The remapping of cortical networks after stroke is hypothesized to be one of the mechanisms subserving functional recovery. Our understanding of cortical remapping remains limited due to the inability to resolve which cells are active while performing motor tasks with high temporal and spatial specificity. The experiments presented in the first chapter of this thesis evaluate the ability of the inducible Arc-CreERT2:Rosa-YFPf/f model to label cells in the motor cortex activated by a motor-related behaviour. Through the modification of previously published 4-hydroxytamoxifen treatment paradigms, this model can differentiate between animals that performed the rotarod task at two time points and home cage controls. In addition, 65% of cells active at the first behavioural time point are reactivated. Taken together, these data suggest that the Arc-CreERT2:Rosa-YFPf/f model is able to reliably label networks used to perform the same behavioural task at two time points. The second chapter of this thesis details a pilot study in which the Arc-CreERT2:Rosa-ChR2:YFPf/f model was used to test the effect of daily optogenetic stimulation of the contralateral cortex on functional recovery. The results of this chapter suggest that stimulating the contralesional motor cortex may impair functional recovery. Overall, the results of this thesis lay the foundation to use this model to investigate motor networks in both naïve and pathological conditions, such as stroke.

Reusability and reliability of published data are fundamental requirements for translating results from animal experiments into reliable clinical biomarkers. Current success rates for biomarker discovery are poor, and we need to develop new tools to collate, integrate and analyse datasets, such as knowledge bases, that would enable more effective translation from mouse to human or across disciplines. However, concerns about the validity, reproducibility and replicability of existing data must be addressed first. Here, I interrogate the quality of methods reporting in experimental models of infection and inflammation. Despite evidence that most of the assessed parameters, such as sex and age, can influence the experimental results, the quality of methods reporting was poor. Inadequate methods reporting means that it is not always possible to confirm whether findings were due to improper experimental conditions that biased the results. Thus, such inaccuracies would have an impact on the construction of knowledge base tools that require appropriate annotation. However, I provide reusable checklists that could improve the quality of methods reporting prior to publication and can be used to verify papers post-publication to enable researchers from different fields to interrogate published data. Another reason that biomarkers may fail is that it can be difficult to determine the causative pathways that will better predict disease outcome in a chronically inflamed tissue where multiple pathways are happening simultaneously. I conducted novel research to identify and verify whether investigating animal models long before onset of colitis would identify potentially causative biomarkers for colitis in an animal model. Previous studies have shown early influx of dendritic cells are associated with resistance to Trichuris muris-induced colitis in mice, suggesting early biomarkers may be detectable that might predict disease outcome in inflammatory diseases, such as inflammatory bowel disease (IBD).In the T. muris colitis model, I identified differences in gene expression of multiple components of the receptor for advanced glycation end-products (RAGE) activation pathway between colitis-resistant and colitis-susceptible mice, occurring just 24 hours post infection; before any observable clinical symptoms were present. RAGE is a receptor that binds products of damage, such as calprotectin, and initiates pro-inflammatory cascades. However, RAGE can be cleaved from the cell membrane to form a soluble receptor (sRAGE) that cannot mediate proinflammatory signals, yet can bind to damage products, effectively rendering them harmless. During a longer infection timecourse, colitis-resistant mice produced significantly more sRAGE during infection, consistent with an increased ability to prevent inflammatory ligands from activating membrane-bound RAGE. These findings were also supported by additional experiments using T. muris infection in Il-10-/- mice. In summary, I have carried out an analysis of methods reporting quality in immunology research that can help improve the reliability of existing data relevant to the further study of IBD and beyond. I have also identified sRAGE as a potential biomarker for the onset of colitis in the T. muris infection model, with implications for diagnosis and treatment of IBD in a clinical setting.

This thesis describes an attempt to increase the life span of mice after injection of a leukemic tumor. Fatty acids were used as a protecting agent against the leukemic tumor.

Diabetic wounds frequently undergo impaired and prolonged wound healing due to a multitude of factors including hypoxia, impaired angiogenesis, hyperglycaemia, formation of ROS and AGEs, and infection - all of which may lead to cellular dysfunction. To date, however, treatment options for individuals suffering from impaired diabetic wound healing are limited, non-specific, and generally unsuccessful. The search for new and effective treatment strategies is severely hampered by the availability of adequately characterized screening models which comprehensively mimic the complexity of the diabetic wound healing process. In order to explore natural products as potential therapeutics to treat diabetic wounds and to encourage more research on this topic, this study sought out to develop and characterize a more convenient and cost effective in vitro screening assay which mimics the effects of protein glycation on the healing process of diabetic wounds. As proof of principal, this model was subsequently used to screen the potential of five wild mushroom species (P. tinctorius, R. capensis, B. badius, P. ostreatus and G. lucidum) as suitable diabetic wound healing therapies. The glycated gelatin model developed during this study was found to suitably mimic the diabetic state as it successfully simulated the major cellular dysfunctions in macrophages (NO production, phagocytosis, macrophage polarization, NF-ĸB translocation and COX-2 expression) and fibroblasts (proliferation and migration) documented during diabetic wound healing. Together these findings provide confidence that the model may serve as a valuable tool to study the poorly understood mechanisms which characterize cellular dysfunction in response to AGE accumulation and also to aid the identification of novel therapeutic agents to treat this pathology. Screening a number of mushroom extracts revealed that the ethanol extracts of R. capensis and P. ostreatus had the greatest potential for attenuating chronic inflammation due to their ability to promote macrophage phagocytosis, increased M2 activation (R. capensis) and decreased M1 activation (P. ostreatus) as well as reduced COX-2 expression while the water extract of G. lucidum proved to be the most promising candidate for stimulating fibroplasia as it was the most successful at promoting both fibroblast proliferation and migration. Different mushroom species were thus shown to promote different stages of the wound healing process providing sufficient evidence to support further studies related to the use of macrofungi as therapeutic agents in the search for more cost-effective and efficient treatment strategies for impaired diabetic wound healing.

The nonrecurring-items delayed nonmatching-to-sample (DNMS) task is an integral part of contemporary monkey models of brain-damage-produced amnesia. This thesis began the development of a comparable rat model of brain-damage-produced amnesia. First, a DNMS task for rats was designed by adapting key features of the monkey task. Then, the rat DNMS task was studied in three experiments; each assessed the comparability of the rat DNMS task to the monkey DNMS task. Experiment 1 determined the rate at which the rat DNMS task is learned and the asymptotic level at which it is performed, Experiment 2 assessed the memory abilities that it taps, and Experiment 3 investigated the brain structures that are involved i n its performance. In Experiment 1, rats were trained on the DNMS task and their performance was assessed at retention delays of 4, 15, 60, 120, and 600 s. All of the rats learned the DNMS task, and their performance was comparable to that commonly reported for monkeys in terms of both the rate at which they acquired the nonmatching rule at a brief retention delay and their asymptotic accuracy at delays of up to 120 s. These results establish that rats can perform a DNMS task that closely resembles the monkey DNMS task and that they can approximate the level of performance that is achieved by monkeys. Experiment 2 examined the effects of distraction during the retention delay on the DNMS performance of rats. Rats were tested at retention delays of 60 s. On half of the trials, the rats performed a distraction task during the retention delay; on the other half, they did not. Consistent with findings from monkeys and humans, distraction during the retention delay disrupted the DNMS performance of rats. This suggests that similar memory abilities are involved in the DNMS performance of rats, monkeys, and humans. Experiment 3 investigated the effects of separate and combined bilateral lesions of the hippocampus and the amygdala on DNMS performance in pretrained rats. Rats were tested both before and after surgery at retention delays of 4, 15, 60, 120, and 600 s. Each experimental rat received bilateral lesions of the hippocampus, amygdala, or both. There were no significant differences among the three experimental groups, and the rats in each of the three experimental groups were significantly impaired, in comparison to no-surgery control rats, only at the 600-s delay. In contrast, rats that had sustained inadvertent entorhinal and perirhinal cortex damage during surgery displayed profound D N M S deficits. These results parallel the results of recent studies of the neural basis of DNMS in monkeys. They suggest that, in contrast to one previously popular view, neither the hippocampus nor the amygdala play a critical role in the DNMS of pretrained animals and that the entorhinal and perirhinal cortex are critically involved. On the basis of these findings, it appears that the rat DNMS task may prove to be a useful component of rat models of brain-damage-produced amnesia. This conclusion is supported by the preliminary results of several experiments that are currently employing the task. / Arts, Faculty of / Psychology, Department of / Graduate

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.

No description available.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder for which no disease modifying treatment exists. Inheritance of the apolipoprotein E (APOE) ε4 allele strongly increases the risk of developing the sporadic form of AD, whereas the ε2 allele is protective. Though the precise role of the different APOE alleles remains unclear, in vitro and in vivo studies have demonstrated that each isoform differentially modulates the deposition, clearance, and degradation of amyloid-beta (Aβ) peptides that form the extracellular plaques of AD. Strategies directed toward increasing the levels of APOE2 could be useful in reducing plaques and alleviating disease progression. Additionally, advances in gene transfer using adeno-associated vectors (AAV) enable efficient and safe expression of therapeutic genes like APOE2. This dissertation, describes experiments that (1) test if intracerebroventricular gene delivery of APOE isoform can modulate amyloid pathology in transgenic animals that accumulate beta-amyloid, (2) evaluate the efficacy of a novel approach by intravenous infusion of AAV, and (3) characterize oligomeric Aβ (oAβ) in rhesus monkeys as a potential translational target. Experiment 1 explored how each APOE isoform impacts the progression of AD pathology using intracerebroventricular injection of AAV4. This demonstrated isoform-dependent effects on Aβ-related neuropathology using multi-photon imaging, in vivo microdialysis, post-mortem immunohistochemistry, and array tomography. Experiment 2 demonstrated the efficacy of intravenous delivery of AAV9 and increased efficiency of self-complementary AAV9 vectors compared to single-stranded AAV9. Furthermore, expression of transgene was restricted to CNS astrocytes by utilizing a restrictive promoter. Finally, the presence of oAβ in rhesus macaque monkeys and its possible association with age-related cognitive decline was explored using fresh samples of monkey cortex. Soluble oAβ was detected in multiple cortical areas but was not significantly associated with age. Nevertheless, a significant positive correlation was observed between oAβ in frontal pole cortex and impairment on a behavioral test of executive function. Taken together, these data suggest that gene transfer of APOEε2 may be useful for modulation APOE expression and beta-amyloid accumulations for the treatment of AD. Furthermore, the rhesus monkey may serve as a model system for future preclinical studies.

No description available.

The improving of our understanding of cancer development still depends on cancer research at the molecular level. In his project, bigenic vectors for animal models of breast and prostate cancer are created. Bigenic constructs are useful because they create animals expressing two genes of interest at a time, with one injection step and no need for crossings. In order to produce these vectors, previous animal models have been analyzed, and the elements that worked successfully in previous models were gathered in a new arrangement for the creation of an improved model. In order to create a bigenic vector, the viral internal ribosomal entry site was utilized, as a means of obtaining two protein products from one transcript. One vector, the MMTV-Neu1842-IRES-Cre was successful in generating a line of transgenic mice. Female founders of this line already express the expected phenotype, tumors of the mammary tissue. Once this line is established, it can be crossed with the Rosa26 line, to determine the pattern of Cre expression. Other vectors were created for models of prostate cancer, using the probasin promoter and the MT oncogene. While transgenic mice were attempted, there were no phenotype differences between wild type and transgenic mice. All created vectors were tested for expression ofthe two genes carried in tissue culture experiments. All the experiments were successful, indicating a working oncogene (by means of a focus assay) and Cre activity (by excission assay). The new breast cancer animal model carrying the MMTV-Neu1842-IRESCre construct is promising and can be used in combination with existing models to answer some of the remaining questions regarding breast cancer signaling pathways. / Thesis / Master of Science (MSc)