Scholarship & Research
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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.