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There is an inherent conflict over land use between humans and wildlife. Human activities can alter habitat, creating pressure on North American large carnivore populations. Traditional wildlife techniques can be slow to show population declines, especially in long lived species with slow reproduction rates and high mortality of young, such as grizzly bears (Ursus arctos), which leads to delayed information for land managers trying to find the balance between human use of land and preservation of wildlife. Concern about population health of grizzlies in Western Alberta, Canada has lead to investigation of the impacts of current land use within grizzly bear habitat. The objective of this work was to develop a protein microarray that could detect patterns of physiological stress in a rapid manner with small samples of grizzly bear tissue. Sampling from four regions in the foothills of the Rocky Mountains in Alberta resulted in the capture of 133 bears. During the developmental phase, proteins involved with mitochondrial function were found, using two dimensional gel electrophoresis, to be altered in situations of increased stress. Limited cross-reactivity was found when evaluating grizzly bear stress protein expression using commercially available protein microarrays. The protein microarray developed in this thesis consists of 31commercial antibodies validated for grizzly bears. These antibodies recognize proteins associated with different aspects of the stress response, including the hypothalamic-pituitary-adrenal axis, apoptosis/cell cycle, cellular stress, and oxidative stress and inflammation. Skin was selected as the tissue for evaluation of protein expression. Strong correlations were found between many of the proteins within functional categories. Model selection for the protein categories revealed variation that corresponded with region, serum markers of stress (total cortisol and hsp60), growth, the density of roads in the habitat and the amount of anthropogenic change in the bears home range. Regional trends of expression found bears in Swan Hills and bears from North highway 16 having elevated expression of the proteins measured by the microarray. The protein microarray was thus able to detect expression patterns reflecting physiological and environmental markers. The array shows great promise for future use in detection of potential distress in wildlife populations due to alterations of their habitat.

There is an inherent conflict over land use between humans and wildlife. Human activities can alter habitat, creating pressure on North American large carnivore populations. Traditional wildlife techniques can be slow to show population declines, especially in long lived species with slow reproduction rates and high mortality of young, such as grizzly bears (Ursus arctos), which leads to delayed information for land managers trying to find the balance between human use of land and preservation of wildlife. Concern about population health of grizzlies in Western Alberta, Canada has lead to investigation of the impacts of current land use within grizzly bear habitat. The objective of this work was to develop a protein microarray that could detect patterns of physiological stress in a rapid manner with small samples of grizzly bear tissue. Sampling from four regions in the foothills of the Rocky Mountains in Alberta resulted in the capture of 133 bears. During the developmental phase, proteins involved with mitochondrial function were found, using two dimensional gel electrophoresis, to be altered in situations of increased stress. Limited cross-reactivity was found when evaluating grizzly bear stress protein expression using commercially available protein microarrays. The protein microarray developed in this thesis consists of 31commercial antibodies validated for grizzly bears. These antibodies recognize proteins associated with different aspects of the stress response, including the hypothalamic-pituitary-adrenal axis, apoptosis/cell cycle, cellular stress, and oxidative stress and inflammation. Skin was selected as the tissue for evaluation of protein expression. Strong correlations were found between many of the proteins within functional categories. Model selection for the protein categories revealed variation that corresponded with region, serum markers of stress (total cortisol and hsp60), growth, the density of roads in the habitat and the amount of anthropogenic change in the bears home range. Regional trends of expression found bears in Swan Hills and bears from North highway 16 having elevated expression of the proteins measured by the microarray. The protein microarray was thus able to detect expression patterns reflecting physiological and environmental markers. The array shows great promise for future use in detection of potential distress in wildlife populations due to alterations of their habitat.

Two influenza viruses, the virulent clone 7a (H3N2) and the attenuated A/Fiji/15899/83 (H1NI) (A/Fiji), induce different level ofapoptosis in both MDCK and HeLa cells. Apoptosis induced by these two viruses could be partially inhibited by two NA inhibitors (GGI67 and DANA), when present during virus entry, but not subsequently. GGI67, which cannot enter cells, did not affect the level of infection. These results suggest that NA plays a role in influenza virus-induced apoptosis and that it acts early in the replication cycle. However, ammonium chloride, which prevents virus entry reduced the level of apoptosis induced by both viruses. In addition, amantadine, which prevents virus uncoating, inhibited apoptosis induced by the amantadine-sensitive strain A/Udorn/307/72 (H3N2). Therefore, other viral components acting intracellularly may also be involved in influenza virus-induced apoptosis. Clone 7a, A/Fiji or A/WSN/33 (H1N1)(A/WSN) proteins were expressed in HeLa cells, fused to the Herpes simplex tegument coat protein VP22. This protein has the ability to translocate from the cell in which it is synthesised to surrounding cells. Therefore, VP22- fusion proteins are delivered to a high number of cells, regardless of the level of transfection. A/WSN PB1, PB2 and HA induced significant levels ofapoptosis as did the NA, M1, M2 and NS1 proteins from clone 7a and the M1, M2, NSI and NS2 proteins from A/Fiji. Conversely, clone 7a and A/Fiji NP and A/Fiji PB2 and NA did not induce apoptosis. Confocal microscopy revealed that apoptosis was associated with fusion protein expression and that the location of the proteins corresponded with that expected during influenza virus infection. In addition, the mechanism of clone 7a NA-induced apoptosis was investigated. GGI67 inhibited apoptosis induced by the expression of clone 7a NA. Hence, NA activity is required for the induction of apoptosis. In addition, apoptosis was completely abrogated in the presence of an anti-transforming growth factor (TGF)-B antibody suggesting the activation of TGF-B during the translocation of the fusion protein from one cell to another is involved in clone 7a NA-induced apoptosis. Furthermore, clone 7a and A/Fiji NS1 proteins induced apoptosis when expressed alone, but they inhibited double-stranded (ds) RNA-induced apoptosis. However, the level of apoptosis observed in the presence of dsRNA was not reduced to background levels suggesting a pro and anti-apoptotic action of NS1 in influenza virus-infected cells. The potential mechanisms involved in the induction of apoptosis by each protein and the relevance to infection is discussed.

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