Graduate Theses & Dissertations

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Carbon and Nitrogen Isotope Changes in Streams along an Agricultural Gradient
Nitrogen is a major constituent of agricultural fertilizers, and nitrogen inputs to stream water via runoff and groundwater lead to a variety of negative environmental impacts. In order to quantify the movement of nitrogen through aquatic food webs, fourteen streams with varying land uses across South-Central Ontario were sampled for two species of fish, freshwater mussels, and water for measurement of isotope ratios of δ15N and δ13C. I found that nitrogen isotopes in fish, water, and mussels were related to the percentage of riparian monoculture, and that carbon isotopes were unrelated to monoculture. Though all species were enriched as monoculture increased, the rate of δ15N enrichment as monoculture increased did not vary between species. This study has improved our understanding of how monoculture affects nutrient enrichment in stream food webs, and assesses the validity of using nitrogen isotopes to measure trophic positions of aquatic organisms across an environmental gradient. Author Keywords: agriculture, fish, food webs, nitrogen, stable isotopes, streams
Changes in Forms of Uranium in Anoxic Lake Sediments and Porewaters Near an Abandoned Uranium Mine, Bancroft, Ontario
Soluble uranium (U) has been observed continuously in the porewaters of Bentley Lake, a lake with semi-permanent anoxic sediments, despite the fact that reduced U(IV) is known to be insoluble. To be able to predict the fate and mobility of U that has been deposited in lake sediments, it is very important to understand the factors that determine soluble uranium in anoxic environments. Understanding soluble U species is crucial for predicting its behavior in natural systems as well as for the development of U remediation schemes. To explore the factors affecting soluble U in natural environments, anoxic lake sediments and porewaters were tested using two analytic methods, ICP-MS and ESI-HR-MS. Reduced uranium (U(IV)) can be precipitated as U(IV)-NdF3. Using this method revealed that most of the uranium in porewater is not able to be co-precipitated with NdF3. In addition, UO2+ was found using ESI-HR-MS, showing uranyl ions exist in reduced porewater. However, the UO2+ might be attached to some organic groups rather than present as free ions. Seasonal variation and air exposure experiments on the mobility of U between sediments and porewater were observed to test for changes of the redox state of U as a function of sample collection and storage. The results of this study will contribute to better remediation strategies for U tailings and will help U mining operations in the future. Author Keywords:
Characterisation of the Giardia Tata-Binding Protein - Preparation for an in vivo approach
The aim of this work was to identify the DNA sequences recognized by the Giardia TBP (gTBP) in vivo by using a chromatin immunoprecipitation assay (ChIP). Since a specific antibody for the protein of interest is required for this assay, a company was contracted to produce and purify a custom polyclonal antibody from the immunization of rabbits. Recombinant GST-gTBP was produced at a suitable yield and purity and used as the immunogen. The antibody was then tested for reactivity to the native protein in our laboratory. By Western blot analysis, it was possible to observe the enrichment of the gTBP within the nuclear fraction compared to a cytoplasmic fraction extracted from Giardia cells. However, the antibody could not be successfully used in an immunoprecipitation assay - suggesting that the antibody is unable to bind to the native structure of gTBP. Therefore, the focus of this work was changed to analyse gTBP via multiple sequence alignments, homology modelling and BLAST to identify any unique regions that may contribute to its unusual binding characteristics. These techniques were also used to identify specific regions of gTBP that may be used to generate synthetic peptides as immunogens for future antibody production. Author Keywords: ChIP, Giardia intestinalis, Homology modelling, Immunoprecipitation, TATA-binding protein, Western Blotting
Characterization of Synthetic and Natural Se8 and Related SenSm Compounds by Gas Chromatography-Mass Spectrometry
Elemental selenium has been extensively quantitatively measured in sediments; however, its physical composition is largely unknown, despite it being the dominant selenium species in some reducing environments. Here, for the first time, it is shown that small, cyclic selenium compounds can account for a quantitatively-relevant fraction of the total elemental selenium present. A new method was developed to analyze for cyclooctaselenium (Se8) in both synthetic samples and selenium-impacted sediments. Despite some analytical limitations, this gas chromatography-mass spectrometry (GC-MS) method is the first GC-MS method developed to identify and quantify Se8 in sediments. Once this method was established, it was then applied to more complex systems: first, the identification of compounds in mixed selenium-sulfur melt solutions, and then the determination of SenSm in selenium-impacted sediments. Despite complications arising from pronounced fragmentation in the ion source, assignment of definitive molecular formulae to chromatographically-resolved peaks was possible for five compounds. Developing a fully quantitative method to obtain elemental ratio information can aid in the assignment of molecular formulae to chromatographically-resolved SeS-containing chromatographic peaks. Coupling the existing gas chromatography method to an inductively coupled plasma-mass spectrometer (ICP-MS) system should accomplish this. However, due to a number of complications, this was not completed successfully during the duration of this thesis project. High detection limits for sulfur, retention time discrepancies, and inconsistent injection results between the GC-MS and GC-ICP-MS system led to difficulties in comparing results between both analytical methods. Despite these limitations, GC-ICP-MS remains the most promising method for the identification and quantification of SenSm compounds in synthetic melt mixtures and selenium impacted sediments. Author Keywords: gas chromatography-mass spectrometry, sediments, selenium
Characterization of a Zn(II)2Cys6 transcription factor in Ustilago maydis and its role in pathogenesis
Ustilago maydis (D.C.) Corda is a biotrophic pathogen that secretes effectors to establish and maintain a relationship with its host, Zea mays. In this pathosystem, the molecular function of effectors is well-studied, but the regulation of effector gene expression remains largely unknown. This study characterized Zfp1, a putative U. maydis Zn(II)2Cys6 transcription factor, as a modulator of effector gene expression. The amino acid sequence of Zfp1 indicated the presence of a GAL4-like zinc binuclear cluster as well as a fungal specific transcription factor domain. Nuclear localization was confirmed by tagging Zfp1 with enhanced green fluorescent protein. Deletion of zfp1 resulted in attenuated hyphal growth, reduced infection frequency, an arrest in pathogenic development, and decreased anthocyanin production. This phenotype can be attributed to the altered transcript levels of genes encoding predicted and confirmed U. maydis effectors in the zfp1 deletion strain during pathogenic growth. Complementation of zfp1 deletion strain with tin2, an effector involved in anthocyanin induction, suggested this effector is downstream of Zfp1 and its expression is influenced by this transcription factor during in planta growth. When wild-type zfp1 was ectopically inserted in the zfp1 deletion strain, pathogenesis and virulence were partially restored. This, coupled with zfp1 over-expression strains having a similar phenotype as the deletion strains, suggested Zfp1 may interact with other proteins for full function. These findings show that Zfp1, in conjunction with one or more binding partners, contributes to U. maydis pathogenesis, virulence, and anthocyanin production through the regulation of effector gene expression. Author Keywords: effector, pathogenesis, transcription factor, Ustilago maydis, Zea mays, zinc finger
Characterization of frog virus 3 and its binding partner LITAF
Iridoviruses are large (120-200nm) double stranded DNA viruses that contain an icosahedral capsid. The iridoviridae family is composed of five genera that infect a wide range of poikilothermic vertebrates (Lymphocystivirus, Ranavirus and Megalocyivirus) and invertebrate hosts (Iridovirus, Chloriridovirus). Frog virus 3 (FV3) is a member of the Ranavirus genus, and is commonly used as a model system to study iridoviruses. I was interested in understanding virus-host interaction in FV3. I studied two viral genes, FV3 97R and FV3 75L. Here I demonstrate that 97R localizes to the endoplasmic reticulum (ER) at 24 hours post-transfection. However, at 35 hours post-transfection 97R localizes to the ER but also begins to form concentrated pockets, continuous with the nuclear membrane This study found that 97R possess a unique phenotype and that its localization to the ER is mediated through its C-terminus transmembrane domain. FV3 75L encodes an 84 amino acids protein. I showed that FV3 75L localizes to the early endosomes, while its cellular binding partner, LITAF, localizes to late endosome/lysosome. Interestingly, when FV3 75L and LITAF are co-transfected into cells, LITAF can alter the subcellular localization of FV3 75L to late endosome/lysosomes. A physical interaction between LITAF and FV3 75L was demonstrated through a pull-down assay and that a highly conserved domain found in both proteins may mediate the interaction. LITAF has been proposed to function in protein degradation, but there is still uncertainty on LITAF's specific role. I was interested in further characterizing LITAF and its implications in protein degradation and a neurodegenerative disorder. At least 9 mutations of LITAF are associated with Charcot-Marie-Tooth disease type 1C (CMT1C), which belongs to the group of most common heritable neuromuscular disorders, affecting approximately one in 2500 people. We show that LITAF mutants G112S and W116G mislocalize from the late endosome/lysosome to the mitochondria while the T49M and P135T mutants show partial mislocalization with a portion of the protein present in the late endosome/lysosome and a portion of the protein localized to the mitochondria. Since LITAF is believed to play a role in protein degradation, it is possible that the specific characteristics of CMT1C may occur though impaired degradation of Schwann cell membrane proteins, such as PMP22. I was able to show that when WT LITAF is present, there is a decrease in the PMP22 intracellular levels, which suggest that LITAF plays an important role in protein degradation, and also in other types of CMT. Insight into how mutations in LITAF cause CMT1C may not only help better understand cellular pathways, but also further elucidate the role LITAF's viral homolog FV3 75L during viral infection. Author Keywords: 75L, Charcot-Marie-Tooth, CMTC1, ER, FV3, LITAF
Characterizing the demographic history and prion protein gene variation to infer susceptibility to chronic wasting disease in a naïve population of white-tailed deer (Odocoileus virginianus)
Assessments of the adaptive potential of natural populations are essential for understanding and predicting responses to environmental stressors like climate change and infectious disease. The range of stressors species face in a human-dominated landscape, often have contrasting effects. White-tailed deer (Odocoileus virginianus, deer) are expanding in the northern part of their range following decreasing winter severity and increasing forage availability, caused by climate change. Chronic wasting disease (CWD), a prion disease affecting cervids, is likewise expanding and represents a major threat to deer and other cervids We obtained tissue samples from free-ranging deer across their native range in Ontario, Canada which has yet to detect CWD in wild populations of cervids. High throughput sequencing was used to assess neutral genomic variation and variation in the gene responsible for the protein that misfolds into prions when deer contract CWD, known as the PRNP gene. Neutral variation revealed a high number of rare alleles and no population structure, consistent with an expanding population of deer. Functional genetic variation revealed that the frequencies of variants associated to CWD susceptibility and disease progression were evenly distributed across the landscape and the frequencies were consistent with deer populations not infected with CWD. These findings suggest that an observable shift in PRNP allele frequencies likely coincides with the start of a novel CWD epidemic. Sustained surveillance of genomic and genetic variation can be a useful tool for CWD-free regions where deer are managed for ecological and economic benefits. Author Keywords: Canadian wildlife, population genetics, prion, PRNP, RADseq, ungulate
Chemical characterization of dissolved organic matter in relation with hydrography in the Arctic Ocean
In this thesis, water mass distribution of dissolved organic matter (DOM) characteristics (i.e. molecular weight, fluorescent components, thiols and humic substances concentration) was observed in the Arctic Ocean. For the first time, DOM molecular weight (MW) in Beaufort Sea was assessed using asymmetrical flow field-flow fractionation, as well as the first monitoring of thiols and humic substances (HS) using cathodic stripping voltammetry (CSV) in the Arctic Ocean. Based on fluorescence property, DOM characterization was carried out using parallel factor analysis – excitation-emission matrices. Pacific winter waters in the Canada Basin showed higher MW DOM associated with higher fluorescence intensity. High HS was associated with the Arctic outflow waters in top 300 m of the Canadian Arctic Archipelago. Interestingly, maximum thiol concentration was associated with the subsurface chlorophyll-a maximum at most sites, but not universal along the study area. Comparable distributions of CSV-based HS and humic-like fluorescent components suggest similar sources/ processes in the Arctic Ocean. The findings in this thesis suggested DOM characteristics could be used as fingerprints in tracing water masses in the Arctic Ocean. Author Keywords: Asymmetrical flow field-flow fractionation, Cathodic stripping voltammetry, DOM, Metal-binding ligands, Molecular weight, PARAFAC-EEMs
Clonal structure and mating patterns in a natural population of Sagittaria latifolia
Increased plant size is expected to have negative consequences for mating by increasing pollen transfer among the same plant. However, recent theoretical studies have demonstrated that this may not be true for clonal plants. Instead, clonal expansion could enhance outcrossing opportunities without increasing selfing by reducing distances to potential mates. I investigated how the spatial structure of clones influences patterns of pollen dispersal, selfing rates and siring success in a natural population of Sagittaria latifolia. I found that pollen dispersal distances typically exceeded the spatial extent of clones and there was a positive association between clone size and the likelihood that clones were intermingled. Together, this resulted in a weak positive association between clone size and selfing rates, and a strong positive association between clone size and outcross siring success. This is the first empirical support for the theoretical expectation that any negative effects of selfing in large clones might be offset by increased siring success. Author Keywords: clonal growth, fitness gain curve, geitonogamy, plant mating, plant reproductive ecology, sex allocation theory
Combining Line Transect Sampling and Photographic-Identification Surveys to Investigate the Abundance and Distribution of Cetaceans
Line transect sampling and photographic-identification (photo-ID) are common survey techniques for estimating the abundance and distribution of cetaceans. Combining these approaches in the field (‘combined LTPI’ surveys) and using data from both components has the potential for generating comprehensive ecological knowledge that can be far more valuable than when these techniques and their data are used independently. In this thesis, I evaluated the results and conclusions from these two methods, used singly and in tandem, by investigating the population dynamics of two humpback dolphin (Sousa chinensis spp.) populations: the large and widely distributed Chinese white dolphin (S. c. chinensis) of the Pearl River estuary (PRE), and the small and geographically isolated subspecies of Taiwanese white dolphin (S. c. taiwanensis) in the eastern Taiwan Strait. Data from combined LTPI surveys in Hong Kong waters, at the eastern edge of the PRE, revealed a shift in space use with individuals spending less time in these waters than at the start of surveys. Data from combined LTPI surveys in Taiwan provided further support for a subspecies restricted to the central western waters, and identified a commonly used area at the northern part of their limited range. These two case studies demonstrated an overall efficacy of combined LTPI surveys in ecological studies of cetaceans. However, a multi-criteria analysis revealed that combined LTPI surveys with a line transect focus (e.g., Hong Kong) performed better than a LTPI survey with a photo-ID focus (e.g., Taiwan) when considering ecological aspects of the study populations, labour and data requirements, and ecological output. Even so, the photo-ID focus of Taiwan’s monitoring program led to better assessments of individual space use patterns, likely helped by the Taiwanese white dolphin population’s smaller size and intensive photographic effort. In both cases, the ecological output of combined LTPI surveys could be improved by expanding the study area or extending the field season or frequency of surveys. Overall, I showed that by following a set of general guidelines, different iterations of the combined LTPI approach (i.e., photo-ID focus or LT focus) can serve as powerful tools for uncovering multi-dimensional ecological information on cetaceans. Author Keywords: abundance, cetacean, distribution, line transect sampling, multi-criteria analysis, Photo-ID
Comparative Evaluation of Effective Population Size Genetic Estimation Methods in Wild Brook Trout (Salvelinus fontinalis) Populations
Effective population size (Ne) is a key concept in population genetics, evolutionary biology and conservation biology that describes an important facet of genetic diversity and the capacity of populations to respond to future evolutionary pressures. The importance of Ne in management and conservation of wild populations encouraged the development of numerous genetic estimators which rely on a variety of methods. Despite the number and diversity of available Ne methods, however, tests of estimator performance have largely relied on simulations, with relatively few tests based on empirical data. I used well-studied wild populations of brook trout (Salvelinus fontinalis) in Algonquin Park, Ontario as a model system to assess the comparative performance of multiple Ne estimation methods and programs, comparing the resultant Ne estimates against demographic population size estimates. As a first step, the genetic diversity and ancestry of wild brook trout populations was determined using 14 microsatellite loci. Genetic structure of brook trout populations showed variable contributions from historical supplemental stocking and also identified localized gene pools within and between watersheds, reflecting variable levels of connectivity and gene flow. Once the genetic ancestry and connectivity of populations had been resolved, single sample (point) and two samples (temporal) genetic estimators were used to estimate Ne of populations with pure native ancestry. Values obtained from genetic estimators utilizing both methods were variable within as well as among populations. Single sample (point) estimators were variable within individual populations, but substantially less than was observed among the temporal methods. The ratios of Ne to the estimated demographic population size (N) in small populations were substantially higher than in larger populations. Variation among estimates obtained from the different methods reflects varying assumptions that underlay the estimation algorithms. This research further investigated the effect of sampling effort and number of microsatellite loci used on Ne values obtained using the linkage disequilibrium (LD) estimation method. Ne estimates varied substantially among values generated from subsets of loci and genotyped individuals, highlighting the necessity for proper sampling design for efforts aiming to measure Ne. Despite the variation observed among and within estimation methods, the Ne concept is a valuable for the conservation and management of both exploited and endangered species. Author Keywords: Brook Trout, Effective population size, Genetic Diversity, Genetic Structure
Comparative efficacy of eDNA and conventional methods for monitoring wetland anuran communities
Identifying population declines and mitigating biodiversity loss require reliable monitoring techniques, but complex life histories and cryptic characteristics of anuran species render conventional monitoring challenging and ineffective. Environmental DNA (eDNA) detection is a highly sensitive and minimally invasive alternative to conventional anuran monitoring. In this study, I conducted a field experiment in 30 natural wetlands to compare efficacy of eDNA detection via qPCR to three conventional methods (visual encounter, breeding call, and larval dipnet surveys) for nine anuran species. eDNA and visual encounter surveys detected the greatest species richness, with eDNA methods requiring the fewest sampling events. However, community composition results differed among methods, indicating that even top performing methods missed species detections. Overall, the most effective detection method varied by species, with some species requiring two to three methods to make all possible detections. Further, eDNA detection rates varied by sampling season for two species (A. americanus and H. versicolor), suggesting that species-specific ecology such as breeding and larval periods play an important role in eDNA presence. These findings suggest that optimized monitoring of complex anuran communities may require two or more monitoring methods selected based on the physiology and biology of all target species. Author Keywords: amphibian, anuran, conventional monitoring, eDNA, environmental DNA, species richness

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