Studies of oxidative damage during the progression of Alzheimer's disease (AD) suggest its central role in disease pathogenesis. To investigate levels of nucleic acid oxidation in both early and late stages of AD, levels of multiple base adducts were quantified in nuclear and mitochondrial DNA from the superior and middle temporal gyri (SMTG), inferior parietal lobule (IPL), and cerebellum (CER) of age‐matched normal control subjects, subjects with mild cognitive impairment, preclinical AD, late‐stage AD, and non‐AD neurological disorders (diseased control; DC) using gas chromatography/mass spectrometry. Median levels of multiple DNA adducts in nuclear and mitochondrial DNA were significantly (p ≤ 0.05) elevated in the SMTG, IPL, and CER in multiple stages of AD and in DC subjects. Elevated levels of fapyguanine and fapyadenine in mitochondrial DNA suggest a hypoxic environment early in the progression of AD and in DC subjects. Overall, these data suggest that oxidative damage is an early event not only in the pathogenesis of AD but is also present in neurodegenerative diseases in general.

The isolation of high-purity cellular biomacromolecules and sub-cellular organelles is an essential aspect to mass spectrometry based studies. Mitochondria are sub-cellular organelles that perform a central role in cellular energy production. Mitochondria are of great interest due to their potential to generate reactive oxygen species (ROS) and susceptibility to oxidative damage and subsequent functional impairment. Current methods of mitochondria isolation are optimized for respiratory-based studies that favor viability. Whereas, proteomic and lipidomics studies of mitochondria require procedures that optimize for purity and enrichment. We describe a procedure derived from previously established methods for the isolation of mitochondria, nuclear and cytosolic fractions from a neurological tissue sample. In addition to the isolation being of significant purity for mass spectral based ‘-omics’ analysis, mitochondrial yields were routinely 500μg per tissue wet weight, allowing multiple studies to be conducted from a single isolation procedure.

OF DISSERTATION MICHAEL DOUGLAS TIMMONS The Graduate School University of Kentucky 2010 EXPLORATIONS IN HOMEOVISCOUS ADAPTATION AND MASS SPECTRAL ANALYSIS OF MEMBRANE LIPIDS ABSTRACT OF DISSERTATIONOF DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Arts and Sciences at the University of Kentucky By Michael Douglas Timmons

Clostridium thermocellum is a candidate organism for consolidated bioprocessing of lignocellulosic biomass into ethanol. However, commercial use is limited due to growth inhibition at modest ethanol concentrations. Recently, an ethanol-adapted strain of C. thermocellum was produced. Since ethanol adaptation in microorganisms has been linked to modification of membrane lipids, we tested the hypothesis that ethanol adaptation in C. thermocellum involves lipid modification by comparing the fatty acid composition and membrane anisotropy of wild-type and ethanol-adapted strains. Derivatization to fatty acid methyl esters provided quantitative lipid analysis. Compared to wild-type, the ethanol-adapted strain had a larger percentage of fatty acids with chain lengths >16:0 and showed a significant increase in the percentage of 16:0 plasmalogens. Structural identification of fatty acids was confirmed through mass spectral fragmentation patterns of picolinyl esters. Ethanol adaptation did not involve modification at sites of methyl branching or the unsaturation index. Comparison of steady-state fluorescence anisotropy experiments, in the absence and presence of ethanol, provided evidence for the effects of ethanol on membrane fluidity. In the presence of ethanol, both strains displayed increased fluidity by approximately 12%. These data support the model that ethanol adaptation was the result of fatty acid changes that increased membrane rigidity that counter-acted the fluidizing effect of ethanol.