Date of Award

2014

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Clore, Amy

Keywords

Biochemistry, Cancer, Genetics, Methylation, DNA

Area of Concentration

Biology

Abstract

In the body, macrophage cells phagocytize foreign prokaryotic cells. Once phagocytosed, these prokaryotic cells are sequestered in vesicles called phagosomes that fuse with lysosomes. The resulting phagolysosome contains the enzyme NADPH oxidase which releases reactive oxygen species that denature the encapsulated prokaryotes’ RNA. Damaged mRNAs serve as flawed translation templates and result in the synthesis of abnormal proteins. These proteins are not able to maintain homeostasis. As a counter to reactive oxygen species, the prokaryotes express the exoribonuclease polynucleotide phosphorylase (PNPase). PNPase binds to and subsequently neutralizes damaged RNA. Activators, including interferon- gamma (INF-Y), lipopolysaccharides (LPS) and chitin microparticles (CMPS), are often used to activate macrophage cells. This results in elevated levels of free radical species, phagocytosis and bacterial elimination rates. The first purpose of this study was to determine which combination of the aforementioned activators would result in greater macrophage activation. Activation was measured by quantifying the nitric oxide and tumor necrosis factor alpha (TNFα) levels in each type of activated macrophage culture. The combination of IFNY (20 ng/mL) and LPS (100 ng/mL) proved to be the most effective. In addition, murine macrophage cells and two strains of K12 MG1655 Escherichia coli (E. coli) were cocultured to observe PNPase’s efficacy in protecting phagocytosed E. coli from oxidative stress. The wild-type strain E. coli, MG1655, and the second lacked the PNPase encoding cassette and was thus PNPase(-/-). The combination of LPS and INFY resulted in the greatest macrophage activation. It was found that there was little statistical evidence supporting the hypothesis that elimination of PNPase in bacteria lower their overall survival rate.

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