Theses and Dissertations at Montana State University (MSU)
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Item Structural characterization of the Csa3/cA4 complex - a nexus for class 1 CRISPR-Cas immune response coordination & establishing a cure for highly efficient galectin expression(Montana State University - Bozeman, College of Letters & Science, 2024) Charbonneau, Alexander Anthony; Chairperson, Graduate Committee: C. Martin Lawrence; This is a manuscript style paper that includes co-authored chapters.Though Class I CRISPR-Cas systems, primarily Type I and Type III, are the most abundant CRISPR systems in archaea and bacteria, mechanisms driving their immune response regulation are not well understood. Csa3 family transcription factors, composed of N-terminal CARF and C-terminal winged helix-turn-helix domains, are frequently encoded within Type I CRISPR-Cas systems. Csa3 transcription factors are hypothesized to bind cyclic oligoadenylate (cOA) second messengers produced by Type III interference complexes, likely modulating their DNA-binding activity. Therefore, we investigated the interaction between Csa3a and cyclic tetra-adenylate (cA4). Isothermal titration microcalorimetry showed S. solfataricus Csa3a binds cA4 at biologically relevant concentrations in an entropically driven interaction. Ring nuclease assays revealed Csa3a lacks self-regulatory phosphodiesterase activity exhibited by other CARF domain proteins. We crystallized and solved the structure of the Csa3/cA4 complex, which revealed conserved motifs are responsible for cA4 binding and illuminated significant conformational changes induced by the interaction. We also identified an 18-bp palindromic motif, which we designated CAPPa, that is conserved in the 27 sequenced members of the order Sulfolobales, and shows synteny with Csa3a and acquisition genes in these genomes. We found Csa3a binds CAPPa in a nonspecific, cooperative, and cA4-independent manner. These characteristics suggest a more complex method of transcriptional regulation than previously hypothesized. However, the interaction between Csa3a and cA4 confirmed here signifies a nexus between Type I and Type III systems; we thus propose a model in which this interaction coordinates the two arms of an integrated immune system to mount a synergistic, highly orchestrated, adaptive immune response. We applied the workflow designed to produce significant protein quantities for crystallographic studies of Csa3a to the study of Homo sapiens galectin proteins, a family of beta-galactoside-binding proteins. Here, we identified a putative autoinhibitory mechanism affecting traditional IPTG-induction methods by characterizing IPTG-binding capabilities of galectins and quantifying basal protein expression over various IPTG concentrations. To bypass this predicted feedback loop, we employed a highly efficient and approachable autoinduction method, resulting in a 7-fold increase in protein expression. Much of this work was done in the context of a course-based undergraduate research experience with great success.Item Sepsis bundle evaluation for quality improvement(Montana State University - Bozeman, College of Nursing, 2019) O'Connor, Christine Elizabeth; Chairperson, Graduate Committee: Susan LuparellSepsis is a common diagnosis in the acute care setting. Left untreated, sepsis can result in many long-term complications including permanent organ damage and death. Sepsis has become such a common diagnosis that the Centers for Medicare & Medicaid (CMS) have implemented core measures that are meant to aid in quickly diagnosing and treating septic patients. Because sepsis requires prompt treatment, these guidelines have been divided into three- and six-hour bundles to assure prompt treatment after diagnosis. If hospitals fail to follow these core measures, the institution is not reimbursed for the cost of medical care for that patient. Implementation of the three and six-hour bundles have been shown to improve patient outcomes, decreasing mortality associated with sepsis. Compliance rates with these core measures in a rural hospital in Northwest Montana, which will be called Hospital X, have been consistently below the goal of 80% compliance. This quality-improvement project (QIP) utilized interventions to identify where non-compliance was occurring and interventions to improve overall institution compliance rates. Chart review and process flow observation were used to identify which parts of the bundle were not being implemented according to CMS guidelines. Use of a newly created sepsis handoff tool and implementing nurse education on the core measures were interventions used in an effort to increase overall institution compliance. Results: Overall compliance rates improved from 57% in May, 2018 to 87% in June, 2018 after implementation of interventions. For the months of June, 2018 - September, 2018, compliance rates remained >70%. Conclusion: The two interventions that were implemented during the course of this project seemed to improve compliance based off a significant improvement in overall compliance rates during months where the interventions were implemented. There are many recommendations for future research and interventions based off the findings from this project.Item The Staphylococcus aureus two component system, SaeR/S, modulates monocyte production of TNF-alpha to influence neutrophil functions(Montana State University - Bozeman, College of Letters & Science, 2018) Sward, Eli Winfield; Chairperson, Graduate Committee: Jovanka Voyich-Kane; Elizabeth M. Fones, Russel R. Spaan, Kyler B. Pallister, Brandon L. Haller, Fermin E. Guerra, Oliwia W. Zurek, Tyler K. Nygaard and Jovanka M. Voyich were co-authors of the article, 'Staphylococcus aureus SaeR/S-regulated factors decrease monocyte-derived tumor necrosis factor-alpha to reduce neutrophil bactercidal activity' in the journal 'Journal of infectious diseases' which is contained within this thesis.; Kyler B. Pallister and Jovanka M. Voyich were co-authors of the article, 'Staphylococcus aureus inhibits tumor necrosis factor-alpha in monocyte subsets to influence neutrophil functions' submitted to the journal 'Journal of infectious diseases' which is contained within this thesis.Staphylococcus aureus (S. aureus) is a commensal organism that colonizes the anterior nares of more than half the population. Although most individuals colonized with S. aureus remain asymptomatic, showing no signs of complications, colonization is associated with a predisposition to infection. S. aureus infections include skin- and softtissue infections as well as life-threatening infections, such as necrotizing fasciitis, necrotizing pneumonia, and sepsis. To date, it is not clearly understood how S. aureus transitions from a commensal organism to a deadly pathogen but evidence highlights that this capacity is largely dependent on two-component gene-regulatory systems that control expression of cytolytic and immunomodulatory virulence factors. The SaeR/S twocomponent system (SaeR/S TCS) of S. aureus is critical for the regulation of virulence factors that enables immune evasion and attenuates killing of S. aureus by human neutrophils. However, the precise SaeR/S-dependent mechanisms used by S. aureus to overcome and effective neutrophil response remains incompletely define. To advance our understanding, we studied SaeR/S-dependent immunomodulation of TNF-alpha. TNF-alpha is an important inflammatory mediator because it can recruit neutrophils to the site of infection and promote increased neutrophil killing of S. aureus. Using primary human cells, we demonstrated that the SaeR/S system reduced early monocyte production of TNF-alpha and showed that this modulation influenced the neutrophil priming and subsequent staphylocidal activity. These results demonstrated that S. aureus could reduce TNF-alpha early during infection to diminish neutrophil production of reactive oxygen species. As increased TNF-alpha is associated with morbidity and mortality during systemic infections, we propose that SaeR/S modulation of monocyte-derived TNF-alpha is important for reducing protective immune responses during localized infections. These data add to our understanding of how S. aureus disrupts early inflammatory responses to initiate infection.Item The role of mast cells during influenza A virus infection(Montana State University - Bozeman, College of Letters & Sciences, 2015) Graham, Amy Catherine; Chairperson, Graduate Committee: Josh Obar; Rachel M. Temple and Joshua J. Obar were co-authors of the article, 'Mast cells and influenza A virus: association with allergic responses and beyond' submitted to the journal 'Frontiers in immunology' which is contained within this thesis.; Kimberly M. Hilmer, Julianne M. Zickovich and Joshua J. Obar were co-authors of the article, 'Inflammatory response of mast cells during influenza A virus infection is mediated by active infection and RIG-I signaling' in the journal 'The journal of immunology' which is contained within this thesis.; Julianne M. Zickovich, Kimberly M. Hilmer and Joshua J. Obar were co-authors of the article, 'Differential role of influenza A virus binding preference in mast cell activation' submitted to the journal 'The journal of virology' which is contained within this thesis.Influenza A virus (IAV) is a major cause of seasonal viral respiratory infections and causes ~36,000 deaths and ~1.7 million hospitalizations each year in the United States alone. Moreover, IAV has the potential to cause global pandemics, which have significantly greater morbidity and mortality. Morbidity and mortality associated with IAV infections is thought to be the result of significant pulmonary immunopathology from the inflammatory response rather than viral replication. The initial lines of defense against pathogens in the lungs include alveolar epithelial cells, endothelial cells, tissue resident alveolar macrophages, dendritic cells, and mast cells. Additionally, recruitment of neutrophils and macrophages is required for optimal clearance of IAV. Recent global analysis of lungs from mice infected with highly pathological IAV strains demonstrated enrichment of a mast cell transcriptional response, but the role of mast cells during severe pulmonary viral infections has been under studied. We found that A/WSN/33 causes significant immunopathology in C57Bl/6 mice and viral-induced pathology is mast cell-dependent. A/WSN/33 is able to directly activate bone marrow cultured mast cells (BMCMC) to produce histamine, leukotrienes, inflammatory cytokines, and anti-viral chemokines. Moreover, human H1N1, H3N2, and influenza B virus isolates can activate murine BMCMC in vitro suggesting this pathway could play a role during human infections. BMCMC activation requires infection of mast cells by IAV, which is dependent on the viral hemagglutinin specificity for alpha2,6-linked sialic acids. Cytokine and chemokine production from BMCMC occurs in a RIG-I-dependent fashion that requires the de novo production of vRNA. Conversely, degranulation occurs through a RIG-I-independent mechanism. Reconstitution of mast cell deficient mice with RIG-I -/- BMCMC generates lung pathology similar to wild-type BMCMC, suggesting that mast cell degranulation, rather than production of cytokines, causes A/WSN/33 induced lung pathology. Using recombinant A/WSN/33 strains, we found an association between binding of the A/WSN/33 hemagglutinin to alpha2,6-sialic acids and subsequent interactions with neuraminidase is important for degranulation. Thus, we have identified a unique inflammatory cascade that could be therapeutically targeted to limit morbidity following infection with IAV.Item Insights into the reciprocal communication between Neutrophils and Staphylococcus aureus(Montana State University - Bozeman, College of Letters & Science, 2015) Zurek, Oliwia Wiktoria; Chairperson, Graduate Committee: Jovanka Voyich-Kane; Tyler K. Nygaard, Robert L. Watkins, Kyler B. Pallister, Victor J. Torres, Alexander R. Horswill and Jovanka M. Voyich were co-authors of the article, 'The role of innate immunity in promoting SAER/S-mediated virulence in Staphylococcus aureus' in the journal 'Journal of innate immunity' which is contained within this thesis.; Kyler B. Pallister and Jovanka M. Voyich were co-authors of the article, 'Staphylococcus aureus inhibits neutrophil-derived IL-8 to promote cell death' in the journal 'Journal of Infectious Diseases' which is contained within this thesis.Staphylococcus aureus (S. aureus) is a highly adaptable pathogen that can cause endocarditis, skin abscesses, tissue necrosis, and sepsis. S. aureus success can be partially attributed to its ability to colonize and subsequently infect a wide variety of host tissues. This capacity is dependent on elaborate two-component gene-regulatory systems that control expression of virulence and immunomodulatory factors. The S. aureus exoprotein expression (SaeR/S) system is recognized as a major regulator of virulence that significantly contributes to the pathogen's ability to evade killing by the human neutrophil. However, it is unclear how this system becomes activated and how the SaeR/S system modulates neutrophil function. In this study, we elucidated how S. aureus evades neutrophil killing by studying the reciprocal communication between the host and pathogen. We demonstrated that only select SaeR/S-regulated genes (as opposed to all targets) were transcriptionally up-regulated in response to stimulation by neutrophils as well as alpha-defensin and show that the mouse skin environment (that lacks alpha-defensin) promoted transcription of specific saeR/S-targets, different from the expression profile elicited following neutrophil interaction or alpha-defensin. These results were unexpected and demonstrated differential activation of saeR/S targets was dependent on specific stimuli. Furthermore, we studied the influence of SaeR/S on neutrophil function and showed that this system promoted accelerated cell death by decreasing NF-kB activity, and in-turn IL- 8 production, to promote neutrophil lysis. These findings underscored the importance of neutrophil signaling demonstrating that neutrophil-derived production of IL-8 was necessary for this cell to kill S. aureus effectively. It follows that treatment of human neutrophils with recombinant IL-8 significantly increased neutrophil staphylocidal activity. Finally, we propose that both timing and magnitude of inflammation in neutrophils play major roles in dictating the outcome of staphylococcal disease and that alteration in the innate ability of neutrophils to produce IL-8 may increase susceptibility to S. aureus infections. Taken together, the findings define novel pathogen- and host-derived factors that play pivotal roles in the course of S. aureus infection.Item Cobalt supplementation affects humoral immune response in beef calves(Montana State University - Bozeman, College of Agriculture, 2015) Sager, Robert Bascom; Chairperson, Graduate Committee: Glenn Duff; Glenn C. Duff and Carl J. Yeoman were co-authors of the article, 'Cobalt supplementation in pre-weaned calves affects humoral immune response and feedlot health' in the journal 'Proceedings, western section, American Society of Animal Science' which is contained within this thesis.Economic losses from morbidity and mortality associated with bovine respiratory disease (BRD) in beef cattle are approaching $2 billion annually in the United States. Incidence and severity of BRD is increasing despite advances in animal health programs in prevention and treatment compared to twenty years ago. Mineral supplementation during pre-weaning has potential to reduce sickness and improve health. Cobalt (Co) is used by rumen-inhabiting microbes for the production of vitamin B 12. Vitamin B 12 is a cofactor for vital metabolic pathways in tissue carbohydrate and lipid metabolism required for maintenance and growth. Vitamin B 12 is also vital for B-cell proliferation to form plasma cells that secrete antibodies. National Research Council (NRC) recommendations for Co are 0.1 ppm (0.1 mg/kg; DM dry matter basis). Beef production has changed tremendously since NRC recommendations were set in the 1950's. The hypothesis of these three studies is NRC Co concentrations need to be increased to meet today's beef cattle metabolic requirements and production needs. The objectives of these studies were to evaluate if an orally-supplied Co dosed at nursing, pre-weaning, or post weaning affects humoral immune response during the post-weaning feeding period and reduces the incidence of BRD. Mannheimia haemolytica is a major pathogen of BRD which causes increased pathophysiological pulmonary tissue severity, increased treatment time, and increased mortality in beef calves. Calves were vaccinated with M. haemolytica in all studies as an indicator of immune response. Different dosages and forms of Co were administered to evaluate humoral immune response. Results indicate increased NRC Co concentrations affect humoral immune response and potentially improve beef calf health. Study results suggest current NRC Co concentrations should be increased to improve post-weaning health in beef calves.Item Regulation of the immune response to polyvinylpyrrolidone(Montana State University - Bozeman, College of Agriculture, 1977) Lake, Jeffrey PeterItem A critical analysis of bone marrow-spleen cell interaction in the immune response(Montana State University - Bozeman, College of Agriculture, 1970) Sieckmann, Donna GailItem Dark areas and the immune response to the cestode, Hymenolepis diminuta in mice and rats(Montana State University - Bozeman, College of Agriculture, 1978) Larson, Michael AllenItem The influence of lipopolysaccharide (LPS) on cellular activities in LPS-unresponsive C3H/HeJ mice(Montana State University - Bozeman, College of Agriculture, 1976) Rampy, Patricia Anne Nelson