Digitized Honors Theses (2002-2017)

Date of Award

5-2003

Document Type

Undergraduate Thesis

Degree Name

BS

Department

Biomedical Sciences

Faculty Mentor

Mark Gillespie

Advisor(s)

Cindy Stanfield, Steve Itaya

Abstract

Hypoxia generates a wide variety of cell responses in many different regions of the vascular system's endothelium, including microvascular endothelial cells (MVECs) and pulmonary artery endothelial cells (PAECs). The signaling mechanism during hypoxia is a current topic of debate. Reactive oxygen species seem to play an important role throughout this process. It has been shown that oxidants are produced in response to hypoxia in PAECs, but this has not been proven in MVECs. Furthermore, it is unclear if the mitochondria are solely responsible for the generation of the reactive oxygen species or if the oxidants could also be cytoplasmic in origin.

Fluorescent microscopy techniques were employed to help elucidate the location and time course of oxidant production after exposure to hypoxia. Cells were stained with molecular probes targeting both mitochondria and the cytoplasm and then exposed to hypoxia in order to study the oxidant production of the cells. Mitotracker Red CMH2XROS, which fluoresces when oxidized, was used to detect mitochondrial oxidant stress. DCF, the second fluorescent probe, was employed to determine overall cytosolic oxidant stress.

We hypothesize that both MVECs and PAECs produce oxidants in response to hypoxia. Furthermore, we hypothesize that these oxidants are mitochondrial in nature and may cause an oxidant stress in the vicinity of the nucleus. Accordingly, the goal of this study is to produce a quantifiable time course of oxidant production in both cell types and determine the location of oxidant production.

Difficulties in creating a hypoxic environment while the cells are imaged have hindered the experiments. Menadione was used to test the oxidant detection and co­ localization capabilities of the experimental setup. The setup was successful in detecting oxidant production and determining where in the cell the oxidants were being produced (i.e in the vicinity of the nucleus). The methodology developed will also be useful in compartmentalizing oxidant production from other physiological sources.

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