converts the satellite cell reversible
G0 arrest into an irreversible G0 arrest that abrogates cell activation
upon muscle injury, thus precluding efficient regeneration and stem cell
self-renewal in aged mice. We have established that satellite cells from
sarcopenic geriatric, unlike those from non-sarcopenic old mice, enter
into a point of no-return, and lose their reversible quiescence state in
homeostatic conditions. We have also found this loss of quiescence in
satellite cells of progeric mice with accelerated aging and in samples from
aged humans.
Mechanistically, we have found that geriatric and progeric satellite cells
express senescence markers like p16INK4a. Indeed, p16INK4a, which is
never expressed in adult or old muscle stem cells in homeostatic conditions,
becomes de-repressed in geriatric and progeric satellite cells, correlating
with loss of reversible quiescence. Genetic silencing of p16INK4a in
geriatric and progeric satellite cells reverses G0 irreversible senescence into
a G0 reversible quiescence, thus allowing stem cell activation and muscle
regeneration in aged mice. Our results further show that in conditions of
muscle injury, in which satellite cells need to proliferate extensively to form
new myofibers, geriatric satellite cells are unable to proliferate and undergo
instead an accelerated entry into a deep senescence state (geroconversion).
We propose that satellite cell geroconversion arises from two opposed
forces: the proliferative pressure of the damaged muscle local environment
and the persistent cell cycle arrest induced by a p16INK4a -regulated Rb/
EF2 anti-proliferative axis on aged satellite cells.
P18r-18
Function of C3G as a regulator of cell migration/

invasion and tumour growth
Celia Sequera1, Neibla Priego1, Mara Arrechederra1, Ana Vzquez1,
Sara Ortiz-Rivero2, Arnzazu Snchez1, Carmen Guerrero2,
Almudena Porras1
1
Farmacia, UCM, IdISSC, Madrid, ES, 2Centro de Investigacin del
Cncer IBMCC (USAL-CSIC), IBSAL, Salamanca, ES
C3G is a guanine nucleotide exchange factor (GEF) for Rap1 and R-Ras,
but it also acts through mechanisms not dependent on its GEF activity. It
is essential for embryonic development due to its role in integrin-mediated
adhesion and migration. In addition, C3G inhibits migration of highly
invasive breast carcinoma cells, although the mechanisms involved remain
unknown. The function of C3G in human cancer is controversial acting as
either a tumour suppressor or mediator.
p38 MAPKs regulate different cellular functions, including migration.
p38a MAPK is the most abundant isoform and essential for embryonic
development. p38a plays also a dual role in cancer depending on the tumour
type and stage and it can promote migration and invasion of tumour cells.
We have previously described that C3G through inhibition of p38a MAPK
regulates cell death. We have now analyzed if C3G also regulates migration
and invasion through mechanisms dependent on p38a in non-tumour and
tumour cells. By knocking-down/out C3G and/or p38a, we have found that
C3G inhibits migration and invasion through a mechanism that involves
down-regulation of p38a activity. This was confirmed by using a selective
p38a/b inhibitor. In addition, we have demonstrated that Rap-1 activation is
not required for these C3G actions. MMPs might be important mediators of
C3G/p38 effects on invasion. We are further characterizing the mechanisms
used by C3G and/or p38a to regulate these processes.
We have also found that both C3G and p38a promote tumour growth of
HCT116 cells, as determined by in vitro and in vivo assays.
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P18-19
P18r-21 (R18-6)
DUSP10 is a cyclooxygenase 2 activity induced

gene and it regulates colon cancer cell stress
responses
P2X7 receptor stimulation in neurons mediates

different pathway activation in the presence or
absence of calcium
Marta Jimnez Martnez, Konstantinos Stamatakis, Alba JimnezSegovia, Beatriz Barrocal, Manuel Fresno
Centro de Biologa Molecular Severo Ochoa (UAM-CSIC), Madrid, ES
Bakarne Urzelai1, Francisco Llavero1, Jos Luis Zugaza2

1
Universidad del Pas Vasco (UPV/EHU)/Centro Vasco de
Neurociencias Achucarro, Leioa, ES, 2Ikerbasque/Centro Vasco de
Neurociencias Achucarro/Universidad del Pas Vasco (UPV/EHU),
Leioa, ES
Colorectal cancer is one of the deadliest types of cancer, due to its high
metastatic capacity and chemoresistance acquisition. Cyclooxygenase 2
(COX-2) elevated expression has been shown to play an important role in
colorectal tumour development and its inhibition can help prevent cancer
death.
We performed gene expression microarray analysis to identify genes
up- or down-regulated by COX-2 overexpression. We identified genes
with altered expression in these conditions, one of them being the Dual
Specificity Phosphatase 10 (DUSP10), specific for JNK and p38 MAPKs.
DUSP10 resulted to be up-regulated by COX-2 activity as well as by PGE2
treatment in a concentration depended manner.
We generated cell lines stably expressing DUSP10 or silencing DUSP10
by infecting with lentivirus the colorectal adenocarcinoma HT29-LucD6
cells. We found that DUSP10 are implicated in cell proliferation and stress
resistance to serum deprivation, osmotic and genotoxic agents and to
confluence arrest. DUSP10 could be regulating the HT29 cell response at
these conditions through the p38 pathway.
These results suggest that DUSP10 expression can confer a growth and
survival advantage in tumor environment to colorectal cancer cells.
P18-20
Identification of PMEPA1 as a cyclooxygenase

2 induced gene and its potential implication in
cancer progression
Alba Jimnez Segovia, Konstantinos Stamatakis, Marta Jimnez,
Beatriz Barrocal, Manuel Fresno
Centro de Biologa Molecular Severo Ochoa (UAM-CSIC), Madrid, ES
Cyclooxygenase 2 (COX-2) elevated expression has been associated
with tumour development especially in colorectal cancer, and it is known
that its inhibition can prevent cancer. In order to study the mechanisms
that mediate the pro-tumorigenic effects of COX-2 we generated cell
lines stably overexpressing this gene and performed gene expression
microarray analysis to identify genes regulated by this overexpression.
One of the up-regulated genes in the COX-2 overexpressing cells is
PMEPA1, identified as an androgen-induced gene, highly expressed in
several cancers. The encoded product is a transforming growth factor-
(TGF-)-induced transmembrane protein overexpressed in breast,
prostate and colorectal cancer. In order to study PMEPA1 effects, we both
overexpressed and silenced, PMEPA1 in human colon adenocarcinoma
(HT29) and human ovarian carcinoma (Skov3) cells, generating stable
cell lines. We studied in vivo and in vitro cell characteristics, noting
significant differences between empty vector (EV) and PMEPA1
overexpressing cells. We observed differences in cell morphology, caused
by a different distribution of the actin cytoskeleton. Those morphological
differences are related to a PMEPA1- induced process of Epithelial
Mesenchymal Transition.
Mouse xenograft experiments, with subcutaneous and intraperitoneal
injection of the mentioned cells in nude mice, showed a differential effect
of PMEPA1 overexpression in the two cell types, HT29 and Skov3,
leading to a final growth delay of HT-29 xenografts, while PMEPA1
overexpressing Skov3 cells had a high initial survival and proliferative
advantage compared with EV cells.
These findings suggest that PMEPA1 may have an important role in cancer
progression.
164
P2X7 belongs to the family of ionotropic ATP-gated receptors, which

function as ion channels mediating calcium influx. P2X7 is one of the
predominant purinergic P2X receptor subtype expressed in neural cells.
It is involved in a variety of processes such as ATP-mediated cell death,
regulation of receptor trafficking, and inflammation. It is largely known
that P2X7 receptor stimulation leads to regulation of the mitogen-activated
protein kinases (MAPK) extracellular regulated kinases (ERK1/ERK2),
c-Jun N-terminal kinase (JNK) and p38. The canonical pathway for the
activation of these MAPKs is mediated by Ras.
Here, we show that P2X7 receptor stimulation triggers not only the MAPK
activation but also the Ras GTPase activation. Besides, we demonstrate that
in the presence of calcium this Ras/MAPK pathway regulation is mediated
by the Ras guanine exchange factor (GEF) RasGRF1. For its activation,
this GEF requires a calcium input from the extracellular medium, which
will intracellularly bind to calmodulin. In this configuration, Ca+2-loaded
calmodulin binds to the RasGRF1 IQ motif and activates it. Surprisingly,
P2X7 also activates Ras in a Ca+2-independent manner. In addition, this
alternative Ras activation leads to the activation of the small GTPase Rap1
and more importantly, triggers neural cell death. Our findings demonstrate
that P2X7 stimulation in neurons activates two different signaling pathways
depending on the presence or absence of extracellular Ca+2, leading to cell
survival versus cell death.
P18-22 (R18-1)
Tetraspanin-enriched microdomains. Translating

signals from the plasma membrane to Rac
GTPase and exosomes
Mara Yez-M
Universidad Autnoma de Madrid, Madrid, ES
Tetraspanin-enriched microdomains (TEM) have unique biophysical
properties suited for the regulation of cell motility and intercellular adhesion.
In this study we demonstrate that TEM play a role in the compartmentalization
of Rac GTPase at the plasma membrane and into exosomes, providing a
molecular mechanism for the regulation of cell motility by tetraspanins.
We characterized the intracellular TEM interactome by high-throughput
mass-spectrometry, and found a specific interaction of tetraspanin CD81
with the GTPase Rac. This interaction was confirmed biochemically and
microscopy cross-correlation analysisthat demonstrated in situ integration of
both molecules into the same molecular complex. Pull-down experiments
revealed that CD81-Rac interaction was direct and independent of Rac
activation status. Knockdown of CD81 resulted in enhanced protrusion rate,
altered focal adhesion formation and decreased cell migration, correlating
with increased active Rac. Re-expression of wild typeCD81, but not
its truncated form lacking the C-terminal cytoplasmic domain, rescued
these effects. The phenotype of CD81 knockdown cells was mimicked
by treatment with a soluble peptide with the C-terminal sequence of the
tetraspanin. TEM-driven compartmentalization was also relevant for protein
sorting into exosomes. Quantitative proteomics showed that TEM ligands
account for a great proportion of the exosome proteome and that a selective
repertoire of CD81-associated molecules, including Rac, is not correctly
routed to exosomes in cells from CD81-deficient animals. Therefore TEMdependent compartmentalization is revealed as a new regulatory mechanism
of Rac activity. Moreover, our data provide firm evidence that insertion into
TEMs is critical for protein inclusion into the exosome structure.
Granada 2014
P18r-23
GRK2 modulates cell proliferation and invasive

migration of the highly metastatic MDA-MB-231
breast cancer cells
Laura Nogus1, Philippe Chavrier2, Federico Mayor Jr.1, Petronila
Penela3
1
Centro de Biologa Molecular Severo Ochoa (CSIC-UAM), Madrid,
ES, 2Membrane and cytoskeleton dynamics department, Institut
Curie, Paris, FR, 3Instituto de Investigacin Sanitaria La Princesa,
Madrid, ES
Breast cancer progression and outcome are conditioned by the acquisition
of two key abilities by tumour cells: the growth and survival capability that
trigger resistance to therapy and the invasion into host tissues resulting
in metastatic dissemination. Although both processes are usually studied
separately, the underlying mechanisms seem to be governed by the same
signalling nodes, including growth factor and chemokine receptors, p53
mutations and components of the Ras/ MAPKs/ PI3K axis. In addition, other
cancer-associated factors can cooperate with these oncogenic-signalling
routes to strength tumoural properties. In this sense, the serine/threonine
kinase GRK2 is emerging as a key signalling node tightly connected to
these cascades, given its participation in relevant biological processes such
as cell cycle, proliferation or migration. Moreover, our recent findings
show a novel interaction of GRK2 with the histone deacetylase HDAC6,
which has also been associated with malignant transformation and invasive
motility in breast cancer. All these evidences strongly suggest a relevant
contribution of GRK2 in breast cancer tumorigenesis. We report herein that
GRK2 subcellular distribution is altered in transformed breast cancer cells.
Moreover, GRK2 modulates in vitro and in vivo proliferation of the triple
negative MDA-MB-231 cells, in a HDAC6- phosphorylation dependent
manner, and protects from SAHA (an HDAC6 inhibitor)-induced cell
death. Furthermore, GRK2 also potentiates MDA-MB-231 cell migration
towards different oncogenic stimuli, whereas GRK2 down-regulation
results in suppression of both cell migration and invasion in 2D and 3D
models. Finally, GRK2 is required for two-dimensional matrix proteolysis,
suggesting a relevant role of GRK2 in the formation of protrusive structures
termed invadopodia. Overall, these results point GRK2 as a possible
contributor towards cell malignancy and as a potential drug target.
P18r-24
K-Ras modula la respuesta fibrtica inducida

por TGF-1 mediante mecanismos independientes
de la va de Smad
Cristina Cuesta Apausa1, Jos Manuel Muoz Fliz1, Nuria Perretta
Tejedor1, Carlos Martnez Salgado2, Isabel Fuentes Calvo1
1
Unidad de Fisiopatologa Renal y Cardiovascular, Instituto
de Investigacin Biomdica de Salamanca-IBSAL, Salamanca, ES,
2
Sofa de Investigacin Nefrolgica, Departamento de Fisiologa y
Farmacologa, Universidad de Salamanca - Instituto de Estudios
de Ciencias de la Salud de Castilla y Len-IECSCYL, Unidad de
Investigacin, Hospital Universitario de Salamanca, Salamanca, ES
La fibrosis es uno de los estadios finales de muchas enfermedades crnicas.
Se caracteriza por la presencia de miofibroblastos y por una deposicin
excesiva de protenas de matriz extracelular (MEC). El factor de
crecimiento transformante-1 (TGF-1) es una citoquina muy importante
en la regulacin de los procesos fibrticos. Sealiza a travs de las protenas
Smad2 y Smad3 gracias a la activacin de su receptor tipo I. Adems puede
activar otras vas de sealizacin, como la va de Ras. Hay 3 isoformas de
las protenas Ras: H-, N- y K-Ras.
Nuestro objetivo es determinar si K-Ras regula la expresin de protenas de
MEC inducida por TGF-1 y a travs de que ruta de sealizacin intracelular.
Psters
Para ello hemos generado una lnea de fibroblastos embrionarios knock-out
de K-Ras (KO). La expresin proteica se ha analizado por western blot.
Las expresiones basales de fosfo-Smad2 y fosfo-Smad3 son similares en
fibroblastos KO de K-Ras y WT; sin embargo la expresin de fosfo-Erk1/2
es menor en fibroblastos K-Ras-/-, mientras que la expresin de fosfo-Akt
es mayor en estas clulas. TGF-1 aumenta la expresin de Ras-GTP y la
fosforilacin de Erk1/2 slo en fibroblastos WT, e induce una fosforilacin
similar de Smad2 y Smad3 en fibroblastos WT y K-Ras-/-. Adems, TGF-1
estimula la expresin de fibronectina, colgeno tipo I y colgeno total slo
en fibroblastos WT. En ausencia de K-Ras la inhibicin de la va de Erk1/2
no reduce la expresin de fibronectina y colgeno tipo I. Sin embargo, la
inhibicin de la va PI3K/Akt disminuye la sntesis de colgeno tipo I y de
fibronectina en fibroblastos WT y KO.
Estos datos sugieren que la isoforma K-Ras regula negativamente la
expresin basal de protenas de MEC (a travs de la va de PI3K/Akt),
regula positivamente la activacin de la va de Erk1/2, y es necesaria para
la activacin de Erk1/2 en respuesta a TGF-1, pero no para la activacin
de la Smad2 y 3. Adems, K-Ras es necesaria para el incremento en la
expresin de protenas de la MEC inducido por TGF-1, fundamentalmente
a travs de la va de Erk1/2.
P18-25
Chemotaxis of Pseudomonas to gammaaminobutyrate (GABA)

Jose Antonio Reyes Darias*1, Vanina Garca*1, Miriam Rico
Jimnez1, Andrs Corral Lugo1, Olivier Lesouhaitier2, Dalia Jurez
Hernndez3, Yiling Yang4, Shuangyu Bi4, Marc Feuilloley2, Jess
Muoz Rojas3, Victor Sourjik4, Tino Krell1
1
Department of Environmental Protection, Estacin Experimental del
Zaidn, Consejo Superior de Investigaciones Cientficas, Granada,
ES, 2Laboratory of Microbiology Signals and Microenvironnement
LMSM, EA 4312, Normandie Universit, Universit Rouen, Evreux,
FR, 3Laboratorio de Ecologa Molecular Microbiana, Centro de
Investigaciones en Ciencias Microbiolgicas-Instituto de Ciencias,
Benemrita Universidad Autnoma de Puebla, Puebla, MX, 4Max
Planck Institute for Terrestrial Microbiology & LOEWE Research
Center for Synthetic Microbiology (SYNMIKRO), Marburg, DE
Bacteria are able to migrate toward a more suitable environment by
detecting chemical and physical stimuli, in a process called chemotaxis.
Environmental stimuli are sensed by chemoreceptors, so called methyl
accepting chemotaxis proteins (Mcp), which is typically composed of
a periplasmic ligand-binding domain (LBD) and a cytosolic signalling
domain. Chemotaxis occurs primarily to growth substrates but was also
reported for neurotransmitters, plant signals or quorum sensing signals and
the physiological relevance of taxis to these compounds is poorly understood.
Gamma-aminobutyrate (GABA) is ubiquitously present in nature and
has many functions like being a neurotransmitter, plant signal or growth
substrate. We show by microcalorimetric analysis that the recombinant LBDs
of the chemoreceptors PctC in the pathogenic Pseudomonas aeruginosa and
McpG in the saprophytic P. putida KT2440 bind GABA. Receptor chimeras
comprising the LBD of either PctC or McpG with the Tar signaling domain
were constructed and the signal output determined using fluorescence
resonance energy transfer (FRET). The resulting EC50 values for GABA
were with 3.6 0.3 nM (PctC-Tar) and 5.7 2 nM (McpG-Tar) extremely
low, indicating that both receptors are highly sensitive for GABA. The EC50
of PctC-Tar for GABA was 3100-fold inferior to the value for L-Pro, the
strongest responding protein amino acid. Experiments with Caenorhabditis
elegans showed that pctC mutation did not alter bacterial virulence. However,
mcpG mutation caused a reduction in tomato root colonization following
chemotaxis. The C. elegans data and the presence of a GABA receptor in a
saprophytic indicate that GABA taxis may not be related to virulence. The
environmental omnipresence of GABA and its multi-functionality suggests
that other bacteria may also show GABA taxis.
* Authors contributed equally to this work.
165
Psters
P18-26
grupo ha demostrado que mientras la sealizacin tumoral por Wnt

conduce al acmulo citoslico del efector de la va: la -catenina, su
retencin nuclear y activacin transcripcional (responsable del aumento
de proliferacin y otras caractersticas tumorales) requiere la existencia
de niveles elevados de glucosa. Esta acumulacin nuclear dependiente de
hiperglucemia est mediada parcialmente por inhibicin de la actividad
desacetilasa de las sirtuinas y, aunque hay evidencia de la implicacin de
SIRT1, se desconoce si hay otras sirtuinas implicadas y cules son los
mecanismos subyacentes.
Recientemente se ha demostrado que el silenciamiento de SIRT6
altera el metabolismo de la glucosa y conduce a una disminucin de la
glucemia y, por consiguiente, de la insulinemia. A diferencia de otras
sirtuinas los niveles de SIRT6 fluctan de unos tipos celulares a otros y
esta sirtuina adems de la actividad desacetilasa posee actividad ADPribosilasa.
Los importantes efectos de SIRT6 sobre el metabolismo de la glucosa nos
han conducido a plantear el Objetivo: identificar estmulos que alteran los
niveles y actividad de SIRT6 y su relacin con el fenotipo tumoral.
Metodologa: clulas tumorales intestinales humanas y murinas
estimuladas con: glucosa, Wnt-3A o LiCl. Se evalan cambios en niveles
o modificaciones por Western Blot e inmunofluorescencia; en expresin
gnica por qPCR y en actividad desacetilasa por luminiscencia. Mediante
citometra determinamos su influencia en proliferacin celular. Anlisis
estadstico por ANOVA y post-test Bonferroni.
Resultados: Las protenas Wnt-3A y LiCl reducen significativamente los
niveles de SIRT6 afectando a la actividad desacetilasa. Las alteraciones
de los niveles de SIRT6 correlacionan con alteraciones de la proliferacin
celular.
Conclusiones: Algunos estmulos como Wnt o LiCl pueden facilitar la
aparicin o progresin tumoral a travs de la deplecin ejercida sobre SIRT
6.
Characterization of a new human oncogene

Brbara de la Pea, Cristian Surez, Angustias Page, Josefa P.
Alameda, Llanos Casanova, ngel Ramrez, Manuel Navarro
CIEMAT, Madrid, ES
Most human tumors have mutations in genes of the Ras small GTPase
protein family. Ras proteins work as binary molecular switches that
control intracellular signaling networks. Inappropriate activation of
ras causes aberrant signal transduction, proliferation and malignant
transformation. Oncogenic mutations in Ras prevent GTP hydrolysis,
locking Ras in a permanently active state. The main Ras isoforms are
H-Ras, N-Ras and K-Ras, mutations of which are found in several types
of human tumors. In spite of this, the human Ras family consists on
some 36 different genes, many of which have been scarcely studied.
One of these relatively unkonwn genes is ERas (Embryonic Ras), which
is a constitutively active Ras protein. ERas is located in chr X, and is
expressed only in embryonic stem cells, being undetectable in adult
tissues.
In the course of a high-throughput genetic screening to identify novel genes
mutated in human cancer, we detected insertional mutations leading to the
activation of ERas in a number of tumors. In this work, we describe the
cloning and introduction of ERas in several non-malignant human cell
lines. Forced expression of a HA-tagged ERas results in drastic changes in
cell shape, mobility and invasivity, as well as in the activation of specific
downstream pathways. Our results suggest that inappropriate expression of
ERas could have a role in human cancer.
P18-27 (R18-5)
Identificacin de Hit1 como un nuevo regulador

de la salida de mitosis en Saccharomyces
cerevisiae
Ana Isabel de los Santos Velzquez, Fernando Monje Casas
(CABIMER), Utrera, ES
En la levadura de gemacin Saccharomyces cerevisiae, la salida de
mitosis se produce gracias a la liberacin de Cdc14 desde el nucleolo hasta
el citoplasma, donde esta fosfatasa revierte los eventos de fosforilacin
determinados por las ciclinas mitticas, lo cual conduce finalmente a la
inactivacin de las mismas. La liberacin de Cdc14 se produce gracias
a dos cascadas de sealizacin: la ruta FEAR (Cdc-Fourteen Early
Anaphase Release) y la ruta MEN (Mitotic Exit Network). Aunque no
es esencial, FEAR juega un papel importante en la correcta segregacin
del ADNr y los telmeros, la dinmica del huso mittico, y la activacin
de MEN. As, la identificacin de nuevos componentes de esta ruta es
importante para entender mejor el proceso de salida de mitosis. En nuestro
grupo de investigacin, hemos identificado la protena Hit1, de funcin
desconocida hasta el momento, como un posible nuevo componente de
la ruta FEAR, y estamos analizando el papel especfico que esta protena
juega en la correcta regulacin de la liberacin de Cdc14 y la salida de
mitosis.
P18r-28
Regulacin de la desacetilasa de histonas SIRT6

en relacin con el fenotipo tumoral
Mara Gutirrez-Salmern1, Valle Montalvo-Romeral1, Ana ChocarroCalvo2, Jos Manuel Garca-Martnez1, Antonio De la Vieja3,
1
Facultad Ciencias de la Salud. Universidad Rey Juan Carlos,
Alcorcn, ES, 2Ludwig Institute for Cancer Research, University of
Oxford, Oxford, UK, 3Instituto de Salud Carlos III, Majadahonda, ES
Introduccin: La hiperglucemia favorece el desarrollo tumoral con
efectos directos sobre las clulas tumorales y efectos sistmicos. Nuestro
166
P18r-29
Interleukin 2 requieres p56Lck/PLCy/nPKCs

(PKC)/PIX pathway to induce Rac 1-PYGM
activation in T cells
Francisco Llavero Bernal1, Bakarne Urzelai Lpez de Abersturi1,
Jos Luis Zugaza Gurruchaga2
1
Universidad del Pas Vasco (UPV/EHU), Leioa, ES, 2Ikerbasque/
Centro Vasco de Neurociencias Achucarro/Universidad del Pas
Vasco (UPV/EHU), Leioa, ES
Small GTPases of the Rho family have been implicated in important
cellular processes such as cell migration and adhesion, protein secretion,
and/or gene transcription. However, little is known about the role that
Rho GTPases play in IL-2-mediated responses in Kit 225 T cells. Our
group has shown that IL-2 induces Rac 1 activation, which interacts
with PYGM and modifies its activity to trigger T cell proliferation.
We have investigated the mechanisms by which PYGM is activated
through Rac 1 in T cells. Our results suggest that IL-2 stimulates
-PIX/Rac 1/PYGM pathway activation through the novel Protein
kinase C theta type (PKC). Furthermore, by constructing PIXS225A y
PIXS488A mutants we show that these residues are essential for PYGM
activation. Moreover, it has been published that subunit of the IL-2receptor activates p56Lck which in turns activates the phosphoinositide
phospholipase C-gamma (PLC). Here, we have observed that p56Lck
and PLC are implicated in the IL-2 signaling cascade that leads to
Rac1/PYGM pathway activation.
These data reveal a novel signaling pathway in which IL-2 transduces
signals through the Lymphocyte cell-specific protein-tyrosine kinase
(p56Lck)/Phosphoinositide phospholipase C-gamma (PLC)/novel Protein
kinase C theta type (PKC)/Rho guanine nucleotide exchange factor PIX
/Rac 1 axis to activate PYGM.
Granada 2014
P18r-30
Novel functional complexes between Galphaq

and PB1 domain-containing proteins
Sofa Cabezudo Violero1, Guzmn Snchez Fernndez2, Catalina
Ribas Nez2, Federico Mayor Menndez2
1
Centro de Biologa Molecular Severo Ochoa (CBMSO), Instituto
de Investigacin Sanitaria La Princesa, Madrid, ES, 2Max-PlanckInstitute for Heart and Lung Research, Centro de Biologia Molecular
Severo Ochoa (CSIC-UAM) and Instituto de Investigacin
Sanitaria La Princesa, Madrid, ES
Introduction: G proteins play an essential role in the initiation of G-protein
coupled receptor (GPCR) signalling. Particularly, the Gq/11 family has
been classically shown to associate and activate PLC. However, additional
effectors are responsible for other Gq functions. We have reported that
GPCR activation promotes a direct interaction between Gq and PKC.
This association involves the basic PB1-type II domain of PKC and a
novel acidic region in Gq different from the classical effector-binding
region. This interaction leads to the stimulation of the ERK5 pathway
and to the development of cardiac hypertrophy, independently of the
canonical effector PLC. In this work, we have described that another PB1
containing protein, such as p62, belong to this Gq/PKC complex and
explored additional downstream pathways for this new Gq interactome.
Methods: Biochemical characterization of the Gq/p62 complex
was performed by mutagenesis and co-immunoprecipitation analysis.
Functionality of the Gq/PKC complex was determined performing
XCELLigence, propidium iodide incorporation and annexinV assays.
Results: We demonstrate that Gq and p62 can be found in the same
functional protein complex. This association is enhanced upon Gq
activation by GPCR. Furthermore, GRK2, a negative regulator of Gq
signalling, abrogates this complex. This association involves the PB1
domain of p62 and the acidic region of Gq, described to be essential for
PKC binding. Interestingly, this association is preserved in PKC deficient
cells, suggesting a direct interaction. Additionally, we show that the Gq/
PKC/ERK5 complex links Gq to an apoptotic cell death pathway which
is autophagy-dependent.
Conclusion: Overall, our study provides concluding evidence showing
that PKC acts as a functional effector of Gq through the engagement of
a novel binding region in the alpha subunit leading to ERK5 activation and
apoptotic cell death in a mechanism dependent on autophagy where p62
could play an interesting role as part of this complex. These results could
help to uncover the identification of new potential pharmacological targets
for the treatment of cardiovascular diseases.
P18-31
IRF2-Binding Protein Like: implication of

the Ser526 phosphorylation on its cellular
localization, transcriptional activity and its novel
role as growth suppressor
Jon Vallejo1, Aintzane Apraiz2, Dorkaitz Basarte3, Asier Fullaondo1,
Ana M. Zubiaga1
1
Departamento de Gentica, Antropologa Fsica y Fisiologa Animal,
Facultad de Ciencia y Tecnologa, Universidad del Pas Vasco UPV/
EHU, Leioa, ES, 2Departamento de Biologa Celular e Histologa,
Facultad de Medicina y Odontologa, Universidad del Pas Vasco
UPV/EHU, Leioa, ES, 3Dpt Gentica, Antropologa Fsica y Fisiologa
Animal, Fac. de Ciencia y Tecnologa, Universidad del Pas Vasco
UPV/EHU, Leioa, ES
Ras proteins are important molecular switches that control intracellular
signaling networks, and their deregulation is one of the key events leading
to cellular transformation. On-going work in the laboratory has identified
IRF2BPL, an interferon regulatory factor family member, as a new
downstream effector of H-Ras. IRF2BPL encodes a transcription factor
that is thought to play a role in transcriptional regulation of hormones
Psters
such as GNRH1 and PENK. It shows both target dependent transcriptional
activator and repressor activities and it has been described to form a
complex together with IRF2BP1 and IRF2BP2. However, the mechanisms
that underlie the regulation and functional activity of IRF2BPL are largely
unknown. The aim of this study is to characterize the regulation and
function of this protein.
We present evidence that IRF2BPL is predominantly nuclear and shows a
strong transcriptional repressor activity in serum-starved cells. Activation
of signaling pathways by the addition of serum leads to translocation of the
protein to the cytoplasm. IRF2BPL contains a nuclear localization sequence
(NLS) that is highly conserved in the interferon regulatory factor family.
Within the NLS of IRF2BPL2 lies a Serine residue (Ser526) that has been
found phosphorylated in proteomic studies, raising the possibility that this
phosphorylation may be critical for its localization and functional activity.
Substitutions of Serine at position 526 to Alanine (non-phosphorylable)
and Aspartic acid (phospho-mimetic) have been generated by site-directed
mutagenesis. Preliminary results suggest that Ser526 phosphorylation is
important for IRF2BPL localization and function. Moreover, we have
found that IRF2BPL overexpression leads to a decreased colony formation
in both non-transformed and HRasV12-transformed NIH3T3 cells, which
is associated with an early accumulation of cleaved caspase-3, suggesting
increased apoptotic cell death. Taken together, our results point to an
important function of IRF2BPL in the negative regulation of transcriptional
activity and cellular proliferation.
P18r-32
Regulation of c-Src tyrosine kinase activity

by calmodulin
Silviya R. Stateva1, Estefana Anguita1, Valentina Salas2, Gustavo
Benaim3, Antonio Villalobo1
1
Instituto de Investigaciones Biomdicas, Consejo Superior de
Investigaciones Cientficas Universidad Autnoma de Madrid,
Madrid, ES, 2Universidad Central de Venezuela, Facultad de
Ciencias, Instituto de Biologa Experimental, Caracas, VE,
3
Universidad Central de Venezuela, Facultad de Ciencias, Instituto
de Biologa Experimental & Instituto de Estudios Avanzados (IDEA),
Caracas, VE
Calmodulin (CaM) is a Ca2+-receptor protein that regulates more than
one hundred target proteins with and without enzymatic activity in Ca2+dependent and independent manners and modulates a myriad of cellular
processes, some of which are vital for tumorigenesis (i.e. cell proliferation,
apoptosis and autophagy) [1]. c-Src is a non-receptor tyrosine kinase
that participates in signaling pathways that transduces events initiated
by different types of cellular receptors and adhesion molecules, and
controls many cellular functions including: cell migration, proliferation,
differentiation, apoptosis and stress-response, among others [2]. It has been
previously demonstrated that CaM directly interacts with c-Src in a partial
Ca2+-dependent manner and that trifluoperazine, a CaM antagonist, inhibits
the phosphorylation of c-Src, suggesting that CaM plays an important
role in survival signals in pancreatic tumor cells [3]. We demonstrate in
this work that recombinant c-Src directly binds CaM in Ca2+-dependent
and independent manners, suggesting that this kinase likely has more
than one CaM-binding domain. A couple of IQ-like motifs identified
in silico in c-Src could be responsible for the binding of CaM to this
kinase in the absence of Ca2+. We also show that CaM enhances the autophosphorylation and tyrosine kinase activity of c-Src in the absence and
presence of Ca2+, most strongly in the former condition. We are testing
whether the CaM antagonist W-7 inhibits or not the hydrogen peroxideinduced phosphorylation of c-Src in human breast adenocarcinoma SKBR-3 cells, and whether or not CaM-dependent protein kinase II and/or the
CaM-regulated protein phosphatase calcineurin, play a role in this process.
Bibliografa
[1] Berchtold MW, Villalobo A (2014) Biochim. Biophys. Acta Mol. Cell
Res. 1843: 398-435.
167
Psters
[2] Thomas SM, Brugge JS (1997) Annu. Rev. Cell Dev. Biol. 13: 513-609.
[3] Yuan K, Jing G, Chen J, Liu H, Zhang K, Li Y, Wu H, McDonald JM,
Chen Y (2011) J. Biol. Chem. 286: 24776-]24784.
Funded by SAF2011-23494, S2011/BMD-2349 and PITN-GA-2011-289033
to AV.
P18m-33
Papel de la AMPK (protena quinasa activada

por AMP) en la amplificacin de la sealizacin
tumoral por glucosa
Ana Chocarro-Calvo1, Jos Manuel Garca-Martnez2, Mara
Gutirrez-Salmern2, Valle Montalvo-Romeral2, Antonio De la Vieja3,
1
Ludwig Institute for Cancer Research, University of Oxford,
Headington, Oxford, UK, 2Facultad Ciencias de la Salud.
Universidad Rey Juan Carlos, Alcrocn, ES, 3Instituto de Salud
Carlos III, Majadahonda, ES
Estudios epidemiolgicos asocian un aumento en la incidencia de cnceres
sitio-especficos con la diabetes. Las clulas tumorales sufren alteraciones
del metabolismo de la glucosa, de lpidos y de protenas por lo que la
reprogramacin del metabolismo energtico celular ha despertado un fuerte
inters clnico como estrategia para combatir la enfermedad. La protena
AMPK permite a la clula adaptarse al estrs metablico por lo que
representa una diana molecular importante en cncer y diabetes. Nuestro
grupo ha demostrado que la hiperglucemia amplifica la sealizacin
tumoral por Wnt/b-catenina en cnceres asociados a diabetes a travs de
aumentos en niveles y actividad de la acetil transferasa p300.
Objetivos: Caracterizar el papel de la AMPK en la amplificacin de la
sealizacin tumoral por glucosa.
Metodologa: Usamos clulas tumorales intestinales murinas (STC1) y humanas (HT-29) en normo o hiper-glucemia. Los tratamientos
incluyen inhibidores/activadores de AMPK, sobreexpresin y deplecin
de AMPK. Las alteraciones en las protenas se miden por western Blot,
inmunofluorescencia e inmunoprecipitaciones.
Resultados: La hiperglucemia aumenta la fosforilacin de AMPK (T172)
correlacionando con un incremento en los niveles, acetilacin y actividad
acetiltrasferasa de p300. Estos efectos son reproducidos por la activacin
de AMPK mediante Metformina/AICAR o la sobreexpresin de una forma
silvestre y son bloqueados por un inhibidor de AMPK (Compuesto C) o su
deplecin mendiante siRNA especfico.
Conclusiones: La AMPK media la amplificacin de la sealizacin
tumoral por hiperglucemia en cnceres asociados a diabetes y sugiere un
nuevo mecanismo de adaptacin de la clula tumoral a estas condiciones
de estrs metablico.
P18-34
Latexin enhances retinoic acid-mediated

differentiation in human neuroblastoma-derived
SH-SY5Y cells
Carla Granados1, Mara Snchez-Osuna2, Josep Vendrell1, Irantzu
Pallars1, Vctor J. Yuste2
1
Departament de Bioqumica i Biologia Molecular, Facultat de
Cincies i Institut de Biotecnologia i de Biomedicina, Universitat
Autnoma de Barcelona , Barcelona, ES, 2Cell Death, Senescence
and Survival Group, Departament de Bioqumica i Biologia
Molecular and Institut de Neurocincies, Facultat de Medicina,
Latexin, the only known endogenous carboxypeptidase inhibitor, was
initially identified in the lateral neocortex of rat brain and has been used
as a marker of neurons in the developing brain. Latexin also plays a
role in regulating hematopoietic stem cells homeostasis, inflammation,
168

and protein aggregation. Interestingly, latexin expression appears downregulated in several cancer cells because of hypermethylation of its
promoter. Thus, the over-expression of latexin in these cells prevents
tumour growth, suggesting a possible involvement of latexin in tumour
suppression. Retinoic acid (RA) is a natural metabolite of vitamin A that
plays an essential role in neural development. RA induces differentiation
through the regulation of genes involved in cell proliferation, cell cycle
and cell death. Among these genes, RA has been found to activate the
expression of a number of tumor suppressors such as retinoic acid receptor(RARB), tazarotene-induced gene 1 (TIG1) or thioredoxin reductase. In
our study, we demonstrate that RA induces the expression of latexin in SHSY5Y neuroblastoma cells. Moreover, SH-SY5Y cells that constitutively
and stably express latexin, present enhanced RA-mediated differentiation,
as shown by both increased levels of the neuron specific enolase (NSE)
differentiation marker, and decreased levels of the DNA-binding protein
inhibitor (ID1) dedifferentiation marker. These first results encourage us
to further characterize the molecular pathways behind latexin-mediated
tumor suppression activity, likely involving cellular differentiation, which
seem of great interest to unveil the initial stages leading to the development
of certain types of cancer.
P18-35 (R18-7)
Development of a luminescence-based
mitocondrial calcium assay optimized
for high-throughput screening
Paloma Navas Navarro1, Javier Garca-Sancho2, Britta Jehle3, Joe
Lewis3, Fabiana Perocchi4, Mara Teresa Alonso Alonso2
1
Instituto de Biologa y Gentica Molecular, Valladolid, ES, 2Instituto
de Biologa y Gentica Molecular (IBGM) - Universidad de Valladolid
- Consejo Superior de Investigaciones Cientficas, Valladolid, ES,
3
Chemical Biology Core Facility - EMBL Heidelberg, Heidelberg, DE,
4
Maximilians-Universitt (LMU) Munich, Munich, DE
Mitochondria are capable of uptaking large amounts of Ca2+ via a
membrane- potential, ruthenium-red-sensitive mechanism recently
identified as the mitocondria calcium uniporter. Mitochondria Ca2+ uptake
controls intracellular Ca2+ signalling, cell metabolism, cell survival and
other celular functions by buffering cytosolic Ca2+ levels and regulating
mitochondrial effectors. Mitochondrial Ca2+ signaling pathways are
considered drug targets, as their regulation can determine cell fate in
disease models such as ischemia-reperfusion injury, neurodegeneration,
cardiomyopathies or metabolic disorders. However, till present, no
selective and specific compounds with therapeutic potential are available
to modulate mitochondrial Ca2+ homeostasis.
Aequorin is a calcium-binding photoprotein that emits bioluminescence
when it is reconstituted with its cofactor coelenterazine. We have developed
an aequorin-based assay to monitor mitocondrial Ca2+kinetics and we
have optimized it for high-throughput screening using a 96-well-platereader platform. A bioluminescence-based assay has several advantages
over a fluorescence-based one: 1) targeting of the aequorin probe to
mitochondria matrix is extremely precise, thus permitting the selective
measurement inside the organelle;2) aequorin can be reconstituted with
different prosthetic groups allowing to cover a large dynamic range of Ca2+
concentration, from 10-7 to 10-3 M; 3) aequorin has a low Ca2+ buffering
effect and it is nearly insensitive to changes in Mg2+ or pH that may occur
inside mitochondria; 4) it has a high signal-to-noise ratio, making it an
ideal Ca2+ sensor for high-throughput screening; 5) the raw luminescent
signal can be calibrated in Ca2+ concentration by discharging all the
remaining aequorin. We have engineered a HeLa monoclonal cell line that
stably expresses a mitochondrial matrix-targeted GFP-Aequorin fusion
protein. Aequorin was reconstituted with coelenterazine n, which reduces
its affinity for Ca2+, it has a slower consumption rate and allows to monitor
Ca2+ kinetics for long periods of time.
Granada 2014
P18-36
Regulation of Zic1 gene expression by nitric

oxide and its role in the regulation of sonic
Hedgehog pathway in mouse embryonic stem
cells
Amparo Beltrn Povea, Carmen Salguero Aranda, Rafael Tapia
Limonchi, Ana Beln Hitos Prados, Irene Daz Contreras, Gladys
Cahuana Macedo, Bernat Soria Escoms, Francisco Bedoya Bergua,
Juan R. Tejedo Huaman
(CABIMER), Sevilla, ES
Our laboratory has developed a protocol for endoderm differentiation
of mouse embryonic stem cells which consists in exposing cells to
high concentrations of a nitric oxide donor for 19 hours. This treatment
suppresses the expression of pluripotency genes such as Oct4 and Nanog,
and induces differentiation events of early acquisition of epithelial
morphology and expression of markers of definitive endoderm such as
Pdx1. We observed in previous studies in mouse embryonic stem cells
(mESC) that nitric Oxide causes an increase of the expression of Zic1, a marker of differentiation toward ectoderm, so we decided to study
this gene in endodermic differentiation process. It has been proved that
Zic1 and Gli proteins physically and functionally interact through their
zinc finger domains, and that Zic1 is essential to the regulation of Sonic
Hedgehog (Ssh) pathway in neural development. The aim objective of
this study is to determine if Zic1 is regulated by NO, and if this gene
has a role in the regulation of Ssh pathway in mESC related with
endoderm development. In our early studies in mouse embryonic stem
cells (mESCs) we have analysed Zic1 gene and protein expression of
cells treated with high concentration of nitric oxide, as well as the DNA
methylation on its promoter region, showing that the expression of Zic1
in presence of Nitric Oxide was increased, without significant changes
on DNA methylation. Furthermore, we found that the transcription
factor Egr1 could active to Zic1 expression after NO treatment. Then
we proved that Zic1 coexpressed with Pdx1 in cells treated with NO, in
adult pancreatic cells and in pancreas tissue, which are enough evidence
to suspect that Zic1 may be involved in the process of differentiation
to endoderm. Finally, we determined that NO treatment suppresses Ssh
signaling pathway, decreasing the expression of it targets genes. To test
whether this modification is promoted by Zic1, we developed gain and
loss of function assays of Zic1, reveling thus the possible role of Zic1
in Ssh signaling pathway inhibition and differentiation process toward
endoderm in mESC.
Subvencionado por:
- Ministerio de Economa y Competitividad-Secretara de Estado de
Investigacin Desarrollo e Innovacin (IPT-2011-1615-900000)
- Junta de Andaluca (CTS- 7127/2011)
P18m-37
Sam68 participa en las vas de sealizacin de

leptina e insulina en clulas de cncer de mama
Flora Snchez-Jimnez1, Antonio Prez-Prez1, Jenifer MartinGonzlez2, Antonio M. Carmona-Fernndez1, Victor SnchezMargalet1
1
Hospital Universitario Virgen Macarena, Montemayor, ES,
2
Departamento de Estomatologa, Facultad de Odontologa,
Universidad de Sevilla, Sevilla, ES
Introduccin: La obesidad y la resistencia a la insulina son factores de
riesgo bien conocidos para el desarrollo del cncer de mama en mujeres
postmenopusicas. La elevacin de los niveles de leptina e insulina modulan
positivamente el crecimiento de estas clulas tumorales. La protena Sam68
es una protena de unin a ARN que tambin interacciona con protenas de
sealizacin. Se ha demostrado previamente la sobreexpresin de Sam68
Psters
en clulas de cncer de mama. Adems, Sam68 puede ser reclutado en
las vas de sealizacin de leptina e insulina como substrato del receptor,
habindose mostrado su participacin en la proliferacin, crecimiento
celular y efectos antiapoptticos mediados por estas hormonas en diferentes
tipos celulares.
Objetivos: Estudiar la expresin de Sam68 y su nivel de fosforilacin
medidos por leptina e insulina en diferentes lneas celulares de cncer de
mama (MCF7, MDA-MB-231 y BT-474), estudiando asimismo el efecto
de la inhibicin de la expresin de Sam68 mediante siRNA.
Resultados: En nuestro trabajo se ha demostrado un incremento en
la cantidad de protena y expresin gnica de Sam68 en respuesta al
estmulo con leptina o insulina en todas las lneas celulares de cncer de
mama estudiadas. Adems, tanto la estimulacin con leptina como con
insulina, promovieron un incremento en la fosforilacin en tirosina de
Sam68, regulando negativamente su capacidad de unin a RNA en estas
lneas celulares. La inhibicin de la expresin de Sam68 result en una
menor activacin de las vas MAPK y PI3K en la estimulacin con ambas
hormonas.
Conclusin: Estos resultados sugieren la participacin de Sam68 en la
sealizacin de los receptores de leptina e insulina en clulas de cncer de
mama, mediando los efectos trficos de estas hormonas en proliferacin y
crecimiento celular.
P18r-38
Leptin prevents apoptosis triggered by increased

temperature in placental explants
Antonio Prez-Prez1, Ayelen Toro2, Flora Snchez-Jimnez1, Jenifer
Martn-Gonzlez3, Teresa Vilario-Garca1, Cecilia Varone2, Vctor
Snchez-Margalet1
1
Departamento de Bioqumica Mdica y Biologa Molecular,
Hospital Universitario Virgen Macarena, 13 Facultad de Medicina,
Sevilla, ES, 2Departamento de Qumica Biolgica, IQUIBICEN,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos
Aires, Buenos Aires, AR, 3Department of Stomatology (Endodontics
section), School of Dentistry, University of Sevilla, Sevilla, ES
Maternal fever is common during pregnancy and and has for many
years been suspected to harm the developing fetus. Whether increased
maternal temperature produces exaggerated apoptosis in trophoblast
cells remains unclear. Since p53 is a critical regulator of apoptosis we
hypothesized that increased temperature in placenta produce abnormal
expression of proteins participating in the p53 pathway and finally
caspase 3 activacion. Moreover, leptin, produced by placenta, has
demonstrated to promote the proliferation and survival of trophoblastic
cells. That is why, we aimed to study the possible role of leptin
preventing apoptosis triggered by high temperature, as well as the
molecular mechanisms underlying this effect.
Fresh placental tissue was collected from normal pregnancies. Placental
explants were exposed to increased temperature (40C and 42C) for
different time points. We employed 10 nM leptin which is the optimal
leptin concentration. Western blotting and real-time PCR were performed
on tissue lysate for protein and mRNA expression of p53 and downstream
effector proteins, p21, Bax, Mdm2 and caspase 3.
Maximal apoptotic effect of high temperature was obserbed after 3 h
incubation of trophoblasts. Protein and mRNA expression of p53, p21,
Bax, Mdm2 and caspase 3 were significantly increased in explants at
40C and 42C in compared with explants to 37C. Conversely, this
increased expression was significantly attenuated by leptin 10 nM at both
40C and 42C.
These data illustrate the potential role of leptin for inhibiting the excessive
apoptosis in villous trophoblast triggered by high temperature. However,
the upstream regulation of p53 with regard to exaggerated apoptosis in
response to elevated temperature merits further investigation.
169
Psters
P18r-39
P18r-41
PTEN mediates the inhibition of c-Src oncogenic

activity promoted by connexin43 in glioma cells
and astrocytes
Transporte ncleo-citoplasmtico del complejo

SNF1 de Saccharomyces cerevisiae
Ana Gonzlez Snchez, Marta Domnguez-Prieto, Sandra HerreroGonzlez, Jose M Medina, Arantxa Tabernero
Castilla y Len), Salamanca, ES
Gliomas are the most common brain tumors and they present a very bad
prognosis. One of their characteristics is a high activity of the oncogen,
c-Src, and a low expression of connexin43 (Cx43), the main protein
forming gap junction channels in astrocytes. In previous works we showed
that restoring Cx43 to glioma cells inhibits c-Src activity. Since c-Src
activity is inhibited by c-terminal Src kinase (Csk) phosphorylation and
PTEN dephosphorylation, we investigated wether Cx43 could regulate
c-Src activity by recruiting or regulating said enzymes.
In this work we show that silencing PTEN expression in C6 glioma cells
stably transfected with Cx43 (C6Cx43) reverted the effect of Cx43 on
c-Src activity and on cell proliferation. These results indicate that PTEN
participates in the inhibition of c-Src activity promoted by Cx43. Using
confocal microscopy and co-inmunoprecipitation studies, we confirmed
the interaction between Cx43, PTEN and c-Src in C6 glioma cells
transfected with Cx43 and astrocytes in primary culture. In addition, we
found Csk in this inmunocomplexes, suggesting that Cx43 can recruit the
machinery required to inactivate c-Src. Finally, we found that restoring
Cx43 expression in C6 glioma cells, in addition to reduce c-Src activity
and proliferation, upregulates PTEN and Csk.
According with the results, it could be concluded that PTEN and Csk
participate in the inhibition of the activity of c-Src promoted by Cx43 in
glioma cells and astrocytes.
Montserrat Vega1, Alejandra Fernndez-Cid2, Alberto Riera2, Pilar

Herrero1, Fernando Moreno1
1
Departamento de bioqumica y biologa molecular, Universidad
de Oviedo, Oviedo, ES, 2MRC Clinical Sciences Centre - Imperial
College Faculty of Medicine, London, UK
La familia de proten quinasas AMPK/SNF1 es esencial para establecer
en todas las clulas eucariotas, incluidas las levaduras, la regulacin
metablica adecuada en respuesta a diferentes tipos de estrs. El complejo
SNF1 de S.cerevisiae est constituido por tres subunidades, una subunidad
cataltica Snf1 y dos subunidades reguladoras, Snf4 y una de las tres
subunidades implicadas en la localizacin subcelular del complejo (Sip1,
Sip2 o Gal83). En S. cerevisiae, este complejo se requiere no solo para la
adaptacin de la levadura a diferentes condiciones ambientales, entre las
que se incluye el estrs nutricional generado por condiciones de limitacin
de glucosa, sino que tambin est implicado en procesos de desarrollo.
En esta comunicacin presentamos datos de la influencia de los niveles
de glucosa en la distribucin subcelular y mecanismo de transporte al
ncleo del complejo SNF1. Experimentos de coinmunoprecipitacin de
cromatina (ChIP) demuestran que las tres protenas que forman parte del
complejo SNF1 (Snf1, Gal83 y Snf4), estn presentes en el ncleo tanto
en condiciones de alta como de baja glucosa. Hasta ahora el sistema de
importacin y exportacin utilizado por estas protenas era desconocido.
Aqu mostramos la interaccin de estas tres protenas con las importinas
del sistema clsico de importacin, Kap95 y Kap60, as como su
interaccin con la exportina Xpo1, en condiciones de alta y baja glucosa.
Experimentos in vitro muestran que la interaccin de Snf4, Snf1 y Gal83
con las importinas Kap60 y Kap95, est regulada por la presencia de
Gsp1GDP y Gsp1GTP. Demostrando que la direccionalidad del transporte
ncleo-citoplsmico viene dada por la GTPasa Gsp1 en funcin de si est
cargada con GDP (citoplasma) o GTP (ncleo).
P18-40
Regulacin de los niveles de PLK1 por la E3

ligasa SKP1-CUL1-F-box
Ana Cristina Rivas1, Servando Girldez1, Joaqun Herrero-Ruiz1, Mar
Mora-Santos1, Mara Cristina Limn-Morts1, Carmen Sez2, Miguel
ngel Japn2, Maria Tortolero1, Francisco Romero1
1
Departamento de Microbiologa, Facultad de Biologa, Universidad
de Sevilla, Sevilla, ES, 2Instituto de Biomedicina de Sevilla (IBiS),
Hospital Universitario Virgen del Roco/CSIC/Universidad de Sevilla
y Departamento de Anatoma Patolgica, Hospital Universitario
Virgen del Roco, Sevilla, ES
PLK1 (Polo-Like Kinase1) es una quinasa de serina/treonina que interviene
en muchos de los eventos que tienen lugar durante la mitosis, como la
entrada en mitosis, la maduracin de los centrosomas, el ensamblaje del
huso mittico, la separacin de las cromtidas hermanas, la activacin del
complejo APC/C y la salida de mitosis y el inicio de la citocinesis. Adems,
PLK1 est implicada en la replicacin del ADN y en el restablecimiento
del ciclo celular tras el punto de control de G2 producido por los daos
en el ADN. Durante el ciclo, PLK1 se degrada va APC/C-proteasoma
comenzando su degradacin al final de la mitosis y continuando a lo largo
de la fase G1. PLK1 tambin se degrada por el APC/C en G2 en respuesta
al dao en el ADN. Por otra parte, recientemente hemos descrito que en
la fase S los niveles de PLK1 estn controlados por la ligasa de ubicuitina
SCFFBXW7. En este caso, PLK1 es ubicuitilada y degradada va proteasoma
en condiciones no estresantes e incrementndose este efecto tras radiacin
ultravioleta. Esta degradacin contribuye a la parada del ciclo celular en
la fase S al impedir la formacin de los complejos de pre-replicacin (preRC), lo que reduce la proliferacin celular. Dado que se ha descrito que las
ligasas de ubicuitina SCFFBXW7 y SCFTrCP pueden regular la estabilidad de
un mismo sustrato, tanto cooperando como interfiriendo en la degradacin
del mismo, hemos estudiado el efecto de SCFTrCP sobre PLK1 y sus
posibles implicaciones en las funciones de la quinasa.
170
P18r-42
Mechanism for the specific targeting of

methyltransferases to chemoreceptors in
Pseudmonas aeruginosa PAO1
Andrs Corral Lugo, Cristina Garca Fontana, Manuel Espinosa
Urgel, Tino Krell
Estacin Experimental del Zaidin, CSIC (EEZ-CSIC), Granada, ES
Chemosensory pathways are major signal transduction mechanisms in
bacteria. Methyltransferases of the CheR family and methylesterases of
the CheB family control chemoreceptor methylation, and this dynamic
posttranslational modification is necessary for proper chemotaxis of bacteria.
Studies with enterobacteria that contain a single CheR or CheB show that,
in addition to binding at the methylation site, some chemoreceptors bind
CheR or CheB through additional high-affinity sites at distinct pentapeptide
sequences in the chemoreceptors. We investigated the recognition of
chemoreceptors by CheR proteins in the human pathogen Pseudomonas
aeruginosa PAO1. Of the four methyltransferases in PAO1, we detected
an interaction only between CheR2 and the chemoreceptor methylaccepting chemotaxis protein B (McpB), which contains the pentapeptide
GWEEF at its carboxyl terminus. Furthermore, CheR2 was also the only
paralog that methylated McpB in vitro, and deletion of the pentapeptide
sequence abolished both the CheR2-McpB interaction and the methylation
of McpB. When clustered according to protein sequence, bacterial CheR
proteins form two distinct families-those that bind pentapeptide-containing
chemoreceptors and those that do not. These two families are distinguished
by an insertion of three amino acids in the -subdomain of CheR. Deletion
of this insertion in CheR2 prevented its interaction with and methylation
of McpB. Pentapeptide-containing chemoreceptors are common to many
bacteria species; thus, these short, distinct motifs may enable the specific
assembly of signaling complexes that mediate different responses.
Granada 2014
P18-43
Singularidad de SUMO en la regulacin de las PHD

Almudena Gerpe-Pita, Amelie Schber, Onintza Carlevaris, Edurne Berra
CIC bioGUNE, Derio, ES
Utilizando el oxgeno como sustrato, las PHD (Prolyl Hydroxylase Domain
containing proteins) son enzimas que catalizan la hidroxilacin de sus
protenas dianas sobre un residuo prolina. Estas dioxigenasas, conservadas
a lo largo de la evolucin y de las que se han descrito tres isoformas en
mamferos (PHD1-3), se identificaron inicialmente como los sensores de
oxgeno responsables de regular la estabilidad de la subunidad del factor
de transcripcin HIF (Hypoxia Inducible Factor), el jefe de orquesta de
la cascada de sealizacin de hipoxia y el mantenimiento de la homeostasis
de oxgeno. No obstante, las PHD no son enzimas redundantes y si bien
PHD1, 2 y 3 comparten ciertos substratos y tareas, es evidente que tambin
muestran ciertas especificidades en cuanto a su funcin y regulacin.
La SUMOilacin es una modificacin postraduccional que conlleva la
conjugacin covalente de SUMO (Small Ubiquitin-like Modifier). El
proceso de SUMOilacin implica una cascada de reacciones enzimticas
en la que intervienen un complejo nico de activacin, Uba2/Aos1 o SAE1/
SAE2, una enzima de conjugacin (Ubc9), una E3 especfica para ciertos
sustratos (PIAS, RanBP2 o Pc2 son algunos ejemplos) y deSUMOilasas,
responsables de la deconjugacin de SUMO. La interaccin dinmica entre
SUMOilacin y deSUMOilacin modula importantes vas de sealizacin
molecular y entre ellas, la cascada de sealizacin de hipoxia. Por ello, nos
planteamos estudiar el papel de SUMO en la regulacin de las PHD. En esta
presentacin discutiremos nuestros resultados ms recientes sobre el papel
singular de SUMO en la regulacin de PHD1, 2 y 3, su implicacin en las
rutas dependientes e independientes de HIF y su relevancia fisiopatologica.
P18-44
La activacin de ERK 1/2 es suficiente para

restaurar la degradacin proteica mediada por
dexametasona en clulas de msculo esqueltico
Jos Dmaso Vlchez Rienda1, Rafael Salto Gonzlez1, Mara Elena
Cabrera Cazorla1, Joan J Guinovart i Cirera2, Mara Dolores Girn
Gonzlez1
1
Departamento de Bioqumica y Biologa Molecular II, Facultad
de Farmacia, Universidad de Granada, Granada, ES, 2Instituto de
Investigacin Biomdica, Universidad de Barcelona y CIBER de
Diabetes y Enfermedades Metablicas (CIBERDEM), Barcelona, ES
La atrofia muscular se desarrolla en situaciones de ayuno prolongado,
enfermedades como cncer, diabetes, SIDA o sepsis y en condiciones como
el envejecimiento, denervacin o tratamiento con glucocorticoides. En
estas situaciones, se produce una disminucin en la sntesis de protenas
conjuntamente con un aumento en su degradacin. La degradacin de
protenas en msculo esqueltico est mediada por la actividad de dos
rutas conservadas: ubiquitina-proteasoma y un aumento de la autofagia
lisosomal. La primera ruta, responsable del recambio de la mayora de
protenas musculares solubles y miofibrilares, se activa como resultado
de una expresin aumentada de ubiquitina y ligasas de ubiquitina, como
ATROGEN1 y MuRF1. La autofagia juega un papel muy importante en la
degradacin proteica en el msculo esqueltico e implica un incremento en
la transcripcin de una serie de genes como LC3, p62 y Bnip3. Ambas rutas
estn moduladas por el factor de transcripcin Forkhead box O3 (FoxO3).
FoxO3 normalmente est fosforilado por Akt y en estado inactivo en el
citosol, pero en ausencia de una represin de Akt, se transloca al ncleo donde
induce la expresin de una serie de genes relacionados con los dos sistemas
de degradacin. En cultivos de clulas musculares de rata L6, el tungstato
sdico, activador de ERK 1/2, es capaz de inhibir la degradacin inducida
por dexametasona. El tungstato promueve la fosforilacin de FoXO3a, lo
que conduce a su inactivacin e inhibicin de su translocacin al ncleo
y por tanto, a una disminucin en la actividad del promotor de ubiquitina
as como una menor expresin de las ligasas de ubiquitina. Asimismo, el
tungstato reduce la formacin de autofagosomas, la expresin de la forma
Psters
lipidada de LC3, la degradacin de p62 y reduce los niveles de mRNA de
Bnip3 en clulas tratadas con dexametasona. Estos resultados obtenidos en
cultivos celulares apoyan la idea de que la activacin de ERK 1/2 disminuye
la degradacin proteica a travs de los dos sistemas celulares, proteasoma y
lisosomal, por lo que el tungstato sdico podra ser un agente til situaciones
fisiopatolgicas en las que se presentase una atrofia muscular.
Proyecto Financiado por la Fundacin Botn.
P18-45
Paralogous chemoreceptors mediate chemotaxis

towards protein amino acids and the non-protein
amino acid gamma-aminobutyrate (GABA)
Miriam Rico Jimnez1, Francisco Muoz Martnez1, Cristina Graca
Fontana1, Matilde Fernandez2, Bertrand Morel3, lvaro Ortega4,
Juan Lus Ramos1, Tino Krell1
1
Estacion experimental del Zaidn, CSIC, Granada, ES, 2Bioilberis
SL, Granada, ES, 3Departamento de Qumica Fsica, Facultad de
Ciencias, Universidad de Granada, Granada, ES,4Universidad de
Murcia, Murcia, ES
The paralogous receptors PctA, PctB and PctC of Pseudomonas aeruginosa
were reported to mediate chemotaxis to amino acids, intermediates of amino
acid metabolism and chlorinated hydrocarbons. We show that the recombinant
ligand binding regions (LBRs) of PctA, PctB and PctC bind 17, 5 and 2
L-amino acids, respectively. In addition, PctC-LBR recognized GABA but
not any other structurally related compound. L-Gln, one of the three amino
acids that is not recognized by PctA-LBR, was the most tightly binding ligand
to PctB suggesting that PctB has evolved to mediate chemotaxis primarily
towards L-Gln. Bacteria were efficiently attracted to L-Gln and GABA, but
mutation of pctB and pctC, respectively, abolished chemoattraction.
The physiological relevance of taxis towards GABA is proposed to reside in
an interaction with plants. LBRs were predicted to adopt double PDC (PhoQ/
DcuS/CitA) like structures and site directed mutagenesis studies showed that
ligands bind to the membrane-distal module. Analytical ultracentrifugation
studies have shown that PctA-LBR and PctB-LBR are monomeric in the
absence and presence of ligands, which is in contrast to the enterobacterial
receptors that require sensor domain dimers for ligand recognition.
P18-46
Protein kinase CK2 affects the stability of the

HB-EGF receptor ErbB4 in 786-O cells
E. Alcaraz1, J. Vilardell1, E. Sarr2, M. Plana1, A. Meseguer2, E. Itarte1
Departament de Bioqumica i Biologia Molecular, Universitat
Autnoma de Barcelona, Cerdanyola del Valls, ES, 2Fisiopatologia
Renal, CIBBIM, Institut de Recerca Hospital Universitari Vall
Hebron, Barcelona, ES
Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF

family, binds and activates ErbB1 and ErbB4. ErbB4 is expressed as different
alternatively spliced isoforms that are characterized by variant extracellular
juxtamembrane (JM) and intracellular cytoplasmic (CYT) domains. The JM
domain of type a (JM-a) can be cleaved by ADAM 17 in response to binding
of ligands to the receptor, or the activation of PKC. The proteolytic fragment
can be further cleaved by -secretase, which releases the intracellular domain
as a soluble fragment of 80kDa (CYT2-ICD) which can translocate into the
nucleus where it functions as a co-activator or co-repressor for a number of
transcription factors. In previous studies we had observed that protein kinase
CK2 was involved in the response to HB-EGF in the renal cell carcinoma 786O cell line. To study the potential role of CK2 in ErbB4 receptor processing,
we generated a stable transfected 786-O cell line that expresses JMa-Cyt2
ErbB4 isoform, characteristic of renal cells. Treatment of these cells with
the CK2 inhibitor CX-4945 resulted in a marked reduction of ErbB4 levels.
Under the same conditions, CX-4945 did not affect the levels of the membrane
171
Psters
protein transferrin receptor or of the CK2 substrate I. Destabilization of
ErbB4 promoted by CX-4945 did not cause to the accumulation of CYT2ICD, as occured in response to HB-EGF or PMA, but to the complete loss
of bands detected with the anti-ErbB4 antibody. Treatment with lysosomal
proteinases inhibitors (leupeptin and pepstatin) attenuated the effect of CX4945 on CYT2-ICD leading to its accumulation. Moreover, interaction
between CK2 (CK2 and, in particular, CK2 subunit) and ErbB4 receptor
has been detected by pull down analysis, and downregulation of CK2 using
siRNAs decreased the expression of ErbB4. To sum up, CK2 seems necessary
to maintain ErbB4 levels within the cell. Supported by grants BFU200910189 (MCINN) and SAF-2011-29506 (MINECO).
P18-47
MDN-0066 a novel inhibitor of VHL/HIF pathway

produced by a new Pseudomonas species
Bastien Cautain, Nuria De Pedro, Javier Pascual, Jesus Martin,
Fernando Reyes, Olga Genilloud, Francisca Vicente
Fundacion MEDINA, Armilla, ES
Throughout recent history, metabolites of microbial origin have had an
extraordinary impact on the welfare of humanity. In fact, natural products have
largely been and still are considered an exceedingly valuable platform for
the discovery of new drugs against diverse pathologies. Such value is partly
due to their higher complexity and chemical diversity as compared to those of
synthetic and combinatorial compounds. Mutations in the Von Hippel-Lindau
(vhl) gene are responsible for VHL disease, congenital polycythemia, and are
found in many sporadic tumor types. The primary cause of morbidity and
mortality for these patients arises from progression of Renal Cell Carcinoma
(RCC) or end-stage renal disease. Inactivation of the Von Hippel-Lindau (vhl)
tumor suppressor gene arises in the majority of Renal Cell Carcinoma (RCC)
as well as in other types of cancer and is associated with a high degree of
vascularization and poor prognosis. Loss of pVHL function thus represents
a pathognomonic molecular defect for therapeutic exploitation. In this study,
renal carcinoma cell lines with naturally occurring vhl mutations (RCC4
VA) and their genetically matched wild-type vhl (RCC4 VHL)counterparts
were seeded onto 96-well plates and treated with a collection of 1,040
organic extracts obtained from 130 unicellular bacteria strains belonging to
at least 25 genera of the phyla Actinobacteria, Firmicutes, Proteobacteria
and Bacteroidetes. This strategy allowed us to identify several extracts
obtained from the bacterial strain F-278,770T showing biological activities
not associated with previously known active metabolites. The fractionation
and structural elucidation of one of these extracts led to the discovery of a new
lipodepsipeptide (MDN-0066) with specific toxicity in pVHL deficient cells
that is not detectable in cells with pVHL expression rescue. This discovery
demonstrated the feasibility of selectively targeting the loss of the vhl tumor
suppressor gene for potential clinical benefit and may have great impact
on the development of new targeted therapies from natural product for the
treatment of cancer and other genetic diseases.
P18m-48
Papel de la protena de unin a ARN Sam68 en

la seal de la leptina y la insulina en clulas de
la granulosa
Teresa Vilario Garca1, Antonio Prez-Prez2, Victor Blasco
Rodriguez3, Nicols Prados Nodd3, Manuel Fernndez Snchez3,
Vctor Snchez Margalet2
1
Departamento Bioquimica Mdica y Biologa Molecular, Facultad
de Medicina de Sevilla, Sevilla, ES, 2Departamento de Bioqumica
Mdica y Biologa Molecular, Hospital Universitario Virgen
Macarena, Facultad de Medicina, Sevilla, ES, 3Instituto Valenciano
de infertilidad de Sevilla, Sevilla, ES
La obesidad es un problema medico no solo porque se asocia a la diabetes
tipo 2 y al riesgo cardiovascular, sino tambin por porque se asocia a ovario
172

poliqustico, resistencia a la insulina y subfertilidad en la mujer en edad
reproductiva.
La protena de unin a ARN Sam68 se expresa en clulas de la granulosa
y las hembras de los ratones knockout son subfrtiles, con problemas
de ovulacin. Ya que hemos encontrado que Sam68 puede reclutarse
a la seal de los receptores de insulina y leptina, planeamos estudiar
la participacin de Sam68 en la seal y la accin de los receptores de
insulina y leptina en clulas de la granulosa. Adems, estudiamos la
expresin de Sam68 en clulas de la granulosa en respuesta a la leptina
y la insulina in vitro.
Las clulas de la granulosa se obtuvieron a partir de la obtencin de
ovocitos para la fertilizacin invitro. La sealizacin se studio por
inmunoprecipitacin e inmunoblot de las protenas fosforiladas. La
expression de Sam68 se inhibe por estrategia antisentido. El nivel de
expression de Sam68, los receptors de leptina e insulina se cuantificaron
por RT-PCR e inmunoblot.
Hemos encontrado que Sam68 se tirosn-fosforila por estimulacin
con insulin o leptina en clulas de la granulosa, reclutando Sam68
a los complejos de sealizacin y disminuyendo la capacidad de
unin al ARN. Adems, tanto la leptina como la insulina aumentan
la expresin de Sam68 en las clulas de la granulosa. Finalmente, la
expresion de Sam68 es necesaria para la activacin completa de las
vas de sealizacin PI3K y MAPK, por insulin y leptina en clulas de
la granulosa.
En conclusion, Sam68 es reclutado a la seal del receptor de leptina e
insulin, su expression se induce por ambas hormonas, y la expression de
Sam68 es necesaria para la completa activacin de la transduccin de la
seal de los receptores de insulina y leptina. Sam68 puede ser un nuevo
elemento en la resistencia a la insulina ovrica y la fertilidad disminuida
en mujeres obesas
P18r-49
3 Integrin is essential for invadopodia

formation in TGF- lung carcinoma treated
cells
Rafael Pelez Cristbal, Xabier Morales, Elisabeth Salvo, Saray
Garasa, Ana Rouzaut
Universidad de Navarra/CIMA, Pamplona, ES
Tumor cell migration and invasion are crucial events during cell metastasis,
which is the main cause of death in patients with cancer. Depending on the
mechanical properties of the tumor stroma, cancer cells develop invasive
actin-rich structures capable of degrading the surrounding extracellular
matrix (ECM) known as invadopodia.
Integrins are transmembrane proteins implicated in cell adhesion and
migration. Integrin activation induces signaling cascades conducive
to the reorganization of cell cytoskeleton. Recent reports have
demonstrated the presence of different integrins surrounding the
invadopodia core in breast and melanoma cancer cells. 3 integrin
and v3 heterodimer have been associated with poor prognosis and
increased metastasis in several carcinoma types. We aimed to determine
whether 3 integrin participates in invadopodia formation in lung
carcinoma cells. We based our hypothesis on our previous findings of
specific TGF- induction of 3 integrin dependent metastasis in lung
carcinoma cells.
In this study, we demonstrated that 3 integrin is temporarily allocated on
the invadopodia of H157 NSCLC cells. 3 integrin silencing or functional
blockade completely abrogates invadopodia formation, even after treatment
with TGF- (a cytokine that significantly induces invadopodia formation)
while 3 integrin re-expression restores the capacity of invadopodia
formation in integrin deficient cells. Moreover, integrin activation induces
different signal pathways (FAK, SRC, ERK and Small GTPases family)
allowing invadopodia formation.
In conclusion, we demonstrated a dual role of the 3 integrin in invadopodia
formation during cancer cell metastasis: it contributes to the initial cell
Granada 2014
adhesion and then it activates signal pathways implicated in invadopodia
formation.
P18r-50
Mecanismos de regulacin de la expresin

de ENA1 en respuesta a pH alcalino: un estudio
cuantitativo
Maria Lpez Malo, Silvia Petrezselyova, David Canadell, Joaqun
Ario
UAB-Instituto Biotecnologa y Biomedicina, Barcelona, ES
La alcalinizacin del medio supone una situacin de estrs para
la levadura Saccharomyces cerevisiae, que conlleva una respuesta
adaptativa que da lugar a un remodelado de la expresin gnica. Uno
de los genes relevantes para la adaptacin a pH alcalino es ENA1, que
codifica una Na+ ATPasa implicada en la detoxificacin de sodio. En la
regulacin del gen ENA1 por pH alcalino estn implicadas diversas rutas
de sealizacin. Una de ellas est mediada por la protena calcineurina
que desfosforila el factor transcripcional Crz1, permitindole la entrada
en el ncleo y por tanto la unin a las secuencias especficas activando
el gen. La respuesta independiente de calcineurina est bajo el control de
dos rutas diferentes, Rim101 y Snf1, en las que est implicado el represor
Nrg1. Si bien este proceso ha sido caracterizado desde un punto de vista
cualitativo, no ha sido investigado desde un punto de vista cuantitativo,
que permita eventualmente el modelado del mismo. Para ello hemos
analizado a lo largo del tiempo en clulas expuestas a estrs alcalino
diversos parmetros como la tasa de transcripcin y la cantidad de mARN
de ENA1, la cantidad de Crz1, su transicin citosol-ncleo y su cintica
de unin al promotor de ENA1, as como la cantidad de Ena1 producida.
Un anlisis similar para el represor Nrg1 est en curso. De esta manera
confiamos definir los parmetros que gobiernan la expresin de Ena1 en
respuesta a estrs alcalino.
P18r-51
Bsqueda de nuevos mecanismos de actuacin

de la protena fosfatasa Ptc1
Laura Tatjer Recorda, Asier Gonzlez Sevin, Joaqun Ario
Carmona
UAB-Instituto Biotecnologa y Biomedicina, Sabadell, ES
Las protenas fosfatasas de tipo 2C (PP2C) conforman una familia
de enzimas monomricas, conservadas a lo largo de la evolucin,
que en S. cerevisiae estn codificadas por 7 genes estructuralmente
relacionados (PTC1-7). La isoforma ms estudiada en levaduras es
Ptc1, ya que sta puede desempear mltiples funciones especficas
no compartidas con las otras PP2C. Como consecuencia, un mutante
ptc1 presenta defectos de crecimiento ante diversas formas de estrs,
como los que activan la va de sealizacin de la integridad de la pared
celular (p. ej., pH alcalino y el blanco de calcoflor) o en presencia
de rapamicina, un inhibidor de la va TOR, la cual sealiza la falta de
nutrientes. Con el objeto de identificar posibles dianas de la accin de
Ptc1, hemos rastreado bibliotecas genmicas para encontrar genes que,
en sobreexpresin, alivien el fenotipo de sensibilidad a pH alcalino,
blanco de calcoflor y rapamicina del mutante. Como resultado
de la bsqueda se han aislado un total de 25 genes que suprimen o
alivian uno o varios fenotipos caractersticos del mutante ptc1. Nos
hemos centrado en el estudio de PPH21 y PPH22, que codifican dos
subunidades catalticas redundantes de la protena fosfatasa de tipo 2A.
Los resultados obtenidos nos indican que la carencia de Ptc1 estara
alterando los diferentes complejos fosfatasa-Tap42, que actan como
efectores de la va TOR cuando sta es inhibida.
Financiado por BFU2011-30197-C3-01.
Psters
P18-52
TGF2 dictates disseminated tumor cell fate

in target organs through TGF-RIII and p38/
signaling
Paloma Bragado Domingo
IDIBAPS, Barcelona, ES
Metastases are thought to arise from disseminated tumor cells (DTCs) that
can either initiate growth immediately or reprogram into dormancy. In this
state they can remain undetected for long periods before resuming growth
and forming overt metastases. The mechanisms driving dormancy of DTCs
remain largely unknown. We hypothesize that via a seed and soil mechanism
the target organ microenvironment where DTCs lodge controls the timing of
DTCs dormancy and thus their fate. Using head and neck and breast cancer
models we have studied the nature of the organ-derived signals that control the
interconversion of DTCs between dormancy and proliferation states. We found
that the bone marrow (BM) represents a growth restrictive micro-environment
in which DTCs persist for long periods of time in a quiescent/dormant state.
In this tissue, host-derived TGF2, but not TGFb1, signaling, activates p38/,
inducing a [ERK/p38] low signaling ratio. This results in induction of DEC2/
SHARP1 and p27, downregulation of CDK4 and dormancy of malignant
DTCs. TGF2-induced dormancy required TGF -receptor-I, TGF -receptorIII and SMAD1/5 activation to induce p27. In contrast, in lungs, which is a
site commonly permissive for metastasis, TGFb2 signals are weaker and
only a short-term dormancy ensues before DTCs start proliferating and fuel
metastases. Importantly, systemic inhibition of TGF-receptor-I or p38/
activities awakened dormant DTCs fueling multi-organ metastasis. Our work
reveals a seed and soil mechanism where TGF2 and TGFRIII signaling
through p38/ regulate DTC dormancy and defines restrictive (BM) and
-permissive (lung) microenvironments for HNSCC metastasis. By identifying
key pathways controlling dormancy we open opportunities for eradication of
the residual disease.
P19. Transgnesis en mamferos

P19r-1
IGF1R (insulin-like growth factor type 1 receptor)

regula la regeneracin del epitelio bronquiolar
induciendo la diferenciacin de las clulas de Clara
Sergio Pieiro-Hermida, Icar P. Lpez, Raquel Torrens, Jos G. Pichel
Centro de Investigacin Biomdica de la Rioja (CIBIR), Fundacin
Rioja Salud, Logroo, La Rioja, ES
Los IGF (insulin-like growth factors) son factores pleiotrpicos que regulan
la proliferacin, diferenciacin y supervivencia celular activando el IGF1R,
su receptor especfico. Controlan el desarrollo y la homeostasis del pulmn,
su regeneracin tras dao y participan en enfermedades pulmonares como el
sndrome de distrs respiratorio, la fibrosis y el cncer. Aunque la expresin
pulmonar de IGF1R es ubicua y sus niveles son elevados en el epitelio, la
accin de IGF1R depende de la disponibilidad de los ligandos, del momento
del desarrollo y del tipo celular. Como los ratones deficientes en IGF1R mueren
al nacer por insuficiencia respiratoria debido a la hipoplasia e inmadurez
pulmonar, para determinar la funcin de IGF1R en el epitelio del pulmn murino
adulto se generaron mutantes condicionales Nkx2.1-Cre; Igf1r fl/fl, que expresan
la recombinasa Cre en el epitelio respiratorio. Estos ratones mutantes expresan
niveles reducidos de mRNA de Igf1r en el pulmn, presentan alteraciones en el
epitelio bronquiolar terminal (adelgazamiento, reduccin de la densidad celular
y aumento de la proliferacin), pero sin impacto morfolgico aparente en el
parnquima alveolar. Durante la recuperacin del dao bronquiolar inducido
por la ablacin selectiva de las clulas epiteliales de Clara con naftaleno, la
deficiencia de Igf1r mantiene los defectos histolgicos, dispara la proliferacin
y retrasa su diferenciacin. In vitro, los IGF inducen la expresin de CCSP
(Clara Cell Secreted Protein) en clulas epiteliales bronquiolares humanas.
173
Psters
Los resultados demuestran que la sealizacin mediada por IGF1R en el
epitelio respiratorio regula la regeneracin del epitelio bronquiolar daado,
induciendo la diferenciacin de las clulas de Clara.
P19-2 (R03_19-2)
Retos y nuevas oportunidades en transgnesis

animal mediante las nucleasas de edicin
genmica CRISPR-Cas9
Llus Montoliu, Davide Seruggia
Centro Nacional de Biotecnologa (CNB-CSIC), Madrid, ES
Las primeras clulas troncales pluripotentes embrionarias (ES) de ratn se
describieron en 1981 por Martin Evans. En 1986, Mario Capecchi integr
el uso de dichas clulas y la experiencia en recombinacin homloga
desarrollada por Oliver Smithies para disear el protocolo de inactivacin
dirigida de genes en el genoma de ratn, proceso por el cual los tres
investigadores recibieron el premio Nobel en 2007. Durante aos esta
poderosa tecnologa ha permanecido relativamente invariable y ha permitido
la generacin de miles de mutaciones en el genoma de ratn. El panorama
cambi a partir de 2009, cuando se publicaron las primeras ratas con genes
inactivados mediante el uso de ZFN. Las nucleasas dirigidas de dedos de
zinc (ZFN) estn constituidas por unas protenas quimricas con un dominio
de unin a DNA, variable, en funcin de la secuencia a inactivar, y un
dominio de corte (endonucleasa) de DNA en doble cadena, derivado del
enzima de restriccin FokI. Un par de aos ms tarde, en 2011, apareci la
segunda generacin de nucleasas de edicin genmica, las TALEN, en los
que de nuevo un dominio de unin a DNA se combinaba con un dominio
endonucleasa. Pero la verdadera revolucin no llegara hasta 2013, cuando
se publicaron los primeros trabajos demostrando el uso de las nucleasas
CRISPR-Cas9, derivadas de bacterias, en las que el reconocimiento no
estaba mediado por una protena sino por una pequea secuencia de RNA
que posteriormente interaccionaba con el dominio endonucleasa (Cas9).
Esta tercera (por el momento) versin de las nucleasas de edicin genmica
ha supuesto una verdadera revolucin en la manera como disear y generar
alteraciones genticas de diverso tipo en prcticamente cualquier organismo.
En el laboratorio hemos realizado la transicin desde las TALEN a las
CRISPR-Cas9 con xito, y por ello comentar las ventajas y las limitaciones
de estas fabulosas nuevas herramientas de modificacin genmica que
estn llamadas a substituir, prcticamente por completo, nuestro trabajo de
mutaciones, tradicionalmente abordado a travs de clulas ES.
P19r-3
Inactivacin de regiones intergnicas mediante

CRISPR-Cas9 en el genoma de ratn
Davide Seruggia, Marta Cantero, Lluis Montoliu
Centro Nacional de Biotecnologia (CNB-CSIC), Madrid, ES
Apenas un 2% del genoma de mamferos est ocupado por regiones gnicas
que codifican para protenas. El resto del genoma est plagado de secuencias de
ADN repetidas y de elementos reguladores de la expresin gnica, esenciales
para el control en tiempo y espacio de la transcripcin. Nuestro laboratorio
est interesado en el estudio estructural y funcional de las secuencias
aisladoras genmicas, capaces de delimitar dominios de expresin e impedir
la interaccin entre loci vecinos. Uno de nuestros modelos experimentales es
el locus de la tirosinasa de ratn, cuya mutacin est asociada con albinismo.
En experimentos anteriores demostramos la existencia de regiones aisladoras
genmicas en posicin 5 y 3 del locus de la tirosinasa de ratn. En particular,
la regin 5 contiene elementos fundamentales para la expresin correcta de
este gen que cuando se eliminan en construcciones transgnicas condicionan
una expresin variegada. Desde hace muchos aos nuestra intencin era
reproducir los experimentos realizados sobre transgenes en el propio locus
endgeno. Sin embargo, la presencia de mltiples secuencias repetitivas
alrededor de estas secuencias intergnicas reguladoras impeda la aplicacin
de aproximaciones tradicionales, basadas en recombinacin homloga en
174

clulas ES. Con la aparicin de las nuevas nucleasas de edicin genmica, en
particular con las CRISPR-Cas9 ha sido posible disear una estrategia para
eliminar estas secuencias reguladoras, intergnicas, directamente del locus
endgeno. Los primeros resultados obtenidos han sido extraordinariamente
positivos, con alteraciones fenotpicas evidentes similares a las obtenidas
anteriormente durante el anlisis de ratones transgnicos. En este trabajo
presentaremos los resultados obtenidos aplicando la tecnologa CRISPRCas9 para la inactivacin especfica de secuencias intergnicas in vivo, en
el genoma de ratn.
P19r-4 (R03_19-4)
Antizyme Inhibitor 2 expression in the adrenal

gland: study with knock-out mice
Ana Lambertos Escudero1, Bruno Ramos Molina2, Carlos Lpez
Garca2, Cristina Peafiel Verd2, Andrs Joaqun Lpez Contreras2,
Asuncin Cremades Campos2, Rafael Peafiel Garca2
1
University of Murcia, Los Alczares, Murcia, ES, 2Department of
Biochemistry and Molecular Biology B and Immunology, Faculty of
Medicine, University of Murcia, Murcia, ES
Polyamines are small organic cations essential for cell proliferation,
differentiation and survival. Ornithine decarboxylase (ODC), the key
enzyme controlling polyamine biosynthesis, is negatively regulated by
proteins termed antizymes (AZs). We recently described a novel antizyme
inhibitor (AZIN2) that stimulates ODC activity and polyamine uptake. To
gain insight on the tissue expression profile of AZIN2 and to explore its
possible physiological role, we generated Azin2 knock-out mice expressing
bacterial -D-galactosidase as a reporter gene, under the control of the
Azin2 endogenous promoter, what allows a very sensitive and specific
detection of the expression of the gene in the tissues of transgenic mice.
Whole mount analysis and histochemical detection of lacZ using X-gal
staining revealed that Azin2 is expressed in the medulla but not in the
cortex of adrenal glands. The majority of chromaffin cells displayed a
granular-like reactivity, with clusters of cells with both cytosolic and a
more intense superimposed granular staining. No sexual dimorphism
was observed. Azin2 mRNA levels were halved in heterozygous and
dramatically reduced in homozygous mice. Adrenal polyamine and
catecholamine content and mRNA levels of genes related with polyamine
metabolism were not affected by Azin2 deficiency. Microarray analysis
comparing gene expression in wild type and knock-out mice identified 28
genes that were down-regulated more than 2-fold in the adrenal gland of
transgenic mice and 67 genes up-regulated. Bioinformatics tools suggest
that both catecholamine secretion and the amount of secretory vesicles may
be reduced in the adrenal gland of knock-out mice. These results support
the previous view that AZIN2 may participate in vesicular trafficking and
secretion. (Supported by SAF2011-29051).
P19-5 (R03_19-5)
Generacin de translocaciones cromosmicas

en clulas humanas asociadas a cncer mediante
el empleo de CRISPR-Cas9
Juan Carlos Ramrez1, Ral Torres1, Sandra Rodrguez-Perales2
Fundacin Centro Nacional de Investigaciones Cardiovasculares,
CNIC, Madrid, ES, 2Centro Nacional Investigaciones Oncolgicas
(CNIO), Madrid, ES
Las translocaciones son un tipo de reordenamiento cromosmico

frecuentemente descrito en el inicio y/o evolucin de muchos tipos de
cncer. Algunas de ellas producen oncogenes que son responsables de la
transformacin maligna. El mecanismo especfico que genera este tipo de
reordenamiento es complejo y an no se conoce en detalle, pero siempre es
desencadenado por la dos roturas de doble hebra (DSB) en el ADN de dos
cromosomas no homlogos. La reparacin errnea de esas roturas al reunir
Granada 2014
de manera ilegtima los dos fragmentos de los cromosomas rotos resulta en
la generacin de translocaciones .
Para poder tener un conocimiento ms completo sobre como afectan las
translocaciones cromosmicas al inicio del cncer es necesario reproducir
de manera precisa estos reordenamientos. Hemos desarrollado una nueva
estrategia para inducir translocaciones cromosmicas especficas basada en
la capacidad del sistema CRISPR-Cas9 de inducir DSBs en localizaciones
especficas.
Como modelos de estudio hemos elegido las translocaciones t(8;21)/
RUNX1-ETO y t(11;22)/EWSR-FLI1, caractersticas de algunos tipos de
leucemia mieloide aguda y del sarcoma de Ewing. Usando el sistema
CRISPR-Cas9 hemos sido capaces de inducir con una elevada frecuencia
estas translocaciones tanto en lneas celulares establecidas, como en
clulas progenitoras humanas (clulas hematopoyticas y mesenquimales).
El anlisis por FISH y la secuenciacin a nivel de ADN, ARN y protena
de los genes de fusin generados revelan una alta fiabilidad y precisin
del sistema CRISPR-Cas9, proporcionando una nueva herramienta muy
poderosa para el estudio del cncer lo que podra llevar al desarrollo de
nuevas terapias basadas en los productos de estas translocaciones.
P20. Transportadores de membrana

P20-1 (R20-1)
Role of Aquaporins in cell proliferation: Functional

inhibition of AQP3 with a gold-based compound
Miriam Echevarra Irusta, Ana Galn-Cobo, Ana Serna, Reposo
Ramrez Lorca, Ismael Snchez Gomar, Juan Jos Toledo-Aral
Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario
Virgen del Roco/CSIC/Universidad de Sevilla (Departamento
de Fisiologa Mdica y Biofsica) - Centro de Investigacin
Biomdica en Red sobre Enfermedades Respiratorias (CIBERES) y
Neurodegenerativas (CIBERNED), Sevilla, ES
Objective: Numerous studies indicated an abnormal AQPs expression in
tumor of different origins and a role for these proteins in angiogenesis,
cell migration and proliferation had been shown. Recently we verified that
the gold (III) complex Auphen significantly inhibits the cell proliferation
of AQP3 expressing cells. Then to better understand the role AQPs may
play in the cell proliferation process we explore the effects that stable
overexpression of these proteins (o-AQPs) produce over the proliferation
and cell cycle of wt-PC12 cells as a cellular model.
Methods: Cell cycle by flow cytometry with propidium iodide and cell
proliferation through cell counting and BrdU staining were used. Using
Nocodazole we evaluated the cell response to arrest its cell cycle and the
resistance to apoptosis by Annexin V staining; and proteomic and transcriptomic
techniques were performed to highlight key molecules implicated in cell
proliferation which expression may be altered by overexpression of AQPs.
Finally, in cells with large expression of AQP3 we explore the effect of Auphen
over cyclins expression and cell cycle progression.
Results: Cells with o-AQPs showed higher cell proliferation rate and
larger percentage of cells in phases S and G2/M. After 24h in the presence
of Nocodazole, o-AQPs cells exhibited less modification of the cell cycle
pattern and lower Annexin V specific staining consistent with a higher
resistance to apoptosis. Additionally, in AQP3-expressing cells treated with
Auphen, strong arrest of the cell cycle in the S-G2/M phases, in concordance
with the analysis of cyclins (A, B1, D1, E) levels was observed. The RTqPCR analysis comparing o-AQPs cells to wt cells validated interesting
changes in the expression of molecules related with cell proliferation, tumor
and cell cycle progression, such as, Zeb2, Jun, JunB, NF-k, Cxcl9, Cxcl10,
TNF, and TNF receptors.
Conclusions: The significant role of AQPs in the cell proliferation process
seems to be connected to increments in the cell cycle turnover. Our results
support the view that larger expression of AQPs confers to the cell a more
Psters
tumor-like phenotype that contributes to explain the presence of these
proteins in much different type of tumors. A potential therapeutic effect of
Auphen in tumors where cell proliferation can be associated with AQP3
seems promising, but more studies are necessary to clarify this issue.
P20-2 (R20-6)
Occurrence of carbon catabolite repressionregulated mechanism involved in the transport

of extracellular ADP-glucose in Escherichia coli
Goizeder Almagro1, Manuel Montero1, Alejandro M. Viale2, Mehdi
Rahimpour1, Abdelhatif Bahaji1, Francisco Jos Muoz1, Edurne
Baroja-Fernndez1, Javier Pozueta-Romero1
1
Mutilva, ES, 2Instituto de Biologa Molecular y Celular de Rosario
(IBR, CONICET), Departamento de Microbiologa, Facultad de
Ciencias Bioqumicas y Farmacuticas, Universidad Nacional de
Rosario, Rosario, AR
ADP-glucose is the precursor molecule for glycogen and starch biosynthesis
in bacteria and plants, respectively. Due to the high abundance of this
nucleotide-sugar in tubers and endosperms present in the normal diet of
many animals, we explored the possible occurrence of an ADP-glucose
transport system in the enterobacterial species Escherichia coli. To avoid
artifacts resulting from the possible conversion of periplasmic ADP-glucose
breakdown products into glycogen, in this work we used glglC and pgm
null mutants unable to catalyze the reversible conversion of ADP-glucose
into glucose-1-P, and glucose-1-P into glucose-6-phosphate, respectively.
Noteworthy, these cells converted the exogenoulsy added ADP-glucose into
glycogen. Uptake of ADP-glucose was confirmed by the disappearance of
this nucleotide-sugar from the culture medium. pgmptsG, pgmptsH,
pgmptsI and pgmcrr cells impaired in the PTS components produced
glycogen from exogenously added ADP-glucose, indicating that the
transport of this nucleotide-sugar is not mediated by PTS. Furthermore,
pgmcyaA and pgmcrp cells, both impaired in the CRP-cAMP carbon
catabolite repression mechanism, neither incorporated ADP-glucose nor
produced glycogen from ADP-glucose, demonstrating that the uptake of
this nucleotide-sugar is CRP-cAMP regulated in E. coli. Finally, using a
systematic and comprehensive gene-disrupted mutant collection of E. coli
we identified some genes belonging to the CRP-cAMP regulon encoding
membrane proteins whose absence impair ADP-glucose transport.
P20-3 (R20-2)
Insights into the functional mechanism of secondary

transporters revealed by X-ray crystallography
Jos Luis Vzquez Ibar
Atomic structures of membrane transport proteins have revealed important
insights regarding the functional mechanism of membrane transport
proteins. In particular, these structures have confirmed the validity of the
alternate-access model, predicted forty years ago, as the general mechanism
used by these proteins to translocate substrates from one side to another side
of the membrane. In addition, questions like how substrate is recognized by
the transporter and how substrate triggers protein conformational changes
are being answered combining these high-resolution crystal structures with
biochemical and functional assays. In this regard, here I am presenting
two examples of substrate induced-fit mechanism in secondary transport
proteins, the initial step for substrate translocation. First, the latest crystal
structure of the lactose permease of E. coli in occluded state with bound
substrate reveals how substrate is able to sculpt the binding site; revealing
some hints about substrate and proton-motive force coupling. Second, the
structure of the onward-facing conformation of the L-arginine/agmatine
exchanger of E. coli with bound substrate reveals the molecular mechanism
of substrate-induced conformational changes in this transporter.
175
Psters
P20r-4 (R20-4)
Bases moleculares del efecto dominante negativo

de una mutacin de GlyT2 asociada
con hiperplexia
Esther Arribas Gonzlez1, Jaime de Juan Sanz2, Carmen Aragn
Rueda3, Beatriz Lpez Corcuera3
1
Centro de Biologa Molecular Severo Ochoa-CSIC-UAM - IdiPAZHospital Universitario La Paz, Universidad Autnoma de Madrid,
Madrid, ES, 2Department of Biochemistry, Weill Cornell Medical
College , New York, US, 3Centro de Biologa Molecular Severo
Ochoa-CSIC-UAM - Centro de Investigacin Biomdica en Red de
Enfermedades Raras, Instituto de Salud Carlos III - IdiPAZ-Hospital
Universitario La Paz, Universidad Autnoma de Madrid, Madrid, ES
La hiperplexia o enfermedad de sobresalto es un sndrome clnico
raro caracterizado por una respuesta exagerada a estmulos triviales
auditivos o tctiles. Este trastorno neurolgico puede producir dao
cerebral y/o muerte sbita en neonatos debido a episodios de apnea y
fallos cardiorrespiratorios. La enfermedad es causada por un dficit en
la neurotransmisin glicinrgica inhibidora. La forma presinptica de
la hiperplexia puede originarse por mutaciones en el gen humano del
transportador neuronal de glicina GlyT2 (SLC6A5). GlyT2 media el
reciclado de la glicina desde la hendidura hasta el terminal presinptico
constituyendo la principal fuente de neurotransmisor liberado en las
sinapsis glicinrgicas. Aunque la mayora de las mutaciones detectadas
son recesivas, produciendo prdida de funcin biallica por inactividad del
transportador o por su ausencia de la membrana, hay un mutante dominante
negativo sobre el trfico de GlyT2. Hemos explorado las propiedades y
alteraciones estructurales de este mutante y analizado el efecto dominante
negativo que retiene a GlyT2 en el retculo endoplasmtico impidiendo su
expresin en superficie. La introduccin de una arginina en lugar de Ser512 altera el plegamiento del transportador y promueve mejor asociacin
con la chaperona calnexina, menor unin a Sec24D y, por tanto, retencin
en retculo. Hemos demostrado la formacin de oligmeros comunes
GlyT2-S512R. La sobreexpresin de calnexina rescata el efecto dominante
negativo incrementando la cantidad de GlyT2 que alcanza la membrana
y reduciendo la interaccin entre GlyT2 y su mutante. El rescate por
chaperonas qumicas ha sido investigado en un sistema heterlogo y en
cultivos primarios de neuronas.
P20r-5 (R20-5)
Regulacin del transportador neuronal de glicina

GlyT2 por el receptor purinrgico P2X3
Luca Villarejo Lpez1, Esperanza Jimenez2, Carmen Aragon3,
Beatriz Lpez-Corcuera3
1
Centro de Biologa Molecular, Madrid, ES, 2Departamento de
Biologa Molecular, Centro de Biologa Molecular Severo Ochoa,
CSIC, UAM - Centro de Investigacin Biomdica en Red de
Enfermedades Raras, Instituto de Salud Carlos III - IdiPAZ-Hospital
Universitario La Paz, UAM, Madrid, ES, 3Centro de Investigacin
Biomdica en Red de Enfermedades Raras, Instituto de Salud
Carlos III - IdiPAZ-Hospital Universitario La Paz, Universidad
Autnoma de Madrid, Madrid, ES
La glicina es el neurotransmisor inhibidor ms abundante en reas
caudales del sistema nervioso central, desempea un importante papel
en la generacin de ritmos motores y permite el procesamiento de
seales nociceptivas, sensoriales y respuestas reflejas espinales. Es,
adems, un activador del receptor N-metil-D-aspartato (NMDA) en vas
glutamatrgicas excitadoras donde funciona como agonista obligado del
glutamato. La neurotransmisin mediada por glicina finaliza cuando es
retirada de la hendidura sinptica por los neurotransportadores (GlyT)
presentes en las membranas plasmticas de la gla adyacente (GlyT1) y en
los botones presinpticos de neuronas glicinrgicas (GlyT2).
Las dos isoformas han sido asociadas a la patognesis de ciertos desrdenes
del sistema nervioso como la esquizofrenia, alteraciones cognitivas,
176

dependencia de alcohol, dolor, epilepsia, desrdenes respiratorios e
hiperplexia o enfermedad del sobresalto, un desorden neurolgico humano
caracterizado por sobresaltos enrgicos y generalizados en respuesta
a estmulos triviales acsticos o tctiles asociado con una deficiente
neurotransmisin glicinrgica inhibidora cuya segunda causa principal son
mutaciones en el gen humano de GlyT2.
Un aspecto de gran inters es el estudio del papel de los transportadores de
glicina GlyT1 y GlyT2 en vas nociceptivas, ya que la red de interneuronas
glicinrgicas localizadas en las astas dorsales de la mdula espinal regulan
la transmisin de la seal dolorosa desde la periferia a regiones superiores
del cerebro. Trabajos de eliminacin gnica han revelado que la actividad
de los GlyTs, puede modular la neurotransmisin glicinrgica, por lo
que conocer la regulacin de los GlyTs en vas nociceptivas puede tener
consecuencias terapeticas.
De hecho, la inyeccin intratecal de inhibidores de GlyTs produce
analgesia en modelos animales de dolor. Estudios previos, han demostrado
que la neurotransmisin glicinergica puede regularse por la actividad de
receptores purinrgicos.
En este trabajo, hemos descrito cmo el receptor P2X3 es capaz de regular
la actividad de transporte de GlyT2 y proponemos las rutas de sealizacin
que median esta comunicacin intercelular entre neuronas sensoriales e
interneuronas glicinrgicas.
P20-6 (R20-3)
Modulacin del trfico de los transportadores de

nuclesidos concentrativos (CNT) a la membrana
plasmtica y sus implicaciones en la accin de
frmacos derivados de nuclesidos
Paula Fernndez-Calotti
Barcelona, Instituto de Biomedicina (IBUB). CIBERehd, Barcelona, ES
Los anlogos de nuclesidos (AN) constituyen una clase importante de
antimetabolitos proapoptticos utilizados en el tratamiento de neoplasias
hematolgicas, tumores slidos y enfermedades infecciosas. El ingreso de
estas molculas a las clulas se produce a travs de los transportadores de
nuclesidos de membrana (TN) concentrativos (hCNT) y equilibrativos
(hENT) contribuyendo a la biodisponibilidad del frmaco y a la citotoxicidad
o capacidad antiviral del mismo. La resistencia a los AN se ha relacionado a
la biodisponibilidad y la entrada a las clulas entre otras causas.
La regulacin de la actividad de los TN de forma especfica es un enfoque
adecuado para modular la biodisponibilidad de un determinado AN.
Demostramos que el cido transretinoico (ATRA) induce el trfico de
hCNT3 a la membrana plasmtica causando un aumento en su actividad
y en la consecuente entrada del AN fludarabina en clulas de leucemia
linfoctica crnica (LLC) causando una reversin de la resistencia al
frmaco. Recientemente hemos observado que el trfico de hCNT3 en
clulas de colon depende de la interaccin directa de la porcin amino
terminal del hCNT3 con Galectina 4.
Por otro lado, la terapia combinada para el tratamiento de la infeccin por
el virus de la hepatitis C implica interfern (IFN) y el AN ribavirina.
Aunque en condiciones basales hENT1 es el principal transportador de
ribavirina, ningn estudio ha evaluado el papel de hCNT2, un excelente
potencial transportador de ribavirina, en presencia de IFN. Demostramos
que IFN induce un rpido y transitorio aumento de la actividad hCNT2 en
clulas HHL5 derivadas de hepatocitos y un consecuente incremento en la
entrada de ribavirina a estas clulas.
Es evidente que la expresin de los TN posee un papel fundamental en la
biodisponibilidad y en la respuesta a frmacos AN. El anlisis del patrn
de expresin de TN podra ser til como biomarcador del metabolismo
y accin de frmacos asi como tambin para predecir la respuesta a una
determinada terapia. Por lo tanto, el estudio de las propiedades regulatorias
de cada TN es fundamental para la mejora de los tratamientos antitumorales
y antivirales.

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LIBRO DE RESMENES

XXXVII Congreso SEBBM

Primera edicin: Septiembre 2014

los autores, 2014

Produccin editorial: Rubes Editorial, S.L.

XXXVII Congreso SEBBM

Junta Directiva de la SEBBM

Socios protectores de la SEBBM

S2 Biotecnologa de plantas y productos de valor aadido

S3 Estructura y funcin de protenas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

P00 II Workshop sobre la Innovacin Docente en la Enseanza de la Bioqumica y Biologa Molecular

P02 Bases moleculares de la patologa

P04 Biologa molecular computacional

P07 Bioqumica perinatal

P08 Bioqumica y biologa molecular de plantas

P10 Estructura y funcin de protenas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

P11 Genmica y protemica

P12 Metabolismo del nitrgeno

P15 Radicales libres y estrs oxidativo

P16 Regulacin de la expresin gnica y dinmica del genoma

P13 Neurobiologa molecular

P19 Transgnesis en mamferos

P20 Transportadores de membrana

P05 Biomembranas y bioenergtica

Ministerio de Economa y Competitividad

XXXVII Congreso SEBBM

Carme Caelles Franch (responsable de Jvenes de la

Joaqun Ros i Salvador (coordinador de la Comisin Asesora

Juan Jos Lzaro Paniagua (Estacin Experimental del

Rafael Salto Gonzlez (secretario, Facultad de Farmacia,

Ana Linares Gil (Facultad de Ciencias, Universidad de

Mara Dolores Surez Ortega (vicepresidenta, Facultad de

Eduardo Lpez-Huertas Len (Estacin Experimental del

Otros miembros del Comit organizador - Junta Directiva de

Esperanza Ortega Snchez (Facultad de Medicina,

Juan P. Bolaos Hernndez (responsable del Portal

Mariam Sahrawy Barragn (Estacin Experimental del

Junta directiva de la SEBBM

Enrique de la Rosa Cano (2010-2014)

Juan Luis Ramos Martn (2010-2014)

Juan Pedro Bolaos Hernndez (2012-2016)

Socios protectores de la SEBBM

XXXVII Congreso SEBBM

Conferencia inaugural Alberto Sols

Selfish genes vs selfish metabolism: how

Conferencia plenaria LOral-UNESCO

Structures and mechanisms of bacterial

Conferencia plenaria Leloir

The brain speaks back to the ear: molecules,

XXXVII Congreso SEBBM

Conferencia plenaria Niemeyer - PABMB

posttranscriptional regulation of gene expression. Examples will be

Caveolin-1, a pleiotropic molecular switch

Conferencia plenaria FEBS National

Dynamics of protein-RNA interactions in

Conferencia plenaria de clausura

Understanding the role of proteolysis in disease

S1.1 Enfermedades moleculares

Pancreatic cancer: From molecular

Assessing the therapeutic role of Vav

XXXVII Congreso SEBBM

Muerte celular, senescencia y autofagia

S1.2 Sealizacin celular cascadas

The loss of memory in Alzheimer disease