Professional Documents
Culture Documents
Multiple Choice: / Multipart Questions: /10 /6 /8 /8 /10 /10
Multiple Choice: / Multipart Questions: /10 /6 /8 /8 /10 /10
Multiple Choice: / Multipart Questions: /10 /6 /8 /8 /10 /10
Final examination
Name: Student #:
Multiple choice: / 25
Multipart questions:
/10
/6
/8
/8
/10
/10
/10
/24
/14
TOTAL: /125
The following defines the honour code expectations of the Faculty of Applied Science and
Engineering. Please write your name above and adhere to the high standards of the Faculty.
The University and its members have a responsibility to ensure that a climate that might
encourage, or conditions that might enable cheating, misrepresentation or unfairness not be
tolerated. To this end, all must acknowledge that seeking credit or other advantages by fraud or
misrepresentation, or seeking to disadvantage others by disruptive behaviour is unacceptable, as
is any dishonesty or unfairness in dealing with the work or record of a student. It shall be an
offence for a student knowingly: (a) to forge or in any other way alter or falsify any document or
evidence required for admission to the University, or to utter, circulate or make use of any such
forged, altered or falsified document, whether the record be in print or electronic form; (b) to use
or possess an unauthorized aid or aids or obtain unauthorized assistance in any academic
examination or term test or in connection with any other form of academic work; (c) to personate
another person, or to have another person personate, at any academic examination or term test or
in connection with any other form of academic work; (d) to represent as one's own any idea or
expression of an idea or work of another in any academic examination or term test or in
connection with any other form of academic work, i.e. to commit plagiarism; (e) to submit,
without the knowledge and approval of the instructor to whom it is submitted, any academic
work for which credit has previously been obtained or is being sought in another course or
program of study in the University or elsewhere; (f) to submit any academic work containing a
purported statement of fact or reference to a source which has been concocted.
1
Answer all questions. All questions are to be answered on the examination paper in the space
provided. State all assumptions.
3) On average, how many ATPs would be made if 4 NADH and 6 FADH2 molecules donated their
high-energy electrons to the mitochondrial electron transport chain?
10
24
12
30
20
4) How do mitochondria generate and store the energy used to produce most of the ATP made
during aerobic respiration?
by producing heat
by generating a heat gradient
by generating a H gradient
by generating a Cl- ion gradient
by generating a Na ion gradient
5) What is formed when electrons reach the bottom of the mitochondrial electron transport chain
and bind to the final electron acceptor?
water
carbon dioxide
carbon monoxide
hydrogen
oxygen
6) All collagen family members consist of chains arranged in a
2, double helix
3, double helix
3, triple helix
3, triple lattice
2, triple helix
7) What substance joins proteoglycans together into gigantic complexes called proteoglycan
aggregates? These complexes can occupy very large volumes.
hyaluronidase
hyaluronic acid
proteoglycase
fibronectin
laminin
8) You coat a Petri dish with fibronectin and proteoglycans and culture cells on the dish. The cells
adhere to the dish. You repeat the experiment but this time add RGD tripeptides to the culture
dish as the cells are added. What happens?
The cells adhere as they normally do.
The cells die immediately.
The cells lyse immediately.
The cells do not adhere to the dish.
The cells immediately change their phenotype.
10) Mesenchymal stem cells were grown on a very stiff substrate. Into what type of cells did they
develop?
skin cells
muscle cells
nerve cells
osteoblasts (bone) cells
pancreatic cells
11) Which cytoskeletal element is described as tough, rope-like fibers composed of a variety of
related proteins like keratin?
microfilaments
microtubules
intermediate filaments
e) macrofilaments
3
12) Which of the following molecular motors is known to travel in a retrograde direction (i.e. toward
the cell body) along microtubules?
kinesins
dyneins
myosins
kinesins and myosins
kinesins and dyneins
13) What is the direct source of energy that powers molecular motors?
hydrolysis of GTP
hydrolysis of ATP
proton gradient
H gradient
condensation of ATP
15) As a fibroblast moves, its leading edge extends from the cell as a broad, flattened, veil-like
protrusion called a
pseudopodium
lamella
lamellipodium
podium
extensor
Answer: c
16) The period in the cell cycle between the end of dell division and the beginning of DNA synthesis
is called the phase.
GI
S
G2
d)M
e) GO
17) The total length of the cell cycle of cells in a cell culture is 24 hours. M phase is found to be 1
hour, S phase is found to be 9 hours and G2 is found to be 4.5 hours. How long is G1 ?
24 hours
9.5 hours
9 hours
1 hour
4.5 hours
4
18) Cells that have stopped dividing and are arrested in a stage preceding the initiation of DNA
synthesis are said to be in a state.
G1 phase
S phase
G2 phase
M phase
Go phase
19) What external stimulation is required by mammalian cells to progress through the cell cycle prior
to reaching the restriction point?
presence of steroid hormones in their culture medium
presence of Na+ ions in their culture medium
presence of growth factors in their culture medium
no external stimulation is required
presence of vitamins in their culture medium
4 -5 2 6 -3 1
- - -
5-4 2 6 - 3 1
- - -
c)4 - 6 2 5 3 -1
- - -
d)4 5 2 -3 1 6
- - - -
e) 1 5 2 4 3 -6
- - - -
21) While bound to phosphorylated GPCRs, to what else can arrestins bind?
G proteins
clathrin-coated pits
other arrestins
hormones
GRKs
22) What event is usually responsible for terminating signal transduction by RTKs?
dephosphorylation of the receptor
degradation of the ligand
receptor internalization
phosphorylation of the receptor
acetylation of the receptor
5
23) is a small protein that is linked covalently to other proteins, thereby marking those
proteins for internalization or degradation.
Chaperonin
Ubiquitin
Proinsulin
Transcriptin
Tubulin
25) Specificity in MAP kinase pathways is sometimes achieved by spatial localization of the
pathway's component proteins. Spatial localization of these components is done by structural
(i.e., nonenzymatic) proteins called
sequestration proteins
partitioning proteins
scaffolding proteins
framework proteins
spatial organization proteins
MULTIPART QUESTIONS
1) Receptor tyrosine kinases (RTK) respond to ligand at the plasma membrane.
(a) What are the basic mechanisms that result in transmission of RTK signal across the plasma
membrane? In your answer, ensure you describe the impact of ligand design on the monomer ->
dimer transition of the receptor. Also contrast the monomer/dimer model with the emerging
model that receptors don't dimerize but instead twist. (4 marks)
(b) How are effector proteins involved in further transmitting this signal? (4 marks)
.N
(c) What are two critical features of a second messenger? (2 marks)
What impact would a drug that shuts off voltage-gated Ca2 channels such as nimodipine
have on resting Ca2 levels? (2 marks)
How does a rise in intracellular Ca2 primarily impact cellular function? (2 marks)
7
(b) Critical proteins involved in the regulation of Epo receptor inactivation are SHP 1
phosphatase and SOCS protein. Describe how these proteins are involved in short and
long-term regulation, respectively. Highlight the role of the SH2 binding domains. (4
marks)
4) You are a tissue engineer in a prestigious institution. However, to be at the top of your
game you need to know some basic things about tissues.
What are four critical components of connective tissue and what do each of these
components do? (4 marks)
You are given a tissue that has a large fibrillar component. The fibers are aligned parallel
to each other and the long axis of the tissue. What can you conclude about the pulling
forces experienced by this tissue? (2 marks)
You study another tissue that also contains a fibrillar component. In this tissue, however,
the fibers are arranged in layers with each layer perpendicular to those above and below
it. In addition, the fibers are uniform in size. What properties would such a tissue have?
(2 marks)
5) Cells migrate towards a chemoattractant gradient with as little as 2% difference in ligand
concentration from the front to rear of the cell.
Describe how the various signaling molecules (receptor, P13K, PTEN) are distributed
across the cell in response to this gradient. (4 marks)
Why would the addition of an irreversibly binding ATP analog to an in vitro system for
monitoring molecular motors stop motor function? (2 marks)
6) Many research labs are focussed on designing new and different fluorescent proteins.
A major focus is in the development of redder fluorescent proteins. Why? (2 marks)
Achieving redder fluorescent proteins through point mutations of the original GFP beta-
barrel has proven to be difficult. Why? (3 marks)
Is there such a thing as the "perfect fluorescent protein"? Please defend your answer (5
marks)
7) The redox exchange pairs NADH/NAD and NADPH/NADP perform very different
functions in a cell.
(a) What are the basic functions of each (i.e. where do they donate their electrons)? (5
marks)
10
Are their ratios similar or different? Why? In your answer indicate the supply and
demand of each. (3 marks)
What do you think would be the major impact on metabolism if a cell is treated with a
drug that inverts the NADH/NAD ratio? (2 mark)
8) The following vector (i.e. plasmid) is used for tagging proteins with EGFP.
pEGFP-NI Vector Information PT3027-5
GenBank Accession #U55762 Catalog #6085-1
Asel
8)
ApaL I SnaB I
(341)
MCS
1591-671)
P UP PCMV 1E
EcoO1O9 I Or)
3856) 7
HSVTK
Poly EGFP
pEGFP-N1
4.7 kb BsrG I (1389
K ann/ SV4O Not l(1402(
Neor
fl
I (1412)
on Xba
sv4oepOIYA
Al! 11 ( 1640)
EGFP
0.OIA.00G CIA CCG GAG TCA OAT GIG GAG GIG AAG Gil CGA All GIG CAG 110 ACG GTA CCG COG 9CC COG OAT CCA CCG GTC 0CC ACC AIG GIG
Nhe7 III BflIXhEIfind W Ec Safl
Sad I Accl Aspll8l 8sp1201 Xmal
EcI136 II Sac It Sma I
11
(a) In this plasmid, what is the purpose of the (3 marks):
MCS:
PCMV:
KanlNeo
(b) If one wanted to tag a protein with EGFP using the above vector, what are three design
concerns to consider when cloning the gene into the vector? (3 marks)
(c) Sketch the general "beta-barrel" structure of GFP indicating (i) the approximate length
and width of the beta-barrel, (ii) the location of the C- and N-terminals, and (iii) the
location of the chromophore. (3 marks)
(d) What constraints does the beta-barrel structure have on FRET between two fluorescent
proteins (e.g. ECFP and EYFP)? Note: the Förster distance for this pairing is 5 nm. (2
marks)
12
(e) The beta-barrel structure of EGFP folds very efficiently. Why is this statement relevant
to: (i) the fact that EGFP rarely affects the protein function, and (2) the quantitation of
protein expression based on fluorescence? (3 mark)
13
(g) Fluorescent proteins are commonly used to create genetically encoded sensors for live
cell imaging. The Rocheleau lab recently designed a genetically encoded sensor for
NADP that depends on a monomer / dimer transition of glucose-6-phosphate
dehydrogenase (GOD) and measuring changes in homoFRET by the steady-state
fluorescence anisotropy.
What did we name the sensor? (full acronym as well please) (2 mark)
What makes the design of this sensor so robust (stable) and amenable to
multiparametric imaging? (4 marks)
9) The following is from a recent paper titled "TRAF4 binds to the juxtamembrane region of
EGFR directly and promotes kinase activation".
Abstract:
The activation of the epidermal growth factor receptor (EGFR) is cru-
cial for triggering diverse cellular functions, including cell proliferation,
migration, and differentiation, and up-regulation of EGFR expression
or activity is a key factor in triggering the development of cancer.
Here we show that overexpression of a scaffold protein, tumor ne-
crosis factor receptor (rNF-R)-associated factor 4 (TRAF4), promotes
EGF-induced autophosphorylation of EGFR (activation) and down-
stream signaling, whereas TRAM deficiency attenuates EGFR activa-
tion and EGF-driven cell proliferation. Using structure-based sequence
alignment and NMR spectroscopy, we identified a TRAM binding site
in the C-terminal half of the juxtamembrane (JIM) segment of EGFR, a
region known to promote asymmetric dimerization and subsequent
activation. Deletion of the TRAM binding site led to dramatic defects
in EGFR activation and EGF-driven cell proliferation. Specific point
mutations in the TRAM binding site also resulted in significant atten-
uation of EGFR activation. Detailed structural examination of the in-
active versus active forms of EGFR suggests that TRAM binding
probably induces a conformational rearrangement of the JM region
to promote EGFR dimerization. These results identify a novel mecha-
nism of TRAF4-mediated EGFR activation and signaling.
14
I '
TRAF4 KO+TRAF4 TRAF4 KO
EGF (nm) 0 0 0.5 1.0 2.5 5.0 0 0 0.5 1.0 2.5 5.0
pEGFR
Y1068
pEGFR
Y992
EGFR 4
Fig. 1. TRAF4 is required for EGFR activation in re-
sponse to EGF stimulation. (A) TRAF4-deficient pAKT
(TRAF4 KO) HeLa cells were transfected with TRAM
'NT or empty vector and, 24 h later, the cells were AKT
starved of serum overnight and stimulated with
2 nglmL EGF for various times. Cell lysates were then TRAF4
analyzed by the Western method with the indicated
antibodies. (B) HeLa cells containing TRAF4-Myc in a
Actin m. 4I
(a) Describe the technique used in this study. In your answer, be sure to indicate what you
are measuring (mRNA, lipids, or proteins), the role of SDS, the polyacrylamide gel,
antibodies, voltage, and blotting paper (8 marks)
(b) How is the data in the blot interpreted? In your description, indicate which rows are
"loading controls" and how they are used in your interpretation. (3 marks)
15
(c) Biologists are fond of cartoon drawings since they distill to a basic idea/model. Attempt
to draw the impact of TRAF4 based on the information given? (3 marks)
16
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