Document Type

Dissertation - Open Access

Award Date

2019

Degree Name

Doctor of Philosophy (PhD)

Department / School

Pharmaceutical Sciences

First Advisor

Xiangming Guan

Abstract

Thiols or sulfhydryl groups (-SH) play a significant role in protein structures and functions. The unique properties of a thiol group are the basis for its roles in cellular functions. Structurally, thiols are divided into protein thiols (PSH) and non-protein thiols (NPSH). NPSH include glutathione, cysteine, and homocysteine. Thiols are distributed in an uneven manner in the cell and subcellular organelles. Disturbance of thiol levels has been associated with serious diseases such as cancer, aging, cardiovascular diseases, Alzheimer disease, liver damage, and edema. The significant roles of thiols in the biological system have stimulated the development of various approaches to monitor and detect thiols. These approaches include enzymatic assays, colorimetric assays, gel electrophoresis, LC/MS, and HPLC. The major drawback of these conventional analytical approaches is that these methods require the cell to be homogenized before analysis. The breakage of cells is timeconsuming and can cause a loss of information. Thus, developing an approach to determine thiol status in intact live cells will provide a unique advantage over the conventional analytical methods. Fluorescence microscopy has been successfully used in determining analytes in intact live cells. The main challenge for using fluorescence microscopy in detecting thiols is to turn thiols into fluorescent molecules for detection. This challenge results in the necessity to develop fluorescent/fluorogenic agents that exhibit selectivity and specificity for thiols and are capable of turning thiols to fluorescent molecules to facilitate detection and quantification. Most of our current knowledge on thiols’ functions/dysfunctions at cellular or subcellular levels were derived from the data obtained from conventional analytical methods that involve breakage of cells. A fluorescence probe that can be used to detect subcellular thiols in live cells will be a valuable tool to provide better understanding of thiols’ roles in the function and dysfunction of the subcellular organelles. In this dissertation, we will present our work related to the development of thiol specific fluorogenic agents for cell surface thiol imaging and lysosomal thiol imaging in live cells. The rational design, synthesis, and determination of TBONES and TBOZEN as thiol specific fluorogenic reagents for cell surface thiol imaging in live cells will be presented. However, TBONES and TBOZEN failed to image cell surface thiols due to their inability to react with PSH. Interestingly, TBONES and TBOZEN turned out to be able to image thiols in lysosomes in live cells. Based on the work of TBONES and TBOZEN, we designed TIMBOS as a thiol specific and fluorogenic agent for lysosomal thiol imaging in live cells. TIMBOS was successfully synthesized and characterized to be thiol specific and fluorogenic. TIMBOS was demonstrated to effectively image NPSH in lysosomes in live cells and to detect NPSH change in lysosomes in a quantitative manner. In summary, we have synthesized and characterized three rationally designed thiol specific fluorogenic reagents. These three reagents were able to image NPSH and to detect NPSH change in a quantitative manner in lysosomes in live cells. These reagents will be useful tools in exploring the roles of lysosomal thiols in cellular function/dysfunction.

Library of Congress Subject Headings

Thiols.
Fluorescence microscopy.
Cell organelles.

Format

application/pdf

Number of Pages

96

Publisher

South Dakota State University

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Rights Statement

In Copyright