Theses and Dissertations at Montana State University (MSU)
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Item The paradoxical role of ATP in viral infections and cellular defense(Montana State University - Bozeman, College of Agriculture, 2024) Zahl, Trevor Ronald; Chairperson, Graduate Committee: Blake Wiedenheft; This is a manuscript style paper that includes co-authored chapters.Conflict between prokaryotes and viruses has driven the evolution of diverse defense systems. The study of these systems has not only expanded our knowledge of the mechanisms underlying genetic conflict, but has also revolutionized biotechnologies. I joined the Wiedenheft lab in early 2021 during the height of the SARS-CoV-2 pandemic. As such, the lab was focused on developing technologies to aid in the identification and study of SARS-CoV-2. I joined a team that was actively repurposing a type III CRISPR system as a molecular diagnostic. Type III CRISPRs are RNA targeting systems that unleash antiviral signaling cascades upon recognition of a target. Previous work in the lab had shown that this activity could be repurposed as a molecular diagnostic, but the first generation technology was not sensitive enough for clinical use. To address this need, we developed a technology termed Capture and Concentrate, which utilized 'CRISPR magnets' to concentrate target RNAs from complex mixtures. Using this technology, we increased diagnostic sensitivity by 1,000 fold. In addition to my work at the bench, I developed an in silico model of the diagnostic to inform future development efforts. While type III CRISPRs are unique due to their ability to generate antiviral signaling cascades, they also contain intrinsic endonuclease activity. In 2023 we demonstrated that this activity could be repurposed as an RNA editing technology. Using the type III system from Streptococcus thermophilus, we introduced precise genomic modifications in a recombinant sindbis virus. This technology removed the need for reverse genetics systems, which hampered the study of the rapidly evolving SARS-CoV-2 virus. In the final year of my graduate studies, I worked with a team focused on determining how, PARIS, a bacterial innate immune system, induced host death upon viral infection. PARIS is a two-component system comprised of the ATPase, AriA, and a nuclease, AriB. Previous studies had shown that PARIS induced abortive infection in response to viral proteins, but the mechanism was not known. We determined that upon antigen recognition, AriB is activated and cleaves host tRNAs to induce cell death and prevent community spread.Item Study of diverse host immune responses to viral and bacterial pathogens(Montana State University - Bozeman, College of Agriculture, 2023) Plewa, Jack Bruno; Chairperson, Graduate Committee: Mark Jutila; This is a manuscript style paper that includes co-authored chapters.Brucella abortus is the bacterium that causes brucellosis, an infection transmitted from cattle to people, often through consumption of raw milk and contact with aborted materials. With antibiotic resistance on the rise, phage therapy for bacterial infection may become a useful approach. The direct effects of phage on mammalian cells is important to understand, yet understudied. In vivo delivery of low phage MOI to the mouse lung was more effective at diminishing Brucella burden than higher doses of phage. In an in vitro model of intracellular Brucella infection, low phage MOI was capable of minimizing human THP-1 monocyte infection, but, unexpectedly, use of higher phage MOI diminished this effect. We hypothesized that recognition of these phage preparations may induce an antiviral immune suppressive response that may counteract their anti-bacterial effects. Indeed, when the type I IFN signaling pathway was disrupted in mice, phage treatment was more effective. However, when attempting to induce type I IFN in vitro using both human monocyte and mouse macrophage cell lines, we were unable to stimulate expression of type I IFN with Brucella phage, including in response to a combination of phage and bacteria. We then examined the effect of phage treatment on macrophage cell surface markers that are indicative of activation/differentiation. Interestingly, while Brucella LPS induced expression of CD71 and CD206, the addition of phage suppressed upregulation of these markers. Our discovery of immune suppressive effects of Brucella bacteriophage is an important consideration for using phage as a treatment.Item Virus-like particle surface toll-like receptor signaling modulates host response to bacterial infection(Montana State University - Bozeman, College of Agriculture, 2022) Hatton, Alexis Alexandria; Chairperson, Graduate Committee: Mark T. Quinn; This is a manuscript style paper that includes co-authored chapters.Innate immune recognition of viruses is critical for the rapid response and subsequent clearance of an infection. The primary focus of virus innate immune recognition is the recognition of viral nucleic acids post-infection. However, innate pattern recognition receptors (PRRs) have been demonstrated to recognize viral proteins independent of infection. One group of PRRs associated with viral recognition are Toll-like Receptors (TLRs). With the discovery of TLRs in the late 1990's, over two decades of research have endeavored to identify if cell surface TLRs recognize viral proteins and if so, understand whether surface TLR-viral recognition benefits the host or the virus. To the benefit of the host, it was previously determined that host-recognition of viral proteins protects mice from bacterial infection early post-virus exposure, independent of viral nucleic acids (virus-like particle; VLP). This suggested that early viral protein recognition could protectively prime the host against bacterial infection. Our investigation here attempts to address the generality of surface TLR-virus recognition independent of infection, how viral protein recognition alters the subsequent signaling response to bacterial infection, and finally, if/how expression system-associated variables interfere with the interpretation of our study. We utilized macrophage deficient in surface TLRs and TLR-associated signaling proteins to address the TLR signaling pathway responsible for the general response to VLPs that results in reduced bacterial burden. We found that different surface TLRs were responsible for reducing bacterial burden, resulting in the activation of different signaling pathways dependent upon the VLP macrophages were exposed to. In addition, our results demonstrate how expression system-associated variables alter the interpretation of signaling pathways activated by surface TLRs.Item Scoping out intestinal epithelium differentiation, proliferation, and homeostasis through the lenses of interleukin-10 and aryl hydrocarbon receptor signaling pathways(Montana State University - Bozeman, College of Agriculture, 2020) Jenkins, Brittany Rene; Co-Chairs, Graduate Committee: Douglas Kominsky and Seth Walk; Nathan A. Blaseg, Heather M. Grifka-Walk, Benjamin Deuling, Steve D. Swain, Eric L. Campbell, Seth T. Walk and Douglas J. Kominsky were co-authors of the article, 'Loss of interleukin-10 receptor disrupts intestinal epithelial cell proliferation and skews differentiation towards the goblet cell fate' submitted to the journal 'The Federation of American Societies for Experimental Biology (FASEB) journal' which is contained within this dissertation.; Heather M. Grifka-Walk, Steve D. Swain, Trevor R. Zahl, Andrew Gentry, Seth T. Walk and Douglas J. Kominsky were co-authors of the article, 'Aryl hydrocarbon receptor modulation of intestinal epithelial cell fate is sex-dependent and exhibits variability among allelic variants' which is contained within this dissertation.; Dissertation contains two articles of which Brittany Rene Jenkins is not the main author.Intestinal epithelial cells (IEC) are crucial for maintaining proper digestion and overall homeostasis of the gut mucosa. IEC proliferation and differentiation are tightly regulated by well described pathways, however, relatively little is known about the influence of interleukin (IL)-10 and aryl hydrocarbon receptor (AHR) signaling pathways on these processes or whether AHR can regulate IL-10R expression in IECs. IL-10 signaling suppresses inflammation. AHR is a ligand activated transcription factor largely known for downstream activation of xenobiotic-metabolizing enzymes but also exerts a diverse range of responses in the host that can be modulated by gut microbe metabolites. Both IL-10 and AHR signaling are shown to promote IEC barrier function, and thus, they may also regulate other critical homeostatic functions like IEC lineage fate and regenerative capacity. These gaps in knowledge were addressed in Chapters 2 and 3. Techniques such as reverse-transcription quantitative polymerase chain reaction (RT-qPCR), western blotting, and histology staining techniques were used to assess changes in expression of target genes and proteins between control and either IL-10R- or AHR-deficient models. Loss of IL-10R or AHR demonstrated substantial impacts exhibiting nearly opposite patterns on lineage fate outcomes and on the proliferative compartment. In Chapter 4, we showed that activation of AHR by microbe-derived tryptophan metabolites increased IL-10R1 expression in IECs, and these metabolites ameliorated disease in a murine model of colitis. Findings from Chapters 2-4 add to a growing body of evidence for the importance of IL-10R and AHR signaling pathways in inflammatory bowel disease (IBD) and gastrointestinal (GI) cancers. Organoid models provide an additional study system to test these gaps in the field and hold great promise for advancing disease research. However, limitations exist for accessing the luminal surface to recapitulate the GI environment. In Chapter 5, we developed the GOFlowChip to solve this problem. This platform applies long-term, steady-state flow through to the organoid and can be modified to serve different research goals. These studies culminate in a deeper understanding of how IEC homeostasis is maintained and how innovative technologies can be developed for advancing this field of research.