Thesis (PhDApSc(MineralsandMaterials))--University of South Australia, 2001.

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.

Introduction of soft ionization techniques, such as electrospray ionization (ESI), has resulted in extensive use of mass spectrometry based techniques to study biomolecules in the gas phase. Despite thorough studies of the gas-phase chemistry of even-electron biomolecules, the examination of their odd-electron counterparts has to this point been much less extensive due to the inefficiency of ESI in generating such species. Among various methods that could be employed to generate and study odd-electron biomolecules in the gas phase, redox processes involving metal ions and homolytic cleavage of metallated amino acid or peptide derivatives would be attractive from a chemical perspective since, in principle, a wide range of metals and biomolecules or biomolecule derivatives could be explored. An important aspect of these approaches is that they can be carried out on a wide range of tandem mass spectrometers equipped with electrospray ionization and collision induced dissociation capabilities. (For complete abstract open document)

The marine bacterium Sphingopyxis alaskensis was isolated as one of the most numerically abundant bacteria from cold (4–10??C) nutrient depleted waters in the North Pacific Ocean. The objective of this study was to examine cold adaptation of S. alaskensis by using proteomics to examine changes in global protein levels caused by growth at low (10??C) and high (30??C) temperatures. Stable isotope labelling-based quantitative proteomics was used, and a rigorous post-experimental data processing workflow adapted from microarray-based methods was developed. The approach included metabolic labelling with 14N/15N and normalisation and statistical testing of quantitative proteomics data. Approximately 400,000 tandem mass spectra were generated resulting in the confident identification of 2,135 proteins (66% genome coverage) and the quantitation of 1,172 proteins (37% genome coverage). Normalisation approaches were evaluated using cultures grown at 30??C and labelled with 14N and 15N. For 10??C vs. 30??C experiments, protein quantities were normalised within each experiment using a multivariate lowess approach. Statistical significance was assessed by combining data from all experiments and applying a moderated t-test using the empirical Bayes method with the limma package in R. Proteins were ranked after calculating the B-statistic and the Storey-Tibshirani false discovery rate. 217 proteins (6% genome coverage) were determined to have significant quantitative differences. In achieving these outcomes a range of factors that impact on quantitative proteomics data quality were broadly assessed, resulting in the development of a robust approach that is generally applicable to quantitative proteomics of biological system. The significantly differentially abundant proteins from the proteomics data provided insight into molecular mechanisms of cold adaptation in S. alaskensis. Important aspects of cold adaptation included cell membrane restructuring, exopolysaccharide biosynthesis, lipid degradation, carbohydrate and amino acid metabolism, and increased capacity of transcriptional and translational processes. A number of cold adaptive responses in S. alaskensis were novel, including a specific cold-active protein folding pathway, a possible thermally-controlled stringent response, and biosynthesis of intracellular polyhydroxyalkanoate reserve material. The overall study provided important new insight into the evolution of growth strategies necessary for the effective competition of S. alaskensis in cold, oligotrophic environments.

The marine bacterium Sphingopyxis alaskensis was isolated as one of the most numerically abundant bacteria from cold (4–10??C) nutrient depleted waters in the North Pacific Ocean. The objective of this study was to examine cold adaptation of S. alaskensis by using proteomics to examine changes in global protein levels caused by growth at low (10??C) and high (30??C) temperatures. Stable isotope labelling-based quantitative proteomics was used, and a rigorous post-experimental data processing workflow adapted from microarray-based methods was developed. The approach included metabolic labelling with 14N/15N and normalisation and statistical testing of quantitative proteomics data. Approximately 400,000 tandem mass spectra were generated resulting in the confident identification of 2,135 proteins (66% genome coverage) and the quantitation of 1,172 proteins (37% genome coverage). Normalisation approaches were evaluated using cultures grown at 30??C and labelled with 14N and 15N. For 10??C vs. 30??C experiments, protein quantities were normalised within each experiment using a multivariate lowess approach. Statistical significance was assessed by combining data from all experiments and applying a moderated t-test using the empirical Bayes method with the limma package in R. Proteins were ranked after calculating the B-statistic and the Storey-Tibshirani false discovery rate. 217 proteins (6% genome coverage) were determined to have significant quantitative differences. In achieving these outcomes a range of factors that impact on quantitative proteomics data quality were broadly assessed, resulting in the development of a robust approach that is generally applicable to quantitative proteomics of biological system. The significantly differentially abundant proteins from the proteomics data provided insight into molecular mechanisms of cold adaptation in S. alaskensis. Important aspects of cold adaptation included cell membrane restructuring, exopolysaccharide biosynthesis, lipid degradation, carbohydrate and amino acid metabolism, and increased capacity of transcriptional and translational processes. A number of cold adaptive responses in S. alaskensis were novel, including a specific cold-active protein folding pathway, a possible thermally-controlled stringent response, and biosynthesis of intracellular polyhydroxyalkanoate reserve material. The overall study provided important new insight into the evolution of growth strategies necessary for the effective competition of S. alaskensis in cold, oligotrophic environments.

Neuropathic pain is defined as "pain initiated or caused by a primary lesion or dysfunction in the nervous system". In contrast to physiological pain that warns of noxious stimuli likely to result in tissue damage, neuropathic pain serves no protective function. Examples of neuropathic pain states include postherpetic neuralgia (shingles) and phantom limb / stump pain. This pain state also exists in the orofacial region, with the possibility of several variants including atypical odontalgia and burning mouth syndrome. There is a paucity of information on the prevalence of neuropathic pain in the orofacial region. One study assessed patients following endodontic treatment and found that approximately 3 to 6percent of patients reported persistent pain. Patients predisposed to the condition atypical odontalgia (phantom tooth pain) include those suffering from recurrent cluster or migraine headaches. Biochemical and neurobiological processes leading to a neuropathic pain state are complex and involve peripheral sensitisation, and neuronal plasticity of the central and peripheral nervous systems. Subsequent associated pathophysiology includes regional muscle spasm, sympathetic hyperfunction, and centralisation of pain. The relevant clinical features of neuropathic pain are: (i) precipitating factors such as trauma or disease (infection), (ii) pain that is frequently described as having burning, paroxysmal, and lancinating or sharp qualities, and (iii) physical examination may indicate hyperalgesia, allodynia and sympathetic hyperfunction. The typical patient complains of persistent, severe pain, yet there are no clearly identifiable clinical or radiographic abnormalities. Often, due to the chronicity of the problem, afflicted patients exhibit significant distress and are poor pain historians, thus complicating the clinician's task of obtaining a detailed and relevant clinical and psychosocial history. An appropriate analgetic blockade test for intraoral sites of neuropathic pain is mucosal application of topical anaesthetics. Other, more specific, tests include placebo controlled lignocaine infusions for assessing neuropathic pain, and placebo controlled phentolamine infusions for sympathetically maintained pain. The treatment and management of neuropathic pain is multidisciplinary. Medication rationalisation utilises first-line antineuropathic drugs including tricyclic antidepressants, and possibly an anticonvulsant. Topical applications of capsaicin to the gingivae and oral mucosa are a simple and effective treatment. Neuropathic pain responds poorly to opioid medication. Psychological assessment is often crucial in developing strategies for pain management. Psychological variables include distress, depression, expectations of treatment, motivation to improve, and background environmental factors. To enable a greater understanding of neuropathic pain, thereby leading to improved treatments, high-performance liquid chromatography-mass spectrometry is one analytical technique that has the potential to contribute to our knowledge base. This technique allows drugs and endogenous substances to be assayed from one sample in a relatively short time. The technique can identify, confirm, and measure the concentrations of multiple analytes from a single sample.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains viii, 144 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (M.S.)--Texas State University-San Marcos, 2007. / Vita. Includes bibliographical references (leaves 52-54).