Graduate Theses & Dissertations

Pages

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
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:
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
Population Genetics and Gut Microbiome Composition Reveal Subdivisions and Space Use in a Generalist and Specialist Ungulate
Natural populations are often difficult and costly to study, due to the plethora of confounding processes and variables present. This is of particular importance when dealing with managed species. Ungulates, for example, act as both consumers and prey sources; they also provide economic benefit through harvest, and as such, are of high ecological and economic value. I addressed conservation and management concerns by quantifying subdivision in wild populations and combined movement with non-invasive sampling to provide novel insight on the physiological drivers of space use in multiple species. This thesis explored biological patterns in ungulates using two distinct approaches: the first used molecular genetics to quantify gene flow, while the second examined the relationship between movement and the gut microbiome using high-throughput sequencing and GPS tracking. The goal of the first chapter was to quantify gene flow and assess the population structure of mountain goats (Oreamnos americanus) in northern British Columbia (BC) to inform management. I used microsatellites to generate genotype data and used a landscape genetics framework to evaluate the possible drivers behind genetic differentiation. The same analyses were performed at both a broad and fine scale, assessing genetic differentiation between populations in all of northern BC and in a case management study area northeast of Smithers BC. The results indicated panmixia among mountain goats regardless of scale, suggesting distance and landscape resistance were minimally inhibiting gene flow. Therefore, management at local scales can continue with little need for genetically informed boundaries, but regulations should be tailored to specific regions incorporating data on local access and harvest pressure. My second chapter aimed to determine the extent to which the gut microbiome drives space-use patterns in a specialist (mountain goat) and generalist (white-tailed deer, Odocoileus virginianus) ungulate. Using fecal samples, we generated genomic data using 16S rRNA high-throughput sequencing to evaluate gut diversity and gut microbiome characteristics. Additionally, individuals were fitted with GPS collars so that we could gain insight into movement patterns. Gut microbiome metrics were stronger predictors of space use and movement patterns with respect to home range size, whereas they were weaker predictors of habitat use. Notably, factors of both the gut microbiome and age of a given species were correlated with changes in space use and habitat use. Ultimately, this research linked high-throughput sequencing and GPS data to better understand ecological processes in wild ungulates. Author Keywords: gene flow, genomics, gut microbiome, home range, population genetic structure, ungulates
Assessment of Potential Threats to Eastern Flowering Dogwood (Cornus florida) in Southern Ontario
In Canada, eastern flowering dogwood (Cornus florida L.) is an endangered tree that occurs only in the Carolinian forest of southern Ontario. Threats to this species include habitat fragmentation and the fungal pathogen dogwood anthracnose (Discula destructiva). I conducted a population genetic analysis using seven nuclear microsatellite markers to determine if fragmented populations are genetically isolated from one another and have low levels of genetic diversity. Genetic comparisons suggest on-going dispersal among sites and relatively high genetic diversity within most sites; however, smaller populations and younger trees were less genetically diverse. I also used linear mixed effects models to assess potential relationships between several ecological variables and the prevalence of dogwood anthracnose. Disease severity was higher in trees on shallow slopes and in larger trees; the latter also had higher likelihood of infection. Insights from this study will be important to incorporate into future management strategies. Author Keywords: Cornus florida, Discula destructiva, dogwood anthracnose, Eastern flowering dogwood, endangered, population genetics
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
Evaluation of silver nanoparticles (AgNPs) and anti-GD2-AgNP antibody-drug conjugates as novel neuroblastoma therapies
Neuroblastoma (NB) has one of the highest mortality rates in pediatric oncology due to relapsed and refractory disease. Current aggressive multi-modal treatments are inhibited by dose-limiting toxicities and are associated with late-effects and secondary malignancies, emphasizing the necessity for novel therapeutics. Uniquely, most NB cells highly express disialoganglioside (GD2) a cell surface glycolipid that can provide a target for tumour-specific delivery. This study demonstrates a comprehensive evaluation of silver nanoparticles (AgNPs) and the first preliminary evaluation of anti-GD2-AgNP antibody-drug conjugates (ADCs) against NB in vitro. This present study validates the potential for AgNPs as an anti-cancer agent against NB as AgNPs demonstrated preferential toxicity towards NB cells through metabolic inhibition and indicative morphological alterations, while a less tumorigenic cell line demonstrated resistance to AgNP treatment. Therefore, this work identified an AgNP cell-type-dependent cytotoxicity effect. Low conjugation efficiency of the anti-GD2 monoclonal antibody, 14.G2a, to NHS-activated AgNPs failed to exert greater toxicity than the AgNPs alone. Collectively, this thesis provides novel information regarding the anti-cancer effects of AgNPs against NB with recommendations for anti-GD2-AgNP ADCs. Author Keywords: ADC, Chemotherapy, GD2, Neuroblastoma, Silver nanoparticles
Frog Virus 3
Understanding the maintenance and spread of invasive diseases is critical in evaluating threats to biodiversity and how to best minimize their impact, which can by done by monitoring disease occurrences across time and space. I sought to apply existing and upcoming molecular tools to assess fluctuations in both presence and strain variation of frog virus 3 (FV3), a species of Ranavirus, across Canadian waterbodies. I explored the temporal patterns and spatial distribution of ranavirus presence across multiple months and seasons using environmental DNA techniques. Results indicate that ranavirus was present in approximately 72.5% of waterbodies sampled on a fine geographical scale (<10km between sites, 7,150 km2), with higher detection rates in later summer months than earlier. I then explored the sequence variability at the major capsid protein gene (MCP) and putative virulence gene (vIF-2α) of FV3 samples from Ontario, Alberta, and the Northwest Territories, with the premise of understanding pathogen movement across the landscape. However, a lack of genetic diversity was found across regions, likely due to a lack of informative variation at the chosen genetic markers or lack of mutation. Instead, I found a novel FV3-like ranavirus and evidence for a recombinant between FV3 and a ranavirus of another lineage. This thesis provides a deeper understanding into the spatio-temporal distribution of FV3, with an idea of how widespread and threatening ranaviruses are to amphibian diversity. Keywords: ranavirus, frog virus 3, amphibians, environmental DNA, phylogenetics, wildlife disease, disease surveillance, major capsid protein, vIF-2α Author Keywords: amphibians, environmental DNA, frog virus 3, phylogenetics, ranavirus, wildlife disease
Using environmental DNA (eDNA) metabarcoding to assess aquatic plant communities
Environmental DNA (eDNA) metabarcoding targets sequences with interspecific variation that can be amplified using universal primers allowing simultaneous detection of multiple species from environmental samples. I developed novel primers for three barcodes commonly used to identify plant species, and compared amplification success for aquatic plant DNA against pre-existing primers. Control eDNA samples of 45 plant species showed that species-level identification was highest for novel matK and preexisting ITS2 primers (42% each); remaining primers each identified between 24% and 33% of species. Novel matK, rbcL, and pre-existing ITS2 primers combined identified 88% of aquatic species. The novel matK primers identified the largest number of species from eDNA collected from the Black River, Ontario; 21 aquatic plant species were identified using all primers. This study showed that eDNA metabarcoding allows for simultaneous detection of aquatic plants including invasive species and species-at-risk, thereby providing a biodiversity assessment tool with a variety of applications. Author Keywords: aquatic plants, biodiversity, bioinformatics, environmental DNA (eDNA), high-throughput sequencing, metabarcoding
De novo transcriptome assembly, functional annotation, and SNP discovery in North American flying squirrels (genus Glaucomys)
Introgressive hybridization between northern (Glaucomys sabrinus) and southern flying squirrels (G. volans) has been observed in some areas of Canada and the USA. However, existing molecular markers lack the resolution to discriminate late-generation introgressants and describe the extent to which hybridization influences the Glaucomys gene pool. I report the first North American flying squirrel (genus Glaucomys) functionally annotated de novo transcriptome assembly with a set of 146,621 high-quality, annotated putative species-diagnostic SNP markers. RNA-sequences were obtained from two northern flying squirrels and two southern flying squirrels sampled from Ontario, Canada. I reconstructed 702,228 Glaucomys transcripts using 193,323,120 sequence read-pairs, and captured sequence homologies, protein domains, and gene function classifications. These genomic resources can be used to increase the resolution of molecular techniques used to examine the dynamics of the Glaucomys hybrid zone. Author Keywords: annotation, de novo transcriptome, flying squirrels, high-throughput sequencing, hybridization, single nucleotide polymorphisms
Cytokinin biosynthesis, signaling and translocation during the formation of tumors in the Ustilago maydis-Zea mays pathosystem
Cytokinins (CKs) are hormones that promote cell division. During the formation of tumors in the Ustilago maydis-Zea mays pathosystem, the levels of CKs are elevated. Although CK levels are increased, the origins of these CKs have not been determined and it is unclear as to whether they promote the formation of tumors. To determine this, we measured the CK levels, identified CK biosynthetic genes as well as CK signaling genes and measured the transcript levels during pathogenesis. By correlating the transcript levels to the CK levels, our results suggest that increased biosynthesis and signaling of CKs occur in both organisms. The increase in CK biosynthesis by the pathosystem could lead to an increase in CK signaling via CK translocation and promote tumor formation. Taken together, these suggest that CK biosynthesis, signaling and translocation play a significant role during the formation of tumors in the Ustilago maydis-Zea mays pathosystem. Author Keywords: Biosynthesis, Cytokinins, Signaling, Translocation, Ustilago maydis, Zea mays
Using DNA Barcoding to Investigate the Diet and Food Supply of a Declining Aerial Insectivote, the Barn Swallow (Hirundo rustica)
Barn Swallow (Hirundo rustica) populations have declined in North America over the past 40 years and they are listed as Threatened in Ontario, Canada. Changes in the food supply have been hypothesized as a potential cause of this population decline. I used DNA barcoding to investigate the diet and food supply of Barn Swallows and to determine if the food supply affects their reproductive performance. In two breeding seasons, I monitored nests, collected fecal samples, and monitored prey availability by collecting insects from the habitat surrounding breeding sites using Malaise traps. I used DNA barcoding to identify insect specimens collected from the habitat and to identify prey items from Barn Swallow nestling fecal samples. I found that Barn Swallow nestlings were fed a very broad range of prey items but were fed larger prey items more frequently. Prey availability was not related to the timing of reproduction, the number of nests at a breeding site, or the reproductive output of individual nests. This study provides information on the diet composition of Barn Swallows in North America and suggests that food limitation during the breeding season may not be a major factor in their population decline. Author Keywords: aerial insectivore, diet, DNA barcoding, Hirundo rustica, metabarcoding, reproductive success

Pages

Description

Graduate student theses produced at Trent University. You may also browse theses and dissertations by year, program, or author.

In collections

Search Our Digital Collections

Query

Enabled Filters

  • (-) ≠ History
  • (-) ≠ Art history
  • (-) ≠ Burness
  • (-) ≠ Ecology
  • (-) ≠ Canadian studies
  • (-) = Genetics

Filter Results

Date

2004 - 2024
(decades)
Specify date range: Show
Format: 2024/05/16

Collection

Author Name

Show more

Name (Any)

Show more

Degree

Degree Discipline

Subject (Topic)

Show more