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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 Application of the thiosulfonate switch technique and a modified biotin switch technique protocols to detect protein S-nitrosothiols in mouse liver lysates in 1D and 2D gel studies(Montana State University - Bozeman, College of Letters & Science, 2015) Miller, Colin Gregory; Chairperson, Graduate Committee: Paul Grieco; Brian Bothner (co-chair)While the role of nitric oxide (NO) in cell signaling and liver growth has been well documented, the identification of S-nitrosylated proteins, one of the major NO transport mechanisms within the cell, remains a challenge. Classically, the implementation of biotin labeling, known as the biotin switch technique (BST), with streptavidin-agarose bead pulldown and subsequent immunoblotting, has offered the best results for identifying S-nitrosocysteine residues within proteins. However, this technique has come under scrutiny for its use of ascorbate as a reducing agent. Numerous published accounts have shown ascorbate's poor reducing potential especially for S-nitrosoproteins. To this end, the Grieco lab has shown that pure S-nitrosylated proteins can be transformed into S-phenylsulfonylcysteine residues, which can be readily converted into mixed disulfides thus allowing for labeling of pure nitrosylated proteins at pH's as low as pH 4. This protocol is referred to as the thiosulfonate switch technique (TST). The Grieco lab has also modified the biotin switch technique to incorporate electrophilic maleimide and orthopyridyl disulfide (OPSS) dyes for fluorescence labeling of the S-nitroso proteome. To examine the scope and limitations of the TST vis-a-vis cell lysates, the TST protocol and the modified BST protocol, both employing novel Z-CyDyes developed in the Grieco Laboratory, have been used to specifically label S-nitrosylated proteins in complex liver lysates. The successful labeling of mouse liver lysates, employing UV and ascorbate SNO knockout negative controls, is demonstrated in both 1D and 2D gel studies. Also reported herein is the creation of novel second generation maleimide dyes (SO3-Cy-Mal) based on Z-CyDyes.