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Document Type

Thesis - University Access Only

Award Date

2016

Degree Name

Doctor of Philosophy (PhD)

Department / School

Pharmaceutical Sciences

First Advisor

Xiangming Guan

Abstract

Thiols (-SH) play important roles in the biological system. They are part of enzyme active sites, involved in signal transduction, cell division, removal of reactive oxygen species (ROS) and nitrogen species (RNS), removal of reactive electrophiles and so forth. Thiols are present inside, outside the cell and also on cell surface. Structurally, thiols are divided into protein thiols (PSH) and non-protein thiols (NPSH). The principal NPSH in the mammalian system is glutathione (GSH), which is present in mM concentration under the normal physiological conditions and serves as a major redox buffer to maintain a reducing cellular environment. The ratio of PSH: NPSH in the biological system is in the range of ~3:1. A decrease in thiol concentration is linked to various cellular dysfunctions, such as aging, neuron degeneration, a change in enzyme activity, membrane permeability, and energy production. Consequently, disturbance of thiol homoeostasis has been associated with a number of diseases, such as cancer, AIDS, Alzheimer's disease, Parkinson's disease, cardiovascular disease and others. Due to the essential role thiol groups play in cellular structure and function, numerous analytical methods have been developed for thiol determination and quantification. Majority of these methods require tissue/cell homogenization before sample analysis, and most of these methods are developed for GSH. Due to a lack of effective thiol specific reagents, limited methods are available to image and quantify thiols in live cells. Determination of thiols through fluorescence imaging in live cells has the advantage of not only providing the information on thiol levels, but also enabling us to visualize the biochemical process in a live cell at the molecular level in real time in its native environment. Therefore, it can reveal valuable information that cannot be revealed by the conventional methods in understanding the biochemical process. We have developed a series of thiol specific fluorogenic agents. These agents are designed based on the benzofurazan sulfide structure. These benzofurazan sulfides can react specifically with thiols to form fluorescent thiol adducts rapidly and completely through a thiol-sulfide exchange reaction. Our data showed that these benzofurazan sulfides exhibited no reaction with amino acids containing -NH2, -OH, -COOH and nucleophilic functional groups other than thiols. These benzofurazan sulfides themselves are not fluorescent, but their thiol adducts are fluorescent. Therefore, they are fluorogenic agents. One of these benzofurazan sulfide thiol specific fluorogenic agents was chosen for the investigation of total thiol imaging and quantification in live cells through fluorescence microscopy. The compound was named GUALY’s reagent. GUALY’s reagent reacts specifically with a thiol, rapidly, and completely to yield a thiol adduct with strong green fluorescence (λex = 430 nm and λem =520 nm). The reagent itself exhibits no fluorescence. GUALY’s reagent was demonstrated to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. GUALY’s reagent is the first reagent that can be used to image and quantify total thiols in live cells. In addition to the application in total thiol imaging and quantification, GUALY’s reagent was also employed to develop the first high-throughput method for simultaneous quantification of cellular PSH, NPSH, and total thiols in a 96-well plate using a fluorescence microplate reader. Unlike most currently employed thiol analytical methods that are time consuming and require high cell density, this high-throughput method can detect thiols with cell concentrations as low as 500 cells/well. The method is convenient, easy to operate with the ability to simultaneously quantify PSH, NPSH, and total thiols in one 96-well plate in 5 minutes. We then extended our work to the development of thiol specific fluorogenic agents for thiol imaging in mitochondria in live cells. Mitochondrion is an important subcellular organelle in eukaryotic cells. Mitochondrion is involved in the energy production through oxidative phosphorylation and involved in both cell programmed death (apoptosis) and necrosis. Mitochondrial thiols play an important role in maintaining mitochondrial normal function. The increase in oxidation of mitochondrial thiols can result in mitochondrial damage, which plays a role in many human diseases, such as neurodegenerative diseases, cardiac dysfunction, inflammation, ischemia-reperfusion injury in heart attack and stroke, etc. Imaging thiol density in mitochondria through fluorescence microscopy in live cells can provide valuable information on the role of thiols in these mitochondrion-related diseases. Based on the mitochondrial membrane potential, TBOP was designed and synthesized to image and quantify mitochondrial thiols. TBOP was characterized as a thiol specific fluorogenic agent with λex = 380 nm and λem = 520 nm respectively. The fluorescence intensity of thiols adducts derived from TBOP was found to be 3 times stronger than the fluorescence intensity from GUALY’s reagent. Although TBOP can be used to successfully image thiols in mitochondria, it was not suitable for mitochondrial thiol quantification due to low fluorescence intensity generated from mitochondrial thiol. The low mitochondrial fluorescence intensity is partially due to the fact that mitochondrial thiols accounts only for 2%~15% of total cellular GSH. Therefore, a much stronger fluorescence reagent is needed to compensate the low thiol concentration in mitochondria. To improve fluorescence intensity, RhoD-2 was designed and synthesized. RhoD-2 effectively imaged mitochondrial thiol in live cells with excellent fluorescence intensity. However, RhoD-2 was found to be sensitive to hydrolysis. The hydrolysis product interfere with mitochondrial thiols quantification by RhoD-2. Consequently, RhoD-2 was not pursued further for mitochondrial thiol imaging. Further work is needed in developing thiol specific fluorogenic agents for imaging and quantifying mitochondrial thiol in live cells. Although the low mitochondrial fluorescence intensity makes TBOP not suitable for mitochondrial thiol imaging, TBOP was unexpectedly found to react with only NPSH not PSH. This finding prompted the investigation of TBOP for NPSH imaging in live cells through fluorescence microscopy. The application of TBOP for NPSH imaging was achieved by extending the TBOP incubation time to allow TBOP to react with all NPSH in cells, not just limited to mitochondrial NPSH. The abundant NPSH in live cells produced enough fluorescence for imaging and quantification by TBOP. In summary, we have developed GUALY’s reagent for imaging and quantifying total thiols in live cells through fluorescence microscopy. This is the first reagent that can image and quantify thiols in live cells. Using GUALY’s reagent, we also developed the first high-throughput method for NPSH, PSH, and total thiols from cell homogenates. Although TBOP was not able to image and quantify mitochondrial thiols in live cells as what it was designed for, the ability to react with only NPSH enable TBOP to image NPSH in live cells. TBOP was developed as the first reagent that can image and quantify NPSH in live cells. TBOP is a complementary reagent of GUALY’s reagent. Together with GUALY’s reagent, TBOP will be able to determine the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions. Reagents developed from this dissertation will be valuable tools in thiol-related pharmaceutical and biomedical research.

Library of Congress Subject Headings

Thiols
Fluorescence microscopy
Benzofurazan sulfides

Description

Includes bibliographical references

Format

application/pdf

Number of Pages

114

Publisher

South Dakota State University

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Rights Statement

In Copyright