Theses and Dissertations at Montana State University (MSU)
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Item Identification of cellulolytic hot spring organisms through bioorthogonal labeling(Montana State University - Bozeman, College of Letters & Science, 2021) Reichart, Nicholas John; Chairperson, Graduate Committee: Roland Hatzenpichler; This is a manuscript style paper that includes co-authored chapters.Microbial physiology is the study of the metabolism and function of microorganisms. The recent expansion of genomic diversity has outpaced the description of physiology. To better understand microbial metabolisms and environmental processes, more detailed research is needed for both novel and undescribed microbes. While many new methods are being developed to describe in situ microbial activity, this dissertation implements bioorthogonal non-canonical amino acid tagging as a proxy to track metabolic activity of microbes under close to environment conditions. Using differential analyses on hot spring microbial communities, we were able to show that certain microbial taxa had preferential activity towards specific incubation amendments. Previous activity-based studies had shown that hot springs were a unique environment for discovering cellulolytic microbes that could be used in industrial processing of plant biomass. Herein, we used computational analysis to screen publicly available metagenomic datasets to identify the enzymatic potential of hot springs worldwide. The wide diversity of taxa and biomass degrading enzymes were investigated and hot springs were further highlighted as a system that could be used to find improvement for the industry of plant biomass degradation and processing. To build upon the cellulolytic potential found in hot spring metagenomic datasets, bioorthogonal non-canonical amino acid tagging coupled with fluorescence-activated cell sorting was applied to the biotechnological relevant field of plant biomass degradation to identify microbes involved in the cellulolytic process. Examination of the active microbes revealed difference in the community when supplemented with cellulose. Taken together, the work in this dissertation served to expand and apply the recent development of activity-based studies used to describe environmental microbial populations, with a focus on plant biomass degradation.Item Quantitative 1 H NMR analyses of immunometabolic modulation in human macrophages(Montana State University - Bozeman, College of Letters & Science, 2019) Fuchs, Amanda Lee; Chairperson, Graduate Committee: Valerie Copie; Sage M. Schiller was an author and Wyatt J. Keegan, Mary Cloud B. Ammons, Brian Eilers, Brian Tripet and Valerie Copie were co-authors of the article, 'Quantitative 1 H NMR metabolomics reveals distinct metabolic adaptations in human macrophages following differential activation' in the journal 'Metabolites' which is contained within this dissertation.; Sage M. Schiller was an author and Isaac R. Miller, Mary Cloud B. Ammons, Brian Eilers, Brian Tripet and Valerie Copie were co-authors of the article, 'Pseudomonas aeruginosa planktonic- and biofilm-conditioned media elicit divergent responses in human macrophages' submitted to the journal 'PLoS pathogens' which is contained within this dissertation.Macrophages are innate immune cells that are found ubiquitously in nearly all human tissues, where they support host innate and adaptive immune responses in an effort to maintain systemic homeostasis. They are inherently plastic in nature and can dramatically modulate their functional phenotype according to pathogen and microenvironmental stimuli. Previous studies have shown that macrophages are particularly important for the resolution of inflammation in acute wound healing, which is marked by a phenotypic transition of wound macrophages from pro-inflammatory to anti-inflammatory. Chronic, or non-healing, wounds, such as diabetic, pressure, and venous leg ulcers, feature a prolonged host inflammatory response due in part to aberrant wound macrophage behavior. Non-healing in chronic wounds has also been shown to be dependent upon the establishment of pathogenic biofilms, which are more resistant to host defense mechanisms than planktonic, or free-floating, bacteria. Therefore, investigating macrophage dysregulation in the presence of bacterial biofilms has gained considerable interest. Here, 1D 1 H NMR-based metabolomics was utilized to identify metabolic pathways that are differentially modulated following primary human monocyte-derived macrophage activation with pro-inflammatory or anti-inflammatory stimuli relative to resting macrophages. Metabolic profiling of inflammatory macrophages indicated a substantial increase in oxidative stress as well as a decrease in mitochondrial respiration. These metabolic profiles also provided evidence that inflammatory macrophages divert metabolites from de novo glycerophospholipid synthesis to inhibit oxidative phosphorylation. In addition, we investigated which metabolic pathways are differentially modulated following primary human monocyte-derived macrophage exposure to Pseudomonas aeruginosa planktonic- and biofilm-conditioned media. Metabolic profiling of PCM- and BCM-exposed macrophages indicated a significant depletion of intracellular glucose without elevation of downstream glycolytic products. These metabolic patterns suggest that PCM- and BCM-exposed macrophages potentially divert glycolytic intermediates towards inositol phosphate metabolism. Overall, our studies provide additional support to previous findings, generate novel results regarding metabolic modulation of human macrophages following activation and exposure to planktonic- vs. biofilm-conditioned media, and contribute new insight to the field of immunometabolism.Item Proteomics analysis of the metabolic transition between aerobic and anaerobic conditions in Escherichia coli(Montana State University - Bozeman, College of Letters & Science, 2019) Refai, Mohammed Yahya; Chairperson, Graduate Committee: Brian Bothner; Nina Paris, Hunter Fausset, Monika Tokmina Lukaszewska were co-authors of the article, 'Proteomics analysis of the transition between aerobic and anaerobic growth conditions in Escherichia coli' submitted to the journal 'Biochimica et biophysica acta' which is contained within this dissertation.As a facultative anaerobe, Escherichia coli has the ability to grow in anaerobic and aerobic environments. Despite detailed characterizations of this model organism in the presence and absence of oxygen, an in-depth understanding of changes to the proteome during transitions from aerobic to anaerobic growth is lacking. This thesis work focuses on elucidating how protein thiol oxidation and reduction change during a facultative anaerobe's transition from aerobic to anaerobic growth conditions, and pathways of thiol-mediated cell signaling. Redox driven changes in cysteine oxidation involved in signaling are referred to as 'thiol switches'. These modulate diverse biological activities ranging from gene expression and protein synthesis to environmental stress response. Surprisingly, little is known about the role of thiol switches during microbial transitions from aerobic and anaerobic growth conditions. To explore this uncharted territory, a mass-spectrometry (MS)-based proteomics workflow was developed and refined. Following extensive protocol optimization for high-throughput MS data processing, normalization, and pattern matching, the analytical pipeline was fine-tuned for the specific proteome-wide analysis of cysteine chemical modifications in E. coli. The approach was based on open-source software and publicly accessible databases, creating a transparent, reproducible, and easily sharable proteomics approach. Herein, the redox state and chemical forms of protein-based thiol switches in E. coli were characterized over time as the bacterium reversibly transitioned between aerobic and anaerobic growth conditions. Unexpectedly, differential alkylation analysis of cysteine-containing E. coli proteins revealed a higher degree of protein thiol oxidation under anaerobic growth conditions, a result not reported for E. coli or any other facultative anaerobe. Our proteome-wide analysis also revealed that cysteine redox potentials vary widely, and several specific E. coli proteins contain highly reactive thiols. These findings provide strong evidence for thiol-based signaling in E. coli in response to environmental changes such as aerobic to anaerobic growth transitions. Characterization of specific redox switches underlying metabolic changes associated with oxygen availability has uncovered a previously unknown E. coli cell signaling mechanism. Since transitioning between aerobic and anaerobic environments is associated with bacterial virulence, this work opens new avenues to target pathogenic facultative anaerobes and to develop novel thiol-based antibacterial therapies.Item Development of a protein-based sensor assay for rapid classification of complex biological samples(Montana State University - Bozeman, College of Letters & Science, 2016) Hamerly, Timothy Kyle; Chairperson, Graduate Committee: Brian Bothner; Joshua Heinemann, Monika Tokmina-Lukaszewska, Elizabeth R. Lusczek, Kristine E. Mulier, Greg J. Beilman and Brian Bothner were co-authors of the article, 'Bovine serum albumin as a molecular sensor for the discrimmination of complex metabolite samples' in the journal 'Analytica chemica acta' which is contained within this dissertation.; Brian Bothner was a co-author of the article, 'Adding metrics to the aging of whiskey using a protein sensor assay' which is contained within this dissertation.; Brian Bothner was a co-author of the article, 'Analysis of wine using the protein sensor assay' which is contained within this dissertation.; Brian Bothner was a co-author of the article, 'Investigations into the use of a protein sensor assay for metabolite analysis' in the journal 'Applied biochemistry and biotechnology' which is contained within this dissertation.Metabolomics, one of the core 'omics' fields within the umbrella of systems biology, is the study of the small molecules which can be used to characterize the state of an organism. Metabolites are constantly being transformed inside a cell in direct response to stimuli around them. This makes the metabolome the most dynamic of all the omics fields and is considered to be a direct readout of the cells state at any given time. Although highly informative, the metabolome is inherently difficult to study, with thousands of known metabolites, any of which could be important for classifying a cell into a healthy or diseased state. Techniques such as mass spectrometry are well suited to study the metabolome and have been used to successfully classify cells by identify markers for a given disease state. However, current methods require lengthy analysis times due in part to the complexity of the metabolome. The research presented in this dissertation highlights a new and promising methodology which improves classification and speeds marker discovery. Making use of a protein found in animals which has evolved to selectively bind metabolites, an assay was developed which better classified samples compared to current methods used in the field of metabolomics. This improved classification was achieved with an overall decrease in analysis time. The implementation of this method in the study of complex biological systems would have an immediate impact in academic and medical research.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 Comparison of early- and late-senescence near-isogenic barley germplasm : proteomics and biochemistry shed new light on an old problem(Montana State University - Bozeman, College of Letters & Science, 2015) Mason, Katelyn Elizabeth; Chairperson, Graduate Committee: Brian Bothner; Jonathan K. Hilmer, Walid S. Maaty, Ben D. Reeves, Paul A. Grieco, Brian Bothner and Andreas M. Fischer were co-authors of the article, 'Proteomic comparison of near-isogenic barley (Hordeum vulgare L.) germplasm differing in the allelic state of a major senescence QTL identifies numerous proteins involved in plant pathogen defense' submitted to the journal 'Journal of experimental botany' which is contained within this thesis.; Timothy Hamerly, Andreas M. Fischer and Brian Bothner were co-authors of the article, 'Metabolomic comparison of post-anthesis barley flag leaves from near-isogenic germplasm differing in its senescence behavior' submitted to the journal 'Journal of metabolomics and systems biology' which is contained within this thesis.Before their death, plant tissues undergo the essential process of senescence. Senescence is characterized by a coordinated recovery of nutrients and their retranslocation to surviving structures, such as seeds of annual plants. In monocarpic crops (e.g., maize, wheat, and barley), timing and efficiency of senescence can impact yield and grain quality. However, our understanding of senescence regulation and nutrient remobilization is limited, and protein- and metabolite-level analyses of the process are scarce, particularly in crops. To improve understanding of physiology in barley (Hordeum vulgare L.) leaf senescence, a systems-level comparison of near-isogenic germplasm, late-senescing/low grain protein content variety 'Karl' and an early-senescing/ high-grain protein content line ('10_11'), was performed. Protein levels in flag leaves (topmost leaves) of 'Karl' and '10_11' were compared at 14 and 21 days past anthesis (dpa) using both two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) and shotgun proteomic approaches. Conspicuously, proteins with roles in plant pathogen defense were present at higher levels in '10_11' as compared to 'Karl'. These included membrane receptors, glucanases, pathogenesis-related and disease resistance proteins. Proteins involved in protein degradation and organic acid/amino acid metabolism were upregulated in line '10_11' as compared to 'Karl', expectedly in early-senescing leaves involved in nitrogen remobilization. Metabolite levels were compared in the same plant material as protein levels except that analyses were also performed at anthesis (0 dpa), using mass spectrometry-based non-targeted metabolic profiling techniques. Metabolites with higher abundance in early-senescing line '10_11' included gibberellin catabolites, Yang cycle intermediates and intermediates of jasmonic acid biosynthesis. These differences were mostly observed at 0 dpa, indicating an early shift in phytohormone metabolism that may be important for senescence regulation and plant disease resistance between 'Karl' and '10_11' during the senescence phase, as jasmonic acid and ethylene have roles in plant pathogen defense. Overall, proteomic and metabolomic analyses performed here shed new light on the regulation of the senescence process, on the importance of plant defense against pathogens during senescence, and possibly on crosstalk between senescence regulation and pathogen defense. Proteins and metabolites identified in this study may become targets for ongoing efforts at improving crop yield, quality and environmental stress resistance.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.Item Characterization of recombinant human kidney diamine oxidase and equine plasma amine oxidase(Montana State University - Bozeman, College of Letters & Science, 2002) Elmore, Bradley OwenItem Studies of acetate and propionate turnover in high and low performance anaerobic digestors(Montana State University - Bozeman, College of Letters & Science, 1983) Craig, Annette Marie HillItem Kinetics and mechanism of the reactions of Cu(II)-N,N'-diglycylethylenediamine with triethylenetetramine and ethylenediaminetetraacetate(Montana State University - Bozeman, College of Letters & Science, 1983) Mitchell, Presley Kirkland