First Advisor

Mark Woods

Term of Graduation

Fall 2024

Date of Publication

10-9-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.) in Chemistry

Department

Chemistry

Language

English

Subjects

Metabolism, Water Exchange, Yeast

Physical Description

1 online resource (xxi, 132 pages)

Abstract

Under conditions of ionic equilibrium with the environment a cell maintains a constant volume yet rapidly exchanges water molecules with the extracellular space. The rate constant for steady state water efflux, kio, can be measured using contrast enhanced magnetic resonance (CE-MR). The active water cycling (AWC) hypothesis is puts forward that kio is primarily influenced by cellular ATP levels and H+ ATPase activity at the cell membrane. Due to the correlation between kio and ATP levels, kio is thought to be a probable MRI biomarker for metabolic diseases such as cancer. Yet, the molecular mechanisms that drive kio remain incompletely understood. The objective of this work is to understand the molecular metabolic origins of differences in kio using yeast (S. cerevisiae) as a model organism. The AWC hypothesis is the main hypothesis in the field and came about when. yeast cells were observed when grown in batch culture. However, batch culture yields an inhomogeneous population of cells and promotes a changing of metabolic profile overtime. A homogenous platform of cells is required to provide support for the AWC hypothesis and further characterize the molecular mechanisms that drive kio.

In this work yeast cells were grown in chemostats, where cell culture of uniform metabolic state was obtained. Low and high glucose concentrations were used to promote distinct metabolisms: respiratory or fermentative. Respiring cells demonstrated higher values of kio in addition to greater ATP levels compared to respiro-fermenting cells. The results indicate that kio can be used to differentiate cell cultures utilizing different metabolic pathways. Tumors are known to be metabolically heterogonous and exhibit regions of both fermentative and hyper-respiratory metabolic state. The experiments presented here presumably suggest that kio could be used to delineate these regions and inform targeted treatment for cancer.

The relationship between kio, cellular ATP concentration, and mitochondrial activity was investigated in yeast as a metabolic model for the Warburg effect. The Warburg effect is defined by upregulation of fermentation, even in the presence of oxygen. Using chemostat cultures of yeast the impact of mitochondrial modulators (2,4-Dinitrophenol (DNP) and oligomycin A) on kio and ATP levels was investigated. Exposure to mitochondrial inhibitors elicited distinct responses: Oligomycin A exposure led to an increase in ATP but a weaker response in kio. This result is likely due to yeast exhibiting oligomycin resistance and requires further investigation. The addition of increasing concentrations of DNP caused a hormetic effect on measured ATP levels. kio was strongly correlated with ATP levels up to a concentration of 750 μM DNP. DNP is known to modulate the membrane potential in the mitochondria. Exposure to DNP caused a twofold change in measured kio values compared to no DNP added. This experiment represents the most significant finding of this work; that kio can be affected by intracellular processes at the mitochondria.

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Persistent Identifier

https://archives.pdx.edu/ds/psu/42890

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