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Graduate Theses & Dissertations
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- Mutation of the B10 Tyrosine and E11 Leucine in Giardia intestinalis Flavohemoglobin
- The flavohemoglobin in Giardia intestinalis (gFlHb) is the only known protozoan member of a protein class typically associated with detoxifying nitric oxide (by oxidation to nitrate) in bacteria and yeast. Mutants of the B10 tyrosine (Y30F) and E11 leucine (L58A), conserved residues thought to influence ligand binding, were expressed and studied using Resonance Raman (RR) spectroscopy. In the wild type protein, RR conducted using a carbon monoxide probe detects two distinct Fe-CO stretches associated with two different active site configurations. In the open configuration, CO does not interact with any polar side chains, while in the closed configuration, CO strongly interacts with one or more distal residues. Analysis of the Y30F mutant provided direct evidence of this tyrosine’s role in ligand stabilization, as it had only a single Fe-CO stretching mode. This stretching mode was higher in energy than the open conformer of the wild type, indicating a residual hydrogen bonding interaction, likely provided by the E7 glutamine (Q54). In contrast the L58A mutant had no effect on the configurational nature of the enzyme. This was unexpected, as the side chain of L58 sits atop the heme and is thought to regulate the access of distal residues to the heme-bound ligand. The similar spectroscopic properties of wild type and L58A suggest that any such regulation would involve rapid conformational dynamics within the heme pocket. Author Keywords: B10 Tyrosine, Catalytic Globin, E11 Leucine, Flavohemoglobin, gFlHb, Giardia intestinalis
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- Mfsd8 regulates growth and multicellular development in Dictyostelium discoideum
- The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are a family of inherited neurodegenerative lysosomal storage disorders. CLN7 disease is a subtype of NCL that is caused by mutations in the MFSD8 gene. MFSD8 encodes a lysosomal transmembrane protein that is predicted to play a role in transporting small substrates across membranes. However, little is known about its role and substrate specificity. Previous work identified an ortholog of human MFSD8 in the social amoeba Dictyostelium discoideum and reported its localization to endocytic compartments. In this study, the effects of mfsd8 loss during Dictyostelium growth and multicellular development were further characterized. Dictyostelium mfsd8- cells displayed increased rates of proliferation and pinocytosis in liquid media. During growth, loss of mfsd8 altered lysosomal enzymatic activities and reduced the intracellular and extracellular levels of autocrine proliferation repressor A. mfsd8- cells grown on a lawn of bacteria formed plaques in a shorter period of time compared to WT cells, providing additional support for the enhanced growth of mfsd8- cells. Upon starvation, the aggregation of mfsd8- cells was delayed, and mfsd8- cells formed more mounds that were smaller in size, which may be attributed to the reduced cell-substrate adhesion and altered lysosomal enzymatic activities observed for mfsd8- cells. Following aggregation, tipped mound formation was delayed, however, loss of mfsd8 did not affect the timing of slug/finger and fruiting body formation. Additionally, slug migration was reduced in mfsd8- cells. These aberrant phenotypes, excluding fruiting body formation, were effectively or partially rescued when Mfsd8-GFP was introduced into mfsd8- cells. Overall, these results show that Mfsd8 plays a role in regulating growth and developmental processes in Dictyostelium via lysosomal-associated functions. Author Keywords: CLN7, Dictyostelium discoideum, Lysosomes, MFSD8, Neuronal Ceroid Lipofuscinoses
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- How Abiotic and Biotic Factors Can Alter the Competitive Landscape in an Aggressive Species Complex (Genus
- Competition is known to impact population dynamics through both indirect and direct interactions, and direct interactions can often lead to injury in one or both parties. As such, response to injury through tissue regeneration can be important for surviving post-competitive interaction. However, the impacts of outside factors like temperature and genome size (e.g. polyploidy) are not well studied, especially in syntopic systems. We addressed this knowledge gap by comparing regeneration rates of diploid Ambystoma laterale and triploid unisexual Ambystoma at two ecologically-relevant temperatures. Environmental factors appeared to have stronger effects on regeneration than ploidy level, but overall mass was impacted more strongly by ploidy level. Interestingly, there was an interaction between temperature and time within unisexuals that was absent when comparing different ploidy levels, implying temperature has a more complex effect on polyploids. This study supports the hypothesis that polyploid organisms are better equipped to respond to shifts in their environments, which can give them a competitive advantage at the northern range limit of this species complex. Author Keywords: Ambystoma, Genome dosage, Hybrid vigor, Polyploidy, Thermal optimum, Tissue regeneration
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- Temporo-spatial patterns of occupation and density by an invasive fish in streams
- Since its introduction to North America in the 1990s, the Round Goby has spread throughout the Great Lakes, inland through rivers and is now moving into small tributary streams, a new environment for this species in both its native and invaded ranges. I explored density and temporal occupation of Round Gobies in four small streams in two systems in south-central Ontario, Canada in order to determine what habitat variables are the best predictors of goby density. Two streams are tributaries of Lake Ontario and two are tributaries of the Otonabee River, and all of these streams have barriers preventing upstream migration. I found that occupation and density differed between the systems. In the Otonabee River system, Round Gobies occupy the streams year round and the most important factor determining adult density is distance from a barrier to upstream movement, with the entire stream occupied but density highest next to the barriers. In the Lake Ontario system, density is highest at mid-stream and Round Gobies appear to occupy these streams mainly from spring to fall. Adult density in Lake Ontario tributaries is highest in sites with a high percentage of cobble/boulder and low percentage of gravel substrate, while substrate is less important in Otonabee River tributaries. Occupation and density patterns may differ due to contrasting environmental conditions in the source environments and distance to the first barrier preventing upstream movement. This study shows diversity in invasion strategies, and provides insight into the occurrence and movement patterns of this species in small, tributary streams. Author Keywords: biological invasion, Generalised Additive Mixed Model, habitat, Neogobius melanostomus, Round Goby, stream
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- Biosynthesis and impact of cytokinins on growth of the oyster mushroom, Pleurotus ostreatus
- The oyster mushroom, Pleurotus ostreatus, is one of the most widely cultivated edible basidiomycetes. It has gained increased attention for its economic, environmental, and medicinal properties. While a lot is known about cytokinins (CKs) and their actions at the molecular and cellular levels in plants, much less is known about the function of CKs in other kingdoms. Cytokinins, which have been detected in several fungal species, play a role in pathogenic attack against plants or during plant growth promotion by plant beneficial microbes; however, the role of CKs in fungal physiology, separate from plant associations remains largely unknown. This thesis focuses on the occurrence of fungal-derived CKs in P. ostreatus when grown in vitro as submerged or aerial mycelium. Cytokinin profiling by UHPLC-HRMS/MS revealed that P. ostreatus produces CKs and that the tRNA degradation pathway is the main source of these molecules. CK dynamics within fungal growth supported previous evidence, which suggested that tRNA degradation products have a role in the physiological development of fungi for which CKs act as fungal growth regulators. A second component of the thesis demonstrated that P. ostreatus responds to exogenous applications of aromatic and isoprenoid CKs and their effects were dependent on the dose and CK type. N6-Benzyladenine (BAP), Kinetin (KIN), N6-isopentenyladenine (iP), and trans-zeatin (tZ) bioassays revealed hormone-type responses (hormesis: biphasic response). At low doses, mycelium growth could be stimulated, whereas, at high doses only inhibitory effects were observed. This stimulation/inhibition was observed whether the measured response was an increase/decrease of aerial mycelium colony diameter, biomass accumulation or a change in mycelium morphology as compared to the controls. Results indicated there is potential to alter mycelium growth and development of P. ostreatus; thus, CKs may play the role of a “mycohormone” and may be specifically helpful for medicinal fungi by increasing growth and efficiency to produce many biologically active substances with valuable medical and environmental applications. Author Keywords: cytokinins, fungal-derived CKs, hormesis, mycelium, mycohormone, Pleurotus ostreatus
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- Relationships between bird densities and distance to mines in Northern Canada
- Increased mining activity in the Canadian Arctic has resulted in significant changes to the environment that may be influencing some tundra-nesting bird populations. In this thesis I examine the direct and indirect effects of mining on birds nesting in the Canadian Arctic. I first perform a literature review of the effects that mining in the Arctic has on northern environments and wildlife and outline several ways in which mines affect Arctic-breeding birds. By using the Program for Regional and International Shorebird Monitoring (PRISM) Arctic plot-based bird survey data from across the Canadian Arctic, collected from 1995 to 2018, I identify the effects of distance to mining operations on the occupancy patterns of Arctic-breeding bird species. Six species’ densities were significantly impacted by mine proximity (Canada/Cackling Goose, Long-tailed Duck, Long-tailed Jaeger, Pectoral Sandpiper, Savannah Sparrow, and Rock Ptarmigan) across five major mine sites. Each species has its own unique relationship to distance from mining activity. Author Keywords: Bird populations, Canadian Arctic, Mining, Mining activities, PRISM, Tundra-nesting birds
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- Behavioural ecology and population dynamics of freshwater turtles in a semi-urban landscape at their northern range limit
- Species are faced with a variety of challenges in the environment, including natural challenges, such as variability in ambient temperature, and anthropogenic threats, such as habitat transformation associated with urbanisation. Understanding how animals respond to these kinds of challenges can advance the field of behavioural ecology and guide management decisions for wild species. Yet, we still have limited understanding of the extent of natural and human-caused impacts on animal behaviour and population dynamics, and lack robust assessment of behaviour in free-ranging animals. Using novel miniaturised biologging technologies, I characterised and validated behaviour in two freshwater turtle species: Blanding’s turtles (Emydoidea blandingii) and Painted turtles (Chrysemys picta). Further, I investigated how these two ectothermic species navigate a thermally heterogeneous landscape near their northern range limit, by comparing selected and available ambient temperatures. I showed that turtles preferred locations that were, on average, warmer and less variable in temperature than the available environment, and that this thermal sensitivity was greatest early in the year, and at fine spatial scales that likely matched the species' perception of the environment. Lastly, I assessed whether urban development was compatible with long-term viability of a Blanding’s turtle population, by monitoring habitat change and turtle survival over one decade of ongoing residential and road development. I found that Blanding’s turtle habitat quantity and connectivity declined in the area, which coincided with high road mortality and severe declines in turtle survival and population size, especially in adult females. I concluded that urban development and current road mortality rates are incompatible with the long-term viability of this at-risk turtle population. Overall, my findings demonstrate the importance of variation in the thermal environment and anthropogenic impacts on habitat in shaping the behaviour and population dynamics of this species-at-risk. Author Keywords: animal behaviour, biologging, ectotherms, habitat selection, temperature, urbanisation
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- Assessing the environmental correlates of a lethal amphibian pathogen, Batrachochytrium dendrobatidis, in Ontario wetlands
- Many emerging infectious diseases are caused by pathogens that possess free-living life stages, in which they interact with the environment directly rather than through the mediation of a host. These diseases represent major impediments to wildlife conservation; however, the dynamics of their interaction with the environment are poorly studied, often due to the difficulty of detecting these microscopic pathogens in environmental samples. One of these pathogens is Batrachochytrium dendrobatidis (Bd), a fungus that has been linked to declines in many amphibian species. In this thesis I use an emerging technique, environmental DNA detection (eDNA), to detect and quantify Bd in the water of southern Ontario (Canada) wetlands and examine its correlation with a variety of aspects of water quality, surrounding habitat, and seasonal timing. My purpose was to inform not only on potential environment-pathogen dynamics for Bd in northern environments, but to provide insight into the use of eDNA as a disease surveillance tool. I found that not only was there high geographic variation in Bd detection and intensity, but also high temporal variation within the same site on time scales as low as two weeks. While Bd prevalence was not strongly correlated with any of the variables tested, intensity showed strong correlation with canopy cover, with greater canopy cover over a waterbody correlating to lower Bd intensity. My results present several promising avenues for further examination of Bd in northern ecosystems, and indicate that, while caution is warranted in its implementation, eDNA may become an important tool in amphibian pathogen surveillance. Author Keywords: amphibian disease, Batrachochytrium dendrobatidis, disease monitoring, environment-pathogen dynamics, environmental DNA, wildlife disease
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- Syrphidae (Diptera) of northern Ontario and Akimiski Island, Nunavut
- Syrphids, also known as hover flies (Diptera: Syrphidae) are a diverse and widespread family of flies. Here, I report on their distributions from a previously understudied region, the far north of Ontario, as well as Akimiski Island, Nunavut. I used samples collected through a variety of projects to update known range and provincial records for over a hundred species, bringing into clearer focus the distribution of syrphids throughout this region. I also analysed a previously un-tested trap type for collecting syrphids (Nzi trap), and report on results of DNA analysis for a handful of individuals, which yielded a potential new species. Author Keywords: Diptera, Ontario, range extension, Syrphidae
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- successful invader in expansion
- Researchers have shown increasing interest in biological invasions for the associated ecological and economic impacts as well as for the opportunities they offer to study the mechanisms that induce range expansion in novel environments. I investigated the strategies exhibited by invasive species that facilitate range expansion. Invasive populations exhibit shifts in life-history strategy that may enable appropriate responses to novel biotic and abiotic factors encountered during range expansion. The spatio-temporal scales at which these shifts occur are largely unexplored. Furthermore, it is not known whether the observed dynamic shifts represent a consistent biological response of a given species to range shifts, or whether the shifts are affected by the abiotic characteristics of the new systems. I examined the life-history responses of female round gobies Neogobius melanastomus across fine and coarser spatial scales behind the expansion front and investigated whether invasive populations encountering different environmental conditions (Ontario vs France) exhibited similar life-history shifts. In both study systems, I found an increase in reproductive investment at invasion fronts compared to longer established areas at coarse and fine scales. The results suggest a similar response to range shifts, or a common invasion strategy independent of environmental conditions experienced, and highlight the dynamic nature of an invasive population’s life history behind the invasion front. The second part of my research focused on the development of an appropriate eDNA method for detecting invasive species at early stages of invasion to enable early detection and rapid management response. I developed a simple, inexpensive device for collecting water samples at selected depths for eDNA analysis, including near the substrate where eDNA concentration of benthic species is likely elevated. I also developed a protocol to optimise DNA extraction from water samples that contain elevated concentration of inhibiters, in particular near-bottom samples. Paired testing of eDNA and conventional surveys was used to monitor round goby expansion along its invasion pathway. Round gobies were detected in more sites with eDNA, permitting earlier, more accurate, upstream detection of the expansion front. My study demonstrated the accuracy and the power of using eDNA survey method to locate invasion fronts. Author Keywords: Age-specific reproductive investment, DNA extraction, Energy allocation, Fecundity, Invasion front, Range expansion
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- Assessing Canada Lynx Dispersal Across an Elevation Barrier
- Mountain ranges are often thought to restrict movement of wildlife, yet previous studies evaluating the role of the Rocky Mountains as a dispersal barrier for Canada lynx (Lynx canadensis) have been contradictory. Our study uses neutral microsatellite loci to evaluate the role of the Rocky Mountains as a barrier to gene flow for lynx. Although lynx exhibited low genetic differentiation, we detected a limited effect of the mountains. Furthermore, we inferred the role played by landscape variables in gene flow (genetic differentiation predicted by landscape resistance). Limited gene flow most strongly related to resistance from physical factors (low snow cover and elevation), rather than other topographic and ecological factors (high terrain roughness, low forest cover, low habitat suitability, and geographic distance). Structural connectivity was a relatively poor predictor of functional connectivity. Overall, the Rockies represent an area of reasonably high functional connectivity for lynx, with limited resistance to gene flow. Author Keywords: Canada lynx, connectivity, gene flow, genetic structure, landscape genetics, Rocky mountains
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- Bank Swallow (Riparia riparia) Breeding in Aggregate Pits and Natural Habitats
- I examined Bank Swallow (Riparia riparia) colony persistence and occupancy, in lakeshore, river and man-made aggregate pit habitat. Habitat persistence was highest on the lakeshore and lowest in aggregate pits, likely due to annual removal and relocation of aggregate resources. Bank Swallow colonies in aggregate pit sites were more likely to persist if a colony was larger or if burrows were located higher on the nesting face. I also compared nest productivity and health factors of Bank Swallows in lakeshore and aggregate pit habitats. While clutch size was the same in both habitat types, the number of fledglings from successfully hatched nests was significantly higher in aggregate pit sites than from lakeshore sites. Mass of fledgling Bank Swallows did not differ significantly between habitat types, however mass of adults from aggregate pits decreased significantly over the nesting season. Parasite loads on fledgling Bank Swallows were significantly lower in aggregate pits than in lakeshore sites. According to these indicators, aggregate pits appear to provide equivalent or higher quality habitat for Bank Swallows than the natural lakeshore sites, making them adequate and potentially key for this species’ recovery. Aggregate pit operators can manage for swallows by (1) creating longer, taller faces to attract birds and decrease predation, and (2) supplementing their habitat with water sources to encourage food availability. Author Keywords: Aerial insectivore, aggregate pits, Bank Swallow, colony persistence, ectoparasites, substitute habitat