Graduate Theses & Dissertations


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
Geochemistry and Toxicity of a Large Slag Pile and its Drainage Complex in Sudbury, Ontario
This study was designed to determine the geochemistry and potential toxicity of water draining a large slag pile in Sudbury, Ontario, which runs through a pond complex prior to entering Alice Lake. Slag leaching experiments confirmed slag is a source of sulphate, heavy metals (including Fe, Al, Ni, Co, Cu, Zn, Pb, Cr, Mn) and base cations (Ca, K, Mg, Na). Concentrations of most metals draining through slag in column experiments were similar to metal concentrations measured at the base of the slag pile, although base cations, S and pH were much higher, possibly because of water inputs interacting with the surrounding basic glaciolacustrine landscape. The increase in pH rapidly precipitates metals leading to high accumulation in the surface sediments. Away from the base of the pile, an increase in vegetation cover leads to an increase in DOC and nutrients and transport of metals with strong binding affinities (Cu). Total metal concentration in water and sediment exceed provincial water quality guidelines, particularly near the slag pile, however WHAM7 modeling indicated that the free metal ion concentration in water is very low. Nevertheless, toxicity experiments showed that water with greater concentrations of solutes collected close to the slag pile negatively impacts D. magna suggesting that water draining the slag pile can adversely impact biota in nearby drainage areas. Author Keywords: geochemistry, heavy metals, leaching, non-ferrous slag, precipitation, toxicity
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
Mercury and Persistent Organic Pollutants in Remote Acid Sensitive Irish Lake Catchments
A catchment-based study was carried out at three remote acid sensitive Irish lakes to determine concentrations of Hg and POPs and to investigate the factors governing the partitioning of these pollutants in various environmental matrices. Both Hg and POPs are an environmental concern due to their ability to travel long distances via atmospheric transport and their tendency to accumulate in biota and in various environmental compartments. Concentrations of POPs and Hg measured in this study were relatively low and consistent with concentrations measured at background levels around the world. Mercury concentrations appeared to be influenced by various site characteristics, specifically organic matter. Many of the POPs examined in this study appeared to be present as a result of long-range transport and more specifically; the physico-chemical properties of POPs appeared to dictate their distribution within soils, moss and sediment at each of the study catchments. Author Keywords:
Time-dependent effects of predation risk on stressor reactivity and growth in developing larval anurans (Lithobates pipiens)
The predator vs. prey dynamic is an omnipresent factor in ecological systems that may drive changes in life history patterns in prey animals through behavioural, morphological, and physiological changes. Predation risk can have profound effects on the life history events of an animal, and is influenced by the neuroendocrine stress response. Activation of the hypothalamic-pituitary-adrenal/interrenal axis, and the induction of stress hormones (e.g., corticosterone (CORT)) have been shown to mediate the onset of inducible anti-predator defensive traits including increased tail-depth, and reduced activity. The predator-prey relationship between dragonfly nymphs and tadpoles can be a powerful model system for understanding mechanisms that facilitate changes in the stress response in accordance with altered severity of risk. It has been well demonstrated early in tadpole ontogeny that increased corticosterone (CORT) levels, observed within three weeks of predator exposure, are correlated with increased tail depth morphology. However, the reactivity of the stress response in relation to the growth modulation in developing prey has yet to be fully explored. Accordingly, this thesis assessed the stress and growth response processes in tadpoles that were continuously exposed to perceived predation risk later in ontogeny. Continuous exposure of prey to predation risk for three weeks significantly increased CORT levels, and tail depth. However, tadpoles exposed to six weeks of predation risk acclimated to the presence of the predator, which was observed as a significant reduction of stressor-induced CORT levels. In addition, although increased tail depth has been attributed to predator defense, predator-naïve tadpoles began to display similar tail depth morphology as treated tadpoles at the six week time point. Thus, this thesis suggests that the stress response in lower vertebrate systems (e.g., tadpoles) may operate in a similarly complex manner to that observed in higher vertebrates (e.g., rats), for which severity of risk associated with the stressor aids in defining activity of the stress response. Moreover, the lack of morphological difference between treatments among tadpoles exposed later in ontogeny suggests that the mechanisms for inducing defenses are normal morphological traits in the development of the animal. This thesis paves the way for future research to elucidate the relationship between the neuroendocrine stress response and hormonal pathways involved in growth modulation in the presence of environmental pressures. Author Keywords: Acclimation, Corticosterone, Growth Modulation, Predation Risk, R. pipiens, Tadpole
Executive Function as a Predictor of Emotional, Behavioural, and Social Competence Problems in Children with Epilepsy
The study aimed to examine the association between different components of executive function (EF) and emotional, behavioural, and social competence problems (EBSP) in children with epilepsy. Although there is evidence of an association between EBSP and EF in typically developing children, little research has examined this relation in children with epilepsy. The sample comprised of 42 children with epilepsy, aged 6.0 to 18.1 years old. Results showed that EBSP were associated with EF in these children; however, different components of EF were related to different EBSP. Shifting was a significant predictor of emotional, behavioural, and social competence problems in children with epilepsy, whereas inhibition was a significant predictor of behavioural problems. This suggests that children with epilepsy, with different EF profiles may be at-risk for developing different types of problems. These results may aid researchers and clinicians with the development of new techniques to identify and treat children with EBSP. Author Keywords: behavioural problems, emotional problems, epilepsy, executive function, social competence
third wheel
Population cycles are regular fluctuations in population densities, however, in recent years many cycles have begun to disappear. With Canada lynx this dampening has also been seen with decreasing latitude corresponding to an increase in prey diversity. My study investigates the role of alternate prey on the stability of the lynx-hare cycle by first comparing the functional responses of two sympatric but ecologically distinct predators on a primary and alternate prey. I then populated a three species predator-prey model to investigate the role of alternate prey on population stability. My results showed that alternate prey can promote stability, though they are unlikely to “stop the cycle”. Furthermore, stability offered by alternate prey is contingent on its ability to increase intraspecific competition. My study highlights that population cycles are not governed by a single factor and that future research needs to be cognizant of interactions between alternate prey and intraspecific competition. Author Keywords: alternate prey, Canis latrans, functional response, Lepus americanus, Lynx canadensis, Tamiasciurus hudsonicus
Stopover Movement Patterns by Blackpoll and Canada Warblers Across Southeastern Canada During Fall Migration
Stopover ecology is a topic that surges in relevancy as choices made by migrants during stationary periods (stopover sites) may not only have important individuals’ fitness consequences but also can affect population dynamics. I used MOTUS automated telemetry array to study fall stopover duration of Blackpoll Warbler (BLPW) and departure decisions of BLPW and Canada Warbler (CAWA) in relation to various predictors. I affixed radio-transmitters on 55 BLPWs and 32 CAWAs at two banding stations in Ontario in September-October 2014-2015. Radio-tagged individuals were tracked through the MOTUS network across southeastern Canada. I developed models relating age class, fat score, Julian date and stopover movement types to Blackpolls’ stopover duration. I also examined whether there were species-related differences of wind selectivity when resuming migration. No explanatory variable significantly influenced BLPW’s stopover duration. Both species tended to depart under increased tailwind assistance, but with no difference in the effect of wind conditions between the two species. This study provides further evidence supporting the relevance of local wind conditions as a key factor affecting the departure likelihood, especially when migrating birds face an ecological barrier. Author Keywords: Cardellina canadensis, departure decisions, minimum stopover length, MOTUS, overland fall migration, Setophaga striata
Investigation of a Model for Stain Selection and a Robust Estimation for Area of Origin in Bloodstain Pattern Analysis
Bloodstain pattern analysis is fundamental to crime scene investigations by providing insight into the estimation of the area of origin of a blood source. The selection of bloodstains from an impact pattern is the foundation from which the estimation of the area of origin is derived. The purpose of this research was to develop a theoretical model for stain selection and establish a more robust estimation for the area of origin in the analysis of impact patterns. This model was developed by using quantitative data from marked zones on the impact pattern target surface. The minimum distance between the known blood source location and a single bloodstain line of trajectory was calculated for every bloodstain within each pattern investigated. It has been determined that the zone location of a bloodstain is relevant for improving accuracy, and that stains with all alpha angles can be used to calculate an area of origin estimation. Finally, research within this thesis also validated directional analysis and investigated the number of stains that may be required for a precise area of origin estimation. Keywords: Forensic science, Bloodstain, Impact pattern, Bloodstain pattern analysis, Statistics, Forensic Physics
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
Electrochemical Characterization of Giardia Intestinalis Cytochromes b5
Giardia intestinalis is a protozoan parasite that causes waterborne diarrheal disease in animals and humans. It is an unusual eukaryote as it lacks the capacity for heme biosynthesis; nonetheless it encodes heme proteins, including three cytochrome b5 isotypes (gCYTB5s) of similar size. Homology modelling of their structures predicts increased heme pocket polarity compared to mammalian isotypes, which would favour the oxidized state and lower their reduction potentials (E°’). This was confirmed by spectroelectrochemical experiments, which measured E°’ of -171 mV, -140 mV and -157 mV for gCYTB5-I, II, III respectively, compared to +7 mV for bovine microsomal cytochrome b5. To explore the influence of heme pocket polarity in more detail, five gCYTB5-I mutants in which polar residues were replaced by nonpolar residues at one of three positions were investigated. While these substitutions all increased the reduction potential, replacement of a conserved tyrosine residue at position-61 with phenylalanine had the most significant effect, raising E°’ by 106 mV. This tyrosine residue occurs in all gCYTB5s and is likely the greatest contributor to their low reduction potentials. Finally, complementary substitutions were made into a bovine microsomal cytochrome b5 triple mutant to lower its reduction potential. These not only lowered the E°’ by more than 140 mV but also weakened the interaction of heme with the protein. The lower reduction potentials of the gCYTB5s may indicate that these proteins have different roles from their more well-known mammalian counterparts. Author Keywords:
Comparison of the Optical Properties of Stratiotes aloids and the Local Plant Community
As part of a mandate to control the spread of Stratiotes aloides (WS; water soldier) in the Trent Severn Waterway, the Ministry of Natural Resources (MNR) created a management plan to eradicate WS. However, one of the biggest challenges in eradicating WS or any invasive aquatic plant is the ability to estimate the extent of its spread and detect new populations. While current detection methods can provide acceptable detection, these methods often require extensive time and effort. The purpose of this thesis was to assess the use optical properties of WS and WS exudates for detection, in order to improve on current detection methods. The optical properties of WS were sampled at three different sites during three different seasons (spring, summer, and fall) by a) randomly sampling tissue from WS and the local plant community at each site, and recording the reflectance properties in a laboratory setting b) collecting dissolved organic matter (DOM) samples from plant incubations and river water in the field. Significant differences in the reflectance properties of WS were observed among samples from different sites and different sampling times; however, changes in fluorescence properties were only seasonal. Despite spatial differences in WS reflectance; WS was detectable using both hyperspectral and multispectral reflectance. When hyperspectral reflectance was used, significant differences between WS and the local plant community were found in June using two bands (i.e. bands 525 and 535, R 2 = 0.46 and 0.48, respectively). Whereas multispectral reflectance was significant different in October using the coastal and blue band. While WS produced a unique signal using both reflectance types, multispectral reflectance had a greater potential for detection. Its greater potential for detection was due to the reduced noise produced by background optical properties in October in comparison to June. DOM derived from WS was also characterized and compared with whole-river DOM samples in order to find unique markers for WS exudates in river samples. While similarities in DOM concentrations of WS exudates to Trent River water limited the ability to detect WS using compositional data, the ratio of C4/C5 components were compared in order to find components that were proportionally similar. Based on the results of this study multispectral and fluorescence techniques are better suited for the detection of a unique WS signature. The results derived from this work are intended to have practical applications in plant management and monitoring, DOM tracing, as well as remote sensing. Author Keywords: Dissolved organic matter, Hyperspectral reflectance, Invasive species management, Multispectral reflectance, PARAFAC, Stratiotes aloides


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