Date of Award

2014

Degree Type

Thesis

Degree Name

M.Sc.

Department

Chemistry and Biochemistry

First Advisor

Boffa, Michael

Keywords

Alternative Splicing, CPB2, Metabolic Labeling, Minigene, Real Time RT-PCR, TAFI

Rights

CC BY-NC-ND 4.0

Abstract

Thrombin Activatable Fibrinolysis Inhibitor (TAFI), encoded by CPB2, cleaves carboxyl-terminal lysine residues from partially degraded fibrin, thus assisting in the attenuation of fibrinolysis. The finding of several new alternatively spliced (AS) CPB2 variants within numerous cell types suggests that AS is an important mechanism regulating CPB2 gene expression. This thesis investigated the extent of AS events occurring in various vascular and immune cells, and identified proteins translated from AS CPB2 mRNA. To determine whether a minigene approach could be used to model the cell-specific AS pattern that occurs endogenously, two minigene constructs were created. Real time RT-PCR analysis revealed that the pattern and extent of AS varies in a cell-specific manner. However, the minigenes were found to be unable to recapitulate the cell-specific splicing pattern of CPB2 that occurs endogenously. Metabolic labeling in conjunction with immunoprecipitation resulted in the detection of the Delta7 TAFI variant retained within HepG2 cells.

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