Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 85-95). Also available via the World Wide Web.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (M.S.)--University of Montana, 2008. / Title from title screen. Description based on contents viewed May 8, 2008. Includes bibliographical references.

Thesis (Ph. D.)--University of New South Wales, 1999. / Also available online.

Thesis (Ph. D. in Neuroscience)--Vanderbilt University, Dec. 2005. / Title from title screen. Includes bibliographical references.

The physiologically important free radicals, nitrogen monoxide and superoxide, can combine to form the reactive intermediate peroxynitrite. Peroxynitrite can react with proteins and their constituent amino acids, such as tyrosine, resulting in protein peroxidation, oxidation and nitration. The nitration of proteins, assessed by the analysis of 3-nitrotyrosine, is a proposed index of pathophysiological activity of peroxynitrite. The aim of the work was to investigate the reaction products between peroxynitrite and protein, develop an assay for 3-nitrotyrosine and measure its levels in biological samples. To study the amino acid products arising from the reaction of peroxynitrite and protein, both liquid chromatography (LC) and gas chromatography (GC) combined with mass spectrometry (MS) were adopted. Approaches to 3-nitrotyrosine assay development were first, to take advantage of the intrinsic sensitivity of electron capture negative ionization GC-MS. Secondly, to avoid possible artefactual problems associated with the derivatisation step in GC-MS, an assay for 3-nitrotyrosine based on combined LC-MS-MS was developed. When a selection of peptides was exposed to peroxynitrite under physiological conditions in vitro, the hydrolysis products showed that 3-nitrotyrosine was the major product. Detectable minor products were 3,5-dinitrotyrosine and DOPA. The GC-MS assay was found to be fraught with difficulty due to artefactual formation of 3-nitrotyrosine. In order to quantify and correct for artefact formation, this complication was approached by incorporating a second isotopomer. This method, however, was confounded by large errors that reduced the overall sensitivity. Either negative or zero levels of endogenous 3-nitrotyrosine were found in tested samples after correction for artefact formation. The LC-MS-MS assay was then used to analyse 3-nitrotyrosine levels in a range of biological samples, including human plasma from healthy volunteers, synovial fluid samples from arthritis patients and tissue extracts from a mouse model of amyotropic lateral sclerosis. In contrast to published data, 3-nitrotyrosine levels were found to be below the limit of detection (1 pg/????L, 10 pg o/c) for all samples - a result somewhat consistent with the negative GC-MS data. It is suggested that the high 3-nitrotyrosine levels previously reported in the literature might reflect artefactual generation of 3-nitrotyrosine and that other approaches to assessing pathophysiological nitration should be sought in future.

Doctor of Philosophy (PhD) / This thesis primarily details the application of high-level ab initio quantum chemistry techniques in order to understand aspects of free-radical mediated protein oxidation. Traditionally, product analysis and electron paramagnetic resonance (EPR) spectroscopy are the primary means for elucidating the chemistry of protein oxidation. However, in experiments involving relatively small proteins reacting with a controlled radical-flux, a vast array of compounds can be produced, which are often difficult to analyse. Quantum chemical techniques on the other hand, can calculate the properties of any particular species directly, without suffering from the problems associated with experiment, such as side-reactions and chain processes. The results presented in this thesis are aimed at elucidating mechanistic details of protein oxidation, which might otherwise be difficult to probe experimentally. Chapter 1 gives an overview of the free-radical hypothesis of disease and ageing. Protein-derived radicals can undergo a variety of reactions, with the particular reaction that occurs depending on numerous aspects. Many types of reactions have been identified through radiolysis experiments of amino acids, and these are detailed in this chapter. In addition, the key reactive species are characterized and their different chemistries explained. Chapter 2 details the theoretical tools used throughout this thesis. Species with unpaired electrons (radicals) present unique problems for quantum chemistry to handle, thus an appropriate choice of theoretical technique is needed. The approach taken in this thesis is to use high-level compound methods, many of which have been directly formulated to give improved results for radical species, to provide benchmark quality results by which other less demanding techniques can be assessed. During the course of this study, it became apparent there was a void in the armoury of tools that could be used for the theoretical chemistry calculations. Chapter 3 details the formulation of a new tool in an attempt to fill this gap. Historically, the formulation of this new procedure came after much of the work in this thesis had been carried out. Thus, for the study of many of the reactions of this thesis the new method has not been used. However, it is most appropriate to place its formulation after summarizing the current status of techniques in common use today. Chapters 4 and 5 detail computations carried out on models of peptides containing backbone carbon- and nitrogen-centered radicals. A number of different theoretical techniques are used in these chapters, ranging from the highly accurate and computationally intensive to the less reliable and less demanding. The highly accurate techniques are used to gauge the accuracy of the other less demanding theoretical techniques so that the latter can be used with confidence in larger systems. Not only is the choice of theoretical technique important but also the judicious choice of model is essential. With this in mind, models are incrementally built until convergence of the particular property of interest is reached. Chapters 6 and 7 detail the calculations of β-scission reactions of alkoxyl radicals, which are a particular class of reaction known to occur on peptide backbones. Alkoxyl radicals are particularly difficult for theory to describe correctly. Therefore, Chapter 6 extensively assesses and then identifies the theoretical methods needed to portray them. Chapter 7 uses the techniques identified in the previous chapter in order to predict how the preference for a particular type of β-scission reaction changes.

The physiologically important free radicals, nitrogen monoxide and superoxide, can combine to form the reactive intermediate peroxynitrite. Peroxynitrite can react with proteins and their constituent amino acids, such as tyrosine, resulting in protein peroxidation, oxidation and nitration. The nitration of proteins, assessed by the analysis of 3-nitrotyrosine, is a proposed index of pathophysiological activity of peroxynitrite. The aim of the work was to investigate the reaction products between peroxynitrite and protein, develop an assay for 3-nitrotyrosine and measure its levels in biological samples. To study the amino acid products arising from the reaction of peroxynitrite and protein, both liquid chromatography (LC) and gas chromatography (GC) combined with mass spectrometry (MS) were adopted. Approaches to 3-nitrotyrosine assay development were first, to take advantage of the intrinsic sensitivity of electron capture negative ionization GC-MS. Secondly, to avoid possible artefactual problems associated with the derivatisation step in GC-MS, an assay for 3-nitrotyrosine based on combined LC-MS-MS was developed. When a selection of peptides was exposed to peroxynitrite under physiological conditions in vitro, the hydrolysis products showed that 3-nitrotyrosine was the major product. Detectable minor products were 3,5-dinitrotyrosine and DOPA. The GC-MS assay was found to be fraught with difficulty due to artefactual formation of 3-nitrotyrosine. In order to quantify and correct for artefact formation, this complication was approached by incorporating a second isotopomer. This method, however, was confounded by large errors that reduced the overall sensitivity. Either negative or zero levels of endogenous 3-nitrotyrosine were found in tested samples after correction for artefact formation. The LC-MS-MS assay was then used to analyse 3-nitrotyrosine levels in a range of biological samples, including human plasma from healthy volunteers, synovial fluid samples from arthritis patients and tissue extracts from a mouse model of amyotropic lateral sclerosis. In contrast to published data, 3-nitrotyrosine levels were found to be below the limit of detection (1 pg/????L, 10 pg o/c) for all samples - a result somewhat consistent with the negative GC-MS data. It is suggested that the high 3-nitrotyrosine levels previously reported in the literature might reflect artefactual generation of 3-nitrotyrosine and that other approaches to assessing pathophysiological nitration should be sought in future.

Doctor of Philosophy (PhD) / This thesis primarily details the application of high-level ab initio quantum chemistry techniques in order to understand aspects of free-radical mediated protein oxidation. Traditionally, product analysis and electron paramagnetic resonance (EPR) spectroscopy are the primary means for elucidating the chemistry of protein oxidation. However, in experiments involving relatively small proteins reacting with a controlled radical-flux, a vast array of compounds can be produced, which are often difficult to analyse. Quantum chemical techniques on the other hand, can calculate the properties of any particular species directly, without suffering from the problems associated with experiment, such as side-reactions and chain processes. The results presented in this thesis are aimed at elucidating mechanistic details of protein oxidation, which might otherwise be difficult to probe experimentally. Chapter 1 gives an overview of the free-radical hypothesis of disease and ageing. Protein-derived radicals can undergo a variety of reactions, with the particular reaction that occurs depending on numerous aspects. Many types of reactions have been identified through radiolysis experiments of amino acids, and these are detailed in this chapter. In addition, the key reactive species are characterized and their different chemistries explained. Chapter 2 details the theoretical tools used throughout this thesis. Species with unpaired electrons (radicals) present unique problems for quantum chemistry to handle, thus an appropriate choice of theoretical technique is needed. The approach taken in this thesis is to use high-level compound methods, many of which have been directly formulated to give improved results for radical species, to provide benchmark quality results by which other less demanding techniques can be assessed. During the course of this study, it became apparent there was a void in the armoury of tools that could be used for the theoretical chemistry calculations. Chapter 3 details the formulation of a new tool in an attempt to fill this gap. Historically, the formulation of this new procedure came after much of the work in this thesis had been carried out. Thus, for the study of many of the reactions of this thesis the new method has not been used. However, it is most appropriate to place its formulation after summarizing the current status of techniques in common use today. Chapters 4 and 5 detail computations carried out on models of peptides containing backbone carbon- and nitrogen-centered radicals. A number of different theoretical techniques are used in these chapters, ranging from the highly accurate and computationally intensive to the less reliable and less demanding. The highly accurate techniques are used to gauge the accuracy of the other less demanding theoretical techniques so that the latter can be used with confidence in larger systems. Not only is the choice of theoretical technique important but also the judicious choice of model is essential. With this in mind, models are incrementally built until convergence of the particular property of interest is reached. Chapters 6 and 7 detail the calculations of β-scission reactions of alkoxyl radicals, which are a particular class of reaction known to occur on peptide backbones. Alkoxyl radicals are particularly difficult for theory to describe correctly. Therefore, Chapter 6 extensively assesses and then identifies the theoretical methods needed to portray them. Chapter 7 uses the techniques identified in the previous chapter in order to predict how the preference for a particular type of β-scission reaction changes.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 140-155).