Theses and Dissertations at Montana State University (MSU)
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Item Studies of novel glycine-containing lipids that differ greatly in type 2 diabetes(Montana State University - Bozeman, College of Letters & Science, 2014) White, Autumn Brooke; Chairperson, Graduate Committee: Edward DratzType 2 Diabetes (T2D) is a growing problem that affects hundreds of millions of people. This work focused on understanding the pathogenesis of T2D by studying novel glycine-containing lipids found greatly changed in T2D with glycine linked via the carboxyl end to a hydrophobic moiety (Bowden, 2011). Different forms of these novel lipids were found in fasting Type 2 Diabetes (F-T2D), fasting healthy control (F-HC), and non-fasting healthy control (NF-HC) plasma by separation on Reverse Phase-High Performance Liquid Chromatography (RP-HPLC). The ratio in F-T2D/F-HC differed by approximately fifty-fold, but both were present in NF-HC plasma, along with several additional forms with different RP retention. We isolated compounds of interest from the plasma from NF-HC volunteers and pooled enough plasma in hopes for structural elucidation using Nuclear Magnetic Resonance (NMR). Since these compounds were found to be carried by Human Serum Albumin (HSA), HSA was purified from human plasma. A 2:1 Dichloromethane (DCM):Methanol (MeOH) modified Folch-extraction (Folch, 1957) was used to extract the hydrophobic metabolites from HSA. The lipid mixture was separated by RP-HPLC and a small portion of each fraction was derivatized by pentafluorobenzyl-bromide (PFB-Br) and analyzed by LC-MS. A portion of each underivatized fraction, based on the results of the PFB-Br reaction, was analyzed by LCMS. Each prominent peak was fragmented using collision-induced dissociation tandem mass spectrometry (CID-MS/MS). Peaks of the most interest showed neutral loss masses corresponding to glycine and phosphate. This evidence led to the hypothesis that the glycine moiety is attached to a phosphate via a mixed acyl-phospho anhydride linkage. This linkage is thought to be consistent with the rapid hydrolysis of the compounds of interest under mild conditions during sample preparation. Compounds were synthesized that could be followed in the HSA work-up procedure to determine the stability of the anhydride linkages at different steps of the work-up. It is also possible that the linkage could be a mixed-acyl anhydride and this linkage was also synthesized and studied by mass spectrometry (MS) and CID-MS/MS. Understanding these structures could provide new insights into the mechanisms of T2D and perhaps lead to enhanced prevention and treatment.Item Cyclopentannulation : novel ring formation mediated by allylsilane functionality(Montana State University - Bozeman, College of Letters & Science, 1998) Ryter, Kendal TroyItem NMR investigation of non-local effects in a temperature sensitive mutant of the 25 kD tryptophan repressor protein(Montana State University - Bozeman, College of Letters & Science, 2003) Tyler, Robert Charles; Chairperson, Graduate Committee: Valerie CopieItem Multinuclear association of tetraethylisocyanideplatinum (II) tetracyanoplatinate (II) and the oxidized solutions of tetracyanoplatinate (II)(Montana State University - Bozeman, College of Letters & Science, 1998) Martellaro, Phillip JohnItem In situ conversion of methyl bromide to methyl iodide : a new technique for the measurement of atmospheric methyl bromide by GC-ECD(Montana State University - Bozeman, College of Letters & Science, 1998) Hughes, Richard AllenItem Reactive scattering of oxygen and chlorine atoms on hydrocarbon surfaces(Montana State University - Bozeman, College of Letters & Science, 1998) Garton, Donna JoanItem Application of proton transfer reaction mass spectrometry to measure hydrocarbon emissions in engine exhaust(Montana State University - Bozeman, College of Letters & Science, 2007) Rogers, Todd Michael; Chairperson, Graduate Committee: Walter B. Knighton; Patrik Callis (co-chair)Megacities around the world face air quality problems due to exhaust emissions from large vehicle fleets. Chemical reactions involving volatile organic compounds (VOC) from automobile exhaust are known to produce ground level ozone which poses a threat to human health and plant life. Additionally, some of these organic compounds are known carcinogens. While the dangers of VOC are well known, their production from in use vehicles and aircraft are relatively poorly understood. This dissertation will describe how a Proton Transfer Reaction Mass Spectrometer (PTR-MS) was altered to make the first on-road, speciated VOC measurements of its kind and how the response of the instrument in such a complex sample matrix was interpreted. The developed experimental technique was then used to observe aircraft exhaust plumes from in-use commercial aircraft, and to further characterize aircraft exhaust in a ground based experiment. PTR-MS was an appropriate technique for these measurements due to its ability to perform rapid, quantitative measurements. A commercial PTR-MS instrument was modified to operate in a mobile laboratory with capabilities of sampling and analyzing intercepted air in real-time.Item Investigating host response to viral infection through proteomics : a study of murine norovirus(Montana State University - Bozeman, College of Letters & Science, 2008) Furman, Linnzi Marie; Chairperson, Graduate Committee: Brian Bothner; Michele Hardy (co-chair)Norovirus causes roughly 23 million cases of foodborne illnesses in the United States each year. While this virus was characterized over 30 years ago, it remains non-cultivatable in human cells, resulting in an incomplete understanding of the host cell's response to infection. However, in 2004 murine norovirus (MNV) was found to be cultivatable in mice and has since been successfully cultured in RAW 264.7 cells. MNV has become an important model system for studying norovirus, as it is structurally and genetically similar to human norovirus. A global proteomics approach using fluorescently tagged, activity-based probes and 2D differential gel electrophoresis analysis was used to study MNV infection. Specifically, the process of cell death was investigated to determine if apoptosis, or programmed cell death, occurred in response to infection. Through the 2D differential gel analysis, 27 differentially regulated proteins were identified at 4 hours post infection, and 22 differentially regulated proteins were identified at 12 hours post infection; a strong majority of these proteins have been related to apoptosis in the literature. Using fluorescently-labeled activity-based probes and fluorimetric assays, we have monitored the activation of several caspases induced by viral infection. Infected samples show a significant increase in caspase activity over control samples within the first few hours post infection, indicating a virally induced activation of caspases. Cells were also infected in the presence of a pan-caspase inhibitor, Boc-D(OMe)-fmk, which led to caspase-independent cell death. Using propidium iodide and Hoechst staining, it was concluded that infected cells undergo necrosis in the presence of the caspase-inhibitor, while those infected in the absence of the inhibitor undergo apoptosis. From these studies it can be concluded that cells infected with MNV undergo a caspase-mediated, apoptotic cell death, while the caspase-independent cell death can be classified as necrosis. This study provides significant insight to norovirus-induced cell death.