Graduate Theses & Dissertations

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Immunotherapies Targeting the Amyotrophic Lateral Sclerosis-Associated Protein TDP-43
Transactive response (TAR) DNA-binding protein of 43 kDa (TDP-43) pathology, including fibrillar aggregates and mutations, develops in amyotrophic lateral sclerosis (ALS) and is characterized by hyperphosphorylation and aggregation patterns, a mechanism largely understudied. In addition, ALS remains without a cure. Herein, in vitro aggregation of phosphorylated TDP-43 was explored, and the anti-TDP-43 antibodies tested for their inhibitor efficacies. Additionally, in vitro phosphorylation of TDP-43 by protein kinases was conducted to identify which protein kinases catalyze phosphorylation. The aggregation of phosphorylated and unphosphorylated full-length TDP-43 protein (pS410) was monitored by transmission electron microscopy (TEM), turbidity absorbance, and thioflavin (ThT) fluorescence spectroscopy. The protein aggregates were largely insoluble, ThT-positive and characterized with heterogeneous morphologies. Antibodies specific to epitopes within the RNA-recognition motifs and the C-terminal domains reduced the formation of β-sheets and insoluble aggregates, with outcomes highly dependent on the type of antibodies, indicating dual functionality. The only protein kinase able to phosphorylate TDP-43 at S410 was MARK4, indicating its role in the onset of PTMs in the protein. Thus, targeting TDP-43 epitopes for inhibition of aggregation and in vitro phosphorylation represent viable biochemical assays for screening protein kinase inhibitors as potential drugs against ALS. Author Keywords: aggregation, ALS, antibody-based inhibition, phosphorylation, protein kinase, TDP-43
Beyond Habitat
My objective was to understand how individual variation, in conjunction with variation in habitat, can affect individual and population-level variation in animal space use. I used coyotes (Canis latrans) as a model species to investigate the roles of hybridization, an inherited intrinsic factor, and spatial memory, a learned intrinsic factor, on space use. I used a diversity of methods and approaches, including meta-regression, multiple imputation, simulations, resource selection functions, step selection functions, net-squared displacement analysis, and survival analysis. A major contribution was my investigation of the performance of multiple imputation in a meta-regression framework in Chapter 2. My simulations indicated that multiple imputation performs well in estimating missing data within a meta-regression framework in most situations. In Chapter 3, I used published studies of coyote home range size in a meta-regression analysis with multiple imputation to examine the relative roles of hybridization and environmental variables on coyote home range size across North America. I found that hybridization with Canis species was a leading factor driving variation in coyote space use at a continental scale. In Chapter 4, I used telemetry data for 62 coyotes in Newfoundland, Canada, to investigate the influence of cognitive maps on resource use. I found that resident coyotes used spatial memory of the landscape to select or avoid resources at spatial scales beyond their immediate sensory perception relative to transient coyotes, presumably increasing their fitness. Taken together, my dissertation demonstrates that intrinsic factors, such as genetic ancestry and spatial memory, can have substantial influences on how animals use space at both individual and population levels, and at both a local and a continental scales. Author Keywords: canis latrans, hybridization, meta-regression, multiple imputation, Newfoundland, spatial memory
Potential for Greenhouse Gas Emissions from Sediments of Agricultural Streams and Lake Erie
This thesis examines how dissolved organic matter quantity and quality and nutrients influence the flux potential of greenhouse gases (GHG) from sediments collected from streams across southern Ontario as well as Lake Erie. Sediments were collected and incubated in a laboratory setting where headspace gases and interstitial waters were analyzed. Results demonstrated that nutrients (dissolved organic carbon (DOC), total dissolved phosphorous (TDP) and total dissolved nitrogen (TDN)) were commonly significant predictors of CO2, CH4, and N2O fluxes, but spatial discrepancies were observed for the significance of DOM quality and sedimentary characteristics. Land use was not found to be directly related to gas flux potential. Different relationships were observed between the streams and lake, and between the basins of Lake Erie. Overall, results from this study suggest that sediments from freshwater systems have the potential to be sources of GHG, the degree of which depends on nutrient concentrations and DOM structure from watershed inputs. Author Keywords: dissolved organic matter, greenhouse gases, Lake Erie, nutrients, sediment, streams
Fingerprinting of dissolved organic matter and copper ligands in the Canadian Arctic and North Pacific Ocean
Dissolved organic matter (DOM) in oceans provides nutrients and ultraviolet radiation protection to microbes. Some DOM compounds can chelate with metals, including copper, controlling their transport and bioavailability in marine systems. As copper functions as both a nutrient and toxicant, studies into the chemical structures of Cu-ligands is important, however currently limited. In this thesis, the chemical composition of total and Cu-binding DOM is investigated using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in the Canadian Arctic and North Pacific. Chapter 2 reveals chemical differences in DOM composition between the southern and northern Canada Basin, revealing the influence of terrestrial and biological sources. Chapter 3 shows the uniqueness of Cu-binding ligands found in the Canadian Arctic and North Pacific Ocean. Studying the composition of DOM gives insight into the chemical diversity of marine DOM, helping to predict the effects of a changing climate on marine ecosystems. Author Keywords: biological, dissolved organic matter, fluorescence, immobilized metal-ion affinity chromatography, mass spectrometry, terrestrial
Genome annotation, gene characterization, and the functional analysis of natural antisense transcripts in the fungal plant pathogen Ustilago maydis
Ustilago maydis (DC) Corda is the causal agent of 'common smut of corn'. Completion of the U. maydis lifecycle is dependent on development inside its host, Zea mays. Symptoms of U. maydis infection include chlorosis and the formation of tumours on all aerial corn tissues. Within the tumours, thick-walled diploid teliospores form; these are the reproductive and dispersal agent for the fungus. U. maydis is the model to study basidiomycete biotrophic plant-pathogen interactions. It holds this status in part because of the completely sequenced 20.5 Mb genome; however, thorough genome annotation is required to fully realize the value of this resource. The research presented here improved U. maydis genome annotation through the analysis of cDNA library sequences and comparative genomics. These analyses identified and characterized pathogenesis-related genes, and identified putative meiosis genes. This enabled the use of U. maydis as a model for investigating 'host-induced' meiosis. Further, the cDNA library analyses identified non-coding RNAs (ncRNAs) and natural antisense transcripts (NATs). NATs are endogenous RNA molecules with regions complementary to a protein-coding transcript. Although NATs have been identified in a wide variety of mammals, plants, and fungi, very few have been functionally characterized. Over 200 U. maydis NATs were annotated by analyzing full-length cDNA sequences. NAT structural features were characterized. Strand-specific RT-PCR was used to detect NATs in U. maydis and in a related smut fungus, U. hordei. The data supported a common role for NATs in smut teliospore development, independent of the RNA interference pathway. Analysis of the expression of one U. maydis NAT, as-um02151, in haploid cells, led to a model for NAT function in U. maydis during teliospore dormancy. This model proposed NATs facilitate the maintenance of stored mRNAs through the formation of double-stranded RNA. In testing this model, it was determined that the deletion of two separate upstream regulatory regions, one of which contained a ncRNA (ncRNA1), altered NAT levels and decreased pathogenesis. These studies strengthened U. maydis as a model organism, and began the functional investigation of NATs in U. maydis, which identified a new class of fungal pathogenesis genes. Author Keywords: cDNA library analysis, genome annotation, mRNA stability, natural antisense transcripts, pathogenesis, Ustilago maydis
Disease ecology of ophidiomycosis in free-ranging snakes
Ophidiomycosis (snake fungal disease) is caused by the pathogen Ophidiomyces ophiodiicola. Infected snakes exhibit dermal lesions, occasional systemic infections, and, in some cases, mortality. We studied snakes at Rondeau Provincial Park, Ontario, Canada, to explore whether ophidiomycosis develops during brumation or year-round. Throughout their active season, we quantified the prevalence of clinical signs of the disease on snakes and conducted qPCR of skin swabs to determine the prevalence of O. ophiodiicola on snakes. Prevalence of O. ophiodiicola and disease symptoms were highest on eastern foxsnakes (Pantherophis vulpinus) and very rare on other snake species. In P. vulpinus, pathogen and clinical sign prevalence was highest, directly after emergence from overwintering, with the majority of P. vulpinus being able to resolve clinical signs of ophidiomycosis by the return of winter. When we analyzed the survivorship of P. vulpinus we determined that the likelihood of a snake dying with ophidiomycosis is similar to a snake dying without ophidiomycosis. Given that P. vulpinus were the most affected species at our study site, ophidiomycosis does not appear to pose an imminent threat to our study population of P. vulpinus under current conditions. Author Keywords: Eastern Foxsnake, Fitness, Ophidiomycosis, Pantherophis vulpinus, Seasonal trends, Snake fungal disease
EVALUATION OF HAYFIELD MANAGEMENT STRATEGIES AND BOBOLINK TERRITORIAL HABITAT IN SOUTHERN ONTARIO
I implemented three hayfield management regimens in southern Ontario (a typical schedule at the farmer`s discretion, a delayed first harvest after July 14, and an early first harvest before June 1 with 65 days before second harvest), and evaluated the costs/benefits to farmers regarding hay quality and feasibility, and to Bobolinks (Dolichonyx oryzivorus) regarding reproductive activity and phenology. Typical management resulted in little to no Bobolink reproductive success, and early harvested sites were not (re)colonized. On delayed harvest sites Bobolinks experienced high reproductive success, but hay quality fell below ideal protein levels for most cattle before harvest. I also examined the habitat features Bobolinks use as the basis for establishing territories and associations between Bobolink territory size and habitat quality. I compared vegetation structure, patch size, and prey abundance between small and large territories. Small territories typically occurred on smaller fields with more preferred vegetation characteristics and greater prey abundance. Author Keywords: agro-ecosystem, Bobolink, Dolichonyx oryzivorus, grassland birds, hayfield management
Comparing Biological Responses to Contaminants in Darters (Etheostoma spp.) Collected from Rural and Urban Regions of the Grand River Watershed, Ontario
Urban and agricultural activities may introduce chemical stressors, including contaminants of emerging concern (CECs) and current use pesticides (CUPs) into riverine systems. The objective of this study was to determine if fish collected from sites in a river show biomarkers of exposure to these classes of contaminants, and if the biomarker patterns vary in fish collected from urbanized and agricultural sites. The watershed selected for this study was the Grand River in southern Ontario, which transitions from areas dominated by agricultural land use in the north to highly urbanized locations in the southern part of the watershed. Rainbow darters (Etheostoma caerluem) and fantail darters (Etheostoma flabellare) were collected from the Grand River in June, 2014 for biomarker analysis from two urbanized sites and three agricultural sites (n=20 per site). Over the same period of time, Polar Organic Chemical Integrative Samplers (POCIS) were deployed for 2 weeks at each site to monitor for the presence of CUPs and CECs. The amounts of the target compounds accumulated on POCIS, determined using LC-MS/MS were used to estimate the time weighted average concentrations of the contaminants at each site. Data on the liver somatic index for darters indicate site-specific differences in this condition factor (p<0.05). Significant differences in the concentrations of thiobarbituric acid reactive substances (TBARS) in gill tissue (p<0.05) indicate differences in oxidative stress in fish collected from the various sites. Measured concentrations of ethoxyresorufin-O-deethylase (EROD) in liver tissue were significantly different between sites (p<0.05), indicating differences in CYP1A metabolic activity. Finally, acetylcholinesterase (AChE) activity in brain tissue was significantly different between fish from rural and urban sites (p<0.05). The analysis of these biomarkers indicates that fish may be experiencing different levels of biological stress related to different land uses. These data may be useful in developing mitigation strategies to reduce impacts on fish and other aquatic organisms in the watershed. Author Keywords: AChE, Biomarker, Darter, EROD, POCIS, TBARS
Stress Axis Function and Regulation in New World Flying Squirrels
Across vertebrate taxa, the hypothalamic-pituitary-adrenal axis (or the stress axis) is highly conserved, and is central to vertebrate survival because it allows appropriate responses to psychological stressors. Habitat shapes successful physiological and ecological strategies, and to appreciate how individual species respond to stressors in their environment, it is essential to have a thorough knowledge of the basic stress physiology of each species. In this dissertation, I studied the functioning and evolution of the stress physiology of New World flying squirrels. I showed that baseline, circulating cortisol levels in northern (Glaucomys sabrinus) and southern (G. volans) flying squirrels are some of the highest ever reported for mammals, indicating that their stress axes operate at a higher set point than most other species. I also assessed other aspects of their acute stress response, including free fatty acid and blood glucose levels, and indices of immune function, and showed that the flying squirrels’ physiological reaction to stressors may differ from that of other mammals. Using immunoblotting, I found that corticosteroid-binding globulin (CBG) expression levels in flying squirrels appeared to be higher than previously reported using alternative methods. I also concluded however, that these levels did not appear to be high enough to provide their tissues with the protective CBG-bound buffer from their high circulating cortisol concentrations experienced by the majority of vertebrates. Thus, this arm of cortisol regulation within the flying squirrel stress axes may be weak or non-existent. Following this, I focused on southern flying squirrels and showed evidence that the second arm of cortisol regulation — the negative feedback mechanism at the level of the brain — functions effectively, but that this species is glucocorticoid resistant. Their tissue receptors appear to have a reduced affinity for cortisol, and this affinity may change seasonally to allow for the onset of other biological processes required for survival and reproduction. Due to their distinctive stress physiology, northern and southern flying squirrels may provide comparative physiologists with model systems for further probing of the function and evolution of the stress axis among vertebrates. Author Keywords: corticosteroid-binding globulin, flying squirrel, Glaucomys, glucocorticoids, physiological ecology, stress physiology
Effects of wood ash addition on soil chemical properties and sugar maple (Acer saccharum, Marsh.) seedling growth in two northern hardwood forest sites in central Ontario
One possible solution to acidification and losses of base cations in central Ontario forest soils may be the application of wood ash. Wood ash is generally high in pH and contains large amounts of calcium (Ca) and other nutrients essential for ecosystem health, however it also contains trace metals. Understanding the chemistry of soils following ash application to forests is crucial for future policy recommendations and remediation efforts. In this study, soil and soil water chemistry was measured at two acidic forest sites in central Ontario. Sugar maple (Acer saccharum, Marsh.) seedling growth and chemistry, as well as understory vegetation composition, were also measured. At site one, plots (2 m x 2 m) were established with sugar maple, white pine (Pinus strobus L.) and yellow birch (Betula alleghaniensis Britt.) residential wood ash treatments and applied at rates of 0 and 6 Mg ha-1. The effects of residential wood ash on soil and understory vegetation were measured three- and 12-months following ash addition. At site two, plots (5 m x 5 m) were established with both fly and bottom industrial grade bark ash treatments of 0, 4 and 8 Mg ha-1 (n=4), and tension lysimeters were positioned in each plot at 30, 50, and 100 cm depths. The effects of industrial grade wood ash on soil, soil water and understory vegetation were measured four years following ash addition. Metal concentrations in the ashes were generally low but were higher in the fly ash and yellow birch ash types. At site one, significant increase in soil pH, and Ca and magnesium (Mg) concentrations were observed after three months, however changes varied by treatment. Some metal concentrations increased in the upper organic horizons, but metals were likely immobilized in the soil due to increases in soil pH, electrical conductivity (EC) and high organic matter content of the soil. After one year, changes to metal concentrations in soils could be seen in mineral horizons, and a few metals (aluminum (Al), zinc (Zn), copper (Cu), chromium (Cr), strontium (Sr)) increased in treatment plots. At site two, the effects of industrial-grade bark ashes on soil pH could still be seen after four years and soil water metal concentrations were not elevated relative to controls. Changes to understory vegetation composition following ash application were observed, but ash addition had no significant effect on sugar maple seedling growth (root:shoot ratio) and did not lead to significant increases in foliar metal concentrations. There were significant differences in root chemistry, suggesting metal translocation and uptake could be restricted. Mass balance estimates indicate that the organic horizon is a sink for all metals and simulated drought in this horizon led to a decrease in soil pH and increase in soil water metal concentration, but this occurred in all treatments including control. These results suggest that application of industrial and residential wood ash in moderate doses with trace metal concentrations below or near regulatory limits will increase soil pH and base cation concentrations, as well as increase seedling tissue nutrient concentrations in northern hardwood forest soils. However, depending on the parent material of the ash, increased metal availability can also occur. Author Keywords: Acer saccharum, calcium decline, forest soil amendment, Haliburton Forest and Wildlife Reserve, heavy metal, wood ash
Demographic history and conservation genomics of caribou (Rangifer tarandus) in Québec
Genetic variation is the raw material and basis for evolutionary changes in nature. The loss of genetic diversity is a challenge many species are facing, with genomics being a potential tool to inform and prioritize decision making. Whole genome analysis can be an asset to conservation biology and the management of species through the generation of more precise and novel metrics. This thesis uses whole genome re-sequencing to characterize the demographic history and quantify genomic metrics relevant to conservation of caribou (Rangifer tarandus) in Québec, Canada. We calculated the ancestral and contemporary patterns of genomic diversity of five representative caribou populations and applied a comparative population genomics framework to assess the interplay between demographic events and genomic diversity. When compared to the census size, NC, the endangered Gaspésie Mountain caribou population had the highest ancestral Ne:NC ratio which is consistent with recent work suggesting high ancestral Ne:NC is of conservation concern. These ratios were highly correlated with genomic signatures (i.e. Tajima’s D) of recent population declines and explicit demographic model parameters. Values of contemporary Ne, estimated from linkage-disequilibrium showed Gaspêsie having among the highest contemporary Ne:NC ratio. Importantly, classic conservation genetics theory would predict this population to be of less concern based off this metric alone. Inbreeding measures suggested nuanced patterns of inbreeding and correlated to the demographic models. This study suggests that while the Québec populations are all under decline, they harbour enough ancestral genetic variation to replenish any lost diversity, if conservation decisions are made in favour of these populations, specifically supporting NC. Author Keywords:
Aquatic Invertebrate Studies from Two Perspectives
Leaf litter decomposition represents a major pathway for nutrient cycling and carbon flow in aquatic ecosystems, and macroinvertebrates play an important role in the processing of this material. To assess the causes of variable leaf breakdown and nutrient fluxes, I measured decomposition rates and the nutrient release ratios of decomposing leaf material across a broad latitudinal gradient in Ontario boreal lakes which varied in nutrients, temperature, and pH. I examined the effects of macroinvertebrates using inclusion and exclusion bags. Generally, leaves decomposed faster in nutrient-rich, warmer lakes. Macroinvertebrates increased decomposition rates but their effects were relatively small compared to regional effects of nutrients and temperature. In addition, we found differential effects of nutrients and temperature on nutrient release ratios, which were partially determined by the release and retention of N and P. These results indicate that changes in these important environmental lake variables could alter decomposition dynamics in Ontario lakes, with implications for nutrient cycling and the storage of this important external carbon source. I studied the biogeography of predaceous diving beetles (Coleoptera: Dytiscidae) in two remote and understudied regions: the Far North of Ontario, and Akimiski Island, Nunavut. I identified 35 species from northern Ontario, including three first provincial records for Ontario, Acilius athabascae Larson (1975), Hygrotus unguicularis (Crotch 1874), and Nebrioporus depressus (Fabricius 1775). I also documented three significant range extensions and six gap-infills for this region. I collected and identified 16 species from Akimiski Island, Nunavut, which include several first time reports for these species for the Nunavut territory. My collections also extend the known ranges of five species into the Hudson Plains Ecozone. This work provides important baseline information on the distribution of diving beetles for these regions. Author Keywords: biodiversity, Boreal Shield, decomposition, Dytiscidae, ecological stoichiometry, macroinvertebrates

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