Cell Signaling and Molecular Biology Questions

Question 1. (300-500 words)

You wish to grow human fibroblasts in culture to study their signal transduction pathways. Explain your answers to the following questions.  

a) (5 pts) You obtain a sample of fibroblasts from a human volunteer and place them on a tissue culture dish. What might you include, or not include, in a serum-free media for these cells? Why?

b) (5 pts) You wish to characterize these cells and determine what subunits of integrins that may be expressed. Which integrin subtype would you predict to be the predominant class of integrin in these cells? Why? (which alpha and beta subunits?).

c) (5 pts) What ligand would you "coat" (incubate the culture dish with a solution of this protein ligand to allow association with the plastic) your tissue culture dishes with to provide your fibroblasts with the most likely ligand they interact with in vivo? Why?

d) (5 pts) Predict and explain what would happen if you reduced the calcium concentration in the media 100-fold. 

e) (5 pts) Predict and explain what might happen if you included anti-RGD sequence antibodies in your fibroblast culture?

f)  (5 pts) Describe the signal transduction pathway is activated by the predominant integrin in your cells?

Question 2. (300-500 words) 

You believe that the cytosolic protein p007 is involved in mediating growth hormone-stimulated proliferation in cells. You wish to establish a cell model using your cultured fibroblasts. Provide clear explanations to the following, each worth 5 pts each.

a) Describe how you would transfect the growth hormone receptor DNA into your cells to create stable cell lines, and include a description of the benefits of using stable cell lines versus transiently transfected cell line.  

b) Describe how you would measure specific receptor binding of GH to your stable cell line.

c) Describe how you would measure growth hormone-induced proliferation in your stable cell line.  Make sure to include controls.

d) Describe how you would use RNAi to demonstrate the requirement for p007 in growth-hormone stimulated proliferation.  Make sure to include controls.

Question 3. (300-500 words)

You work for a large biotechnology company that is studying viruses, vaccines, and small molecule inhibitors.  You are asked to give an overview of cell signaling as it relates to the coronavirus and young adults. Provide clear explanations to the following, each worth 10 pts each.

a) Describe cell signaling pathways (e.g., at least 3) that are likely manipulated during a coronavirus infection.  In your description, include how the pathways are involved in antagonizing the host antiviral response. (12.5 points)

b) Describe a molecular drug (i.e., either an existing one, or one you come up with through your own analysis) that would interfere and inhibit a coronavirus infection.  What signaling pathways does the drug target, and why are these pathways the best to target? (12.5 points)

Question 4. (300-500 words) 

Explain, in detail, how tyrosine kinase proteins are involved in one signal transduction pathway of your choice. (10 pts) Make sure you describe the complete pathway in which it is involved.

Then, describe how you would experimentally demonstrate the requirement of a tyrosine kinase protein in your chosen pathway. (10 pts)

Question 5. (300-500 words) 

Explain, in detail, how heterotrimeric G proteins are involved in one signal transduction pathway of your choice. (20 pts)Make sure you describe the complete pathway in which it is involved.

Then, describe how you would experimentally demonstrate the requirement of a heterotrimeric G alpha protein in your chosen pathway. (10 pts)

How would you experimentally demonstrate that the G alpha subunit required is Gi alpha or Go alpha? (10 pts)

Question 6 (300 words) 

Explain how each of the following is a signaling molecule, an effector molecule or an adaptor molecule (300 words) 

a) calcium
b) Protein kinase A
c) cAMP
d) beta-catenin
e) phospholipase D

Question 7 (300-500 words) 

When activated, the following proteins (enzymes or receptors) can phosphorylate which amino acid residues on cellular protein targets (including themselves)?

Then..... give an example of one target protein modified by each enzyme or receptor. (4 pts each) 

a) TGF-beta receptor
b) cyclin-dependent kinase   
c) MEKK
d) epidermal growth factor receptor
e) protein kinase C

Question 8 (300-500 words)

Explain, in detail, how GTP switch proteins (such as ras) are involved in one signal transduction pathway of your choice. (10 pts)
Make sure you describe the complete pathway in which it is involved.

Then, describe how you would experimentally demonstrate the requirement of a GTP switch protein in your chosen pathway. (10 pts)

Question 9 (300-500 words) 

Explain why you chose your answers and why you did not choose the other answers. Which of the following mutations would promote malignancy in cells whose proliferation is inhibited by TGFBeta?  (5 pts each)

a)  Dominant negative SMAD4
b) Loss of TGF Beta receptor II 
c) Overexpression of Smad2 
d) Dominant negative Skp1
e) Loss of Smur1 (note: some publications interchange Smur1 with Smurf1; so this can read equivalently as "Loss of Smurf1")