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Item Solution-phase dynamics of the Hepatitis B virus capsid : kinetics-based assays for study of supramolecular complexes(Montana State University - Bozeman, College of Letters & Science, 2009) Hilmer, Jonathan Kyle; Chairperson, Graduate Committee: Brian BothnerViruses are the most abundant form of life on the planet. Many forms are pathogenic and represent a major threat to human health, but viruses can also be useful nanoscale tools: as adjuvants, gene therapy agents, antimicrobials, or functionalized nanoscale building blocks. Viruses have historically been viewed as static and rigid delivery vehicles, but over the last few decades they have been recognized as flexible structures. Their structural dynamics are a crucial element of their functionality, and they represent a substantial target for antiviral strategies. To overcome the inherent problems of characterizing the biophysics of supramolecular complexes, we have developed a set of kinetic assays to probe capsid motion at several different amplitudes. The first assay, kinetic hydrolysis, works via the differential cleavage of folded versus unfolded proteins, and reports on large-scale conformational changes. The second assay, hydrogen-deuterium exchange, is a probe of small-amplitude dynamics. Both of these assays were used to study the solution-phase dynamics of the hepatitis B virus (HBV) capsid under the influence of assembly effectors and temperature. The results of these assays indicate that the HBV capsid adopts multiple conformations in response to the external environment. The dimeric subunit becomes primed for assembly via an entropically-driven process, but once assembled the capsid has reduced dependence on hydrophobic contacts. Depending on the assembly state, the subunit protein has varying response to assembly effectors, with changes to both small-amplitude and large-amplitude motions. The sum of the assay results indicate that the HBV capsid protein is capable of rotational translocations of the alpha-helices, while maintaining most of the secondary structure. Concerted structural shifts are implied, consistent with an allosteric model, which helps to explain previously observed allostery of capsid assembly and response to drugs.Item The role of rodents as a potential reservoir for Pasteurella multocida on the National Elk Refuge, Wyoming(Montana State University - Bozeman, College of Letters & Science, 2005) Swanekamp, Leatrice June; Chairperson, Graduate Committee: Lynn IrbyHemorrhagic septicemia (HS) is a fatal disease affecting domestic and wild ruminants caused by the bacterium Pasteurella multocida. Although uncommon in the U.S, outbreaks of HS in elk (Cervus elaphus) occurred on the National Elk Refuge (NER) in the winters of 1986, 1987, 1993, 1995, 1999, and 2001. DNA fingerprinting of P. multocida from the 1987 and 1993 outbreaks (B:3,4 HhaI 036/HpaII 001) revealed the same organism was responsible for mortality in both years. However, testing has failed to find this genetic variant in healthy elk on the NER, suggesting reservoirs other than elk might play a role in HS epidemiology. I investigated the potential for rodents to serve as biological reservoirs for bacteria responsible for HS on the NER. Rodents are known to harbor P. multocida, may be carriers of variants capable of causing HS, and have been observed at sites where elk are fed during winter on the NER. I used mark-recapture techniques to determine densities of rodents on feedgrounds, feed-storage areas and other sites and removal trapping to collect tissues to determine prevalence of P. multocida in rodents on the NER.Item Investigating host response to viral infection through proteomics : a study of murine norovirus(Montana State University - Bozeman, College of Letters & Science, 2008) Furman, Linnzi Marie; Chairperson, Graduate Committee: Brian Bothner; Michele Hardy (co-chair)Norovirus causes roughly 23 million cases of foodborne illnesses in the United States each year. While this virus was characterized over 30 years ago, it remains non-cultivatable in human cells, resulting in an incomplete understanding of the host cell's response to infection. However, in 2004 murine norovirus (MNV) was found to be cultivatable in mice and has since been successfully cultured in RAW 264.7 cells. MNV has become an important model system for studying norovirus, as it is structurally and genetically similar to human norovirus. A global proteomics approach using fluorescently tagged, activity-based probes and 2D differential gel electrophoresis analysis was used to study MNV infection. Specifically, the process of cell death was investigated to determine if apoptosis, or programmed cell death, occurred in response to infection. Through the 2D differential gel analysis, 27 differentially regulated proteins were identified at 4 hours post infection, and 22 differentially regulated proteins were identified at 12 hours post infection; a strong majority of these proteins have been related to apoptosis in the literature. Using fluorescently-labeled activity-based probes and fluorimetric assays, we have monitored the activation of several caspases induced by viral infection. Infected samples show a significant increase in caspase activity over control samples within the first few hours post infection, indicating a virally induced activation of caspases. Cells were also infected in the presence of a pan-caspase inhibitor, Boc-D(OMe)-fmk, which led to caspase-independent cell death. Using propidium iodide and Hoechst staining, it was concluded that infected cells undergo necrosis in the presence of the caspase-inhibitor, while those infected in the absence of the inhibitor undergo apoptosis. From these studies it can be concluded that cells infected with MNV undergo a caspase-mediated, apoptotic cell death, while the caspase-independent cell death can be classified as necrosis. This study provides significant insight to norovirus-induced cell death.