Graduate Theses & Dissertations

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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
Fungal pathogen emergence
The emergence of fungal hybrid pathogens threatens sustainable crop production worldwide. To investigate hybridization, the related smut fungi, Ustilago maydis and Sporisorium reilianum, were selected because they infect a common host (Zea mays), can hybridize, and tools are available for their analysis. Hybrid dikaryons exhibited filamentous growth on plates but reduced virulence and limited colonization in Z. mays. Select virulence genes in the hybrid had similar transcript levels on plates and altered levels during infection of Z. mays relative to each parental dikaryon. Virulence genes were constitutively expressed in the hybrid to determine if its pathogenic development could be influenced. Little impact was observed in hybrids with increased expression of effectors known to modify host response and metabolism. However, increased expression of transcriptional regulators of stage specific pathogenic development increased the hybrid’s capacity to induce symptoms. These results establish a base for investigating molecular aspects of fungal hybrid pathogen emergence. Author Keywords: effectors, hybrid pathogenesis assays, Sporisorium reilianum, transcription factors, Ustilago maydis, virulence factors
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
Immunogenetic Responses of Raccoons and Skunks to the Raccoon Rabies Virus
Interactions between hosts and pathogens play a crucial role in their adaptation, evolution and persistence. These interactions have been extensively studied in model organisms, yet it is unclear how well they represent mechanisms of disease response in primary vectors in natural settings. The objective of my thesis was to investigate host-pathogen interactions in natural host populations exposed to raccoon rabies virus (RRV). RRV is endemic to North America, that causes acute encephalopathies in mammals and is commonly regarded as 100% lethal if untreated; however variable immune responses have been noted in natural reservoirs. In order to further understand variable immune responses to RRV, my thesis examined (i) potential immunogenetic associations to RRV using genes intimately associated with an immune response, (ii) the nature of immune responses triggered in the host after infection, and (iii) viral expression and genetic variation, to provide insight into factors that may influence RRV virulence. Immunogenetic variation of RRV vectors was assessed using major histocompatibility complex (MHC) DRB alleles. Associations were found between specific MHC alleles, RRV status, and viral lineages. Further, similarities at functionally relevant polymorphic sites in divergent RRV vector species, raccoons and skunks, suggested that both species recognize and bind a similar suite of peptides, highlighting the adaptive significance of MHC and contemporary selective pressures. To understand mechanisms of disease spread and pathogenesis, I screened for variation and expression of genes indicative of innate immune response and patterns of viral gene expression. RRV activated components of the innate immune system, with transcript levels correlated with the presence of RRV. These data indicate that timing of the immune response is crucial in pathogenesis. Expression patterns of viral genes suggest they are tightly controlled until reaching the central nervous system (CNS), where replication increases significantly. These results suggest previous molecular mechanisms for rabies host response derived from mouse models do not strictly apply to natural vector populations. Overall my research provides a better understanding of the immunological factors that contribute to the pathogenesis of RRV in a natural system. Author Keywords: immune response, major histocompatibility complex, rabies, raccoons, skunks, virus
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:
Hormonal Algae
Based on an endogenous hormone study, three cytokinin type phytohormones; benzyladenine (BA), trans-zeatin (tZ) and methylthiol trans-zeatin (MeSZ), as well as abscisic acid (ABA) were exogenously added at three concentrations (10-7, 10-6 and 10-5 M) to cultures of Chlorella vulgaris in an attempt to alter growth rate, total lipid and fatty acid yields and fatty acid profile. Growth stimulation was highest at 10-6 M for BA, MeSZ and ABA and 10-5 M for tZ. All treatments caused changes in total lipid and fatty acid content, with BA causing an increase to lipid content. The most significant change in the fatty acid profile was observed with the addition of MeSZ at 10-7 and 10-6 M causing increases of 204% and 457% in linolenic acid respectively above the control. These results are novel and potentially highly impactful, as MeSZ has never been added exogenously to algae and may be used to stimulate overproduction of linolenic acid for pharmaceutical or industrial purposes. Author Keywords: Abscisic Acid, Chlorella vulgaris, Cytokinin, Fatty acid, Linolenic Acid, Methylthiol trans-Zeatin
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
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
Regulation of Cytokinins During Kernel Development in High and Low Yielding Oat and Barley Lines
Cytokinins (CKs) are a family of plant phytohormones responsible for many areas of plant growth and development. There are four free base types of CKs found in higher plants, trans-zeatin (tZ), N6-(∆2-isopentenyl)adenine (iP), cis-Zeatin (cZ) and dihydrozeatin (DZ). CK biosynthesis is regulated by adenosine phosphate-isopentenyltransferase (IPT), which is encoded by a multi-gene family in many plant species. There are two types of IPT pathways responsible for CK production, the tRNA pathway and the AMP (ATP/ADP) pathway. The tRNA pathway putatively produces cZ and the latter predominantly produces iP type nucleotides. CKs have long been studied for their role in stress tolerance, signal transduction, and involvement in many areas of plant growth and development. This study focuses on the role of CKs and CK biosynthesis by IPT during kernel development and comparisons of its regulation in high and low yielding barley and oat lines. The sequence of a putative IPT encoding gene in barley and oat was identified by a blast search of other known IPT gene fragments in closely related species. Quantitative Real time PCR results based on primers designed for the putative barley and oat IPT gene revealed changes in expression of IPT during different stages of kernel development, but no significance difference was associated with yield. Correlation of IPT gene expression in barley with cZ CK profiles measured by HPLC-MS/MS, confirms a putative IPT gene is a tRNA- IPT. HPLC-MS/MS results reveal some CK types, such as benzyladenine, are more predominant in higher yielding lines. This suggests different types of CKs play a role in yield production. Future studies on more IPT genes in the barley and oat IPT gene family will outline a more clear representation of the role of IPT in barley kernel development. Author Keywords: Benzyladenine, Cereal grain, Cytokinin, Isopentenyl Transferase, Mass Spectrometry, Real Time PCR
Differential expression of cytochrome b5s in Giardia intestinalis during nitrosative stress and encystation
The waterborne protozoan Giardia intestinalis cycles between the environmentally-resistant and infectious cyst and the metabolically-active trophozoite that adheres to the epithelial lining of the small intestine. Adhesion can trigger the innate immune response in epithelial cells, including the synthesis of the free radical nitric oxide (NO) that inhibits cell proliferation and encystation of trophozoites. In this work changes in protein expression of three Giardia isotypes of the redox heme protein cytochrome b5 (gCYTb5 I, II and III) were studied in response to either nitrosative stress or induction of encystation. Two nitrosative stressors, sodium nitrite and the NO donor DETA-NONOate, were used at sub-lethal concentrations (0.5 mM and 0.05 mM, respectively) that do not affect cell proliferation until later time points so that subtle changes in protein expression could be observed in the absence of other confounding factors. Nucleolar gCYTb5-I and nucleoplasmic gCYTb5-III expression patterns were similar in trophozoites exposed to either stressor, showing gradual increases in expression with peaks between 4 and 12 hours, which indicates these cytochromes respond to nitrosative stress and possibly to potential DNA damage in Giardia. In contrast, gCYTb5-II of the peripheral vacuoles, which are part of the endocytic pathway of Giardia, showed little change in expression in response to either stressor. However, changes in gCYTb5-II expression were observed in encysting trophozoites, with a 1.4-fold increase in protein levels at seven hours after induction of encystation, followed by a gradual decrease in expression. These changes are consistent with previous mRNA analysis done in our laboratory and suggest a role for gCYTb5-II in the increase in nutrient uptake during early encystation. Author Keywords: cytochrome, encystation, Giardia, heme, nitrosative, parasite
Fungi and Cytokinins
Cytokinin biosynthesis in organisms aside from plant species has often been viewed as a byproduct of tRNA degradation. Recent evidence suggests that these tRNA degradation products may actually have a role in the development of these organisms, particularly fungi. This thesis examines the importance of cytokinins, a group of phytohormones involved in plant cell division and differentiation as well as the phytohormone abscisic acid, involved in plant response to environmental factors, and their presence and role in fungi. An initial survey was conducted on 20 temperate forest fungi of differing nutritional modes. Using HPLC-ESI MS/MS, cytokinin and abscisic acid were detected in all fungi regardless of their mode of nutrition or phylogeny. The detection of the same seven CKs across all fungi suggested the existence of a common CK biosynthetic pathway and dominance of the tRNA pathway in fungi. Further, the corn smut fungus Ustilago maydis is capable of producing CKs separate from its host and different U. maydis strains induce disease symptoms of differing severity. To determine if CK production during infection alters disease development a disease time course was conducted on cob tissue infected with U. maydis dikaryotic and solopathogenic strains. Dramatic changes in phytohormones including an increase in ABA followed by increases in cisZCKs were detected in tumour tissue particularity in the more virulent dikaryon infection, suggesting a role for CKs in strain virulence. Mining of the U. maydis genome identified a sole tRNA-isopentenyltransferase, a key enzyme in CK biosynthesis. Targeted gene deletion mutants were created in U. maydis which halted U. maydis CK production and decreased pathogenesis and virulence in seedling and cob infections. CK and ABA profiling carried out during disease development found that key changes in these hormones were not found in deletion mutant infections and cob tumour development was severely impaired. These findings suggested that U. maydis CK production is necessary for tumour development in this pathosystem. The research presented in this thesis highlights the importance of fungal CKs, outlines the dominant CK pathway in fungi, identifies a key enzyme in U. maydis CK biosynthesis and reveals the necessity of CK production by U. maydis in the development of cob tumours. Author Keywords: abscisic acid, cytokinins, high performance liquid chromatography-electrospray ionization tandem mass spectrometry, tRNA degradation pathway, Ustilago maydis, Zea mays
Development of genetic profiles for paternity analysis and individual identification of the North Atlantic right whale (Eubalaena glacialis)
The endangered North Atlantic right whale (Eubalaena glacialis) has been internationally protected from whaling since 1935 but recovery has been slow compared to the southern right whale (Eubalaena australis) due to anthropogenic mortalities and poor reproduction. Prey availability, genetic variability, and alleles of genes associated with reproductive dysfunction have been hypothesized to contribute to low calf production. The North Atlantic Right Whale DNA Bank and Database contains 1168 samples from 603 individuals. I added 115 new genetic profiles to the database which now contains profiles for 81% of individuals alive since 1980. Paternity assignments using these profiles resulted in 62% of sampled calves being assigned a father and only 38% of candidate males being assigned a paternity. This may suggest false exclusion due to genotyping errors or the existence of an unknown group of males. The use of the DNA database allowed for the identification of 10 deceased individuals which has implications for identifying cause of death and reducing mortalities. However, genetic identification is dependent on the time of post-mortem sample collection which influences DNA quantity and quality. An assessment for variations in methylenetetrahydrofolate reductase, a candidate gene associated with reproductive dysfunction, revealed six females heterozygous for a synonymous A/T variant in exon four which may influence reproductive success through changes in enzyme production, conformation or activity. Author Keywords: Eubalaena glacialis, Forensic Identification, Genetic Profiling, North Atlantic Right Whale, Paternity, Reproductive Dysfunction

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

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