Department Chair

I. Martha Skerrett, Ph.D.

Date of Award

12-2018

Access Control

Open Access

Degree Name

Biology, M.A.

Department

Biology Department

Advisor

Gregory J. Wadsworth, Ph.D.

Department Home page

http://biology.buffalostate.edu/

First Reader

Gregory J. Wadsworth, Ph.D.

Second Reader

I. Martha Skerrett, Ph.D.

Third Reader

Douglas P. Easton, Ph.D.

Abstract

Chaperones maintain cellular homeostasis by facilitating protein folding. However, under standard cultured conditions, Caenorhabditis elegans strains genetically deficient for the ER chaperone GRP170B (encoded by the T14G8.3 locus) seems unaffected by the loss of this large chaperone. For most ER chaperones, the loss of GRP170B chaperone increases sensitivity to toxins that disrupt protein folding in the ER. I hypothesized that GRP170B’s role may involve mediating the nematodes response to ER protein folding stress. To investigate a stress role for GRP170B chaperone, I analyzed the effect of toxins that are known to effect ER protein folding on nematodes deficient for the chaperone. The three toxins tested I tested were: 1. tunicamycin (TM), an ER glycosylation inhibitor, 2. dithiothreitol (DTT), a disrupter of disulfide bond formation and 3. thapsigargin (TG) an inhibitor of an ER calcium pump which disrupts protein folding by interfering with the activity of calcium dependent ER chaperones. Eggs of nematodes deficient for GRP170B (strain BSC06) and the control strain (N2) were cultured at varying doses of each toxin, and the development of the nematodes was monitored. At 80-320 mg/μl of TM, the GRP170B deficient worms were less sensitive to the toxin than the control strain. This was similar to a previous study which found that nematodes deficient for GRP170B were less sensitive to 3 μg/ml TM. At the highest dose of TM tested (640 μg/μl), development was similar in nematodes with and without GRP170B. Results showed that at low doses of the second toxin DTT (80-320 mg/μl), development was similar in nematodes with and without GRP170B. However, at highest dose (640 mg/μl) GRP170B deficient worms were more sensitive to the toxin and none of them developed to L4/adult stages. For the third toxin tested, TG there was a notable experimental variation across all treatments from 0.5 mM to 1.25 mM. The effect of TG on development of nematodes deficient for GRP170B was also tested. Thapsigargin doses(0.5 nM to 5 nM) did not affect the development of either strain. These data demonstrate that GRP170B deficient worms are not being affected specifically with one toxin versus the other. These data revealed that GRP170B has a critical role during normal C. elegans physiology role and during stress physiology with the use TM. However, the role of GRP170B could not be determined using DTT and TG at the tested doses.

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