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Item Pharmaceutical biomarkers to inform public and environmental health law and policy(Montana State University - Bozeman, College of Agriculture, 2020) Margetts, Miranda Lee; Chairperson, Graduate Committee: Robert K. D. Peterson and Deborah Keil (co-chair); Aparna Keshaviah, Cindy Hu, Victoria Troeger, Jordan Sykes, Nicholas Bishop, Tammy Jones-Lepp, Marisa Henry and Deborah E. Keil were co-authors of the article, 'Using wastewater-based epidemiology with local indicators of opioid and illicit drug use to overcome data gaps' submitted to the journal 'Journal of the American Medical Association' which is contained within this dissertation.; Terri Mavencamp, Jordan Sykes, Tammy Jones-Lepp, Nicholas Bishop, Victoria Troeger, Robert K. D. Peterson and Deborah E. Keil were co-authors of the article, 'The environmental impact of substance use in Montana's waterways: investigation of prescription, illicit, and recreational drug metabolite concentrations into receiving waters' which is contained within this dissertation.; Trent McCallson and Deborah E. Keil were co-authors of the article, 'Wastewater testing to support new environmental health compliance obligations in the healthcare industry' which is contained within this dissertation.The increasing awareness of the prevalence of prescription and illicit drug metabolites in wastewater is affecting changes to public and environmental health laws and policies. Drug takeback laws have been enacted to limit environmental pollution from drugs flushed into sewers; however, these laws only apply to legally prescribed drugs. Wastewater-based epidemiology, which relies on the measurement of drug concentrations in untreated wastewater, is also emerging as a complementary drug-use data tool to estimate drug consumption patterns by a community in near real-time. We sampled both the untreated influent and treated effluent at two locations in Montana over three months from April to June, 2019, to ascertain the concentrations of certain prescription and illicit drugs of abuse. The concentrations of drugs obtained from the untreated influent were used to inform a wastewater-based epidemiology study that compared drug-dose estimates from our wastewater samples against existing local drug-use sources (emergency medical services calls, drug seizures, and prescription dispense data). We also measured the treated effluent to determine the concentration at which drugs of abuse are persisting through the wastewater-treatment process and potentially affecting aquatic life exposed to those concentrations in receiving waters. We undertook a risk assessment whereby measured drug concentrations were assessed against corresponding ecotoxicology thresholds. Our results indicate that both codeine and morphine concentrations were above predicted no-effect concentrations. The overall results indicate that (1) wastewater-based epidemiology may be an effective tool to better describe substance abuse in communities and (2) drugs are persisting at levels above ecotoxicological thresholds from wastewater treatment plants into receiving waters. To our knowledge, these investigations are the first of their kind to have been conducted in Montana.Item Human gut phages in health and disease(Montana State University - Bozeman, College of Letters & Science, 2018) Manrique Ronquillo, Maria del Pilar; Chairperson, Graduate Committee: Mark J. Young; Michael S. Dills and Mark J. Young were co-authors of the article, 'The human gut phage community and its implications for health and disease' in the journal 'Viruses' which is contained within this dissertation.; Benjamin Bolduc, Seth T. Walk, John van der Oost, Willem M. de Vos and Mark J. Young were co-authors of the article, 'Healthy human gut phageome' in the journal 'Proceedings of the National Academy of Sciences of the United States of America' which is contained within this dissertation.; Mark J. Young was a co-author of the article, 'Interactions of the healthy gut phage community (HGP) with the core gut bacterial community' submitted to the journal 'PLOS computational biology' which is contained within this dissertation.; Yifan Zhu, John van der Oost, Willem M. de Vos and Mark J. Young were co-authors of the article, 'Gut bacteriophages and fecal microbial transplantation outcome in subjects with metabolic syndrome' which is contained within this dissertation.; Seth T. Walk and Mark J. Young were co-authors of the article, 'Bacteriophage-enriched filtrates: a potential tool to modify the structure of the gut-associated bacterial community' which is contained within this dissertation.The human body is colonized by a diverse microbial community known as the human microbiota. Most of these microbes, reside in the human intestinal tract. The gut microbiota has coevolved with humans and has become essential for multiple physiological functions that range from digestion, to development of the immune system, protection for pathogens, and even behavior. The gut microbial community is primarily dominated by Bacteria and their viruses- bacteriophages (or phages for short). Even though our knowledge of the contribution of the former to human health is extensive, the role of bacteriophages in human health and disease has been explored considerably less. Study of bacteriophages in other microbial environments has highlighted their importance in influencing the structure and function of their host community. Therefore, understanding the role of bacteriophages in the human gut ecosystem, and overall, in human health, has become a focus of current research. The main overarching hypothesis of this thesis is that human gut bacteriophages contribute to human health. To test this hypothesis, viral metagenomic surveys of healthy and disease individuals, together with experiments in a gnotobiotic mouse model system were performed. A group of bacteriophages shared among healthy individuals and significantly depleted in individuals with IBD was identified. Moreover, a host reservoir for these phages was identified in the core gut bacterial community of healthy subjects. Study of phage dynamics during an FMT treatment in patients with metabolic syndrome further highlighted the association of bacteriophages with human health. Patients that showed significant clinical improvement harbored a richer community, and a community more similar to healthy donors than patients that did not respond to the treatment. Moreover, a set of potential phage biomarkers associated with health and treatment outcome were identified. Lastly, experiments in gnotobiotic mice demonstrated the ability of bacteriophage-enriched filtrates to modify the microbial community structure. This result highlights the potential use of bacteriophages to manipulate the human gut microbiota, and potentially restore human health.Item Transduction of antigens into amplifiable DNA signals using structure switching aptamers(Montana State University - Bozeman, College of Engineering, 2019) Kayalar, Canberk; Chairperson, Graduate Committee: Stephanie McCallaDetection of specific antigens has one vital step in common: detection of biomarkers. Diagnostic testing that is rapid and reliable is unavailable in limited resource and rural settings. The solution to this need must be simple, inexpensive, robust, rapid and not require highly trained personnel to operate. Aptamers are capable of delivering those needs when matched with a novel high gain amplification method. This thesis focuses on important aspects of a novel protein detection assay that uses aptamers. Aspects that play an important role on the assay's success were investigated; aptamer selection and design of structure switching aptamers, designing DNA templates that will transduce the signal created by the aptamers, solid phase selection, aptamer immobilization on the solid phase, protein capture, and amplification of the signal. The first step was to find aptamers that were proven to specifically target clinically relevant targets and modify them to suit the needs of the assay. It is important to validate the aptamers' performance. The second important step was finding a solid phase that is compatible with the novel nucleotide amplification reaction that will be used to amplify the signal produced by the aptamers. Paramagnetic microbeads, membranes and polyacrylamide hydrogels were potential candidates for solid phases. Non-specific interaction of the target protein with the solid phase surface will not have negative effects while running the assay due to the structure switching of the aptamers however, it prevented the accurate quantification of the protein capture by aptamers. There is a need for the development of a blocking buffer that is specific to the solid phase. Washing of the excess DNA templates that are not bound to target-bound aptamers plays an important role in the assay's accuracy. The results presented here show the preliminary work that has been done for the novel protein detection assay that uses structure switching aptamers. This assay has the potential to detect diseases at point-of-care in low resource settings.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 A metabolomics approach for the study of long-term progesterone in domestic sheep and physiological processes in domestic and bighorn sheep(Montana State University - Bozeman, College of Agriculture, 2017) Herrygers, Melissa Rashelle; Chairperson, Graduate Committee: James G. Berardinelli; J. M. Thomson, K. A. Perz, K. B. Herrygers and J. G. Berardinelli were co-authors of the article, 'Effect of long-term progesterone on feed efficiency, body compostition, non-esterified fatty acids, and metabolic hormones in mature Rambouillet ewes' submitted to the journal 'Journal of animal science' which is contained within this thesis.; J. M. Thomson, K. A. Perz, K. B. Herrygers, V. Copie, B. Tripet, and J. G. Berardinelli were co-authors of the article, 'Using nuclear magnetic resonance spectroscopy (NMR) metabolic profiling to study the effect of long-term progesterone on metabolic profiles in Rambouillet ewes' submitted to the journal 'Journal of metabolomics' which is contained within this thesis.; J. White, C. Butler, R.A. Garrott, V. Copie, B. Tripet, and J. G. Berardinelli were co-authors of the article, 'Potential identification of metabolic biomarkers using nuclear magnetic resonance spectroscopy (NMR) metabolic profiling for nutrition status, season, and location of bighorn sheep (Ovis canadensis) in Montana and Wyoming' submitted to the journal 'Journal of metabolomics' which is contained within this thesis.Metabolomics allows for a snapshot of global metabolisms by studying metabolic intermediates and products of cellular metabolism. Experiments 1 and 2's objectives were to evaluate the effects of long-term P4 treatment, independent of the influence of the placenta and fetus, on changes in feed efficiency, BW, body composition, NEFA, metabolic hormones, and metabolites identified through nuclear magnetic resonance (NMR) metabolic profiling in mature Rambouillet ewes. Thirty, multiparous, 5- and 6-yr-old Rambouillet ewes were stratified by age and metabolic BW and assigned randomly to receive long-term P4 administration using controlled intravaginal releasing devices (CIDR) or no P4 (CIDRX; CIDR backbone only). Sera samples and body weights were collected every 14-d, along with CIDR/CIDRX replacement. Sera samples were assayed for metabolic hormones, NEFA, and metabolites. There were no differences in BW, RFI, STDMI, body composition, or temporal patterns of T3, T4, NEFA, or metabolites between CIDR- and CIDRX-treated ewes. Insulin concentrations were greater in CIDR-treated ewes than in CIDRX-treated ewes. Long-term P4 did not affect metabolism or body composition, independent from the presence of a fetus or placenta. Progesterone may increase tissue sensitivity to INS. In Experiment 3, the primary aim was to determine if NMR metabolic profiling has the potential to serve as a management tool for evaluating herds of bighorn (Ovis canadensis) sheep. Bighorn sheep herds were sampled between December of 2014 to March of 2015 in Montana and Wyoming. The sampling included 240 bighorn sheep ewes from 13 herds from geographically distinct locations at different times of the year. Metabolites identified by NMR in bighorn sheep serum were analyzed by pathway enrichment analyses, PLS-DAs, and biomarker analyses to determine if bighorn sheep herds can be distinguished by pregnancy status, geographic location, or time of year. NMR metabolic profiling could not distinguish between pregnant and non-pregnant bighorn sheep. Metabolic profiling did differentiate bighorn sheep herds and identified a subset of potential biomarkers that discriminated distinct geographic locations and time of year. Thus, NMR metabolic profiling has the potential to develop a suite of metabolites that wildlife managers can use to assess bighorn sheep nutrition and overall health.Item Fossil viruses, redox paradigms and predictive metabolism from a systems biology perspective(Montana State University - Bozeman, College of Letters & Science, 2014) Heinemann, Joshua Vance; Chairperson, Graduate Committee: Brian Bothner; Walid S. Maaty, George Gauss, Narahari Akkaladevi, Susan K. Brumfield, Vamseedhar Rayaprolu, Mark Young, C. Martin Lawrence and Brian Bothner were co-authors of the article, 'Fossil record of an HK-97-like provirus' in the journal 'Virology' which is contained within this thesis.; Timothy Hamerly, Walid S. Maaty, Navid Movahed, Joseph D. Steffens, Benjamin D. Reeves, Jonathan K. Hilmer, Jesse Therien, Paul A. Grieco, John W. Peters and Brian Bothner were co-authors of the article, 'Expanding the paradigm of thiol redox in the thermophilic root of life' in the journal 'Biochimica et biophysica acta' which is contained within this thesis.; Aurélien Mazurie, Monika Tokmina-Lukaszewska, Greg J. Beilman and Brian Bothner were co-authors of the article, 'Application of support vector machines to metabolomics experiments with limited replicates' in the journal 'Metabolomics' which is contained within this thesis.; Brigit Noon, Mohammad J. Mohigmi, Aurélien Mazurie, David L. Dickensheets and Brian Bothner were co-authors of the article, 'Real-time digitization of metabolomic patterns from a living system using mass spectrometry' submitted to the journal 'Journal of the American Chemical Society' which is contained within this thesis.One of the goals of systems biology is to develop a model which encapsulates the molecular, structural and temporal complexity of a living organism. While modern omics experiments can deliver a high resolution view of an organism's molecular complexity, methods for correlating the information from multiple biomolecular systems (i.e. genes, proteins and metabolites) and their changes over time remain greatly underdeveloped. Presented in this research are: (1) methods for understanding the inter-relation of multiple biomolecular systems correlating genomics, proteomics and metabolomics experiments; (2) techniques for machine learning based metabolic biomarker selection; (3) robotics technology for real-time measurement of changes in metabolism. The methods for correlating information from multiple biomolecular systems have provided a new perspective of biomolecular adaptation and evolutionary relationships in the thermophilic archaea. The techniques for biomarker selection have provided a method to assess the reliability of biomarkers in experiments where limited samples are available. The new technology has provided an engineered system for automated analysis of metabolic patterns and how they change over time. Together, these results have created a framework for future improvement of our understanding of biology through the use of molecular biology, machine learning and robotics.