Digitized Honors Theses (2002-2017)

Date of Award

5-2012

Document Type

Undergraduate Thesis

Degree Name

BS

Department

Biomedical Sciences

Faculty Mentor

Richard Honkanen

Advisor(s)

Robin Mockett, Ph.D., Julio Turrens, Ph.D.

Abstract

Coronary vascular disease (CVD) is a complex, progressive disease that is the leading cause of death in most developed nations. CVD is associated with a wide array of clinical manifestations, including elevated plasma levels of low-density lipoproteins (LDLs), decreased plasma levels of high-density lipoproteins (lIDLs), anginas, myocardial infarctions, and strokes. Existing therapies focus on lowering LDL levels and/or increasing HDL levels in hopes of preventing serious disease complications. However, the onset and progression of atherosclerosis is fundamentally linked to the inability of macrophages to eliminate excessive cholesterol intake. Recent studies reveal that Mycobacteriumtuberculosis have enzymes that allow them to feed on cholesterol, while trapped in phagosomes. We propose that the genes that code for these enzymes, namely KSTD and Ks can be expressed in macrophages to enable the catabolism of cholesterol. In order to introduce KSTD and KshA to human cells, we optimized the GC­ rich codon sequences of both bacterial genes to allow for greater translation in eukaryotic cells. Recent published data suggest that KshA cannot use cholesteryl esters as a substrate and that the activity ofKSTD and KshA may deplete intracellular levels of free cholesterol. Therefore, cholesteryl ester hydrolysis may become a rate-limiting step. Accordingly, we designed and created a plasmid that can be used to express Lysosomal Acid Lipase (LIPA), an enzyme responsible for hydrolyzing cholesteryl esters, producing cholesterol and a free fatty acid, in human cells. Gateway cloning techniques were utilized for the production of expression plasmids, and restriction digestion in combination with gel electrophoresis was used to confirm the integrity of the plasmid construct. The data indicate that we have indeed produced a plasmid that will be useful to express LIPA in human cells. Currently, we are developing an assay to characterize the efficacy of cholesteryl ester hydrolysis after transfecting cells with the LIPA-expression plasmid.

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