Theses and Dissertations at Montana State University (MSU)
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Item An interrogation of herpes simplex virus type-1 gene expression during neuronal infection(Montana State University - Bozeman, College of Agriculture, 2024) Domanico, Luke Frank; Chairperson, Graduate Committee: Matt Taylor; This is a manuscript style paper that includes co-authored chapters.Herpes Simplex virus-type-1 (HSV-1) is a ubiquitous human pathogen casually referred to as "the gift that keeps on giving". The seemingly benign recurring herpetic lesions caused by acute HSV-1 infection are an obnoxious reminder of an incurable infection. HSV-1 maintains lifelong persistence in the infected host through a unique form of infection in peripheral neurons, conventionally termed latency. The latently infected neuron acts as a viral reservoir and is the focal point of herpetic disease. The latent HSV-1 infection represents a brilliant orchestration of viral gene regulation, manipulation of highly polarized cells, and seamless evasion of immunological clearance. Though, the viral mechanisms and cellular factors that govern the establishment, maintenance and reactivation from latency are elusive and challenging to study. The work included here aims to uncover the cryptic factors involved in and supporting the latent HSV-1 infection. Authored publications include the demonstration of a recombinant HSV-1 that enables temporal discretion of viral gene expression, and the revelation of a stunning, yet obscure phenotype of neuronal infection. Next is the implementation of a single-cell culturing method using drop-based microfluidic technology to resolve HSV-1 infection in isolated neurons. Together, this work reveals that the early events of neuronal infection are critical to determining the lytic or latent outcome of infection. Inoculating dose impacts the kinetics of viral replication, and the establishment of lytic or latent HSV-1 infection. Furthermore, evaluation of viral gene expression during latent HSV-1 infection suggests that the distinction between lytic and latent HSV-1 infection is less mutually exclusive than is historically appreciated. Finally, I present preliminary and ongoing research suggesting that a cellular transcription factor called nuclear factor-kappa B (NF-kB) differentially engages in HSV-1 infection. NF-kB supports efficient lytic gene transcription in epithelial cells, while promoting the establishment of latent HSV-1 infection of neurons.Item Honey bee antiviral defense mechanisms at the individual and cellular level(Montana State University - Bozeman, College of Agriculture, 2021) Parekh, Fenali Mukesh; Chairperson, Graduate Committee: Michelle Flenniken; Katie F. Daughenbaugh and Michelle L. Flenniken were co-authors of the article, 'Chemical stimulants and stressors impact the outcome of virus infection and immune gene expression in honey bees (Apis mellifera)' in the journal 'Frontiers in immunology' which is contained within this dissertation.; Alexander J. McMenamin was an author and Verana Lawrence and Michelle L. Flennikenwas were co-authors of the article, 'Investigating virus-host interactions in cultured primary honey bee cells' in the journal 'Insects' which is contained within this dissertation.; Katie F. Daughenbaugh and Michelle L. Flenniken were co-authors of the article, 'Honey bee antiviral response to flock house virus infection' which is contained within this dissertation.; This dissertation contains an article of which Fenali Mukesh Parekh is not the main author.Honey bees are important pollinators of fruit, nut, and vegetable crops that constitute a large proportion of the human diet. Unfortunately, annual honey bee colony losses are high, averaging 38% from 2008-2018 in the United States. Honey bee colony losses are attributed to multiple factors, including pathogens and chemical exposure. Virus incidence and abundance have been associated with colony losses. The majority of honey bee viruses are positive-sense single stranded RNA viruses. Honey bees antiviral defense include RNA interference (RNAi), a double-stranded RNA (dsRNA) triggered sequence-specific post-transcriptional silencing mechanism and a non-sequence specific dsRNA-triggered pathway. In addition, signal transduction cascades include the Toll, Imd, and Jak/STAT pathways that promote the expression of honey bee immune response genes that are also induced in response to virus infections. To investigate the impact of chemical exposure on honey bee immune responses and virus infections, we infected bees with a panel of viruses including two model viruses (i.e., Flock House virus (FHV) and Sindbis-GFP) and a naturally infecting honey bee virus, deformed wing virus (DWV) and fed them sucrose syrup containing either thyme oil, a beekeeper applied fungicide Fumagilin-B ®, or the insecticide clothianidin. We determined that bees fed thyme oil augmented sucrose syrup exhibited greater expression of key immune genes, i.e., ago2, dcr-like, abaecin, hymenoptaecin, and vitellogenin and reduced virus abundance compared to virus-infected bees fed sucrose syrup. Whereas, virus-infected honey bees fed diets containing fumagillin or clothianidin exhibited reduced expression of key immune genes and higher virus abundance suggesting that chemical stressors act as immunosuppressors in honey bees. To understand the interplay of viruses and host cell gene expression more precisely, we cultured primary honey bee cells derived from larvae (i.e., hemocytes, immune cells) or pupae (i.e., mixed cell population including epithelial cells, adipocytes, muscle cells, hemocytes) and demonstrated that these cells supported replication of sacbrood virus, DWV, and FHV. Expression of select immune genes, including bap1, ago2, and dcr-like, in virus-infected honey bee cells was similar to expression in individual bees and varied for each virus. Together, these data further our understanding of the honey bee antiviral defense network and provide new tools for studying honey bee host-virus interactions.Item Comparing network models of gap gene interaction during Drosophila melanogaster development(Montana State University - Bozeman, College of Letters & Science, 2021) Andreas, Elizabeth Anne; Chairperson, Graduate Committee: Tomas GedeonEarly development of Drosophila melanogaster (fruit fly) facilitated by the gap gene network has been shown to be incredibly robust, and the same patterns emerge even when the process is seriously disrupted. In this thesis we plan to investigate this robustness using a previously developed computational framework called Dynamic Signatures Generated by Regulatory Networks (DSGRN). The principal result of this research has been in extending DSGRN to study how tissue-scale behavior arises from network behavior in individual cells, such as gap gene expression along the anterior-posterior (A-P) axis of the Drosophila embryo. Essentially, we extend DSGRN to study cellular systems where each cell contains the same network structure but operates under a parameter regime that changes continuously from cell to cell. We then use this extension to study the robustness of two different models of the gap gene network by looking at the number of paths in each network that can produce the observed gap gene expression. While we found that both networks are capable or replicating the data, we hypothesize that one network is a better fit than the other. This is significant in two ways; finding paths shows us that the spatial data can be replicated using a single network with different parameters along the A-P axis, and that we may be able to use this extension of DSGRN to rank network models.Item Mechanisms of CRISPR-mediated immunity in Escherichia coli(Montana State University - Bozeman, College of Letters & Science, 2019) van Erp, Paul Bertram Geert; Chairperson, Graduate Committee: Blake Wiedenheft; Gary Bloomer, Royce Wilkinson and Blake Wiedenheft were co-authors of the article, 'The history and market impact of CRISPR RNA-guided nucleases' in the journal 'Current opinion in virology' which is contained within this thesis.; Ryan N. Jackson and Joshua Carter were authors and Sarah M. Golden, Scott Bailey and Blake Wiedenheft were co-authors of the article, 'Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli' in the journal 'Nucleic acids research' which is contained within this thesis.; Angela Patterson was an author and Ravi Kant, Luke Berry, Sarah M. Golden, Brittney L. Forsman, Joshua Carter, Ryan N. Jackson, Brian Bothner, and Blake Wiedenheft were co-authors of the article, 'Conformational dynamics of DNA binding and CAS3 recruitment by the CRISPR RNA-guided cascade complex' in the journal 'ACS chemical biology' which is contained within this thesis.; Tanner Wiegand, Royce A. Wilkinson, Laina Hall, Dominick Faith and Blake Wiedenheft were co-authors of the article, 'Protein overexpression reduces specific phage infectivity in prokaryotic argonaute screen' which is contained within this thesis.; Dissertation contains three articles of which Paul Bertram Geert van Erp is not the main author.Prokaryotes are under constant threat from foreign genetic elements such as viruses and plasmids. To defend themselves against these genetic invaders prokaryotes have evolved extensive defense mechanisms. In this thesis I explore two such defense systems: prokaryotic Argonautes and CRISPR-systems. CRISPR-systems acquire short sequences derived from foreign genetic elements and store them in the CRISPR locus. In subsequent rounds of infection these stored sequences are used as guides by Cas proteins to target the invaders. Escherichia coli K-12 contains a type I-E CRISPR system, consisting of two CRISPR loci and eight cas genes. five of these cas genes, together with and 61-nucleotide CRISPR-RNA guide form the RNA-guided surveillance complex Cascade. This complex finds and binds foreign DNA targets that are complementary to its RNA guide. After target binding the helicase/nuclease Cas3 is recruited to the Cascade-DNA complex for destruction of the target. The goal of this research is to understand the molecular mechanisms that lead to target recognition and destruction in the type I-E CRISPR systems. Atomic resolution structures of the proteins involved in these CRISPR systems provide the blueprints of these proteins machines. Structure guided mutational analysis coupled with in vivo and in vitro biochemical experiments are used to investigate the underlying molecular mechanisms of this CRISPR system. Together, these results explain the rules of target recognition and Cas3 recruitment. Prokaryotic Argonautes have been hypothesized to defend against mobile genetic elements such as plasmids and viruses through guided nuclease activity. To test this hypothesis, we overexpressed 8 phylogenetically diverse prokaryotic Argonautes proteins in Escherichia coli and challenged them with seven bacteriophages. This resulted in robust protection against phage Lambda and phage P1 by four of the tested Argonautes, while little impact on phage infectivity was observed for the other phages tested. However, control experiments with a nuclease inactive Argonaute mutant and expression of an unrelated control protein showed similar protection against phage Lambda and phage P1. Collectively, our data suggest that protein overexpression in general, rather than Argonaute expression in particular, results in protection against 2 specific phages.Item Insights into the molecular mechanisms of sensing and responding to the host by Staphylococcus aureus(Montana State University - Bozeman, College of Agriculture, 2019) Meishery-Patel, Delisha; Chairperson, Graduate Committee: Jovanka Voyich-Kane; K. B. Pallister and Jovanka Voyich were co-authors of the article, 'Role of SaeR phosphorylation in regulation of Staphylococcus aureus virulence genes' which is contained within this thesis.; Dissertation contains an article of which Delisha Meishery-Patel is not the main author.Two-component systems (TCSs) are highly conserved across bacteria and are used to rapidly sense and respond to changing environmental conditions. The human pathogen Staphylococcus aureus uses the S. aureus exoprotein expression (sae) TCS to sense host signals and activate transcription of virulence factors essential to pathogenesis. Despite its importance, the mechanism by which the sensor kinase (SaeS) recognizes a stimulus and activates its cognate response regulator (SaeR) to regulate transcription of virulence genes is incompletely defined. However, findings from our lab suggest that SaeR/S mediated transcription is unique-to and dependent-on specific host stimuli. Studies outlined in this dissertation suggest that residues in the extracellular loop may be involved in refinement of the sae regulated targets at the single amino acid level. By generating single amino acid replacement mutants in the response regulator SaeR, we identified a key aspartate residue at position 46 (D46) on SaeR to be important in SaeR mediated signaling as mutation D46A prevented the recombinant protein from binding promoter recognition sequence and subsequently influenced virulence regulation. Current studies are aimed to define the phosphorylation patterns in SaeR using SDS-PAGE analysis and mass spectrometry. Overall, these structure-function studies provide insight into the Sae- signal transduction mechanism and raise some new questions regarding the role the Sae system in the larger regulatory network S. aureus uses to control expression of its secreted virulence factors.Item Utilizing gene suppression technology and hay storage techniques to improve forage quality and animal performance(Montana State University - Bozeman, College of Agriculture, 2017) Staudenmeyer, Danielle Marie; Chairperson, Graduate Committee: Emily Glunk; J.G.P Bowman, R.L. Mack, A.L. Mack and E.C. Glunk were co-authors of the article, 'The effects of feeding reduced-lignin alfalfa on growing beef cattle performance' submitted to the journal 'Journal of animal science' which is contained within this thesis.; J.G.P Bowman, R.L. Mack and E.C. Glunk were co-authors of the article, 'Dry matter and forage quality losses associated with storing large round bales outside at varying geographic locations in the state of Montana' submitted to the journal 'Journal of animal science' which is contained within this thesis.Utilizing technologies such as genetic modification and forage management techniques are two ways to improve forage quality. The objective of the first study in this thesis was to determine the differences in forage quality between reduced-lignin and conventional alfalfa. To test these differences, twenty-four Crossbred Angus heifers were selected to participate in this study and their performance was evaluated based on changes in BW, ADG, DMI, and G:F. In situ digestibility was determined using four ruminally cannulated Hereford cows. Hay samples were collected and used to determine forage quality and leaf-to-stem ratio. There were no differences (P > or = 0.05) in forage quality between treatments, except for DM (P = 0.01). Means did not differ by treatment for percent leaf (P = 0.06) but did differ for leaf-to-stem ratio (P = 0.04). There were no treatment or treatment by day interactions (P > or = 0.05) for BW, ADG, DMI, or G:F. There were no treatment or treatment by time interactions (P > or = 0.05) for in situ digestibility. Overall, the results of this study suggested no difference in forage quality between reduced-lignin and conventional alfalfa. Additionally, animal performance did not differ for crossbred Angus heifers consuming reduced-lignin or conventional alfalfa. The objective of the second study in this thesis was to quantify DM and forage quality losses associated with three different methods of outdoor round bale hay storage at two different sites in Montana. Large round bales consisting of 100% grass hay wrapped in plastic net wrapping were placed into one of four storage systems at both the Bozeman Agricultural Research and Teaching farm (BART) and the Northern Agricultural Research Center (NARC). The four storage systems were: single-stack (SS), pyramid (PYR), mushroom (MSH), and inside stored bales (INSIDE). Results indicated that DM and forage quality losses differed based on geographic location in Montana. This study suggested that DM and forage quality losses differ by location and that bale placement, rather than hay storage formation, is more important for changes in DM and quality for bales stored in Montana over the winter months.Item Genetics of laccase in Gaeumannomyces graminis, the take-all fungus(Montana State University - Bozeman, College of Agriculture, 2002) Litvintseva, Anastasia PavlovnaItem Molecular studies of dicamba-resistant Kochia scoparia L.(Montana State University - Bozeman, College of Agriculture, 2002) Kern, Anthony JohnItem Developmental gene expression in Eimeria bovis : characterization of stage specific genes of sporozoites and merozoites(Montana State University - Bozeman, College of Agriculture, 1995) Clark, Timothy GriffinItem The role of NSP1 in the regulation of rotavirus gene expression(Montana State University - Bozeman, College of Agriculture, 2003) Mitzel, Dana Nicole; Chairperson, Graduate Committee: Michele Hardy.