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Graduate Theses & Dissertations
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- Mutation of the B10 Tyrosine and E11 Leucine in Giardia intestinalis Flavohemoglobin
- The flavohemoglobin in Giardia intestinalis (gFlHb) is the only known protozoan member of a protein class typically associated with detoxifying nitric oxide (by oxidation to nitrate) in bacteria and yeast. Mutants of the B10 tyrosine (Y30F) and E11 leucine (L58A), conserved residues thought to influence ligand binding, were expressed and studied using Resonance Raman (RR) spectroscopy. In the wild type protein, RR conducted using a carbon monoxide probe detects two distinct Fe-CO stretches associated with two different active site configurations. In the open configuration, CO does not interact with any polar side chains, while in the closed configuration, CO strongly interacts with one or more distal residues. Analysis of the Y30F mutant provided direct evidence of this tyrosine’s role in ligand stabilization, as it had only a single Fe-CO stretching mode. This stretching mode was higher in energy than the open conformer of the wild type, indicating a residual hydrogen bonding interaction, likely provided by the E7 glutamine (Q54). In contrast the L58A mutant had no effect on the configurational nature of the enzyme. This was unexpected, as the side chain of L58 sits atop the heme and is thought to regulate the access of distal residues to the heme-bound ligand. The similar spectroscopic properties of wild type and L58A suggest that any such regulation would involve rapid conformational dynamics within the heme pocket. Author Keywords: B10 Tyrosine, Catalytic Globin, E11 Leucine, Flavohemoglobin, gFlHb, Giardia intestinalis
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- Mfsd8 regulates growth and multicellular development in Dictyostelium discoideum
- The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are a family of inherited neurodegenerative lysosomal storage disorders. CLN7 disease is a subtype of NCL that is caused by mutations in the MFSD8 gene. MFSD8 encodes a lysosomal transmembrane protein that is predicted to play a role in transporting small substrates across membranes. However, little is known about its role and substrate specificity. Previous work identified an ortholog of human MFSD8 in the social amoeba Dictyostelium discoideum and reported its localization to endocytic compartments. In this study, the effects of mfsd8 loss during Dictyostelium growth and multicellular development were further characterized. Dictyostelium mfsd8- cells displayed increased rates of proliferation and pinocytosis in liquid media. During growth, loss of mfsd8 altered lysosomal enzymatic activities and reduced the intracellular and extracellular levels of autocrine proliferation repressor A. mfsd8- cells grown on a lawn of bacteria formed plaques in a shorter period of time compared to WT cells, providing additional support for the enhanced growth of mfsd8- cells. Upon starvation, the aggregation of mfsd8- cells was delayed, and mfsd8- cells formed more mounds that were smaller in size, which may be attributed to the reduced cell-substrate adhesion and altered lysosomal enzymatic activities observed for mfsd8- cells. Following aggregation, tipped mound formation was delayed, however, loss of mfsd8 did not affect the timing of slug/finger and fruiting body formation. Additionally, slug migration was reduced in mfsd8- cells. These aberrant phenotypes, excluding fruiting body formation, were effectively or partially rescued when Mfsd8-GFP was introduced into mfsd8- cells. Overall, these results show that Mfsd8 plays a role in regulating growth and developmental processes in Dictyostelium via lysosomal-associated functions. Author Keywords: CLN7, Dictyostelium discoideum, Lysosomes, MFSD8, Neuronal Ceroid Lipofuscinoses
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- Altered Hippocampal Regulation of Immediate Early Genes after Pentylenetetrazol-Induced Seizures
- Seizures induce long-term changes in gene expression in the hippocampus. Experimental evidence has demonstrated a significant effect of epileptic activity on the activity of neurons that participate in complex cognitive and behavioural processes. The present series of experiments involving kindling with subconvulsive doses of PTZ demonstrates a link between seizures and altered immediate early gene expression within the hippocampus and dentate gyrus. In addition, newborn hippocampal neurons were shown to have decreased induction of plasticity-related genes, suggesting deficits in activity-dependent recruitment. These findings may shed light on the mechanisms underlying epileptogenesis and epilepsy-related hippocampal dysfunction in human patients. Author Keywords: hippocampus, IEGs, kindling, neurogenesis, seizures
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- Effect of Nitrosative Stress on Heme Protein Expression and Localization in Giardia Intestinalis
- The parasitic protist Giardia intestinalis has five heme proteins: a flavohemoglobin and several isotypes of cytochrome b5. While the flavohemoglobin has a role in counteracting nitric oxide, the functions of the cytochromes (gCYTb5s) are unknown. In this study, the protein level and cellular localization of three gCYTB5 isotypes (gCYTb5-I, II and III) and flavohemoglobin were examined in Giardia trophozoites exposed to three nitrosative stressors at two different concentrations: nitrite (20 mM, 0.5 mM); GSNO (2 mM, 0.25 mM) and DETA-NONOate (2 mM, 0.05 mM). An increase in protein levels was observed for gCYTb5-II with all stressors at both concentrations. However, the effects of these nitrosative stressors on gCYTb5-I and III were inconclusive due to the variation among the replicates and the poor detection of gCYTb5- III on western blots. The protein level of the flavohemoglobin also increased in response to the three stressors at the low concentrations of stressors that were tested. Only the cellular localization of gCYTb5-I changed in response to nitrosative stress, where it moved from the nucleolus to the nucleus and cytoplasm. This response was extremely sensitive and occurred at the lower doses of the three stressors, suggesting that gCYTb5-I may be involved in a nucleolar- based stress response. Author Keywords:
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- Understanding Historical and Contemporary Gene Flow Patterns of Ontario Black Bears
- Consequences of habitat loss and fragmentation include smaller effective population sizes and decreased genetic diversity, factors that can undermine the long-term viability of large carnivores that were historically continuously distributed. I evaluated the historical and contemporary genetic structure and diversity of American black bears (Ursus americanus) in Ontario, where bear habitat is largely contiguous, except for southern regions that experience strong anthropogenic pressures. My objectives were to understand gene flow patterns in a natural system still largely reflective of pre-European settlement to provide context for the extent of genetic diversity loss in southern populations fragmented by anthropogenic influences. Phylogeographic analyses suggested that Ontario black bears belong to a widespread "continental" genetic group that further divides into 2 subgroups, likely reflecting separate recolonization routes around the Great Lakes following the Last Glacial Maximum. Population genetic analyses based on individual genotypes showed that Ontario black bears are structured into 3 contemporary genetic clusters. Two clusters, located in the Northwest (NW) and Southeast (SE), are geographically vast and genetically diverse. The third cluster is less diverse, and spatially restricted to the Bruce Peninsula (BP). Microsatellite analyses revealed that the NW and SE clusters are weakly differentiated from each other relative to mitochondrial DNA findings, suggesting male-biased dispersal and isolation by distance across the province. I also conducted simulations to assess competing hypotheses that could explain the reduced genetic diversity on the BP, which supported a combination of low migration and recent demographic bottlenecks. I showed that management actions to increase genetic variation in BP black bears could include restoring landscape connectivity between BP and SE; however, the irreversible human footprint in the area makes regular translocations from SE individuals a more practical alternative. Overall, my work suggests that: 1) historical genetic processes in Ontario black bears were likely predominated by isolation by distance, 2) large mammalian carnivores such as black bears can become isolated and experience reduced diversity in only a few generations, and 3) maintaining connectivity in regions under increased anthropogenic pressures could prevent populations from becoming small and geographically and genetically isolated, and should be a priority for conserving healthy populations. Author Keywords: American black bear, carnivore, conservation genetics, Ontario, phylogeography, population genetics
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- 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
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- Daphnia pulicaria responses to temperature and nutrients stress
- Warming climates have had various consequences on terrestrial and aquatic food webs that are expected to persist. There is evidence suggesting that certain organisms are better equipped to handle changing climates compared to others. Therefore, the purpose of my thesis was to study the adaptability of Daphnia under temperature stress and nutrient limitation. First, to examine the effects of dietary phosphorus limitation and temperature on daphniid life-history and population growth, a series of experiments were conducted in the laboratory. In general, I found that Daphnia body growth rates and life-history traits to food carbon to phosphorus (C:P) ratios change with temperature. Next, I identified a protocol to limit the genomic DNA (gDNA) from ribonucleic acid (RNA) extractions. I found that using a modified phenol-chloroform extraction protocol was the most effective way to remove gDNA from extracted Daphnia RNA samples. Overall, results from this study show that temperature and food quality interactions are more complicated than previously thought. Furthermore, the RNA extraction protocol developed will be useful in future studies examining gene expression responses in Daphnia. Author Keywords: ecological stoichiometry, gene expression, life-history, nutrient limitation, RNA puritiy, temperature
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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
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- Impacts of Cover Crops on Soil Health, Soil Nitrogen Dynamics, and Cytokinin Profiles
- In Ontario, the dominant cash crop rotations consist of soybean (SB), which is a leguminous crop grown in rotation with maize (MZ) and winter wheat (WW). In addition to these crops, some farmers integrate cover crops (CC) into crop rotation, especially during the fallow period and winter seasons, to reduce nitrogen (N) losses via nitrate (NO3-) leaching and emission of N2 and the greenhouse gas nitrous oxide (N2O). This thesis focused on understanding the impact of crop phases in a MZ-(SB-WW)-CC rotation on the abundance of N-cycling bacterial communities that mediate nitrification and denitrification pathways. In addition, the influence of CCs on soil cytokinin (CK) profiles, which are plant growth-promoting hormones, were studied in a greenhouse trial to assess their potential impacts when integrating CCs into crop rotations. In particular, the relationship between traditional soil health parameters and the soil CK profiles was studied to understand how CKs might reflect biotic interactions and soil vitality. Results indicate N fertilizer application mono ammonium phosphate (MAP) and starter N:P: K (24:6:24) during WW planting in fall largely supported nitrifying bacterial communities (amoA) and potentially contributed to NO3- leaching. Management of MZ, which included spring-applied MAP resulted in larger denitrifying (nirK) bacterial communities, increasing the potential risk of N-loss via emission of dinitrogen gas (N2) and greenhouse gas N2O. However, CC soils had significantly lower nirK than MZ, reflecting the importance of strong and deep root systems of CCs, which have a higher ability to scavenge the substrates for denitrifying communities (NO3-). This highlights the importance of growing CCs in reducing the potential risk for N-loss via leaching and denitrification. Additionally, in the greenhouse trial, the ability of CCs to affect CK was detected, highlighting the importance of integrating CC in crop rotations. This is particularly noteworthy, given that total CK profiles showed strong associations with traditional soil health parameters such as labile or active carbon and soil microbial community diversity. It was concluded that total soil CK can be used as a novel and dynamic soil health measure. Future research on quantifying N2O fluxes and levels of NO3- in leachates would provide a more precise understanding of the impact of different crop rotation phases on N-dynamics in these fields. Further studies on single or combined measures of soil CKs are warranted to develop its potential as a practical and effective soil health parameter. Author Keywords: Cover crops, Crop rotations, Cytokinin hormone, Nitrogen Cycle, qPCR, Soil health
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- Genome annotation, gene characterization, and the functional analysis of natural antisense transcripts in the fungal plant pathogen Ustilago maydis
- Ustilago maydis (DC) Corda is the causal agent of 'common smut of corn'. Completion of the U. maydis lifecycle is dependent on development inside its host, Zea mays. Symptoms of U. maydis infection include chlorosis and the formation of tumours on all aerial corn tissues. Within the tumours, thick-walled diploid teliospores form; these are the reproductive and dispersal agent for the fungus. U. maydis is the model to study basidiomycete biotrophic plant-pathogen interactions. It holds this status in part because of the completely sequenced 20.5 Mb genome; however, thorough genome annotation is required to fully realize the value of this resource. The research presented here improved U. maydis genome annotation through the analysis of cDNA library sequences and comparative genomics. These analyses identified and characterized pathogenesis-related genes, and identified putative meiosis genes. This enabled the use of U. maydis as a model for investigating 'host-induced' meiosis. Further, the cDNA library analyses identified non-coding RNAs (ncRNAs) and natural antisense transcripts (NATs). NATs are endogenous RNA molecules with regions complementary to a protein-coding transcript. Although NATs have been identified in a wide variety of mammals, plants, and fungi, very few have been functionally characterized. Over 200 U. maydis NATs were annotated by analyzing full-length cDNA sequences. NAT structural features were characterized. Strand-specific RT-PCR was used to detect NATs in U. maydis and in a related smut fungus, U. hordei. The data supported a common role for NATs in smut teliospore development, independent of the RNA interference pathway. Analysis of the expression of one U. maydis NAT, as-um02151, in haploid cells, led to a model for NAT function in U. maydis during teliospore dormancy. This model proposed NATs facilitate the maintenance of stored mRNAs through the formation of double-stranded RNA. In testing this model, it was determined that the deletion of two separate upstream regulatory regions, one of which contained a ncRNA (ncRNA1), altered NAT levels and decreased pathogenesis. These studies strengthened U. maydis as a model organism, and began the functional investigation of NATs in U. maydis, which identified a new class of fungal pathogenesis genes. Author Keywords: cDNA library analysis, genome annotation, mRNA stability, natural antisense transcripts, pathogenesis, Ustilago maydis
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- 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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- 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