Final Exam Study Guide

Below, you will find a list of learning objectives to emphasize from the cumulative material that
will be covered on the remaining portion of the final exam. They are listed in order as they were
presented in lecture and are broken down into categories. You will find that some of these learning
objectives bridge multiple sections and are redundant or related to each other. In addition, you will
find that the material we are learning in this final section will apply to several of these earlier
learning objectives as well, with the goal of helping you tie together many of the concepts we have
discussed across the semester! Think about how you can continue to integrate what we have
learned.
Study suggestions for the final exam
Prepare the new material as you would for a midterm, reviewing lecture notes, slides, relevant book
chapters and articles (from the reading assignments), and in-class activities (including the non-
graded ones too!). The emphasis, as it has been in the past, will be on material covered in lecture.
For reviewing the cumulative material from previous sections here are a few study ideas and
suggestions to consider (many of which will overlap with what you have been doing already!):
Review lecture slides and notes
Use the textbook and articles as resources where you need them: consider re-reading
sections where you need clarification for your notes/slides and sections that you found most
challenging for you on previous exams
Write out information relevant to the learning objectives to help you assess which learning
objectives you need to spend more time reviewing
Use the figures and graphs in the lecture slides and text book to practice explaining and
describing what they show
Go back and review any in-class activities
Flash cards for terminology are a great way to review and practice recalling information if
you find it challenging to remember details from previous sections
Form a study group and quiz each other on relevant material
Concept map and draw out and detail pathways
Think about how you can make connections from one section to another, compare and
contrast processes

Techniques
List several uses of antibodies in scientific research
o Allows for precise visualization of selected proteins among the many thousands that
each cell typically produces
o Individual antibody-producing B lymphocytes from an immunized mouse or rat,
when fused with cells derived from a transformed B lymphocyte cell line, can give
rise to hybrids that have both ability to make a particular antibody and the ability to
multiply indefinitely in culture.
o A monoclonal antibody can be made against any protein in a biological sample.
Once an antibody has been made, it can be used to localized the protein in cell and
tissues, to follow its movement, and to purify the protein to study its structure and
function.
Compare and contrast methods of protein identification
o Use of protein tags for purification- using standard genetic engineering techniques, a
short peptide tag can be added to a protein of interest. If the tag is itself an antigenic
determinant, or epitope, it can be targeted by an appropriate commercially available
antibody. The antibody, suitably labeled, can be used to determine the location of the
protein in cells or to purify it by immunoprecipitation or affinity chromatography. In
immunoprecipitation, antibodies directed against the epitope tag are added to a
solution containing the tagged protein; the antibodies specifically cross-link the
tagged protein molecules and precipitated them out of solution as antibody-protein
complexes.

o Separation by SDS-PAGE- Type of electrophoresis used to separate proteins by size.
The protein mixture to be separated is first treated with powerful negatively charged
detergent (SDS) and with a reducing agent (B mercaptoethanol), before being run
through a polyacrylamide gel. The detergent and reducing agent unfold the proteins,
free them from association with other molecules, and separate the polypeptide
subunits.

o Western Blotting (Immunoblotting)- technique by which proteins are separated by
electrophoresis and immobilized on a paper sheet and then analyzed, usually by
means of a labeled antibody.

o Mass Spectrometry- Technique for identifying compounds on the basis of their
precise mass-to-charge ratio. Powerful tool for identifying proteins and sequencing
polypeptides.

Describe the major methods used to analyze gene expression and alter gene function
o Analyze gene expression
RNA isolation and cDNA production- total mRNA is extracted from a
particular tissue, and the enzyme reverse transcriptase produces DNA copies
(cDNA) of the mRNA molecules. A short oligonucleotide complementary to
the poly-A tail at the 3 end of the mRNA is first hybridized to the RNA to
act as a primer for the reverse transcriptase, which then copies the RNA into
a complementary DNA chain, thereby forming a DNA/RNA hybrid helix.
Treating DNA/RNA hybrid with RNase H creates nicks and gaps in the RNA
strand. The enzyme DNA polymerase then copies the remaining single-
stranded cDNA into double-stranded cDNA. The fragment of the original
mRNA is the primer for this synthesis reaction, as shown. Because the DNA
polymerase used to synthesize the second DNA strand can synthesize through
the bound RNA molecules, the RNA fragment that is base-paired to the 3
end of the first DNA strand usually acts as the primer for the final product of
the second strand synthesis. This RNA is eventually degraded during
subsequent cloning steps. As a result, the nucleotide sequences at the extreme
5 ends of the original mRNA molecules are often absent from cDNA
libraries.

Polymerase Chain Reaction (PCR)- technique for amplifying specific regions
of DNA by the use of sequence-specific primers and multiple cycles of DNA
synthesis, each cycle being followed by a brief heat treatment to separate
complementary strands.
Conventional PCR-

Quantitative PCR-

Red has higher gene expression. It amplifies sooner
Microarray analysis- a large array of short DNA molecules (each of known
sequence) bound to a glass microscope slide or other suitable support. Used
to monitor expression of thousands of genes simultaneously: mRNA isolated
from test cells is converted to cDNA, which in turn is hybridized to the
microarray.

o Alter gene function
pg. 567


RNA molecules being used to inhibit gene expression (siRNA)
Use knowledge of established methods to suggest how to test a research question analyzing
regulation of gene expression or protein function
Protein Function
Explain how proteins containing similar protein domains have diversified function
o In addition to gene duplication, domain shuffling has diversified proteins containing
similar domains. Shuffling of blocks of protein domains has occurred during protein
evolution. Processes such as recombination and splicing have lead to diversified
function (s).

o
Describe the significance of binding specificity for antibody recognition of antigen
o The more compatible they are, the more non-covalent interactions will form which
means that longer associations will occur. If binding is not specific, very short or no
interaction will occur.
Outline two ways that phosphate groups can be used to regulate protein function
o Can turn on/off protein function by covalent addition of a phosphate group to an
amino acid side chain.

o Can induce a conformational changes to more tightly regulate signaling pathway

Activation of Src kinase- Messing up this cascade would lead to cell proliferation
uncontrollably.
Membrane Transport and Intracellular Trafficking
List the transport mechanisms by which molecules can pass through the cell membrane
Track the pathway of a membrane protein to the cell surface and identify several points of
regulation in the pathway
Determine what guides vesicles to their target location
Identify and describe how pathogens usurp the normal immune function in place to protect
the host from these pathogens
Cell Signaling
Suggest how GPCRs may have evolved to regulate taste perception in cells on the tongue
and provide protective responses in the airways
Taste receptor signaling- from tongues to lungs (Kinnamon, Acta Physiol.
2012)
Taste receptor signaling is not confined to the taste buds on the
tongue, found so far in several other locations
o Airways
o Pancreas
o Brain
o Gastrointestinal tract
Bitter receptors
o Evolved to help the organism avoid toxic (bitter) substances
o Detected in the lungs by used of GFP-reporter mice
E.g. GFP under control of a-gust and T1R3
promoters
o Cell types in airways expressing Bitter receptors: Solitary
chemosensory cells (SCCs), ciliated epithelial cells, and
smooth muscle cells
SCCs mostly restricted to vertebrates
o In the tongue, most buds express one set/kind of receptor, but
in airways, the cells express both types

Nuclear export
Describe the known mechanisms regulating nuclear export of mRNAs
NPCs allow bidirectional transport
Diffusion
o Water, sugar, ions, small molecules
Facilitated Transport
o Larger molecules (e.g. mRNPs, rRNA, proteins)
4 NPC-mediated export pathways have been described
CRM1-dependent; 4E;SE
CRM1-dependent; ARE
o CRM: Ran GTP DEPENDENT
CRM1-dependent; NXF3-mediated
NXF1-dependent
o NXF1/NXT1: Ran GTP independent
o Most mRNAs are thought to use this pathway

Suggest a potential function for the newly described nuclear envelope budding model
Export of mRNA by nuclear envelope budding
Budding of inner membrane into lumen of the nuclear envelope, then
vesicular fusion with the outer nuclear membrane
o Herpesvirus (HSV): nuclear egress
Virons in this case use a similar process thought to
be virus specific
Perhaps HSV was able to mimic or take advantage of a
process that already existed in the host cell.
Proposed budding-mediated mRNA export PROCESS:
o Phosphorylation of nuclear lamins is suspected to be involved
in this process (atyplical PKC)
o Identified by a study investigating development of Drosophila
larval synapse formation
Wnt-dependent neuromuscular junction synapse
formation
RNP complexes that function in neuromuscular
junction formation export the nucleus through budding
of the nuclear membrane
DFrizzled (Wnt receptor) in muscle cells binds
to ligand (Wingless, Wg) released from motor
neuron, to form neuromuscular synapse during
development
Upon binding Wg, the receptor is internalized
and the C-term cleavage product (DFz2C) is
released, then imported to the nucleus
RNP granules buds from nucleus and exocytose
into the cytosol in the muscle cells via lamin C
thru perinuclear space
o DFz2C foci formation is dependent on
Lamin C
Experimenters used PABP-GFP fusion protein
to determine if RNA was present in the
granules
o PABP-GFP is a Poly(A) binding protein
aPKC plays in role in phosphorylation of lamin
o HINT from HSV
LamC mutant cells do not have postsynaptic
Par6 protein
Cell Death
Suggest how cells in the human body may utilize necroptosis to defend against intracellular
pathogens
Compare and contrast apoptotic and necroptotic cell death pathways
Cell Adhesion
Describe the role of cell-cell adhesion during an immune response to mediate inflammation
o Selectins and integrins involved in tethering and rolling, the remainder of the
pathway is complicated, but involves a variety of interactions between the
lymphocytes and the endothelial cells (heterophilic interactions)
o Tethering and rolling occurs between leukocytes and endothelial cells until firm a
point of firm adhesion is reached. After this, diapedesis occurs which is a process of
cells attaching and going through 2 endothelial cells.
Describe the basic events that occur during an inflammatory response to infection and how
this leads to increased vascular permeability
o 1) Tissue Damage Occurs
o 2) Tissue damage causes release of vasoactive and chemotactic factors that trigger a
local increase in blood flow and capillary permeability.
o 3) Permeable capillaries allow an influx of fluid (exudate-complement, antibody, C-
reactive protein) and cells
o 4)Phagocytes migrate to site of inflammation (chemotaxis)
o 5) Phagocytes and antibacterial exudate destroy bacteria
o 6) Tissue Repair occurs
o General properties:
The bodys defense mechanism in response to tissue damage and/or infection
Initiators: infection, trauma (physical or chemical), and pathologic immune
responses
Purpose: identify the area affected and call into play mechanisms that lead to
(a) elimination of the inflammatory stimulus and (b) healing
o Selectins and integrins important here
White blood cells and endothelial cells interact with surface receptors
(selectins)
Once rolling and strongly adhered (integrins), diapedesis/extravasation
occurs

Suggest how decreasing vascular permeability may be advantageous for combat severe flu
infections
o Decreasing vascular permeability may be advantageous for combat severe flu
infections because one wouldnt have the issue of dealing with leukocytes which
have been shown to release cytokines, ROS, elastase, and amino acids which damage
the endothelium barrier.
Identify cell adhesion/junction components that may serve as promising drug targets for
development of future therapeutics
o Slit2N: increases retention of VE-cadherin at the cell-cell endothelial junctions
o Doxycycline: increases expression of VE-cadherin
o Both of these have been tested for these functions in mouse models and shown
improvement increased survival and/or decreased symptoms
o Can target ENaC- agonist which would lead to help clear fluid from lungs

Cell Cycle
Describe at least three techniques that can be used to study cell division and/or cell cycle
progression and suggest when these techniques may be useful
o Count cells to determines cell density
Dead cell exclusion by Trypan Blue staining
Trypan blue stains dead cells. Dying cells allow access of the outside to the
inside.
How would dying cells affect interpretation of your cell counts?
How quickly cells divide (rate) and rate at which theyre dying
influences count.
This approach is not good because cells could replicate rapidly and
then die instantly.
o BrdU
Artificial thymidine analog incorporated into replicating DNA. Instead of
thymidine, BrdU is added to interact with adenine.
Short vs. long pulse
Short pulse to figure out how many cells are dividing in short period
of time. Long pulse to determine how many cells are dividing over a
longer period of time
Visualize with anti-BrdU antibody
Can determine what % of cells are proliferating and timing of progression
through cell cycle stages with flow cytometry
o CFSE
(Carboxyfluorescein succinimidyl ester)
Fluorescent dye that binds to proteins in the cell
Can monitor the number of cell divisions
of CFSE will go to a daughter cell and to the other. Each round of
division you half the amount of CFSE
o DNA can be labeled with a dye too- allowing analysis of DNA content in cells by
flow cytometry. Example: using propidium iodide

Are these cells proliferating? Yes. All cells in G1 phase peak have a relative amount
of 1 DNA. Second peak shows # of cells whose DNA has been replicated already.
Level of dye detected is equivalent to amount of DNA present.
Cells in second peak are closer to getting duplicated.
Would not be able to tell difference between cells in G1 and G0
o Can follow cell cycle in vitro
Following two proteins that function to regulate interphase in human
fibroblasts by time-lapse live cell imaging:
Cdt1 is labeled red: protein expressed during G1 and early S phase
Geminin is labeled green: S/G2 expression
So, cells are red during G1, yellow during S, and green in G2 -> then
the cells enter M phase to divide. Yellow seen if there is overlapping
Cdt1 and Geminin proteins are localized in the nucleus. Once
accumulated, cells stopped dividing and were red (stuck in G1). Cells
ran out of space.

Define the roles of cyclins, Cdks, and Cdk inhibitors and determine how alteration in
expression or function of any of these regulators will influence cell cycle control
o Cyclins- protein that periodically rises and falls in concentration in step with the
eukaryotic cell cycle. Cyclins activate crucial protein kinases(Cdks) and thereby help
control progression form one stage of the cell cycle to the next.
o Cdks- Protein kinase that has to be complexed with cyclin protein in order to act.
Different Cdk-cyclin complexes trigger different steps in the cell-division cycle by
phosphorylating specific target proteins.
o Cdk inhibitors- protein that binds to and inhibits cyclin-Cdk complexes, primarily
involved in the control of G1 and S phases.
NEW MATERIAL

Ovarian Cancer Case Study
Determine ways to test if a cancer marker serves a function in the development or
maintenance of the cancer or if it is simply a byproduct
Describe two factors associated with prognosis for ovarian cancer patients undergoing
surgical removal of their tumor and chemotherapy
Discuss the types of mutations and changes to the cell that must occur to develop an ovarian
cancer, including the role of oncogenes, tumor suppressor genes, and EMT/MET transitions
Suggest why ovarian cancer treatments are not successful in preventing cancer
reoccurrences in some patients
Compare and contrast several current treatments/therapies in use and in the pipeline

Hematopoiesis
Explain how blood cells are derived from hematopoietic stem cells in the bone marrow
Hematopoietic stem cells (HSC) become Multipotential Stem Cells (MSC) which
then differentiates into Lymphoid Progenitor Cells (CLP-common lymphoid
progenitor) OR Myeloid Progenitor Cell (MPC- common myeloid progenitor). CLP
later differentiates into Natural Killer Cells (NK), T lymphocytes, B lymphocytes or
dendritic cells. MPC goes on to differentiate into Granulocytes (Neutrophils,
Basophils, Eosinophils), platelets, monocytes, mast cells, dendritic cells, or red blood
cells (erythrocytes).
Describe how bone marrow reconstitution was used to identify blood cell precursors
Mice were injected with IVs that were constituted of common lymphoid progenitor
(CLP) cells and HSC. This graph showed that more CLPs showed reconstitution of B
and T cells faster in the spleen because theyre already on their way there. In
contrast, HSC showed more reconstitution at a later time. The experiment tells us
that CLP is not prolonging the production the way that HSC is. CLP is not going to
be able to fully reconstitute the way the stem cell can in terms of the duration. HSC
differentiate and renew. CLP cant renew.
The second graph shows how T cells develop from observing CLP and HSC cells. In
CLP, by 6 weeks, you have all mature T cells. Takes at least 3 weeks to detect
progenitor cells in the thymus. CD4 cells develop faster. Once again, CLP are
showed to differentiate faster. In end results, both will have pretty similar outcome
(more CD4). The graph shows density of cells and percentages of each type. Bottom
left quadrant = DN = double negative. Top right= DP= double positive. Other two
are SP= single positive (for either CD8 or CD4). Top left is CD4, bottom right CD8.

Identify two important features of hematopoietic stem cells that allow for life long
production of new blood cells
HSC must be able to self- renew and differentiate.
Stromal cells in bone marrow support HSC maintenance: survival and self-renewal.
Stromal cells will interact with HSC to keep them alive and in their undifferentiated
state.
The dependence of HSCs on stromal cell signals will limit the availability of HSC
niches in the bone marrow.

Describe the role of signals from the extracellular environment and the role of transcription
factors during the stepwise differentiation of the blood cells lineages
HSC and its progenitors know whether to go down myeloid/lymphoid pathway
depending on signals it receives from the environment (colony stimulating factors).

There are specific receptors that will bind to cytokines and then they take use of
common/shared subunits to put together final signaling complex. If a progenitor cell
has ability to give rise to multiple lineages, it may express a variety of receptors on
its surface and then together with a common secondary binding partner depending on
which ligand is present. The signal that results from activation is what tells the cell to
do something.

The role of signals from extracellular environment is to cause a change in gene
expression in order to produce cells that are needed at the moment.
Transcriptional regulation of hematopoiesis
Lineage fate decisions
Developmental checkpoints
ex: lineage and stage specific recombination of Ig (surface antibody
on surface of B cell) and TCR genes in B and T cells, respectively
Establishment of gene expression profiles
Determines cell specific function. Transcriptional regulation is a
process of how we get cell specific functions.
Transcription factors determine lineage fate and drive cell specific
characteristics and functions
Suggest why hematopoietic progenitor cells can be the source of leukemias when regulatory
pathways are disrupted
Enhancing what a stem cell already does is going to be compatible with developing a
cancer.
Deregulated pathways leading to leukemia. Although leukemias are heterogeneous in
terms of phenotypes, there are general mechanisms underlying leukemic
transformation such as increased cell survival, increased proliferation capacity,
increased self-renewal capacity, genomic instability, and prevention of
differentiation. Examples of such deregulated mechanisms and/or signaling pathways
that have been found in various types of leukemias are indicated.

Innate and Adaptive Immunity
List the main cellular and molecular components of innate and adaptive immunity and the
general functions of each component
Innate, non-specific defenses
Physical and/or chemical barriers, e.g., epithelia
Cellular - phagocytic cells, natural killer (NK) cells (patrol for virally
infected or cancer forming cells)
Molecular - cytokines, chemokines, acute phase proteins, complement
Acquired, adaptive, specific defenses
Molecular antibodies (secreted by B cells)
Cellular lymphocytes (naive, effector, and memory lymphocytes), help
from phagocytic cells
Innate and Acquired are NOT mutually exclusive
Explain how the innate immune response functions to elicit an adaptive response when
necessary
Recognition of potential dangera pathogen and/or its products--by host cells and
molecules.
Recruitment and/or generation of destructive effector mechanisms for containment or
elimination of the potential danger.
Example: Dendritic cells (antigen presenting)
Describe the main features of the adaptive immune response, including specificity and
memory
For INNATE Immune Response:
A limited number of binding specificities (distinct receptors) are expressed
by cells of the innate defense system
Recognition and response are rapid--measured in minutes to hours
There is no specific memory generated as a consequence of recognition and
response
For ADAPTIVE Immune Response:
Specific responses expressed by cells of adaptive defense system
Recognition and response are slow in 4-14 days.
Time after Infection Type of Immunity Defenses Used
Immediate Innate Physical and chemical barriers
0 -4 hours Innate Molecular and cellular
recognition and response
4 96 hours Innate Molecular and cellular
recruitment of cells from
circulation and their activation
4 14 days Adaptive Lymphocytes: Specific responses
(memory cells are generated)

Determine how the genome can encode for the diverse antigen receptor repertoire utilized by
B and T cells to detect a wide array of pathogens
Stem cells in bone marrow are the source of T- and B- cells.
T cells undergo further maturation in the thymus and are responsible for cell-
mediated immunity and regulation of immune responses.
B cells, when activated, are responsible for the production of antibodies.
In B cells, clonal expansion method is used. Proliferation and diversification in bone
marrow first occurs. Then, antigen binds to specific B cell in peripheral lymphoid
organ. Last, proliferation (clonal expansion) and differentiation of B cells occurs.
Benefit of clonal expansion is that you dont create cells that you dont need
When antigen is present, only B cell that is specific for antigen will be
activated. Once activated, it will make lots of copies of itself.

Memory also plays a role to detect wide array of pathogens.
Memory cells hang out for some time.
Nave cell forms effector cells (they go out and neutralize or destroy
pathogen) (also form memory cells).
Memory cells can now generate more memory cells and effector cells upon
another encounter with antigen. This is only seen in innate response.

Alternative splicing gets a lot of products from same gene.
Non homologous end-joining
In each cell specific to certain pathogen, the DNA will be different among cells

Describe two mechanisms that support immunological tolerance to self antigens
Central- Maturing B cells in the bone marrow and T cells in the thymus are screened
for expression of potentially auto-reactive receptors. Auto-reactive cells are
deletedclonal deletion.
Peripheral- In the periphery, auto-reactive B and T cells are inactivated (anergized)
clonal inactivation, or are prevented from being activated by inhibitory regulatory T
lymphocytes (T
reg
).
Antigen Presentation
Explain how T cells see antigen, and how this is different from how antibodies detect
antigen
T cells only see the processed peptide loaded on the MHC
The structure of TCRs is analogous to immunoglobulins, but it is always cell-bound
and never secreted (like antibodies from B cells are)
TCRs must recognize antigen presented by specialized cells; antibodies recognize
antigens in the milieu (or environment)
TCRs recognize a complex of an antigen-derived peptide epitope AND portions of
the presenting, self MHC molecule

T cells see antigenic determinants via their antigen specific T cell receptor (TCR)
TCRs recognize linear antigenic peptide epitopes
These epitopes do not exist in the native antigen structure
T cell receptors recognize antigenic peptide epitopes presented by MHC molecules
processed polypeptide epitopes are brought to and expressed at the surface of
antigen presenting cells by MHC class I or MHC class II presenting
molecules
T cell sees chewed up peptide of bacteria. Doesnt see whole bacteria. Antibodies see
epitope on surface of bacteria in its native form.
Define "major histocompatibility complex (MHC)" and its role in the immune response.
They were originally defined as a major barrier to organ and tissue transplantation,
since they are all encoded by extremely polymorphic genetic loci in humansthe
major histocompatibility complex
Describe the difference between Class I and Class II MHC molecules - what cells express
them and the role they play in defense.
MHC class I molecules expressed on antigen presenting cells are required for
presentation of antigen to and activation of CD8+ cytotoxic T cells
MHC class I molecules are expressed on all cells of the body except red
blood cells.
MHC class II expressed on antigen presenting cells are required for presentation of
antigen to and activation of CD4+ helper T cells
MHC class II molecules are only expressed by antigen presenting cells
(dendritic cells, monocytes/tissue macrophage and B cells)
Both classes of MHC molecules bind and present antigenic peptides to T cell
antigen receptors
Suggest why CD8 T cells tend to respond more to intracellular antigens whereas CD4 T
cells tend to respond to extracellular antigens

Autoimmunity Case Study Discussion: Celiac Disease
Define, draw, and explain what an antibody is
An antibody is a large protein that consists of four interlinked peptides. The antigen
binding sites work in a ligand-receptor or lock-and-key fashion with antigens that
accompany microbial invasion.
Two identical light chains
2 or 2.
Two identical heavy chains
2m, 2d, 2g1, 2g2, 2g3, 2g4, 2a1, 2a2, or 2e.
The heavy chain type defines the class
Two 1 chains would define an IgG antibody, and in this case, the subclass
IgG1
Two m heavy chains would define an IgM antibody, etc.

Define autoimmune disease and the role of autoantibodies
Describe the symptoms, diagnosis, risk factors, and treatments for celiac disease
Discuss possible hypotheses for the development of autoimmune disease
Explain, at a cellular and molecular level, how celiac disease is thought to be triggered and
maintained, including the role of gluten, TG2, B cells, and T cells, and Dendritic cells
Suggest why celiac disease challenges previous hypotheses for the development of
autoimmunity