Theses and Dissertations at Montana State University (MSU)
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Item Drug development for tuberculosis: design and synthesis of cytochrome bd oxidase inhibitors(Montana State University - Bozeman, College of Letters & Science, 2022) Hopfner, Sarah Marie; Chairperson, Graduate Committee: Mary J. Cloninger; This is a manuscript style paper that includes co-authored chapters.In 2019, more than 10 million people worldwide became ill with Tuberculosis (TB) according to the World Health Organization 2020 Global Tuberculosis Report. More disturbing is the continued rise in cases of drug resistant TB. Thus, there is an immediate need for the development of new antimicrobials and treatment options that can quickly eliminate resistant variants of Mycobacterium tuberculosis (Mtb) infection. The oxidative phosphorylation pathway in Mtb is an attractive target for drug development because only two terminal oxidases are present: cytochrome bcc : aa 3 (cyt-bcc:aa3) and cytochrome bd (cyt-bd). Q203, a small-molecule inhibitor, targets cyt-bcc:aa3 in the oxidative phosphorylation pathway. However, Q203 is bacteriostatic and does not inhibit respiration in Mtb. Thus, innovative drugs and effective drug combinations which target the oxidative phosphorylation pathway are still needed. In this dissertation, I report the synthesis and characterization of new cytochrome bd inhibitors that can be used in conjunction with a cytochrome bcc : aa 3 inhibitor such as Q203 to rapidly kill mycobacterium tuberculosis. This combination of drugs is expected to shut down oxidative phosphorylation in Mtb, thereby removing both the primary (cyt-bcc : aa 3) and the backup (cyt-bd) power sources of energy for Mtb. Additionally, the syntheses of molecules with a thieno[3,2-d]pyrimidine-4-amine core and substituted phenylethyl substituents are described. IC 50 values of these compounds against three mycobacterial strains are presented using M. bovis BCG, M. tuberculosis H37Rv, and M. tuberculosis clinical isolate N0145 strains. Since the structure of the Mtb cyt-bd oxidase has only very recently been reported, these molecules are important targets not only to study the efficacy of a dual drug therapy but also to study membrane association of thienopyrimidine molecules. Therefore, the membrane partitioning of thienopyrmidine molecules into 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was studied using time-correlated single photon counting and correlated with activity against Mtb. Finally, the syntheses of molecules with substitutions on a quinazoline core and substituted phenylethyl substituents are described. Through focused structure-activity relationships, activity against all 3 bacterial strains was improved with 2 compounds showing greater activity than the naturally derived cyt-bd inhibitor aurachin D.Item Development and analysis of lipidomics procedures for the causal investigation of Alzheimer's disease(Montana State University - Bozeman, College of Letters & Science, 2022) Koch, Max Richard; Chairperson, Graduate Committee: Edward Dratz; This is a manuscript style paper that includes co-authored chapters.Uncovering sets of molecular features which cause a healthy metabolic state to transition to one of disease, requires extensive experimentation and often presents a difficult analysis. In the case of neurodegenerative diseases, such as Alzheimer's Disease, simply obtaining suitable samples can be a challenging endeavor. Many current 'Omics' techniques excel at profiling a vast array of molecules, such as water-soluble metabolites, lipids, and proteins, in order to compare groups of samples from healthy and diseased organisms. Such approaches primarily use various associations between molecules and disease to identify biomarkers. However, these 'omics' experiments frequently result in intriguing biological hypotheses, but to date have rarely provided mechanistic explanations. How then, can mechanistic explanations be recovered from metabolite or lipid profile data? In our work, we applied these methods to 6 Alzheimer's diseased brain samples and 6 age matched controls. When analyzed via mass spectrometry, lipids which differed significantly between control and disease were identified, but this information was not able to'provide mechanistic insight. The beginning of any 'omics' based experiment starts with the extraction of the desired molecules. In order to assess the efficiency of three different lipid extraction methods, a lipid standard was extracted from a matrix composed of rat liver tissue and analyzed by mass spectrometry. The classic Folch extraction was found to be best at reproducibly extracting a wide range of lipids. Several of the lipids identified from human brains showed oxidative damage. Lastly, 5 statistical measures of dependence and 3 network algorithms were investigated for their ability to reconstruct mechanistic relationships in a dynamic model of arachidonic acid metabolism. Many of the metabolites of arachidonic acid are oxidation products. Under conditions of high noise and relatively few samples, standard measures of correlation, such as Pearson's correlation, Spearman's correlation and Kendall's Tau were found to perform the best. Metrics which incorporate nonlinear metabolic relations and network algorithms were found to be applicable, when sample size is large and the signal to noise ratio is close to l.Item Complexation of lipids with cyclodextrin carriers for fully defined supplementation of cell culture(Montana State University - Bozeman, College of Letters & Science, 2019) Corbin, Elizabeth Dale; Chairperson, Graduate Committee: Edward Dratz and Renee Reijo Pera (co-chair)Induced Pluripotent Stem Cells (iPSCs) hold great promise for revolutionizing medicine and research. Scientists are currently able to reprogram adult cells of almost any type to a genetically 'open' state, pluripotency, wherein they lose the characteristics of their original cell type, and revert to a state that can reproduce indefinitely, and can be differentiated to many different cell types. IPSCs are currently grown in 'chemically defined' media that contains no animal derived components. This media eliminates animal and human sera because these tend to be quite variable and confound the reprogramming process, but the chemically defined media in use has almost no lipid content. The central goal of this project was to develop methods for chemically defined addition of lipids to cell culture media. The methods developed promise to be an advance in stem cell and general cell culture methodology, providing more reproducible experimental results, and supporting cells in culture with optimized lipid contents. In order to facilitate the addition of lipids to cell culture media without animal serum or serum albumin, complexation of individual lipids with a methyl beta-cyclodextrin starch was accomplished without addition of other molecules or oxidation of delicate lipids at a 1:1 stoichiometry. The lipid/MBCD complexes are soluble in aqueous media, and can be added individually or as a mixture to cell cultures. Application of complexed lipids to stem cells and fibroblasts revealed significant differences in lipid responses. Supplementation of human fibroblasts with a mixture of complexed lipids and other elements caused a 60% increase in proliferation over a 10 day period. Supplementation of stem cells with complexed lipids significantly increased proliferation, without reduction of pluripotency. Differences in lipid responses were also found between iPSC and embryonic stem cells, that may help elucidate differences between genetic or metabolic states which may point the way for more effective reprogramming of adult cells to pluripotency.Item Organic enrichment at aqueous interfaces studied with non-linear spectroscopy: cooperative adsorption of soluble saccharides to lipid monolayers(Montana State University - Bozeman, College of Letters & Science, 2019) Link, Katie Ann; Chairperson, Graduate Committee: Robert Walker; Chia-Yun Hsieh, Aashish Tuladhar, Zizwe Chase, Zheming Wang, Hongfei Wang and Robert A. Walker were co-authors of the article, 'Vibrational studies of saccharide-induced lipid film reorganization at aqueous/vapor interfaces' in the journal 'Chemical physics' which is contained within this thesis.; Gabrielle N. Spurzem, Aashish Tuladhar, Zizwe Chase, Zheming Wang, Hongfei Wang and Robert A. Walker were co-authors of the article, 'Organic enrichment at aqueous interfaces: cooperative adsorption of glucuronic acid to DPPC monolayers studied with vibrational sum frequency generation' submitted to the journal 'Journal of physical chemistry B' which is contained within this thesis.; Gabrielle N. Spurzem, Aashish Tuladhar, Zizwe Chase, Zheming Wang, Hongfei Wang, and Robert A. Walker were co-authors of the article, 'Cooperative adsorption of trehalose to DPPC studied with vibrational sum frequency generation' which is contained within this thesis.Field measurements of sea spray aerosols have reported high concentrations of soluble organic material that are in excess of the concentration of soluble organics in the ocean. The studies described in this dissertation investigated a possible mechanism for this increase deemed cooperative adsorption. The cooperative adsorption mechanism describes an interaction between an insoluble Langmuir monolayer at the aqueous/vapor interface and soluble organic molecules that would not normally be enriched at the surface. In this model, the soluble organics are drawn to the surface through non-covalent interactions with the lipid surfactant. This mechanism was investigated with the surface specific nonlinear optical technique, vibrational sum frequency generation spectroscopy. These optical measurements were coupled with surface tension measurements and differential scanning calorimetry measurements. To study cooperative adsorption, model systems were used; these were composed of a phosphatidylcholine lipid surfactant, DPPC, and soluble saccharides including glucosamine, glucuronic acid, and trehalose. Glucosamine, in both a positive and neutral state, induced ordering in both expanded and condensed DPPC monolayers, supporting cooperative adsorption as a mechanism. Glucuronic acid, an anion, ordered lipid monolayers in the limits that the lipid DPPC was moderately packed and there were no competing ions in solution. Trehalose, a larger, uncharged saccharide showed, through ordering the DPPC monolayer, indications of cooperative adsorption in moderately packed DPPC when the trehalose concentration was sufficiently high. These results support cooperative adsorption as a mechanism for the accumulation of soluble organics in sea spray aerosols with some limitations.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.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 Structure and metabolism of cuticular lipids of the grasshopper melanoplus sanguinipes(Montana State University - Bozeman, College of Letters & Science, 1973) Blomquist, Gary James