Final Exam Study GuideBelow, 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 examPrepare 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 Allows for precise visualization of selected proteins
among the many thousands that each cell typically produces
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. 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 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.
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. 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. 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 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.
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
functionProtein Function Explain how proteins containing similar
protein domains have diversified function 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).
Describe the significance of binding specificity for antibody
recognition of antigen 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 Can turn on/off protein
function by covalent addition of a phosphate group to an amino acid
side chain.
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 Airways Pancreas Brain
Gastrointestinal tract Bitter receptors Evolved to help the
organism avoid toxic (bitter) substances Detected in the lungs by
used of GFP-reporter mice E.g. GFP under control of a-gust and T1R3
promoters Cell types in airways expressing Bitter receptors:
Solitary chemosensory cells (SCCs), ciliated epithelial cells, and
smooth muscle cells SCCs mostly restricted to vertebrates 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 Water,
sugar, ions, small molecules Facilitated Transport Larger molecules
(e.g. mRNPs, rRNA, proteins) 4 NPC-mediated export pathways have
been described CRM1-dependent; 4E;SE CRM1-dependent; ARE CRM: Ran
GTP DEPENDENT CRM1-dependent; NXF3-mediated NXF1-dependent
NXF1/NXT1: Ran GTP independent 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 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: Phosphorylation of nuclear
lamins is suspected to be involved in this process (atyplical PKC)
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 DFz2C foci formation is dependent on
Lamin C Experimenters used PABP-GFP fusion protein to determine if
RNA was present in the granules PABP-GFP is a Poly(A) binding
protein aPKC plays in role in phosphorylation of lamin HINT from
HSV LamC mutant cells do not have postsynaptic Par6 proteinCell
Death Suggest how cells in the human body may utilize necroptosis
to defend against intracellular pathogens Compare and contrast
apoptotic and necroptotic cell death pathwaysCell Adhesion Describe
the role of cell-cell adhesion during an immune response to mediate
inflammation 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) 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 1) Tissue Damage Occurs 2) Tissue damage causes
release of vasoactive and chemotactic factors that trigger a local
increase in blood flow and capillary permeability. 3) Permeable
capillaries allow an influx of fluid (exudate-complement, antibody,
C-reactive protein) and cells 4)Phagocytes migrate to site of
inflammation (chemotaxis) 5) Phagocytes and antibacterial exudate
destroy bacteria 6) Tissue Repair occurs 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 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 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 Slit2N: increases retention of VE-cadherin
at the cell-cell endothelial junctions Doxycycline: increases
expression of VE-cadherin Both of these have been tested for these
functions in mouse models and shown improvement increased survival
and/or decreased symptoms 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 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. 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 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 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 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 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. 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. 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 InfectionType of
ImmunityDefenses Used
ImmediateInnatePhysical and chemical barriers
0 -4 hoursInnateMolecular and cellular recognition and
response
4 96 hoursInnateMolecular and cellular recruitment of cells from
circulation and their activation
4 14 daysAdaptiveLymphocytes: 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
(Treg).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