Molecular biology

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

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

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

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.

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

Assessments of the adaptive potential of natural populations are essential for understanding and predicting responses to environmental stressors like climate change and infectious disease. The range of stressors species face in a human-dominated landscape, often have contrasting effects. White-tailed deer (Odocoileus virginianus, deer) are expanding in the northern part of their range following decreasing winter severity and increasing forage availability, caused by climate change. Chronic wasting disease (CWD), a prion disease affecting cervids, is likewise expanding and represents a major threat to deer and other cervids We obtained tissue samples from free-ranging deer across their native range in Ontario, Canada which has yet to detect CWD in wild populations of cervids. High throughput sequencing was used to assess neutral genomic variation and variation in the gene responsible for the protein that misfolds into prions when deer contract CWD, known as the PRNP gene. Neutral variation revealed a high number of rare alleles and no population structure, consistent with an expanding population of deer. Functional genetic variation revealed that the frequencies of variants associated to CWD susceptibility and disease progression were evenly distributed across the landscape and the frequencies were consistent with deer populations not infected with CWD. These findings suggest that an observable shift in PRNP allele frequencies likely coincides with the start of a novel CWD epidemic. Sustained surveillance of genomic and genetic variation can be a useful tool for CWD-free regions where deer are managed for ecological and economic benefits.

Author Keywords: Canadian wildlife, population genetics, prion, PRNP, RADseq, ungulate

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

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

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

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