Candida albicans is an opportunistic fungal pathogen. It is the fourth leading cause of nosocomial infections and can endanger immunocompromised patients. Candida has the ability to form biofilms on plastic medical devices, such as catheters and central nervous system shunts. Two clinical isolate series were profiled using a number of phenotyping assays comprising in vivo, ex vivo, and in vitro tests. These tests shed light on host-pathogen relations as well as offer potential information useful in the treatment of these infections. Fluconazole, an antifungal, is the first line of treatment for fungal infections. The incidence of fluconazole-resistant infections is increasing annually, and there are not many other drugs available to treat infections. In 2013, Fazly et al. discovered the drug Filastatin, which prevents adhesion and filamentation of Candida albicans. In our study, two screens were performed to identify the target of Filastatin. Because there is no complete knockout library for Candida, an available, partial knockout library was screened. This library is enriched for transcription factors. We screened for regulators of biological pathways that may be important for adhesion and filamentation in Candida albicans, to identify potential Filastatin targets. The iron-uptake pathway was chosen as the focus for the remainder of this study.

Bibliography: leaves 155-180.

This inquiry investigated the suitability of the jaw of domestic sheep as an animal model for dental implantology research. Initially, parameters for osseous healing of critical size defects (CSD) in the sheep mandible were established. Pilot studies were conducted using machined-surface implants and a surgical protocol established for dental implant placement in ovine mandibular sites. Subsequent experiments considered the utility of this animal model for examination of techniques designed to enhance osseointegration. Hydroxyapatite-coated implants were compared with titanium plasma-sprayed (TPS) implants, either alone or combined with autogenous bone grafts or a bone graft/collagen vehicle loaded with transforming growth factor-beta (TGF-β). Immunofluorescent bone labelling gave information on the mineral apposition rate (MAR). Implant survival and "acceptability" (likelihood of clinical success) were major output variables, along with histomorphometric analysis of percent bone-implant contact (%BIC) and percent peri-implant bone density (%density). Naturally-occurring "broken-mouth" periodontitis in sheep was identified as a potential confounder. Subsequent experiments considered implants with different surfaces. The model was also extended from a two-stage surgical protocol to include single-stage implants. The effect of pre-existing ovine peridontitis was also examined. A systematic review and meta-analysis of published animal implant experiments was conducted in order to validate the candidate sheep model. Major findings were as follows. The size of non-healing sheep mandibular unicortical CSD is >12mm. Attempts to establish a chronic non-healing CSD were unsuccessful. The sheep diastema proved unsuitable for implant placement. The model was modified to a post-extraction protocol. Implant "acceptability" rates after 3 months integration in the sheep mandible (defined as implant survival with %BIC >10%) ranged from 50% - 100% for different implant surface treatments and placement protocols. Histomorphometriic analyses revealed that %BIC ranged from 11 � 17% to 81 � 29 % for different titanium surfaces and up to 85 � 11% for hydroxyapatite surfaces. Implants with TGF-β plus autogenous bone grafts had %BIC of 36 � 30% compared with 43 � 30% for implants with grafts alone. Bone per unit area (%density) adjacent to, but outside of the implant threads, ranged from 63 � 16% to 86 � 3% and was markedly lower for titanium plasma-sprayed surfaces and for one-stage implants. Within the implant threads, %density varied from 31 � 33% to 73.4 � 8.3%, and was markedly lower for machined titanium surfaces. Sheep periodontitis had little effect on the protocols investigated. The sheep mandibular model was found to be comparable to similar models in other species and merits further development.

The aim of this thesis was to investigate neuronal cellular mechanisms that may underlie the behavioural characteristics of the spontaneously hypertensive rat strain (SHR). The SHR was developed by selective breeding for elevated blood pressure and is also described as having increased levels of locomotor behaviour compared to its normotensive control strain, the Wistar-Kyoto. This hyperactivity and other behaviours, including altered sensitivity to reinforcement, have been used to model aspects of behaviour displayed in attention deficit hyperactivity disorder. In vivo intracellular recording of striatal spiny projection neuron activity in urethaneanaesthetised animals from three genetically related strains: the SHR, Wistar-Kyoto and standard Wistar, was employed to measure basic cellular properties and cellular mechanisms of reward-related learning. This population of neurons was chosen because alterations in their activity can influence behaviour and they are known to show cellular changes (synaptic plasticity) that are associated with learning. Cellular properties were measured in 71 neurons. Comparison between strains revealed a significant difference in action potential amplitude and duration between the SHR and Wistar-Kyoto strains. Interestingly, when measured at a later time, in a different sample of rats, the SUR action potential amplitude and duration were significantly different from the earlier sample. A change in the membrane potential repolarisation rate following action potential firing also occurred over this time. Twenty-nine of these neurons were also used in a study investigating the neuronal responses to a low dose of amphetamine (0.5 mg/kg). Changes were observed in some cellular properties following intraperitoneal administration of amphetamine. Synaptic plasticity at the corticostriatal synapses is sensitive to the timing of dopamine release in relation to cortical input. In anaesthetised preparations the spiny projection neuron membrane potential fluctuates between hyperpolarised (DOWN) and depolarised (UP) states, which reflect the level of cortical input. During the present study the responses of nine neurons to the induction of cortical spreading depression were observed to investigate the suitability of this method for use during synaptic plasticity experiments. Spiny projection neurons showed unpredictable responses to cortical spreading depression, therefore this method was not used further. Corticostriatal synaptic plasticity was induced in sixteen spiny projection neurons from two strains: SHR and Wistar. High frequency stimulation of the dopamine neurons in the substantia nigra, during the DOWN-state, did not induce any significant changes in corticostriatal synaptic efficacy. This was also true when high frequency stimulation of dopamine neurons was applied during the UP-state in neurons from the SHR strain. This thesis represents the first in vivo intracellular study of neuronal physiology in the SHR and Wistar-Kyoto rat strains. Results revealed action potential differences between these two behaviourally distinct rat strains. Synaptic mechanisms thought to underlie reward-related learning were not different between the SHR and Wistar strains, although the observed levels of plasticity were inconsistent with previous literature.

No description available.

Parkinson’s disease (PD) is the most common movement neurodegenerative disease affecting approximately 1% of the population over 60. Though originally thought to be sporadic in nature, a genetic component is increasingly being linked to the disease. Of these genes, mutations in DJ-1 (PARK7) cause early onset autosomal recessive PD. Initial workup of the DJ-1 protein has suggested that it may act in the cell by combatting oxidative stress though the mechanism by which it does so is unclear. Thus, though much work has attempted to elucidate a function at the biochemical, cellular and organismal level, the overt physiological role of DJ-1 remains elusive. In this dissertation, we explore the mechanisms through which DJ-1 confers neuroprotection, particularly in the case of oxidative stress insult. We demonstrate that DJ-1 acts through the pro-survival protein AKT to accomplish its neuroprotective function. Moreover, we note that DJ-1 likely serves its role as an antioxidant through the NRF2 master antioxidant regulator pathway a pathway that is, itself, likely to be regulated by AKT. Together, our results demonstrate that neuroprotection by DJ-1 is done through a signaling pathway involving both AKT and NRF2 and that disruption of the former in PD likely results in abolishing this signaling pathway. Finally, to generate a better animal model of PD, we demonstrate that backcrossing DJ-1 null mice - which originally did not demonstrate any gross histopathological or behavioral phenotypes – display unilateral dopaminergic degeneration that progresses to bilateral degeneration with aging, a feature reminiscent of classical PD progression. Collectively, this thesis takes a two-sided approach to address the biochemical and physiological functions of DJ-1 within the cell and the mouse in hopes of elucidating mechanisms of neuronal death to devise better translational therapies.

Ph.D.

The work described in this dissertation aims to understand how postural instability (PI), a troubling symptom of advanced Parkinson's disease (PD) in humans, develops from the degeneration of nigrostriatal dopamine neurons characteristic of PD. The studies herein (1) outline the development of clinically relevant methods for evaluating PI in experimental rodents, (2) indicate that PI may not result directly from disruption of dopamine systems but may instead arise from non-dopaminergic changes that occur subsequent to dopamine depletion, and (3) search for specific evidence of plasticity or degeneration outside of the damaged nigrostriatal dopamine system that may be linked to the development of PI. It is hoped that this work will help lay the foundation for the development of novel prophylactic treatments aimed at preventing the progression of PD to advanced stages where treatment-resistant symptoms such as PI appear. / text

published_or_final_version / Physiology / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences