Date of Graduation

5-2020

Document Type

Thesis

Degree Name

Bachelor of Science in Biomedical Engineering

Degree Level

Undergraduate

Department

Biomedical Engineering

Advisor/Mentor

Quinn, Kyle

Abstract

Oxidative damage and stress impacts metabolic activity and has been implicated in a number of diseases . These medical conditions have been found to be associated with a lack of glutathione (GSH). There is a need to study the effects that the absence of glutathione has in order to expand knowledge of indicators that cause conditions like cataracts, cancer, or impaired wound healing. L-buthionine-sulfoximine (BSO) has been used in past studies to hinder GSH production and induce oxidative stress within a cell. This study explores the effects of BSO and induced oxidative stress on the metabolic activity and processes within NIH 3T3 fibroblasts. This was done by employing the optical redox ratio which is an emerging tool that quantifies relative autofluorescent molecules, like NADH and FAD, associated with metabolic activities. The effects of oxidative damage were assessed by noting and analyzing the responding changes in concentrations of NADH and FAD. These cofactors are naturally autofluorescent at 755 nm and 855 nm excitation respectively and can be spectrally isolated. This study employed methods to quantify and analyze the presence of these molecules. Two-photon excited fluorescence (TPEF) microscopy was one method employed to assess and quantify the presence of the cofactors, and the optical redox ratio was a tool used to analyze the changes in the presence of the cofactors with changes in the BSO treatment. The changes in the redox ratio were interpreted as the effects of potential changes in the activity of metabolic pathways like the citric acid cycle or the electron transport chain in response to oxidative stress. This study proved BSO impacted and increased the optical redox ratio of cells when compared to untreated cells, and it suggests the changes in metabolic activity may be directly caused by a decrease in citric acid cycle activity, mitochondria structural and organizational changes, or the lack of cell viability.

Keywords

BSO, oxidative stress, redox ratio

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