Theses and Dissertations at Montana State University (MSU)
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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 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 Characterization and embellishment of protein cages for nanomedical and nanomaterial applications(Montana State University - Bozeman, College of Letters & Science, 2014) Servid, Amy Eloise; Chairperson, Graduate Committee: Trevor Douglas; Laura E. Richert, Ann L. Harmsen, Agnieszka Rynda-Apple, Soo Han, James A. Wiley, Trevor Douglas and Allen G. Harmsen were co-authors of the article, 'A virus-like particle vaccine platform elicits heightened and hastened local lung mucosal antibody production after a single dose' in the journal 'Vaccine' which is contained within this thesis.; Paul Jordan, Alison O'Neil, Peter Prevelige and Trevor Douglas were co-authors of the article, 'Location of the bacteriophage P22 coat protein C-terminus provides opportunities for the design of capsid-based materials' in the journal 'Biomacromolecules' which is contained within this thesis.In nature, protein cages are found within the structures of viruses, heat shock proteins, and ferritins. They assemble from subunits into spherical oligomeric structures, which serve to encapsulate, protect, and/or deliver cargo. The fundamental understanding of protein cage structure is a key component in the design of novel nanomaterials that best exploit and expand upon the natural functions of protein cage architectures. By mimicking the re-occurring design strategies employed by natural systems, the protein cages produced during virus infection and/or stress responses can be modified to yield particles that fight disease and/or serve as the building blocks for materials design. In particular, the work described here highlights the design and characterization of protein cages in an effort to develop and uncover new strategies for immunization of the lung against a variety of pathogens. A combination of genetic and chemical engineering approaches is described here in order to better understand the structure of protein cage architectures and the relationship between the structure and in vivo function. This work describes the chemical cross-linking of large antigens and immunomodulatory molecules to the surface of a protein cages, and it shows that intensified and accelerated immune responses result from the display of antigens on a protein cage surface. Genetic incorporation of point mutations within the capsid structure provided unique attachment points for chemical functionalization. In addition, genetic modifications revealed information about the location of the C-terminus of the bacteriophage P22 capsid. The knowledge that this position was displayed on the capsid exterior prompted its use to promote inter-capsid interactions and target nanoparticles to melanoma cells. This research emphasizes that both the protein cage structural design and the local in vivo environment can influence the outcomes of protein cages when administered to the lung environment. It also lays the foundation for the logical design of biomaterials that offer enhanced protection against influenza and other respiratory diseases. Finally, regions of protein cages that are amendable to chemical and genetic modifications are described herein, and these have paved the way for the continued development of protein cage platforms for nanomedical and material applications.Item Grasshopper lectin : cDNA sequence, amino acid sequence and computer-based homology model(Montana State University - Bozeman, College of Letters & Science, 1996) Radke, Jay RichardItem Grasshopper agglutinin : preparation and characterization by MALDI/TOF-MS(Montana State University - Bozeman, College of Letters & Science, 1996) Wenzlick, Donald Lee