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Date of Award

2022

Document Type

Campus Access Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Administrative Home Department

Department of Chemistry

Advisor 1

Tarun K. Dam

Committee Member 1

Paul Goetsch

Committee Member 2

Patricia Heiden

Committee Member 3

Christo Christov

Abstract

Discovery of relevant biomolecules with impact in the realm of human health is fundamental to our progress towards treating many diseases. It is becoming increasing important to investigate plants due to their variability and wide range of potential therapeutic molecules. Our lab has discovered such a biomolecule from a plant traditionally used in African Tribal medicine named Hemolysin X (HelyX), as it displayed vigorous hemolytic activity. Through both aqueous and organic extractions with subsequent purification using size-exclusion chromatography (SEC) and high-performance liquid chromatography (HPLC), identical fractions and bioactivities have been observed within each subset. This has indicated the purified sample’s structure and function as a cytotoxically lytic compound possessing significant glycosylation of a hydrophobic core. Structural information was obtained through MALDI-TOF and ESI. Through binding studies, HelyX has demonstrated dual specificity towards both lipids and glycoproteins, characterizing its mode of function in achieving lytic activity.

Expansion of lysis characterization beyond red blood cells has revealed activity towards cancerous mammalian cells (MCF-7, MDA-231), yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli) demonstrated primarily through field emission electron microscopic analysis. A wide array of HelyX impact upon membranes and cell walls was achieved to better understand how HelyX functions. A preference towards mammalian cell types was observed over microbial ones, while action was noted amongst all tested cell types. Cells were observed to undergo a variety of morphological changes as they transitioned to cell death; one such observed progressive deterioration was of once healthy cells becoming spheroid (stressed), then an observed loss of membrane cohesion and ultimately a completely ruptured membrane.

Currently, through our efforts HelyX is now able to be purified to its near individual components and extensively characterized. Purification of bioactive plant compounds and their characterization is a difficult and time-consuming process. HelyX may potentially serve as a clinically relevant biomolecule. Along with these findings is the development of extensive electron micrographic analysis for cellular changes upon exposure to bioactive molecules. Such cataloguing can serve to better standardize the analysis of cells through such methods to reduce the current variability observed in morphologies across publications of the same cell types.

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