Theses and Dissertations at Montana State University (MSU)
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Item Redox homeostasis and stress in mouse livers lacking the NADPH-dependent disulfide reductase systems(Montana State University - Bozeman, College of Letters & Science, 2019) Miller, Colin Gregory; Chairperson, Graduate Committee: Mary J. Cloninger; Edward E. Schmidt (co-chair); Arne Holmgren, Elias S.J. Arner and Edward E. Schmidt were co-authors of the article, 'Introduction --NADPH dependent and --independent disulfide reductase systems' in the journal 'Free radical biology and medicine' which is contained within this thesis.; Edward E. Schmidt was a co-author of the article, 'Disulfide reductase systems in the liver' in the journal 'British journal of pharmacology' which is contained within this thesis.; Jean A. Kundert, Justin R. Prigge, Julie Amato, Allison E. Perez and Edward E. Schmidt were co-authors of the article, 'Supplemental ascorbate compromises hepatocyte survival and increases risk of acute liver failure during severe oxidative stress' submitted to the journal 'Antioxidant' which is contained within this thesis.; Dissertation contains two articles of which Colin Gregory Miller is not the main author.This thesis includes two reviews that cover the background of cellular disulfide reduction, from its earliest form in hydrothermal vents and its evolution to the current, multifaceted systems that maintain cellular redox homeostasis, to the roles of the disulfide reductase systems in different subcellular compartments, as well as provide a current status for many of the unkown roles disulfide reductase enzymes play. Furthermore, this thesis includes two published research articles, both relating changes in the NADPH-dependent disulfide reductase systems to altered physiology and the possible impacts of these changes to human health (ie cancer, acetaminophen overdose or toxic arsenic exposure.) A third research paper is also included in this thesis, which demonstrates the pro-oxidant effects of administration of the antioxidant ascorbate to TrxR1/Gsr-null livers. This paper is potentially valuable both in a clinical aspect, where ascorbate might be prescribed to counter the effects of excess oxidants, but also to the general public, as ascorbate is one of the most commonly taken over-the-counter supplements. The final chapter of this thesis is fundamental groundwork for future projects aimed at identifying how cells manage accumulation of oxidants/compromised disulfide reductase systems. The two isotopically labled amino acids, L-(^34 S)Met and L-(^34 S)cystine, are valuable tools to monitor S-metabolism, both in the TrxR1/Gsr-null livers but also in other disease states, such as those mentioned above. L-(^34 S)cystine is of particular interest to one of our collaborators, Dr. Gina DeNicola, who plans to use L-(^34 S)cystine to monitor S-metabolism in pancreatic organoids to study pancreatic adenocarcinoma.Item Mass spectrometry based lipidomics as a tool in the search for biomarkers and mechanisms of disease(Montana State University - Bozeman, College of Letters & Science, 2016) Willems, Daniel Lee; Chairperson, Graduate Committee: Edward Dratz; Nicholas E. Goocey and Edward A. Dratz were co-authors of the article, 'A highly reproducible and efficient lipid extraction protocol enhanced using 3D printing of centrifuge adapters for optimum glass vials' submitted to the journal 'Lipids' which is contained within this thesis.; Max Koch, Nicholas E. Goocey, Blaine R. Roberts and Edward A. Dratz were co-authors of the article, 'Lipidomic analysis of human brain cortex in alzheimer's disease reveals aberrant levels of acetylcholine precursor speices' submitted to the journal 'American journal of alzheimer's disease and other dementias' which is contained within this thesis.; Max Koch, Nicholas E. Goocey, Blaine R. Roberts and Edward A. Dratz were co-authors of the article, 'Lipidomics reveals aberrent metabolism of lipid molecules in alzheimer's disease cerebral cortex' which is contained within this thesis.Lipidomics studies a highly diverse class of compounds insoluble in water and soluble in organic solvents. Lipids are a major component of cells and tissues, take part in a rich network of metabolic reactions, and are implicated in many disease mechanisms. Lipidomics complements genomics, proteomics and the more common metabolomic analysis of hydrophilic metabolites and can provide new insights into disease mechanisms. The problem approached in this thesis was to compare different methods of sample preparation for lipidomics and apply lipidomics to the study of two major health problems: Nonalcoholic Fatty Liver Disease (NAFLD) and Alzheimer's Disease (AD). Excessive dietary intake of sucrose and fructose, common in the Western Diet, increases deposition of triacylglycerides in the liver and leads to cognitive decline in experimental animals. NAFLD increases the risk of type 2 diabetes, obesity and AD. The high diversity and hydrophobicity of lipids complicates their separation, detection and analysis. However, modern chromatography and mass spectrometry instrumentation and techniques are greatly improving the capability of lipidomic analysis. A lipid extraction protocol was optimized for reproducibility and yield, and was used to extract lipids from rat liver under sucrose stress in a model of human NAFLD and human brain cortex from Alzheimer's Disease (AD) compared to controls. The samples were analyzed using mass spectrometry. The NALFD study did not yield the expected results, instead these data provided a foundation for designing future experiments in progress and to validate the methods used in the AD study. The AD studies showed that several phosphatidylcholine species are down regulated along with acetyl-CoA, which may be the source of low levels of the neurotransmitter acetylcholine in AD. Two different chromatography methods were used to seek a higher coverage of different lipids. Differences in the lipids in AD and controls were evident in the omega-6 and omega-3 fatty acids. The precursors of long omega-3s synthesis were increased while the products EPA and DHA were decreased. In a similar fashion, precursors to long omega-6s were found to be decreased, while the products were increased. This suggests that the omega-6 synthesis pathway may be outcompeting the omega-3 synthesis.