Date of Award

2022

Document Type

Thesis

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

Doulas Weiser

First Committee Member

Lisa A. Wrischnik

Second Committee Member

Craig Vierra

Abstract

The failure to balance protein synthesis, folding, and degradation in the endoplasmic reticulum (ER) leads to the accumulation of unfolded proteins, leading to ER stress. Cells respond to this stress by activating a response signaling pathway known as the Unfolded Protein Response (UPR). One of the branches of the UPR induces the phosphorylation of eIF2α (Eukaryotic Initiation Factor 2) to attenuate global protein synthesis, allowing for a chance to clear misfolded and unfolded proteins. This phosphorylation of eIF2α is opposed by a phosphatase, containing a catalytic subunit, Protein Phosphatase 1, and a scaffolding protein, either GADD34 or CReP. Inhibition of eIF2α phosphatases has shown to promote survival in cell types by prolonging the effects of the UPR. This research focuses on understanding the gene expression patterns and localization of UPR specific genes with the presence of constant ER stress. Zebrafish are an ideal model for this research because they are a good mimic of what happens in humans and provide the ability to study gene expression and localization patterns at different stages during ER stress and its recovery. The eIF2α phosphatases were shown to have a protective effect on apoptosis when overexpressed in acute ER stress but were shown to have a protective effect on apoptosis when knocked out in chronic ER stress. We sought to determine the flow of gene expression of these phosphatases as well as other UPR specific genes, such as BiP and CHOP, to determine the contradictory effects of acute versus chronic ER-stress induced apoptosis. We studied the changes in gene expression for these genes in zebrafish embryos by isolating RNA and performing RT-qPCR after the induction of ER stress with pharmacological drugs across multiple time points. There was increased gene upregulation and mRNA localization to the fin epidermis and eye of GADD34, CReP, and BiP in acute ER stress from 2 hours to 6 hours, and these genes steadily declined in chronic ER stress from 24 hours to 48 hours. CHOP is a late-phase pro-apoptotic protein whose gene expression was upregulated in chronic ER stress from 12 hours to 48 hours. This data was also supported by mRNA localization studies performed by conducting whole mount in-situ hybridization on zebrafish embryos treated with ER stress inducers for 4 hours and 24 hours. Our results indicate that all UPR genes examined are affected by ER stress and their expression patterns are dependent on the time length of ER stress induction, allowing us to get a more in-depth working model of this branch of the UPR signaling pathway in zebrafish.

Pages

122

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