Document Type

Dissertation - Open Access

Award Date

2021

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmaceutical Sciences

First Advisor

Xiangming Guan

Keywords

cell surface, Fluorescence imaging, lysosomes, mitochondria, subcellular organelles, thiols

Abstract

Thiols (–SH) play vital and irreplaceable roles in various cellular functions in the biological system. These roles include serving as the most important antioxidant to terminate reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive electrophiles; being part of enzyme active sites; and being involved in signal transduction and cell division. Abnormality of thiols’ status has been linked to various cellular dysfunctions and diseases such as cancer, AIDS, Alzheimer's disease, Parkinson's disease, cardiovascular disease and others. In the biological system, thiols can be categorized structurally into two classes: protein thiols (PSH) and non-protein thiols (NPSH). PSH are mainly the cysteine residue in proteins. NPSH are mainly small molecule thiols such as cysteine, homocysteine and glutathione (GSH). The major NPSH is GSH which is present at mM concentration (1–10 mM) in the biological system and serves as the major endogenous antioxidant to maintain the intracellular redox activities, metabolism and intracellular signal transduction. Thiols are present everywhere in the biological system: intracellularly and extracellularly. They distribute at different concentration in different tissues. Intracellularly, thiols are unevenly distributed with different concentration in different subcellular organelles such as mitochondria, lysosomes, nuclei, cytosol, and cell surface. Due to the essential roles thiols play in the biological system, numerous analytical methods have been developed in detecting and quantifying thiols. However, most of these methods detect and quantify thiols through the use of homogenized biological samples. Limited methods are available to detect and quantify thiols in live cells through fluorescence microscopy. Methods for detecting and quantifying subcellular thiols in live cells through fluorescence microscopy are even more limited. Live cell imaging and quantification of thiols through fluorescence microscopy has the advantage of revealing information while thiols are in their native environment - the information cannot be revealed through the analysis of a homogenized biological sample. The major reason for a lack of analytical methods to image and quantify thiols in live cells through fluorescence microscopy is a lack of analytical reagents that can turn thiols into fluorescent specifically and sensitively with low cytotoxicity. The major objective of this dissertation work was to develop reagents that can be used to image and quantify thiols in subcellular organelles in live cells through fluorescence microscopy. This dissertation will present data demonstrating that we have successfully developed thiol specific fluorogenic reagents that can be used to image and quantify thiols in mitochondria, cell surface, and lysosomes in live cells through fluorescence microscopy. These reagents react only with thiols. They exhibit no fluorescence but becomes fluorescent after reacting with a thiol. Chapter 1 describes the design, synthesis, and characterization of thiol specific mitochondria selective fluorogenic reagents and presents the data to show how TBROS effectively imaged and quantified mitochondrial thiols in live cells through fluorescence microscopy. Chapter 2 presents the synthesis and characterization of TROX as a thiol specific fluorogenic agent to image and quantify cell surface thiols. Chapter 3 reports the development of thiol specific fluorogenic reagents for imaging and quantifying thiols in lysosomes. In chapter 4, we present preliminary data to demonstrate the ability of TROX to selectively detect cysteine in the presence of other thiols. An abnormal level of cysteine has been associated with many health issues such as cardiovascular diseases, neurological disorders, liver damage, skin lesions and growth retardation. An analytical reagent that can selectively detect cysteine will be a valuable tool in exploring the roles of cysteine in cysteine-related health issues. In conclusion, the dissertation fills a gap in the field that is a lack of reagents for imaging and quantifying thiols at subcellular level (mitochondria, cell surface, and lysosomes) in live cells through fluorescence microscopy. The dissertation also developed a reagent that can potentially detect cysteine in the presence of other thiols. The reagents developed from this dissertation will be valuable tools to aid our investigation on thiol-related cellular functions and dysfunctions.

Format

application/pdf

Number of Pages

137

Publisher

South Dakota State University

Rights

Copyright © 2021 the Author

Available for download on Wednesday, May 15, 2024

Share

COinS