Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

Enzymes and other related proteins are promising targets as biomarkers for cancer diagnostics, and it is therefore important to be able to quantify intracellular enzyme activity levels, not just presence of enzyme. Thus, there is a great need for the development of methods that offer spatiotemporal quantification of intracellular enzyme activity levels in respiring 2- and 3-dimensional cultures or their sample-processed variants. Previous studies have demonstrated that ß-gal is upregulated in senescence and in select ovarian cancers, and its expression/activity is upregulated when cells are under oxidative stress conditions. To that end, the primary goal of this work was to relate ß-gal activity in ovarian cancer cells to the fluorescence response of an enzyme-activatable ratiometric fluorescence probe, and evaluate the hypothesis that the expression and activity of ß-gal is directly correlated with cellular senescence in cancer cells that experience environmental stress, with there also being a direct relationship between ß-gal expression and activity and human NAD(P)H:quinone oxidoreductase isozyme-1 (NQO1) expression and activity. In order to accurately and reliably quantify ß-gal activity in ovarian cancer cells, a quantitative spectrophotometric assay was developed wherein the solution conditions allow for optimal detection of catalytic activity. This enzyme activity assay was then used with traditional biochemical techniques and an enzyme-activatable ratiometric probe for ß-gal, GalNap, to evaluate ß-gal activity and expression in ovarian cancer cells that are hypothesized to be entering senescence; the ovarian cancer cells were evaluated for senescence by employing a panel of purported senescence-associated biomarkers. Ovarian cancer cell lines were also probed for expression/activity of ß-gal and NQO1 to assess if modulation of NQO1 activity could induce senescence in said ovarian cancer cells.

Date

8-18-2021

Committee Chair

McCarley, Robin L.

Available for download on Monday, August 12, 2024

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