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.

Animal models can contribute significantly to our understanding of human gambling behavior. However, no proposed animal models of human gambling have been tested using human subjects. The purpose of the present paper was to validate an animal model of gambling with human subjects. Twenty undergraduate rehabilitation students (all women) were recruited and participated for extra course credit. Participants were presented with a concurrent choice between two different simulated slot machines; one machine with symbols and one machine without symbols. During the first 50 choice trials, the payout of the two machines was equated at 50% overall. For the remaining 50 choice trials, probability of winning on the machine with symbols was systematically decreased by 10% overall every ten trials until there was no probability of winning for the last ten trails. On average, participants showed a preference for the machine with symbols during choice trials when win rate was equated; allocating significantly more than 50% of responding to this machine. A repeated measures ANOVA indicated that response allocation to the symbol machine only significantly decreased in the final two conditions (10% and EXT) and did not decrease significantly across any other conditions. Results were also interpreted through behavioral economic analyses. Results indicate that conditioned reinforcement may affect the subjective value of probabilistic reinforcers in humans. These results are similar to those obtained with pigeons under similar conditions and may imply that animal models are relevant to the study of human gambling behavior.

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.

Human gut harbors 100 trillion microbial organisms that is intrinsically linked to individual’s health and diseases, including cancer. Food fiber and phytochemicals such as polyphenols are considered as prebiotic-like dietary modifiers. They can influence the gut microbial communities, and in turn to modulate disease outcome and drug responses of the host. Saponins belong to a family of phytochemicals commonly found in many medicinal and edible plants. Herbal saponins have raised keen interest among scientists for their health-promoting effects, but have not been investigated for their potential as prebiotics. Gynostemma pentaphyllum (Gp) is riched in triterpenoid saponins and has been consumed in China and other part of the world as an herbal tea and as a folk medicine. In our lab, we have demonstrated that Gp possesses strong anticancer and anti-inflammatory effects. Whether Gp possesses prebiotic property and whether gut microbiota plays any part of the anticancer effect of Gp are the questions addressed in the present study. Thus, we hypothesized that Gp saponins (GpS) might modulate the gut microbiota, which in turn enhance its anticancer activities. In the study, the gut microbiome analysis were carried out using two main techniques, neamly the enterobacterial repetitive intergenic consensus (ERIC-PCR) and 16S pyrosequencing approaches. Both xenograft nude mice and Apcmin/+ mice were employed as the animal models to investigate the interaction between the herbal saponins and the gut microbiota in the host. Athymic nude mice have been employed for tumorigenic research for decades, however, the relationships between the gut microbiome and host’s response to the grafted tumors and drug treatments are unexplored. For the first part of the thesis, we investigated the relationship between the gut microbiota and grafted tumor in the nude mice under the treatment of Gp saponins. Partial least squared discriminant analysis (PLS-DA) of ERIC-PCR data showed that the microbiota profile of xenograft nude mice departed from that of the nonxenograft mice. However, prolonged treatment of GpS seems to realign the fecal microbiota with the pretreatment control. Pyrosequencing data reiterated the differences in fecal microbiome between the nonxenograft and xenograft animals. GpS treatment had a much stronger impact on the phylotypes of the xenograft than the nonxenograft mice. In addition, GpS treatment markedly induced the relative abundance of Clostridium cocleatum and Bacteroides acidifaciens, for which the beneficial effects on the host have been well documented. ApcMin/+ colorectal cancer mouse model was further employed for the investigation of the association of the gut microbiota and cancer occurred inside the gut, which was a more direct site to interact with the gut microbiota. In the ApcMin/+ mouse model, we found distinct difference of fecal microbiome between the ApcMin/+ and the wild-type littermates. GpS treatment significantly reduced the number of intestinal polyps. GpS also increased the ratio of Bacteroidetes/Firmicutes and reduced the sulfate- and sulfur-reducing bacteria lineage and potential opportunistic pathogens, which might cause certain deleterious effects to the host. The impact of GpS on the gut mucosal environment was also examined. We found GpS treatment improved the gut barrier function by increasing the numbers of Paneth cells, goblet cells, up-regulating the expression of E-cadherin and down-regulating the expression of N-cadherin in the intestine. In addition, GpS treatment down-regulated the protein expression of beta-catenin and p-STAT3. Furthermore, higher levels of anti-inflammatory and tissue repair-related cytokines as well as Arginase I, but lower level of iNOS expression were found in GpS-treated ApcMin/+ mice, indicating increased anti-inflammatory macrophage phenotype M2 (associated with tissue repair) and reduced proinflammatory phenotype M1. Furthermore, in addition to GpS, other herbal saponins also showed prebiotic-like effects in C57BL/6 mice. In summary, this study provides first hand evidence for the impact of herbal saponins on the gut microbial ecosystem and new insight into mechanisms responsible, at least in part, for the activities of GpS. We demonstrate that tumor growth induce intestinal dysbiosis. GpS treatment can inhibit tumor progression and concurrently alter the microbiome by increasing symbionts and/or decreasing pathobionts, which may contribute to its chemopreventive effect against tumorigenesis. Herbal saponins showing prebiotic-like effects may be used for improving the health of the host by manipulation of the gut microbiota.

Irritable bowel syndrome (IBS) is characterized by chronic abdominal pain/discomfort along with altered bowel habits, which accounts for a large proportion of gastrointestinal (GI) disorders worldwide. While psychiatric distress like depression is one of the most frequent comorbidities in IBS patients, which not only influences the quality of life, it also leads to a substantial economic burden and inefficient treatment in IBS patients. Inflammation, altered activities of the HPA axis, aberrant central neuroplasticity and neurotransmission have been highly regarded as pathogenic factors of the depression. Whereas, in recent years, dysfunctions in the gut microbial community has been increasingly discovered to provoke depression disorder. Considering that gut dysbiosis plays causal role in IBS progression, dysfunction of the gut microbiota has been speculated for contributing to the depressive symptoms in IBS (IBS-DP) patients. However, whether and how gut dysbiosis affecting IBS-DP patients remain unclear. We hypothesized that gut microbiota changes contribute to the development of IBS-DP and the change of gut microbiota-driven metabolites induces the structural and/or functional changes of the central nervous system (CNS), thus resulting in the development of IBS-DP. In this thesis, IBS patients with and without depression and animal models of IBS have been systematically studied, to investigate whether gut dysbiosis mediates depressive disorder in IBS. Firstly, we conducted a cross-sectional study involving the distribution of depressive disorder in the IBS population of Hong Kong. According to this survey, we found that there is 36.6% (135/369) of IBS patients showed symptoms of depression. The severity of depressive symptoms was positively associated with the harshness of visceral pain and bloating signatures in IBS patients. Secondly, in comparison to the non-depression of IBS (IBS-ND) group, faecal metagenomic results unveiled the disrupted gut microbiota in IBS-DP patients, mainly with the deficiency of several beneficial bacterial groups, including Akkermansia, Bifidobacterium and Eubacterium, whereas the gut microbiota profile between IBS-ND patients and healthy controls (HCs) showed no significant changes. Compared with HCs, enzyme-linked immunosorbent Assay (ELISA) results showed higher levels of serum IL-1β, IL-6, and TNF-a in IBS-DP patients, indicating a low-grade peripheral inflammation in IBS-DP patients. Moreover, the abundance of Akkermansia muciniphila (A. muciniphila), was negatively correlated with Hamilton Rating Scale for Depression (HAMD) score and Zung Self-Rating Depression Scale (SDS) score, IL-1β, TGF-β, and TNF-a in IBS-DP patients. These findings indicate that gut dysbiosis, especially deficiency in A. muciniphila, is related to the depressive symptoms and inflammation in IBS-DP patients. Thirdly, in a neonatal maternal separation (NMS) rat model, behavioural tests such as colorectal distention (CRD) test, open field test (OFT), forced swimming test (FST), and sucrose preference test (SPT) results showed visceral hypersensitivity and depression symptoms in rats. These results indicate that the model can successfully mimic the visceral hyperalgeisa and the depression-like behaviour of IBS-DP. Immunohistochemical analysis showed an altered morphology and decreased the quantity of astrocytes in the hippocampus of NMS rats when compared with that of controls. More importantly, the mRNA expressions of genes related to Astroglial glutamate transmission including glutamate transporters (GLTs), glutamate receptors, and also glutamate-related exchangers, as well as astrocyte biomarker glial fibrillary acidic protein (GFAP), which are mediated with chronic inflammation and/or stress, were decreased in NMS rats when compared with the control group. These results indicate that impaired astroglial glutamate neurotransmission in NMS rats. Furthermore, pseudo-GF rats with faecal microbiota transplantation (FMT) of NMS microbiota were also conducted, and results showed that the association of A. muciniphila deficiency, depressive-like behaviours and impaired astroglial glutamate neurotransmission were repeated in the rat recipients. These results indicate a causal relationship between NMS microbiota and depressive phenotype, involving dysfunction of the astrocyte- glutamate pathway. Fourthly, to further verify the role of A. muciniphila in NMS microbiota-induced depressive phenotype and impaired astrocytic glutamate pathway, we orally administered live and heat-killed A. muciniphila bacteria in NMS adult rats. A. muciniphila (108 CFU in 1mL PBS) was administered once-daily for four consecutive weeks. Besides, rifaximin and fluoxetine were also separately treated in NMS rats as control groups. Rifaximin is a broad-spectrum GI-specific antibiotic that is commonly used for IBS treatment, and fluoxetine, a selective serotonin re-uptake inhibitor, is one of the most frequently prescribed anti-depressants. The results showed that A. muciniphila efficiently improved depressive-like behaviours, attenuated the impaired astrocytic glutamate neurotransmission, as well as restored the normal morphology and number of astrocytes in the hippocampus of NMS rats. While rifaximin-treated rats only exhibited amelioration of visceral pain, and fluoxetine group mainly performed antidepressant effect, without any significant change in astrocytic glutamate neurotransmission impairment. These results demonstrate that A. muciniphila improves depressive symptoms in IBS phenotype and ameliorate astroglial-glutamatergic pathway dysfunction. Whether and how A. muciniphila modulates astroglial glutamate transmission, therefore leading to the improvement of depressive symptom in IBS, remains to be further investigated. Taken together, the works of this thesis, combining both clinical and animal studies reveal that gut dysbiosis, particularly deficiency of A. muciniphila, contributes to the development of IBS-DP via regulating the astroglial glutamatergic pathway. This study gives a different direction to microbial-guided therapy for the IBS-DP patients in the future

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