Name: ! BME 302 Cellular Engineering HW 2 – Cell Signaling & Assays (112 points) Due in class February 16, 2016 ! Section 1 – Cell Signaling Please provide concise and clear answers – WRITE WELL ! 1. What Kind of Signals Do Cells Receive? List and describe the main types (4) 2. What’s the difference between neurotransmitters and the follicle-stimulating hormone signaling molecules? (4) 3. How Do Cells Recognize Signals? What types of cellular signal-receiver exist? (4) 4. How Do Cells Respond to Signals? (4) 5. What are the second messenger and give an example (4) 6. Why do protein conformational changes affect cell signaling? (4) Sarah Al Ameer Growth factors: factors that promoting growth and tissue repair. Hormones: Long range signal molecules to trigger action of target. Neurotransmitters: Short range signaling molecules that travel between neurons and muscle cells. Extracellular matrix components. 7. What’s the role of protein kinase and protein phosphatase and what is the mechanism of action? (4) 8. How Do Signals Affect Cell Function? (4) 9. Based on the wnt signaling pathways that is schematized below what do you think will happen to the cell if you - knock-out IP3 (2) - overexpress CKIa (2) - knock-out NKD (2) The Calcium ion channels will not open in the cell membrane, therefore cannot release Calcium ions in large amounts to activate protein kinase C. 10. Based on the TGF-beta signaling pathways that is schematized below what do you think will happen to the cell if you - Overexpress Smad7 (2) - Knock-out Smad 2/3 (2) - Inhibit PTHR by using soluble receptor ligands (2) 11. What does CELL PHENOTYPE mean? (4) 12. What’s the difference between quantitative and qualitative assessment of cell phenotype. Prove examples for both. (4) 13. You have cell A and cell B. Cell A changes phenotype after you place it in an in vitro co- Precievable characteristics and traits of gene. culture system with cell B. How do you determine if this is the result of paracrine or contract- dependent signals? (4) 14. You start a culture of stem cells and you use a protocol to differentiate them into beta cells. To make sure they are truly beta cells. First you want to check whether the insulin gene is expressed at higher levels in the differentiated vs. the undifferentiated cells. What methods that we studies would you use and describe the step by step procedure. (4) Then you want to demonstrate that they secrete insulin when you place glucose in their culture media. What methods that we studies would you use and describe the step by step procedure. (4) Finally, you want to show that inside differentiated cells insulin is located within secretory vesicles and quantify in whic.

1. Name:
!
BME 302 Cellular Engineering
HW 2 – Cell Signaling & Assays (112 points)
Due in class February 16, 2016
!
Section 1 – Cell Signaling
Please provide concise and clear answers – WRITE WELL
!
1. What Kind of Signals Do Cells Receive? List and describe
the main types (4)
2. What’s the difference between neurotransmitters and the
follicle-stimulating hormone
signaling molecules? (4)
3. How Do Cells Recognize Signals? What types of cellular
signal-receiver exist? (4)

2. 4. How Do Cells Respond to Signals? (4)
5. What are the second messenger and give an example (4)
6. Why do protein conformational changes affect cell signaling?
(4)
Sarah Al Ameer
Growth factors: factors that promoting growth and tissue
repair.
Hormones: Long range signal molecules to trigger action of
target.

3. Neurotransmitters: Short range signaling molecules that travel
between neurons and muscle cells.
Extracellular matrix components.
7. What’s the role of protein kinase and protein phosphatase and
what is the mechanism of
action? (4)
8. How Do Signals Affect Cell Function? (4)
9. Based on the wnt signaling pathways that is schematized
below
what do you think will happen to the cell if you
- knock-out IP3 (2)

4. - overexpress CKIa (2)
- knock-out NKD (2)
The Calcium ion channels will not open in the cell membrane,
therefore cannot release Calcium ions in large amounts to
activate protein kinase C.
10. Based on the TGF-beta signaling pathways that is
schematized below
what do you think will happen to the cell if you
- Overexpress Smad7 (2)
- Knock-out Smad 2/3 (2)
- Inhibit PTHR by using soluble receptor ligands (2)

5. 11. What does CELL PHENOTYPE mean? (4)
12. What’s the difference between quantitative and qualitative
assessment of cell phenotype.
Prove examples for both. (4)
13. You have cell A and cell B. Cell A changes phenotype after
you place it in an in vitro co-
Precievable characteristics and traits of gene.
culture system with cell B. How do you determine if this is the
result of paracrine or contract-
dependent signals? (4)

6. 14. You start a culture of stem cells and you use a protocol to
differentiate them into beta cells.
To make sure they are truly beta cells.
First you want to check whether the insulin gene is expressed at
higher levels in the
differentiated vs. the undifferentiated cells. What methods that
we studies would you use and
describe the step by step procedure. (4)
Then you want to demonstrate that they secrete insulin when
you place glucose in their
culture media. What methods that we studies would you use and
describe the step by step
procedure. (4)
Finally, you want to show that inside differentiated cells insulin
is located within secretory
vesicles and quantify in which percentage of the cells. What
methods that we studies would
you use and describe the step by step procedure. (4)

7. 15. You hypothesize that a group of tolerogenic cells is located
in the fetal pancreas.
What method would you use to identify their location within the
tissue? (4)
What method would you use to quantify their abundance in the
whole fetal pancreas based on
their surface expression of protein A and Protein B and the
absence of protein C? (4)
16. What is the difference between genomic DNA and cDNA?
(4)
17. You need to determine the effects of the duration of

8. treatment with ethylene vs.
ethylene+A23187 on calmodulin gene expression. You extract
total mRNA from your cells,
generate cDNA by reverse transcription and run end-point PCR.
You run your PCR product on
a gel and you get the following picture. Draw a bar plot to
quantify the following results of
end-point PCR. (4)
18. You need to determine the effects of knocking-down (KO)
the CIZ gene on expression of the
following genes: COL1, ALP, OPN, OSX. You extract total
mRNA from your control cells: wild
type (WT) cells, and from your genetically engineered cells
(CIX-KO), then you generate cDNA
by reverse transcription and run end-point PCR. You run your
PCR product on a gel and you
get the following picture. Draw a bar plot to quantify the
following results of end-point PCR.
(4)

9. 19. You need to quantify expression of the INS gene on several
stem cell derived insulin-
secreting cell products (SCb) that you have generated: SCb1,
SCb2, SCb3, SCb4, SCb5, SCb7
and choose the product that displays the highest INS expression.
You extract total mRNA
from all your seven cell products, then you generate cDNA by
reverse transcription and run
real time PCR and you get the following plot. (8)
Make a table with the Ct for each sample. (4)
Assuming that all seven samples had a Ct of the endogenous
gene of 16 and a Ct of the INS
gene for the control cells (islets) of 10, express the expression
of the INS gene for each cell
product relative to islets. (4)

10. 20. If you run a real time PCR on your cDNA samples and you
get the following plot, what does it
mean and what are the possible causes? (4)
21. You need to characterize the vascularization of an
engineered pancreatic tissue (from mice).
You have prepared the tissue by fixing it in 10% formalin and
by sectioning it into 5µm-thick
sections. Now you decide to perform in situ
immunohistochemistry. (4)
You have the following primary antibodies:
Rabbit anti-mouse CD31 (will stain all endothelial cells in
blood and lymphatic vessels)
Goat anti-mouse Lyve-1 (will stain only endothelial cells in
lymphatic vessels)

11. Guinea pig anti-mouse INSULIN (will stain all beta cells)
Rabbit anti-mouse GLUCAGON (will stain all alpha cells)
And these secondary antibodies:
Chicken anti-rabbit HRP
Chicken anti-rabbit AF488 (green fluorophore)
Chicken anti-rabbit AF594 (red fluorophore)
Chicken anti-rabbit AF546 (red fluorophore)
Chicken anti-rabbit AF605 (cyan fluorophore)
Chicken anti-goat HRP
Chicken anti-goat AF488 (green fluorophore)
Chicken anti-goat AF594 (red fluorophore)
Chicken anti-goat AF546 (red fluorophore)
Chicken anti-goat AF605 (cyan fluorophore)
Rabbit anti-guinea pig HRP
Rabbit anti-guinea pig AF488 (green fluorophore)
Rabbit anti-guinea pig AF594 (red fluorophore)
Rabbit anti-guinea pig AF546 (red fluorophore)
Rabbit anti-guinea pig AF605 (cyan fluorophore)
Chicken anti-guinea pig HRP
Chicken anti-guinea pig AF488 (green fluorophore)
Chicken anti-guinea pig AF594 (red fluorophore)
Chicken anti-guinea pig AF546 (red fluorophore)
Chicken anti-guinea pig AF605 (cyan fluorophore)
Design one or more panels that allow you distinguish between
blood and lymphatic vessels
and that allow you to determine whether alpha or beta cells are
vascularized (the vessels are

12. in contact with the cells)
22. Discuss these FACS plot. In specific, tell me what are the
effects of IL-7 vs. IL-12 vs. IL-12 + IL-
17 addition to the medium compared to the medium on IFNγ
expression on CD4+ cells and on
CD8+ cells (4)

13. 23. Discuss these FACS plot. In specific, tell me what are the
effects of growing A549 cells in
sphere (A549 sphere) vs. control (A549) on expression of
CD133 (as measured by PE-
conjugated antibody) and on CD90 (as measured by FITC-
conjugated antibody) (4)
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