Digitized Honors Theses (2002-2017)

Date of Award

12-2012

Document Type

Undergraduate Thesis

Degree Name

BS

Department

Biomedical Sciences

Faculty Mentor

Diego Alvarez

Abstract

Characteristic respiratory rates of cells and tissues may indicate normal or abnormal cell and tissue physiology. In this project, we aimed to develop protocols for measuring characteristic cellular respiration of cells and tissue samples. Samples were suspended in media and placed into air-tight chambers of an oxygraph which measured the decrease in dissolved oxygen over time. We determined the effects of tissue preservation on mitochondrial respiration. We show that respiration decreases in tissues as a function of time after in vivo excision, most likely due to the breakdown of tissue integrity following biopsy and consequent auto-digestion. We determined basal respiration of pulmonary microvascular endothelial cells (PMVECs) in various media. We show there are no significant differences in respiration of PMVECs in DMEM versus MiR06. This observation can be attributed to the abundance of glucose in culture media (DMEM); exogenous substrates in sample media do not affect respiration under these conditions. In order to develop protocols in cells, we used rat fibroblasts as a model. To stimulate a maximum rate of ADP phosphorylation at Complex V, ADP was added to cells following permeabilization with varying concentrations of saponin. We show that a saponin concentration of 0.01% is ideal for protocols measuring respiration in permeabilized cells. This is an important observation, given that too little saponin causes impartial permeabilization of the cellular membrane while too much causes degradation of the inner-mitochondrial-membrane. The maximum rate of the electron transport chain was also measured using the mitochondrial proton-gradient uncoupler, carbonyl cyanide m-chlorophenyl hydrazone. Information regarding the respiratory states of PMVECs and pulmonary tissues during times of disease and health may give insight into the pathophysiology of acute lung injury and sepsis.

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