Graduate Theses & Dissertations

Expression optimization and NMR spectroscopy of Giardia intestinalis cytochrome b5 isotype III
The parasitic protist Giardia intestinalis does not synthesize heme and lacks many common eukaryotic heme proteins, yet it expresses four cytochrome b5 (gCYTB5) isotypes of unknown function. These have low reduction potentials and distinct subcellular locations that are consistent with structural features and biological functions that differ from their mammalian counterparts. Isotype III (gCYTB5-III) is particularly fascinating for its unusual location in the nuclei of Giardia. This thesis reports the optimization of recombinant gCYTB5-III overexpression for structural studies by NMR spectroscopy. Vital optimization factors for isotope labelling were first identified, finding that auto-induction promotes the optimization of many other conditions, such as colony selection, starter cultures, media components, temperature, pH and aeration. Optimized conditions were then applied to the expression and NMR spectroscopy of isotope-labelled gCYTB5-III and bovine cytochrome b5 as a control. These results can be extended to other heme proteins and will expand our biochemical knowledge of Giardia. Author Keywords: Auto-induction, Cytochrome b5, Giardia intestinalis, Isotope Labelling, Nuclear Magnetic Resonance Spectroscopy, Recombinant Protein
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
Functional Investigation of A Ustilago maydis Xylose Metabolism Gene and its Antisense Transcripts
Ustilago maydis is a biotrophic fungal plant pathogen that causes ‘common smut of corn’ disease. During infection, U. maydis develops a metabolic dependency on its host, relying on uptake of the carbon molecules provided within Zea mays tissues. The research presented indicated a requirement for metabolism of the pentose sugar D-xylose through functional investigation of a U. maydis xylitol dehydrogenase (uxm1), an enzyme involved in the bioconversion of D-xylose. This work is the first to outline the importance of pentose metabolism during biotrophic plant pathogenesis, as U. maydis haploid cells lacking this gene were impaired in their ability to cause disease and grow on medium containing only D-xylose. This thesis also explored the possibility that expression of this carbon-related gene is controlled by antisense RNAs (asRNAs), endogenous molecules with complementarity to mRNAs. Previous investigation of U. maydis asRNAs identified some that are exclusively expressed in the dormant teliospore, suggesting they have a functional role within this cell-type. A subset of these asRNAs at the uxm1 locus were investigated, with the purpose of identifying the mechanism(s) by which they influence U. maydis pathogenesis. This investigation involved the creation and functional analysis of a series of U. maydis deletion and expression strains. Together, these findings provided additional knowledge regarding the possible functions of U. maydis asRNAs, and their involvement in controlling important cellular processes, such as carbon metabolism and pathogenesis. Author Keywords: antisense transcripts, fungal carbon metabolism, non-coding RNAs, pathogenesis, Ustilago maydis, xylitol dehydrogenase
Effect of the neonicotinoid imidacloprid on embryogenesis and anuran survivorship in frog virus 3 infected tadpoles
Exposure of pre-metamorphic amphibians to neonicotinoid insecticides may be contributing to the global decline in amphibian populations. In this study, anuran embryos and tadpoles of the African clawed frog (Xenopus laevis) and the North American leopard frog (Lithobates pipiens) were used to determine the effects of embryonic exposure to neonicotinoids. In addition, Xenopus was used to determine if prolonged exposure to neonicotinoids influenced tadpole sensitivity to frog virus 3 (FV3). Exposure of anuran embryos to concentrations of the neonicotinoid insecticide, imidacloprid, ranging from 1 -20 ppm induced a concentration dependent increase in malformations of the retina in Xenopus embryos. However, similar responses were not observed with embryos of leopard frogs. Exposure of Xenopus tadpoles to 500 ppb concentration of imidacloprid followed by challenge with FV3 showed that pesticide exposure unexpectedly decreased the rates of mortality, although total mortalities by the end of the experiment were not significantly different from controls. This unexpected observation may be attributed to a reduced inflammatory response induced by exposure to imidacloprid. Despite the low acute toxicity of neonicotinoid insecticides to vertebrates, these studies indicate that exposure to this class of insecticides causes sublethal effects in anuran species during early life stages. Author Keywords: embryogenesis, Lithobates pipiens, neonicotinoid, ranavirus, tadpole, Xenopus laevis
Pathogen vs. Predator
Stressors are often an inescapable part of an organism’s life. While the effects of many stressors have been well studied individually, potential interactions between stressors exist that may result in greater than additive negative effects. Stressors may be linked by conflicting demands on energy budgets, interfering with important physiological pathways, or necessitating incompatible adaptive responses. Using Ranavirus (FV3) and larval dragonfly predators (Anax spp.) in a 2x2 factorial experiment on green frog (Lithobates clamitans) tadpoles, I investigate the interactions in behaviour, morphology, and metabolism when both stressors were applied in concert. I demonstrate that activity and feeding are reduced additively by both stressors, and tadpoles increase distance between conspecifics in FV3-exposed tanks, but only in the absence of predators. I also note decreases in mass, and a non-significant marginal increase in metabolic rate of tadpoles exposed to FV3. Interestingly, I provide evidence that FV3 can compromise morphometric responses through antagonistic interactions with perceived predation risk exposure, which may result in significantly elevated mortality even when either stressor is present in sub-lethal quantities. Thus, I conclude that sub-lethal exposure to stressors can nonetheless have substantial impacts on organisms and a more integrative approach to examining the impacts of stressors on individual physiology and fitness is necessary. Author Keywords: Behaviour, Interaction, Morphology, Predation Risk, Ranavirus, Tadpoles
Studies of the Giardia intestinalis trophozoite cell cycle
To study the Giardia intestinalis cell cycle, counterflow centrifugal elutriation (CCE) was used to separate an asynchronous trophozoite culture into fractions enriched for cells at the different stages of the cell cycle. For my first objective, I characterized the appearance of a third peak (Peak iii) in our flow cytometry analysis of the CCE fractions that initially suggested the presence of 16N cells that are either cysts or the result of endoreplication of Giardia trophozoites. I determined that this third peak consists of doublets of the 8N trophozoites at the G2 stage of the cell cycle that were not removed effectively by gating parameters used in the analysis of the flow cytometry data. In the second objective, I tested the use of a spike with RNA from the GS isolate of Giardia as an external normalizer in RT-qPCR on RNA from CCE fractions and encystation cultures of Giardia from the WB isolate. My results showed that the GS RNA spike is as effective as the use of previously characterized internal normalizer genes for these studies. For the third objective, I prepared two sets of elutriation samples for RNA seq analysis to determine the transcriptome of the Giardia trophozoite cell cycle. I confirmed the results of the cell cycle specific expression of several genes we had previously tested by RT-qPCR. Furthermore, our RNA-seq identified many genes in common with those identified from a microarray analysis of the Giardia cell cycle conducted by a collaborator. Finally, I observed an overall <4 fold change in differentially expressed genes during the G1/S and G2/M phase of the cell cycle. This is a modest change in gene expression compared to 10 - 30 fold changes for orthologous genes in mammalian cell cycles. Author Keywords: Cell cycle, Counterflow Centrifugal Elutriation, Flow Cytometry, RNA-sequencing, RT-qPCR
Flavohemoglobin expression in Giardia intestinalis exposed to nitrosative stress
The parasitic protist Giardia intestinalis lacks most heme proteins yet encodes a flavohemoglobin (gFlHb) that converts nitric oxide to nitrate and likely protects the cell from nitrosative stress. In this work an antibody raised against gFlHb was used to examine both changes in gFlHb expression levels and intracellular localization in Giardia in response to nitrosative stress. Giardia trophozoites exposed to stressors which either directly release nitric oxide (diethyltriamine NONOate, 1 mM) or are sources of other reactive nitrogen intermediates (sodium nitrite 20 mM or S-nitrosoglutathione, 1 or 5 mM) exhibited a 2 to 9-fold increase of gFlHb after 24 hours. Increased expression levels of gFlHb were detectable by 8 hours in S-nitrosoglutathione and diethyltriamine-NONOate-treated trophozoites, and by 12 hours after sodium nitrite exposure; these differences were likely due to differences in the rates of release of RNS from these compounds. In addition to a band of the expected size for gFlHb (52 kDa), western blots detected a second, higher molecular weight band (72 kDa) with comparable or higher intensity upon treatment with these RNS donors, which is consistent with sumoylation of gFlHb. Immunofluorescence microscopy of Giardia trophozoites detected gFlHb diffused throughout the cytoplasm and more punctuated staining along the cell membrane and between the nuclei. The punctuated staining may be due to the association of gFlHb with either peripheral vacuoles or basal bodies. Author Keywords: Flavohemoglobin, Giardia intestinalis, Nitrosative stress
Natural antisense transcripts to nucleus-encoded mitochondrial genes are linked to Ustilago maydis teliospore dormancy
Ustilago maydis is a basidiomycete smut fungus and the causal agent of common smut of corn. Disease progression and fungal development in this pathogen occur in planta, terminating in the production of dormant teliospores. Dormant spores of many fungi are characterized by reduced metabolic activity, which is restored during spore germination. The transition out of dormancy requires the rapid translation of stored mRNAs, which may be stabilized through natural antisense transcript (NAT)-mediated mechanisms. Transcript analysis revealed that as-ssm1, a NAT to the mitochondrial seryl-tRNA synthetase (ssm1), is detected in the dormant teliospore and absent in haploid cells. Disruption of ssm1 leads to cell lysis, indicating it is essential for cellular viability. Presented data supports the hypothesis that as-ssm1 has a role in facilitating teliospore dormancy through stabilizing ssm1 transcripts, which reduces mitochondrial function. as-ssm1 expression during in planta development begins 10 days post-infection, coinciding with the first appearance of dormant teliospores. To assess the impact of as-ssm1 expression on cell division, virulence and mitochondrial function, as-ssm1 was ectopically expressed in haploid cells, leading to increased ssm1 transcript levels and the formation of double-stranded RNA. These expression mutants are characterized by attenuated growth rate, virulence, mitochondrial membrane potential and oxygen consumption. Together, these findings support a role for NATs in moderating mitochondrial function during the onset of teliospore dormancy. Author Keywords: Dormant teliospore, Mitochondria, mRNA stability, Natural antisense transcripts, Non-coding RNA, Ustilago maydis
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
Effect of SP600125 JNK Inhibitor on Cadmium-Treated Mouse Embryo Forelimb Bud Cells In Vitro
This study investigated the role of the JNK signaling pathway in cadmium-treated mouse embryo forelimb bud cells in vitro. Primary cultures of forelimb bud cells harvested at day 11 of gestation were pre-treated with JNK inhibitor SP600125, and incubated with or without CdCl2 for 15, 30, 60, 120 minutes and 24, 48 hours or 5 days. Endpoints of toxicity were measured through cell differentiation by Alcian Blue Assay and phosphorylation of JNK proteins by Western blot. The results demonstrated that, in the cell differentiation assay, inhibiting JNK activation by 20 μM SP600125 causes an enhanced toxic effect in limb cells and inhibits cell differentiation, whereas 2 μM decreases differentiated nodule numbers under both cadmium stress and normal conditions. In conclusion, the JNK pathway has an essential role in the differentiation processes of limb bud cells in normal growth conditions. Author Keywords: Cadmium, Cell Signaling, JNK, Limbs, Mouse Embryo, Teratology

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