Graduate Theses & Dissertations

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Expression of Giardia intestinalis flavoenzyme GiOR-1 and characterization of its electron transfer properties
Giardia intestinalis possesses four isotypes of cytochrome b5 (gCYTB-I-IV) that differ from their mammalian counterparts, suggesting different functions in this protozoan parasite. Although the recently discovered Giardia flavoenzyme, GiOR-1, reduces these cytochromes, its properties have not been thoroughly studied, owing to the difficulty in its expression. Here I describe successful conditions for expression of GiOR-1 using autoinduction. GiOR-1 is obtained with flavins bound as indicated by its UV-visible spectrum. Its ability to catalyze electron transfer from donors (NADH, NADPH) to acceptors (oxygen, ferricyanide, cytochrome c, gCYTB5-III) were studied in spectrophotometric rate assays. NADPH is the preferred electron donor, while cytochromes are the preferred electron acceptors. Interestingly, the His-tag used to purify gCYTB5-III decreases its reaction rate with GiOR-1, as an untagged version has slightly faster rates. These findings establish the appropriate conditions for further studies on GiOR-1, including the identification of endogenous electron acceptors. Author Keywords: Autoinduction, Cytochrome b5, Cytochrome P450 oxidoreductase, Giardia intestinalis, GiOR-1, Polyhistidine tag
Prey abundance and habitat during the breeding season for Piping Plovers in the Ontario Great Lakes region
Similar to other shorebird trends around the world, the Piping Plover population (Charadrius melodus circumcinctus) is projected to decline if concerted conservation efforts are relaxed. To date, there is insufficient understanding of the connection between habitat type, prey abundance, and chick behaviour of the Piping Plover breeding population in Ontario. The aim of my thesis was to gain knowledge about prey abundance at recent and historic breeding locations, understanding how habitat influences prey abundance and chick behaviour across the Piping Plover breeding range in Ontario’s Great Lakes. The objective of my first study was to understand prey abundance across the breeding region Great Lakes of Ontario from 2018-2019, including occupied and unoccupied sites, and to quantify variation among habitats and periods of reproduction. To evaluate resources, I sampled 17 locations to compare prey abundance using invertebrate traps (n= 3,507). Sampling took place over the reproductive periods of nest initiation, post-hatch, and fledging and in four habitat types of shoreline, wrack, berm, and back dune. Occupied breeding sites had higher prey abundances, and different assemblages of invertebrate prey than unoccupied sites. Additionally, breeding sites had higher prey abundance during nest initiation and supported higher amounts of prey in shoreline and wrack habitat. The objective of my second study was to understand how habitat types influence chick behaviour. To evaluate behaviour-habitat trends, instantaneous chick observations were recorded at the four nest sites from the post-hatch to fledging stages. In total there were 23 fledged chicks that we observed across the two years. Chicks in this study spent 60.9% of their time foraging, 11.9% of the time displaying alert behaviour, 21.4% of their time resting or being brooded, and 5.9% of their time preening. Chicks spent a large proportion of time foraging in the shoreline, resting in the back dune, and alert in berm habitat. The frequency of these alert, defensive behaviours differed among sites, with Sauble Beach chicks spending more time in defensive behaviours compared to other sites. I concluded that in both nesting and brood-rearing periods, habitat is selected non-randomly by adult and young Piping Plovers to maximize access to invertebrate prey for growth and survival. Access by chicks to the most productive habitats should be considered in local management decisions. Author Keywords: chick behaviour, endangered, Great Lakes Region, habitat, Piping Plovers, prey abundance
Sensitivity of Forest Soils to Acidic Deposition Downwind of an Aluminum Smelter, Kitimat, B.C.
Maximum permitted SO2 emissions from an aluminum smelter in Kitimat, B.C., increased after modernization in 2015. An increase in acidic deposition can potentially acidify forest soils. Monitoring was conducted at two long-term soil monitoring plots at near (7 km) and far (41 km) sites downwind from the smelter. Change in soil properties was assessed between 2015 and 2018: for the near plot, there was significant decrease in pH and exchange acidity; far plot soils exhibited significant decrease of base cations and exchange acidity in the 0–5 cm layer only. The average total SO42- deposition at near and far plots were estimated to be between 8.2–12.1 and 6.7–7.4 kg/ha/yr, respectively. It was concluded no soil acidification was detected. Observed changes were attributed to measured differences in organic matter, likely influenced by sampling difficulty and measurement process discrepancies. Estimated SO42- deposition levels pose no risk to soil base cation depletion. Author Keywords: acid forest soils, acidic deposition, aluminum smelter, exchangeable base cations, long-term monitoring, minimum detectable change
Assessing effects and fate of environmental contaminants in invasive, native, and endangered macrophytes
Macrophytes play an important role in aquatic ecosystems, and thus are integral to ecological risk assessments of environmental contaminants. In this dissertation, I address gaps in the assessments of contaminant fate and effects in macrophytes, with focus on glyphosate herbicide use for invasive plant control. First, I evaluated the suitability of Typha as future standard test species to represent emergent macrophytes in risk assessments. I concluded that Typha is ecologically relevant, straight-forward to grow, and its sensitivity can be assessed with various morphological and physiological endpoints. Second, I assessed effects from glyphosate (Roundup WeatherMAX® formulation) spray drift exposure on emergent non-target macrophytes. I performed toxicity tests with five taxa, Phragmites australis, Typha × glauca, Typha latifolia, Ammannia robusta, and Sida hermaphrodita, which in Canada collectively represent invasive, native, and endangered species. I found significant differences in glyphosate sensitivity among genera, and all species’ growth was adversely affected at concentrations as low as 0.1% (0.54 g/L), much below the currently used rate (5%, 27 g/L). Third, I assessed the potential for glyphosate accumulation in and release from treated plant tissues. I found that P. australis and T. × glauca accumulate glyphosate following spray treatment, and that accumulated glyphosate can leach out of treated plant tissues upon their submergence in water. Finally, I assessed effects of released glyphosate on non-target macrophytes. I found that P. australis and T. × glauca leachate containing glyphosate residues can stimulate the germination and seedling growth of T. latifolia, but can exert an inhibiting effect on A. robusta, although leachate without glyphosate caused similar responses in both plants. Additionally, I found no negative effects in A. robusta when exposed to glyphosate residues in surface water, or when grown with rhizosphere contact to an invasive plant that was wicked (touched) with glyphosate. My results show that non-target macrophytes can be at risk from glyphosate spray for invasive plant control, but risks can be mitigated through informed ecosystem management activities, such as targeted wick-applications or removing plant litter. Integrating contaminant fate and effect assessments with emergent macrophytes into ecological risk assessments can support the protection of diverse macrophyte communities. Author Keywords: Ecosystem management, Ecotoxicology, Glyphosate, Herbicide, Invasive plant, Species at risk
Phytohormone-enhanced heavy metal responses in Euglena gracilis
Phytohormones, Cytokinin (CK) and Abscisic acid (ABA), are best known for controlling plant growth and stress responses; but they also mediate various developmental perspectives in alga. Yet, their mode of action in algal adaptive strategies to heavy metal responses, their involvement in orchestration of phytohormone crosstalk remain largely unknown and a molecular framework of phytohormone-controlled heavy metal uptake is absent. I found that three metals known globally to contaminate aquatic ecosystems, nickel (Ni), lead (Pb), and cadmium (Cd), cause changes in the levels of endogenous CKs, ABA, auxins, and gibberellins (GAs) in the green alga Euglena gracilis. Exogenous ABA or CK (trans-zeatin) alleviated metal toxicity through improved metal uptake efficiency and the regulation of the endogenous CKs activity profiles and GAs activity. This new evidence suggests that E. gracilis possesses functional phytohormone signals and metabolic pathways that are under metal stress response. Exogenously applied ABA or CK provoked the coordinated activation of metal uptake, likely via enhanced accumulation of metal binding compounds (i.e., proline, glycine, cysteine containing peptides), which are effective for metal sequestration. Using untargeted metabolomics analysis and functional annotation, this thesis further established that, CK and ABA modified pathways and metabolites, which were mainly involved in metal acclimation and resistance. These modified metabolites that were under the influence of phytohormones in algal cells growing under metal stress conditions were associated with: lipid pathways, riboflavin metabolism, biosynthesis of cofactors/vitamin, and carbohydrate metabolism. Bioactive secondary compounds (e.g., terpenoids, alkaloids, flavonoids, carotenoids) were also modified in algal cells treated with phytohormones. The present study highlights that ABA and CKs are important regulators of algal metal accumulation/acclimation strategies based on increased metal uptake, enhanced CK metabolism, regulation of hormonal crosstalk and regulation of some core cellular metabolism pathways, all of which improve metal uptake efficiency. Finally, our results suggest that ABA and CK form a novel strategy for metal bioremediation techniques and for sourcing microalgal value-added metabolites. Author Keywords: abscisic acid, cadmium, cytokinin, Euglena gracilis, lead, nickel
Effects of Intensive Agriculture on Stream Nutrient Export in East-Central Ontario, Canada
Recent agricultural land use change in east-central Ontario, including the expansion of intensive agriculture (corn and soybean crops) and tile drainage (TD) infrastructure, may alter the fluxes of both phosphorus (P) and nitrogen (N) to the Lower Great Lakes. Through intensive monitoring of several sub-watersheds that encompassed a range of row crop and TD extents, this study examined differences in stream N and P concentrations both seasonally and during variable flow conditions, to better understand land use/land cover (LULC) relationships with nutrient export. There was no clear relationship between total P (TP; concentration or export) and agricultural LULC, and instead, TP delivery was highly sensitive to flow events, and TP concentrations (especially particulate P; PP) were significantly higher during event flow compared with baseflow. In contrast, the dissolved fraction of P (total dissolved P; TDP) and dissolved nitrogen as nitrate (NO3-N) were not sensitive to flow, but were instead positively related to row crop agriculture, and associations between NO3-N concentrations/export and tile-drained row crop area were particularly strong (concentration r2 = 0.93; export r2 = 0.88). Regression relationships showed that with every 10% increase in row crop area in watersheds, NO3-N and TDP flow-weighted concentrations increased by 0.34 mg/L and 1.5 µg/L, respectively. As well, the same 10% increase in row crop agriculture translated to an increase of NO3-N export of as much as 130 kg/km2. Geospatial records of TD are incomplete in east-central Ontario, which presents challenges for evaluating the contribution of TD to nutrient export. Understanding the response of nutrients to changes in agriculture and agricultural practices is an integral part of watershed management as rapid changes in both urban and agricultural LULC continue to put pressure on water quality in the Lower Great Lakes. Author Keywords: East-Central Ontario, Nutrient Export, Row crops, Streams, Tile Drainage, Water Quality
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
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:
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
evolutionary ecology of Alaska's mountain goats with management implications
The integration of genetic and environmental information can help wildlife managers better understand the factors affecting a species’ population structure and their response to disturbance. This thesis uses genetic techniques to assess the broad and fine scale population structure of mountain goats in Alaska. The first chapter aims to determine the number of genetically distinct subpopulations and model the demographic history of mountain goats in Alaska. The second chapter investigates the population structure and demographic history of mountain goats in Glacier Bay National Park and examines the impact that climate change will have on these mountain goats. My results indicate that Alaska has eight subpopulations which diverged during the Wisconsin glaciation. In Glacier Bay, population structure is reflective of the landscape during colonization, and mountain goat population density and movement corridors are likely to decline due to future climate change. Author Keywords: Alaska, biogeography, gene flow, landscape genetics, mountain goat, population genetic structure
Anthropogenic particles and microplastics in headwater lake catchments in Muskoka-Haliburton, Canada
Microplastics, plastic particles less than 5 mm in diameter, are ubiquitous in the environment. This study estimated the abundance of microplastics (MP) in atmospheric deposition from four background monitoring stations in Muskoka-Haliburton, south-central Ontario, Canada and quantified the fate of microplastics to three background headwater lake catchments in Muskoka-Haliburton. Microplastics were observed across all sample media with polyethylene terephthalate and polyamide being predominant. The average atmospheric deposition of anthropogenic particles was 57 particles/m2/day with a plastic deposition rate of 7 MP/m2/day. Atmospheric deposition represented the highest daily microplastic flux rate to the three headwater lake catchments compared, 1.5 to 4 times greater than the flux rate for the inflow streams suggesting that atmospheric deposition can account for all the inflowing microplastics. A large fraction of the microplastics from atmospheric deposition (41 – 73%) were retained in the terrestrial catchment and there was a high retention of microplastics in each of the study lakes (1.44 – 7.39 million MP/day; 30 – 45%) suggesting that a large fraction of the terrestrial catchment export is retained by the lakes and that lakes are a reservoir for microplastics. Author Keywords: Atmospheric deposition, Microplastics, Ontario, Plastic pollution, Sinks, Sources
Electrochemical Biosensors for Neurodegenerative Disease Biomarkers
The onset of neurodegenerative diseases such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) are typically characterised by the aggregation of protein biomarkers into cytotoxic fibrils. Novel means of analysing these biomarkers are needed to expand the literature toward earlier diagnosis of these conditions. Electrochemical sensors could offer the sensitivity and selectivity needed for specialised analysis, including potential point-of-care applications. The AD biomarker Tau, and ALS biomarker TDP-43 proteins are explored here by using a label-free electrochemical sensors. Tau protein was covalently bound to gold electrode surface to study the in vitro mechanisms of aggregation for this protein. An immunosensor to TDP-43 was developed by covalently binding primary TDP-43 antibodies (Abs) on gold electrode surface. A novel direct ELISA sensor for TDP-43 with visual detection and electrochemical quantification was also developed. The results validated the experimental designs toward specialised and selective analysis of these biomarkers and their aggregation mechanisms. Author Keywords: ALS, Alzheimer's, Biosensors, Electrochemistry, Tau, TDP-43

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