Graduate Theses & Dissertations

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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
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
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
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
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
Comparative phylogeography in conservation biology
Phylogeographic histories of taxa around the Great Lakes region in North America are relevant to a range of ongoing issues including conservation management and biological invasions. In this thesis I investigated the comparative phylogeographic histories of plant species with disjunct distributions and plant species with continuous distributions around the Great Lakes region; this is a very dynamic geographic area with relatively recent colonisation histories that have been influenced by a range of factors including postglacial landscape modifications, and more recently, human-mediated dispersion. I first characterized four species that have disjunct populations in the Great Lakes region: (Bartonia paniculata subsp. paniculata, Empetrum nigrum, Sporobolus heterolepis, and Carex richardsonii). Through comparisons of core and disjunct populations, I found that a range of historical processes have resulted in two broad scenarios: in the first scenario, genetically distinct disjunct and core populations diverged prior to the last glacial cycle, and in the second scenario more recent vicariant events have resulted in genetically similar core and disjunct populations. The former scenario has important implications for conservation management. I then characterized the Typha species complex (T. latifolia, T. angustifolia, T. x glauca), which collectively represent species with continuous distributions. Recent microevolutionary processes, including hybridization, introgression, and intercontinental dispersal, obscure the phylogeographic patterns and complicate the evolutionary history of Typha spp. around the Great Lakes region, and have resulted in the growing dominance of non-native lineages. A broader geographical comparison of Typha spp. lineages from around the world identified repeated cryptic dispersal and long-distant movement as important phylogeographic influences. This research has demonstrated that comparisons of regional and global evolutionary histories can provide insight into historical and contemporary processes useful for management decisions in conservation biology and invasive species. Author Keywords: chloroplast DNA, conservation genetics, disjunct populations, invasive species, phylogeography, postglacial recolonisation
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
Expression and characterization of cytochrome b5 from Giardia lamblia
Giardia lamblia is an intestinal parasite found globally in freshwater systems that is responsible for endemic outbreaks of infectious diarrhea. As a unicellular parasite that lacks mitochondria, a respiratory chain and lives in the anaerobic environment of its host's intestine, Giardia was assumed for decades to lack heme proteins. However, its genome encodes several putative heme proteins, including three with sequence similarity to the cytochrome b5 family, referred to as Giardia cytochromes b5 (gCYTb5). Recombinant expression of one of these genes (gCYTb5-I), results in a protein (17-kDa) that is isolated with noncovalently bound heme. Resonance Raman and UV-visible spectra of gCYTb5-I in oxidized and reduced states resemble those of microsomal cytochrome b5, while sequence alignment and homology modelling supports a structure in which a pair of invariant histidine residues act as axial ligands to the heme iron. The reduction potential of gCYTb5-I measured by cyclic voltammetry is -165 mV vs the standard hydrogen electrode and is relatively low compared to those of other family members. The amino- and carboxy-terminal sequences that flank the central heme-binding core of the gCYTb5 are highly charged and do not occur in other family members. An 11-kDa core gCYTb5-I variant lacking these flanking sequences was also able to bind heme; however, we observe very poor expression of this truncated protein as compared to the full-length protein. Author Keywords: b-type cytochrome, cytochrome b5, electron transfer protein, Giardia intestinalis, heme/heam protein, spectroelectrochemistry
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
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
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
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

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