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Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

The gaseous free radicals, alkaline-earth metal monoalkylamides, monoacetylides, monoformamidates and monopyrrolidates, consisting of a metal atom (Ca or Sr) bonded to a single ligand, were synthesized in a Broida oven. The electronic and vibrational structures of these molecules were studied by low-resolution laser spectroscopy techniques. These inorganic molecules are ionic, well represented by the structure M⁺L⁻ (M = Ca, Sr: L = ligand). Three electronic transitions were identified for the metal monoalkylamides and the metal monoformamidates. The formamidate anion bonds to the metal in a bidentate fashion through the oxygen and nitrogen atoms. Two electronic transitions were observed for the metal monopyrrolidates. The pyrrolide anion ring bonds to the metal to provide these "open-faced sandwich" type molecules with pseudo-C₅ᵥ symmetry. For the metal monoacetylide molecules, only one electronic transition (Ā²Π-Ẋ²Σ⁺) was observed. Several vibrational frequencies were determined for these inorganic molecules from the low-resolution spectra. The Ā²Π-Ẋ²Σ⁺ transition of the calcium monoacetylide molecule was rotationally analyzed at high-resolution using the filtered laser excitation spectoscopy technique. The rotational line positions were fitted to a ²Π-²Σ⁺ Hamiltonian to obtain several rotational constants. The calcium-carbon bond length in CaCCH was calculated for the ground (2.248 Å) and excited (2.200 Å) electronic states. The vibration-rotation spectra of the gaseous bismuth hydride and bismuth deuteride molecules were recorded, using a diode laser system. The 1-0 fundamental band and several hot bands with Δv-1 were rotationally analyzed. The rotational line positions were fitted first, to a Dunham energy expression and then to a ³Σ⁻ Hamiltonian, to obtain ground state rotational constants. The bismuth-hydrogen (deuterium) bond distance was calculated to be 1.809 Å (1.807 Å).

The addition of benzyltrimethylsilane to maleic anhydride mediated by TiO2 photocatalysis was initially investigated. The affect of changing the catalyst, the radical trap loading and the substitution of the benzyltrimethylsilane molecule was assessed. Cyclisation precursors based on benzyltrimethylsilane were prepared, but were found not to cyclise via TiO2 photocatalysis. A number of other systems were assessed for their ability to cyclise under TiO2 photocatalysis; tertiary amines, aminomethyltrimethylsilanes, phenoxymethyltrimethylsilanes and phenoxyacetic acids. Phenoxymethyltrimethylsilane and phenoxyacetic acid were found to add effectively to maleic anhydride under TiO2 photocatalysis conditions, however they were unreactive with regards to cyclisation. EPR spectroscopy has been employed to characterise further the reaction of benzylsilanes with maleic anhydride under TiO2 photocatalytic conditions. A number of EPR active species were observed; trapped holes and electrons, which reside within the TiO2 catalyst. In addition, methyl and benzyl radicals were observed and were found to originate from the oxidation of the benzylsilanes by trapped holes in the TiO2 catalyst. However, no radical species were observed from the maleic anhydride. These observations had the following consequences for the currently proposed reaction mechanism for the addition of benzyltrimethylsilane with maleic anhydride under TiO2 photocatalysis.  The observation of the benzyl radical definitely proved that the reactive intermediate was indeed the proposed benzyl radical  The absence of any maleic anhydride EPR active species cast doubt on the role of maleic anhydride as an electron trap. Moreover when maleic anhydride is removed from the reaction system, interstitial Ti3+ species is absent from the EPR spectra, indicating that maleic anhydride is in fact acting as a hole trap.

o,o???-Dityrosine (dityrosine), an oxidation product of tyrosine produced by reaction between tyrosyl radicals, is becoming established as a biomarker of free radical oxidative protein damage in vivo. Attempts to measure dityrosine concentrations in various physiological and pathological systems have produced varied and often contradictory results. Dityrosine concentrations in urine, plasma, cerebrospinal fluid (CSF) and brain tissue varying over three orders of magnitude have been reported, together with inconsistent claims of significant dityrosine elevation in several ageing-related pathologies. Some of these findings have contributed to the implication of free radical activity in the pathology of several neurodegenerative disorders, vascular and ocular abnormalities and in phagocyte response to infection. The aim of this study was to test the hypothesis that dityrosine levels are elevated in ageing and ageing-related disease. The study also aims to determine the utility of dityrosine measurement as an index of oxidative damage, and elucidate possible explanations for the inconsistent levels reported. An assay for the quantification of dityrosine was developed using capillary HPLC with electrospray tandem quadrupole mass spectrometry (HPLC-MS/MS). The assay was highly specific for dityrosine and has the highest absolute sensitivity for dityrosine of any method reported to date, with a detection limit of 3 femtomoles of dityrosine on-column. Urine samples from volunteers of different age and from hospital patients with various pathologies were analysed. Plasma protein hydrolysates from control, Alzheimer???s and stroke subjects were analysed, together with hydrolysates of post mortem brain tissue from Alzheimer???s and control subjects. Urinary dityrosine level is elevated in states of acute infection and inflammation, but does not correlate with age or chronic disease. Protein dityrosine in four sections of Alzheimer???s brain was not significantly different from control sections. Dityrosine was present in human plasma and tissue proteins at approximately 5-35 residues per million tyrosine residues, and in normal urine at 5-25 micromol/mol creatinine or 20-200 nM. Most of the discrepancies in the literature relate to inadequate specificity of the analytical method. Interpretation of published data with critical appraisal of measurement technology specificity is essential in developing an accurate understanding of the role of free radicals in ageing and disease.

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.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Free radicals are thought to be involved in the destructive process of beta cells in Type 1 diabetes mellitus. Studies were performed to test the hypotheses (1) that malondialdehyde (MDA), a by-product of lipid peroxidation, affects $ beta$-cell function and integrity in vitro and (2) that such effects might be prevented in the BB rat (a model of spontaneous autoimmune diabetes) in vivo by administration of $ alpha$-phenyl-N-tert-butylnitrone (PBN), a free radical spin trap. First, islets of Wistar-Furth rats were studied at 12, 24 and 40 hr of culture in either 5.5, 11 or 16.5 mM glucose, and MDA at a range of concentrations ($6 times10 sp{-12}$-10$ sp{-3}$M). High concentrations of MDA inhibited glucose-stimulated insulin release without corresponding decreases in islet insulin content, suggesting that in situations with high predicted islet free radical content (e.g., autoimmune insulitis) beta cell function may be affected even before the cells are destroyed. Second, 28 diabetes-prone (BBdp) and 13 non diabetes-prone (BBn) rats were given PBN (20 mg/kg) s.c. 2x/day and 27 BBdp and 12 BBn rats received an equal volume of saline. PBN was able to decrease MDA in the absence of the autoimmune process and is remarkably non-toxic. However, it did not prevent diabetes for reasons which may include its concentration at the site of the inflammatory process or specificity to types of radicals trapped. Because it did decrease MDA, either a higher dose or a combination of PBN with other agents may hold promise for disease prevention.