Saville, Barry

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

Iridoviruses are large (120-200nm) double stranded DNA viruses that contain an icosahedral capsid. The iridoviridae family is composed of five genera that infect a wide range of poikilothermic vertebrates (Lymphocystivirus, Ranavirus and Megalocyivirus) and invertebrate hosts (Iridovirus, Chloriridovirus). Frog virus 3 (FV3) is a member of the Ranavirus genus, and is commonly used as a model system to study iridoviruses. I was interested in understanding virus-host interaction in FV3. I studied two viral genes, FV3 97R and FV3 75L. Here I demonstrate that 97R localizes to the endoplasmic reticulum (ER) at 24 hours post-transfection. However, at 35 hours post-transfection 97R localizes to the ER but also begins to form concentrated pockets, continuous with the nuclear membrane This study found that 97R possess a unique phenotype and that its localization to the ER is mediated through its C-terminus transmembrane domain. FV3 75L encodes an 84 amino acids protein. I showed that FV3 75L localizes to the early endosomes, while its cellular binding partner, LITAF, localizes to late endosome/lysosome. Interestingly, when FV3 75L and LITAF are co-transfected into cells, LITAF can alter the subcellular localization of FV3 75L to late endosome/lysosomes. A physical interaction between LITAF and FV3 75L was demonstrated through a pull-down assay and that a highly conserved domain found in both proteins may mediate the interaction. LITAF has been proposed to function in protein degradation, but there is still uncertainty on LITAF's specific role. I was interested in further characterizing LITAF and its implications in protein degradation and a neurodegenerative disorder. At least 9 mutations of LITAF are associated with Charcot-Marie-Tooth disease type 1C (CMT1C), which belongs to the group of most common heritable neuromuscular disorders, affecting approximately one in 2500 people. We show that LITAF mutants G112S and W116G mislocalize from the late endosome/lysosome to the mitochondria while the T49M and P135T mutants show partial mislocalization with a portion of the protein present in the late endosome/lysosome and a portion of the protein localized to the mitochondria. Since LITAF is believed to play a role in protein degradation, it is possible that the specific characteristics of CMT1C may occur though impaired degradation of Schwann cell membrane proteins, such as PMP22. I was able to show that when WT LITAF is present, there is a decrease in the PMP22 intracellular levels, which suggest that LITAF plays an important role in protein degradation, and also in other types of CMT. Insight into how mutations in LITAF cause CMT1C may not only help better understand cellular pathways, but also further elucidate the role LITAF's viral homolog FV3 75L during viral infection.

Author Keywords: 75L, Charcot-Marie-Tooth, CMTC1, ER, FV3, LITAF

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

Interseeding legume cover crops in grain corn may improve the environmental sustainability of corn production system in Southern Ontario. This study aimed to assess the effects of legume species, nitrogen (N) fertilizer rate and arbuscular mycorrhizal fungi (AMF) inoculation on biomass and N requirement in a corn-legume system. Corn was grown with red clover (RCl), microclover (MCl), hairy vetch (HV), or beans at 10 and 80 kg N ha-1 rates with and without AMF inoculation in a greenhouse for 7 weeks. Corn dry matter (DM) and N uptake were reduced by beans and HV (average 35%) compared with control; however, the DM for beans and HV was 7 and 3 times higher than RCl and MCl, respectively. The N2 fixation ability was similar among legume species and no significant N transfer from legume was detected. Overall, species collection was critical to the success of incorporating legumes into grain corn production.

Author Keywords: Arbuscular mycorrhizal fungi, corn, legume cover crop, nitrogen

Moose are an iconic species, known for their large size and impressive antlers. Eight subspecies are classified in circumpolar regions of the planet - four in North America. Two subspecies are similar in shape and size, the north-western moose (Alces alces andersoni) and the eastern moose (Alces alces americana). It was previously believed that these two subspecies meet in northern Ontario. Earlier genetic population studies used a small number of samples from Ontario, primarily in broad studies covering all of North America.

A comprehensive genetic study of moose populations in Ontario has not previously been conducted. We examined the genetic diversity and population structure at 10 polymorphic loci using 776 samples from Ontario, as well as outgroups from representative populations – Manitoba/Cape Breton, representing A. a. andersoni, and New Brunswick/Nova Scotia, representing A. a. americana. Results indicated three genetic populations in the province, in north-western Ontario, north-eastern Ontario and south-central Ontario. RST values, compared against both FST and Jost's D values for phylogenetic analyses, indicated no phylogenetic pattern which suggests no subspeciation present in the province.

Population movement patterns in Ontario were studied. Gene flow was estimated using genetic and spatial data. Isolation by distance was only seen within the first distance class of 100 kilometres and then not seen again at further distances, indicating that moose display philopatry. There were very few migrants travelling across the province, with a greater number moving gradually north and west, towards better habitat and food sources.

A forensic database in the form of an allele frequency table was created. Three loci showed very low levels of heterozygosity across all three populations. Probability of identity was calculated for the three populations and quantified. Samples with known geographic origins were run against the database to test for sensitivity, with identification of origin occurring at an accuracy level between 87 and 100%.

Within Ontario, there are not two different subspecies, as previously believed, but two different populations of the same subspecies meeting in northern Ontario. The genetic data does not support previous research performed in Ontario. The sample sizes in our research also provide a more comprehensive view of the entire province not seen in any previous studies. The comprehensive research enabled the building of a reliable forensic database that can be used for both management and forensic purposes for the entire province.

Author Keywords: Alces alces, Genetic Diversity, Moose, Ontario, Phylogeography, Subspecies

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

Trinucleotide repeats (TNRs) are a class of highly polymorphic microsatellites which occur in neutral and non-neutral loci and may provide utility for individual- and population-identification. Exonic trinucleotide motifs, in particular, offer additional advantages for non-human species that typically utilize dinucleotide microsatellite loci. Specifically, the reduction of technical artifacts, greater separation of alleles and greater specificity of amplification products leading to more efficient multiplexing and cross-taxa utilization. This study aims to identify and characterize polymorphic trinucleotide repeats and conserved primer sequences which are conserved across Cervidae (deer) species and their potential for individual identification in forensic wildlife investigations. Chapter one provides a broad introduction to trinucleotide microsatellites, chapter two deals with data-mining TNRs and chapter three applies the identified TNRs as genetic markers for individual identification. Results demonstrate proof-of-concept that exonic TNRs are capable of giving random match probabilities low enough to be employed in individual identification of evidentiary samples.

Author Keywords: DNA typing, Exons, Genetic Markers, Individual Identification, Trinucleotide, Wildlife Forensics

Phytohormones, Cytokinin (CK) and Abscisic acid (ABA), are best known for controlling plant growth and stress responses; but they also mediate various developmental perspectives in alga. Yet, their mode of action in algal adaptive strategies to heavy metal responses, their involvement in orchestration of phytohormone crosstalk remain largely unknown and a molecular framework of phytohormone-controlled heavy metal uptake is absent. I found that three metals known globally to contaminate aquatic ecosystems, nickel (Ni), lead (Pb), and cadmium (Cd), cause changes in the levels of endogenous CKs, ABA, auxins, and gibberellins (GAs) in the green alga Euglena gracilis. Exogenous ABA or CK (trans-zeatin) alleviated metal toxicity through improved metal uptake efficiency and the regulation of the endogenous CKs activity profiles and GAs activity. This new evidence suggests that E. gracilis possesses functional phytohormone signals and metabolic pathways that are under metal stress response. Exogenously applied ABA or CK provoked the coordinated activation of metal uptake, likely via enhanced accumulation of metal binding compounds (i.e., proline, glycine, cysteine containing peptides), which are effective for metal sequestration. Using untargeted metabolomics analysis and functional annotation, this thesis further established that, CK and ABA modified pathways and metabolites, which were mainly involved in metal acclimation and resistance. These modified metabolites that were under the influence of phytohormones in algal cells growing under metal stress conditions were associated with: lipid pathways, riboflavin metabolism, biosynthesis of cofactors/vitamin, and carbohydrate metabolism. Bioactive secondary compounds (e.g., terpenoids, alkaloids, flavonoids, carotenoids) were also modified in algal cells treated with phytohormones. The present study highlights that ABA and CKs are important regulators of algal metal accumulation/acclimation strategies based on increased metal uptake, enhanced CK metabolism, regulation of hormonal crosstalk and regulation of some core cellular metabolism pathways, all of which improve metal uptake efficiency. Finally, our results suggest that ABA and CK form a novel strategy for metal bioremediation techniques and for sourcing microalgal value-added metabolites.

Author Keywords: abscisic acid, cadmium, cytokinin, Euglena gracilis, lead, nickel