The presence of thiols in living systems is critical for the maintenance of cellular redox homeostasis, the maintenance of protein thiol-disulfide ratios and the protection of cells from reactive oxygen species. In addition to the well studied tripeptide glutathione (??-Glu-Cys-Gly), a number of compounds have been identified that contribute to these essential cellular roles. Many of these molecules are of great clinical interest due to their essential role in the biochemistry of a number of deadly pathogens, as well as their possible role as therapeutic agents in the treatment of a number of diseases. A series of studies were undertaken using theoretical, chemical and biochemical approaches on a selection of thiols, ergothioneine, the ovothiols and mycothiol, to further our understanding of these necessary biological components. Ergothioneine is present at significant physiological levels in humans and other mammals; however, a definitive role for this thiol has yet to be determined. It has been implicated in radical scavenging in vivo and shows promise as a therapeutic agent against disease states caused by oxidative damage. Given the clinical importance of this intracellular thiol, further investigation into the behaviour of ergothioneine appeared warranted. A high level theoretical study was performed to determine the thermodynamic driving force behind the instability of the ergothioneine disulfide, as well as the thermodynamics of the reactions of ergothioneine with a selection of biologically relevant reactive oxygen species. These results were compared to those determined for a glutathione model compound, as well as the related ovothiols. The latter are believed to act as hydrogen peroxide scavengers in vivo and are currently under review as possible therapeutics against oxidative damage. The structural differences between the ovothiols and ergothioneine dramatically affect their reactivity and this study investigates the thermodynamic driving forces behind these differences. Mycothiol is the major thiol found in the Actinomycetales bacteria, which include the causative agent of tuberculosis, and the enzymes which use mycothiol have been identified as important targets for the development of novel antimicrobials. To better understand the in vivo behaviour of mycothiol, a thorough conformational search was performed to determine what, if any, trends exist among the low energy conformers expected to be present in solution. Knowledge of the conformations preferred by mycothiol may aid in the design of substrate-based inhibitors targeted at mycothiol-dependent enzymes. In addition, the efforts towards the identification of a mycothiol-dependent glyoxalase system are described. The glyoxalase system is essential for the detoxification of methylglyoxal, a toxic by-product of glycolysis, and this system would serve as a target for the design of new therapeutics against tuberculosis and other pathogenic Actinomycetales bacteria. In addition to the study of intracellular thiols, this work details a preliminary theoretical study of the thermodynamics of the phosphorylation of proteinaceous serine residues by inositol pyrophosphates in eukaryotic cell-free extracts. It has been postulated that this observed activity may represent a novel signalling pathway in eukaryotes. This study focused on the effect of inositol pyrophosphate structure and overall charge on the thermodynamics of these reactions. This information should contribute to our understanding of this novel cellular phosphorylation process.

There is increasing evidence that hydrogen peroxide (H2O2) can act as a signalling molecule capable of modulating a variety of biochemical and genetic systems. Using Jurkat T-lymphocytes, this study initially investigated the involvement of H2O2 in the activation of a specific signalling protein extracellular signal-regulated protein kinase (ERK). It was found that as a result of H2O2 treatment, mitochondrial complex activities decreased which led to subsequent increase of mitochondrial reactive oxygen species (ROS) production. The increase of ROS resulted in higher cellular H2O2 as well as increased ERK activation. This study demonstrated that in an oxidative stress setting, H2O2 production from the mitochondria was an essential component in maintaining the activation of a signalling protein. One way in which H2O2 could influence protein function is by the oxidation of susceptible thiol groups of cysteine residues. To further understand the variety of signalling pathways that H2O2 may be involved in, an improved proteomics technique was developed to globally identify proteins with susceptible thiol groups. The

Thèse doctorat : Chimie et Electrochimie Analytiques : Nancy 1 : 2005. / Titre provenant de l'écran-titre.

The work described in this thesis investigates the mechanisms by which copper complexes catalyse the generation of reactive oxygen species (ROS), including the highly reactive hydroxyl radical (.OH), and induce oxidative damage to DNA. An ESR study into the copper-Fenton reaction revealed that, in the presence of buffers and other copper chelators, .OH is generated. In contrast, it is suggested that a Cu(III) species may be formed in the reaction of aqueous, unchelated copper ions. The generation of .OH in the copper-Fenton reaction, under biomimetic conditions, was confirmed by analysis of the products formed following the incubation of DNA components with this system. Preferential binding of Cu(II) to guanosine over the other nucleosides was determined and copper redox cycling at GC sites was found to be more facile than at AT sites. Stability constants for the copper complexes with several other biochemically important ligands such as glutathione (GSH), Quin2 and 1,10-phenanthroline (OP) were also measured. The ease of redox cycling for the copper complexes was found to be of the order: OP ~ Quin2 > GSH. However, OP enhanced both the copper-Fenton reaction and copper-induced DNA damage while both GSH and Quin2 were inhibitory. Gel chromatography studies suggested that ternary complex formation occurs between Cu(I)-DNA and both Quin2 and OP. This implies that the ternary complex with OP is more redox active than that with Quin2. Whilst cysteine enhanced copper-mediated DNA damage at early incubation times, it was more protective than GSH and homocysteine at later stages. The effects at early incubation times are attributed to the ease of copper redox cycling in the presence of thiols while the effects over prolonged incubations reflect Cu(II) stabilisation by the respective disulphides or similar products.

Thesis (M.S.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on January 11, 2008) Includes bibliographical references.

Thèse (M.Sc.)--Université Laval, 2008. / Bibliogr.: f. 103-105. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (Ph. D.)--Florida State University, 2003. / Advisor: Dr. Kenneth A. Goldsby, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Aug. 23, 2004). Includes bibliographical references.

Coating of organophobic silver nano-particles with thiols was applied to facilitate their dispersing in aprotic organic solvents and solutions of polyacrylic acid and polylactide. Impact of chemical nature of thiols and dipole moment of the solvents on dispersibility of the modified silver particles was studied by UV-Vis spectroscopy (surface plasmon resonance) and scanning electron microscopy. It was shown that thiols' grafting to the surface of metal nanoparticles is an effective route to improve and stabilize dispersions in organic systems.

Hemerythrin is a respiratory protein found in the erythrocytes of certain marine invertebrates. Structures have been obtained by x-ray crystallography for hemerythrins from Phascolopsis gouldii (P. gouldii) and Themiste dyscritum (T. dyscritum). Since many proteins are inactivated upon binding heavy metals such as mercury, an investigation of mercury-protein interactions in this protein was undertaken. In order to understand the nature of heavy-metal protein interactions in T. dyscritum it was necessary to identify the metalloprotein ligands since neither the liganding amino acid residues nor the geometry of the mercury complexes could be identified in the crystallographic studies.

<p> Biological thiols are a class of labile and redox-active metabolite with significant interest to biomedical research due to their involvement in redox mechanisms of cell signaling and physiological control. As a result of oxidative stress, levels of various reduced thiols and oxidized disulfides are altered, which disrupts major cellular regulation pathways modulating protein function and gene expression. Thus, analysis of thiols in biological fluids is essential for understanding the role of oxidative stress and thiol dysregulation in aging and human diseases. However, reliable ex-vivo thiol determination is challenging due to their low abundance and susceptibility to auto-oxidation and thiol-disulfide exchange reactions. In this thesis, capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) in conjunction with maleimide labeling is developed as an integrative strategy for comprehensive plasma thiol redox status analysis for metabolomics. Maleimide labeling helps to address both major constraints in thiol analysis by stabilizing free sulfhydryl groups as their thioether adducts while improving ionization efficiency and analytical sensitivity. This enhancement in ionization efficiency can be quantitatively predicted based on relative changes in fundamental physicochemical properties of thiols that occur upon covalent derivatization when using multivariate calibration. On-line sample preconcentration together with thiol-selective labeling using a cationic quaternary ammonium maleimide analog allowed for simultaneous analysis of reduced thiols and intact oxidized disulfides by CE-ESI-MS with low nanomolar detection limits of 8-30 nM. Improved identification of unknown low abundance thiols and other classes of polar metabolites is also demonstrated by prediction of relative migration times in CE that is complementary to ESI-MS. Comprehensive plasma thiol speciation together with untargeted profiling of polar metabolites provides a novel platform for holistic understanding of complex changes in metabolic networks associated with thiol dysregulation and/or nutritional intervention for the prevention or treatment of human disorders. </p> / Thesis / Master of Science (MSc)