Graduate Theses & Dissertations

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Exploring the Role of Natural Antisense Transcripts in the Stress Response of Ustilago maydis
Fungal pathogens adapt to environmental changes faster than their hosts, due in part to their adaptive mechanisms exhibited in response to stress. Ustilago maydis was used to investigate potential natural antisense transcript (NAT) RNA-mediated mechanisms that enhance fungal adaptation to stress. Of the 349 NATs conserved amongst U. maydis and two related smut fungi, five NATs were identified as having altered transcript levels in response to multiple stress conditions. Subsequently, antisense transcript expression vectors were created for select NATs and transformed into U. maydis haploid cells. When exposed to stress conditions, two antisense expressing mutant strains exhibited alterations in growth. RT-qPCR analysis of mRNA complementary to expressed NATs revealed no significant change in mRNA levels, which suggests NAT expression may influence stress response through dsRNA formation or other RNA mediated mechanisms. These results establish a basis for further investigations into the connection between NATs and the stress response of fungi. Author Keywords: natural antisense transcripts, non-coding RNAs, stress response, Ustilago maydis
Gene flow directionality and functional genetic variation among Ontario, Canada Ursus americanus populations.
Rapidly changing landscapes introduce challenges for wildlife management, particularly for large mammal populations with long generation times and extensive spatial requirements. Understanding how these populations interact with heterogeneous landscapes aids in predicting responses to further environmental change. In this thesis, I profile American black bears using microsatellite loci and pooled whole-genome sequencing. These data characterize gene flow directionality and functional genetic variation to understand patterns of dispersal and local adaptation; processes key to understanding vulnerability to environmental change. I show dispersal is positively density-dependent, male biased, and influenced by food productivity gradients suggestive of source-sink dynamics. Genomic comparison of bears inhabiting different climate and forest zones identified variation in genes related to the cellular response to starvation and cold. My thesis demonstrates source-sink dynamics and local adaption in black bears. Population management must balance dispersal to sustain declining populations against the risk of maladaptation under future scenarios of environmental change. Author Keywords: American black bear, Dispersal, Functional Genetic Variation, Gene Flow Directionality, Genomics, Local Adaptation
Influence of nitrogen and sulfur on cadmium tolerance in Euglena gracilis
Heavy metal pollution threatens human and ecosystem health. E. gracilis was investigated for its potential use in bioremediation due to its tolerance for heavy metals and ability to sequester them from the environment. E. gracilis can remove metals by producing metal binding compounds enriched in sulfur and nitrogen. In this thesis, E. gracilis cultures that were pretreated with elevated levels of sulfur or nitrogen had increased tolerance to CdCl2 compared to non-pretreated cultures. RNA-sequencing revealed that both pretreatments led to transcript level changes and that exposure to CdCl2 led to further transcript level changes. Gene ontology (GO) enrichment analysis reflected changes in nitrogen and sulfur metabolism as well as physiological processes related to metal binding. The data from this thesis revealed important transcription level changes that occur when E. gracilis is challenged with CdCl2 and helps us understand how organisms adapt to heavy metal pollution in the environment. Author Keywords: bioremediation, Cadmium, Euglena gracilis, GO-enrichment, metal-binding, RNA-Sequencing
Determinants of Breeding Bird Diversity in Ontario's Far North
190 species of birds are known to breed in Ontario’s far north making the region an important nursery for boreal birds. Digital point count data were collected using two different autonomous recording units (ARUs): one model with two standard microphones to detect birds and anurans, and one model with one standard microphone and one ultrasonic microphone for detecting bats. ARUs were deployed either in short or long-term plots, which were four to six days or approximately 10 weeks, respectively. I assessed differences in breeding bird richness detections between ARU and plot types. I also tested the relative impact of the habitat heterogeneity and species-energy hypotheses in relation to breeding birds and created predictive maps of breeding bird diversity for Ontario’s far north. I found no difference in species richness estimates between the two ARU models but found that long-term plots detected about 7 more bird species and 1.5 more anuran species than short-term plots. I found support for both the species-energy and habitat heterogeneity hypotheses, but support for each hypothesis varied with the resolution of the analysis. Species-energy models were better predictors of breeding bird diversity at coarser resolutions and habitat heterogeneity models were better predictors at finer resolutions. Breeding bird diversity was highest in the Ontario Shield Ecozone compared with the Hudson Bay Lowlands Ecozone, but concentrated areas of higher diversity found in the Lowlands were associated with large rivers and the associated coastlines. Author Keywords: boreal birds, breeding birds, habitat heterogeneity, Hill diversity, Ontario, species-energy hypothesis
Forest Roost Use by Little Brown Bats (Myotis lucifugus) in Ontario
Roosts offer bats protection from predators, shelter from external environmental conditions, and a space where sociality, mating, and the rearing of young can occur. However, knowledge gaps still remain for many forest roosting species, such as the little brown bat (Myotis lucifugus) surrounding what roosts are selected, and what variables are influential at differing scales of selection. As a species-at-risk, identifying and predicting roost habitat selection may aid conservation and management. I identified forest roosts in a previously unexamined area of this species’ range using radio-telemetry, and measured tree-scale characteristics of located roosts. I then used a logistic model selection process with stand-scale variables to predict roost presence across forest stands. Height of trees in a given stand was the best predictor of roost presence - which may be linked to solar exposure and other thermal benefits. I then examined roost-level variables effecting the abundance of roosting bats in an artificial roosting environment (bat boxes). I found that temperature and social effects were both significant predictors of bat abundance, with warmer minimum temperatures in the box having a positive effect. These results suggest maternal bats may select roosts with higher minimum temperatures, likely due to the energetic benefits that may be gained over the course of reproduction. Author Keywords: forest roost, habitat selection, little brown bat, Myotic lucifugus, roost choice, stand selection
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
Electrochemical versus Chemical Oxidation of Bulky Phenols
Phenolic compounds are used in industry, such as agriculture and biotechnology, and inevitably end up in our environment. These compounds may serve as a phenolic precursor to produce raw materials for a wide range of applications. Chemical oxidation has been the common synthetic pathway to oxidize phenols and related compounds. However, traditional chemical approaches suffer from use of harsh chemicals, waste generation, and lack of reaction selectivity. Electrochemical synthesis has emerged as an alternative method to mitigate common challenges associated with organic synthesis. Herein, electrochemical oxidation of 2,6-diphenylphenol (DPP) and 2,2-dihydroxybiphenol (DHBP) was carried out and compared to traditional chemical oxidation. Contrasted with chemical oxidation, cyclic voltammetry of DPP resulted in a range of products based on the specific potential ranges used, whereas chemical oxidation of DHBP yield a dark-coloured polymeric product. The electrooxidation and chemical oxidation of DPP and DHBP resulted in a solution colour change, indicative of the formation of new, but different products monitored by UV-vis, and characterized by nuclear magnetic spectroscopy (NMR), X-ray single crystal diffraction, IR spectroscopy, transmission electron microscopy (TEM), and gas chromatography-mass spectrometry (GC-MS). The data indicate that the synthetic outcomes are dependent on the synthetic methodology employed, and that electrooxidation and chemical oxidation can form products unique to the pathway utilized. Author Keywords: chemoselectivity, electrochemistry, phenols, radical, synthesis
Genetic Networks to Investigate Structure and Connectivity of Caribou at Multiple Spatial and Temporal Scales
Understanding genetic structure, connectivity, and movement of a species iscritical to management and conservation. Genetic network approaches allow the analysis of genetic information with flexibility and few prior assumptions. In chapter one, I tested the ability of individual-based genetic networks to detect fine-scale structure and connectivity in relation to sampling efforts. My findings revealed individual-based genetic networks can detect fine-scale genetic structure of caribou when using 15 highly variable microsatellite loci. Sampling levels less than 50% of the estimated population size resulted in highly disconnected networks which did not allow for accurate structure analysis; however community detection algorithms were robust in grouping closely related individuals despite low sampling. In chapter two, I used individual-based and population-based genetic networks to investigate structure, connectivity, and movement of caribou across a large study area in Western Canada. A community detection algorithm partitioned the population-based genetic network at multiple spatial scales which uncovered patterns of hierarchical genetic structure and highlighted patterns of gene flow. The hierarchical population structure results aligned with the known distribution of different caribou Designatable Units (DUs) and additional structure was found within each DU. Furthermore, individual-based networks that were constructed with a subset of samples from the Mackenzie Mountains region of the Northwest Territories revealed patterns of long-distance movement and high connectivity across the region. Author Keywords: Biological Conservation, Caribou, Community Detection, Connectivity, Genetic Networks, Structure
Effects of Agricultural Land Use Change on Nitrogen and Phosphorus in North Shore Lake Ontario Tributaries
Row crop agriculture and associated land use practices including tile drainage and conservation tillage have been cited as a probable cause of re-emerging eutrophication in the lower Great Lakes. In this thesis, I sought to quantify and evaluate the effect of agricultural land cover and land use changes on total phosphorus (TP) and nitrate-nitrogen (NO3-N) concentrations and export in north shore Lake Ontario tributaries. This included (a) a long-term data analyses at 12 large watersheds (47 to 278 km²) using historical land cover and water quality data (1971-2010), and (b) a space-for-time study examining 12 small sub-catchments (< 8 km²) with majority (> 50%) row crop, pasture, or forest cover. Concentrations of TP were greatest in urbanized watersheds and declined particularly during the first decades of the study period, while NO3-N concentrations were greatest and steadily increased in agricultural catchments with increasing row crop cover. The space-for-time approach revealed that TP concentrations were similar across agricultural land uses and that export was most dependent on runoff. Meanwhile, NO3-N concentrations and export were greatest in row crop catchments and were positively related to row crop area. These results suggest that increases in row crop cover and associated agricultural practices including increased nutrient amendments and tile drainage may be responsible for increased NO3-N concentrations and export in northern Lake Ontario tributaries. Author Keywords: agriculture, Lake Ontario, nitrogen, phosphorus, streams, Water quality
Larval lake whitefish (Coregonus clupeaformis) recruitment dynamics in Lake Huron
Lake whitefish (Coregonus clupeaformis) recruitment has declined substantially in several regions of the Laurentian Great Lakes since the establishment of non-native dreissenid mussels in the early 1990’s. In Lake Huron, the reasons for the observed recruitment declines are currently unknown and there is limited knowledge about larval life stage. In our study, we determined whether larval hatching and growth rates have changed before and after dreissenid mussel invasion, and the role of several key environmental variables in influencing annual variation in larval densities. Larval fish were collected in the Fishing Islands spawning shoal during two time periods: a historical period (1976-1986) and a contemporary period (2017-2019). Larval densities and growth were lower in recent years, suggesting that recruitment is being limited at the larval life stage and that reduced food availability may be further limiting the growth during the larval stage. Annual variation of larval densities were influenced by spawning stock biomass, water levels, and dreissenid mussel presence, with higher water levels and the presence of dreissenid mussels being associated with higher larval densities. The direction of the effect of spawning stock biomass was either negative or positive depending on the model. We also found that larval density was a significant predictor of age 4 recruitment, indicating that year-class strength may be partly established at the larval life stage. Author Keywords: Coregonus clupeaformis, Great Lakes, Lake Huron, Lake whitefish, Larval, Recruitment
Using Fluorescent Carbon Dots for Biosensing Applications of Amino Acids
Amino acids make up proteins, which are the building blocks of life. A balance of amino acids is needed to maintain a healthy state. Tyrosine (Tyr) is synthesized from the metabolism of phenylalanine, which is an essential amino acid, meaning it can only be obtained from the diet. It is related to many metabolic and neurodegenerative diseases. Tyr can undergo post-translational modifications such as phosphorylation and nitration, which are implicated in cancer and nitrative stress, respectively. Although there are many methods to detect Tyr and its analogues, phosphotyrosine (pTyr) and nitrotyrosine (nTyr), these methods are time-consuming, involve expensive instruments and involve tedious process. This research proposes a new type of nanomaterials, carbon dots (CDs), to detect these amnio acids. Data indicate that CDs can be used to detect nTyr with a limit of detection of 34 μM in the linear range of 20 - 105 μM. The amenability of CD-nTyr assay was also tested in various biological matrices and biological molecules and was shown to be sensitive to nTyr. Nitration of Tyr was carried out in the presence of sodium nitrite and hydrogen peroxide catalyzed by either Cu(II) or Fe(III) to mimic biological reactions and CDs were tested as both inhibitors and indicators of Tyr nitration. Although CDs did not inhibit the nitration reaction of Tyr, they did not serve as indicators of Tyr nitration due to the quenching of CDs by the nitrating agents. This shows the importance of using CDs to detect nTyr and further use it for biological applications to detect diseased states. Author Keywords: amino acids, carbon dots, nanomaterials, sensor, spectroscopy, tyrosine
Interactome study of the Giardia intestinalis nuclear localized cytochrome b5
Giardia intestinalis is a waterborne enteric parasite that lacks mitochondria and the capacity for heme biosynthesis. Despite this, Giardia encodes several heme proteins, including four cytochrome b5 isotypes (gCYTB5-I – IV) of unknown function. The aim of this thesis is to gain insight into the function of the Giardia cytochrome b5 isotype III (gCYTB5-III) that is found in the nucleus, as first reported by our laboratory using immunofluorescence microscopy experiments with an isotype-III specific antibody. Nuclear localization of isotype-III is supported by two of my experiments: i) immunoblot analysis of crude cytoplasmic and nuclear enriched fractions of Giardia trophozoites; ii) association of gCYTB5-III with the insoluble fraction of Giardia lysates crosslinked with formaldehyde is reversed by DNase I treatment. To gain an understanding of the possible roles of gCYTB5-III, I performed immunoprecipitation (IP) experiments on lysates from Giardia trophozoites to identify its protein partners. Mass spectroscopy analysis of the immunoprecipitate identified proteins localized to the nucleus (RNA polymerase, DNA topoisomerase, histones, and histone modifying enzymes). Intriguingly, over 40% of the known mitosomal proteome, which functions in iron-sulfur (Fe-S) cluster assembly was also associated with gCYTB5-III. One of these proteins, the flavoenzyme GiOR-1, has been shown to mediate electron transfer from NADPH to recombinant gCYTB5-III. These IP results provide evidence that GiOR-1 and gCYTB5-III interact in vivo, and furthermore, suggest that some proteins in the mitosome could interact with those in the nucleus. I also found that DNA stress, caused by low concentrations of formaldehyde (0.1 – 0.2%) resulted in the increased expression of gCYTB5-III. Collectively these findings suggest a role of gCYTB5-III in Giardia's response to DNA stress and perhaps the formation of Fe/S clusters. Author Keywords: cluster, cytochrome, heme, iron, mitosome, nuclear

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2003 - 2033
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