Hydrogen sulfide (H2S) has emerged as an important mediator of host function. In the gastrointestinal tract H2S is enzymatically produced and plays a vital role in cytoprotection, inflammation, and tissue repair. During a bout of colitis, the ability of the colon to produce H2S is markedly increased and drives the resolution of colitis. However, little is known about how the production of H2S is regulated in the colon and how dysregulated production can affect the course of colitis in vivo. Additionally, the mechanisms through which H2S can promote the resolution of colitis remain to be fully investigated. In Chapter 3 of this dissertation, the regulation of H2S production in the colon was explored by examining the contributions of three enzymatic pathways to colonic H2S synthesis. The largest source of the H2S synthesis was from a pathway previously unrecognized in the GI tract involving the enzyme 3-mercaptopyruvate sulfurtransferase (3MST). Additionally we found that the upregulation of H2S production during colitis occurred specifically at sites of mucosal ulceration. At the same time H2S inactivation via the enzyme sulfide quinone reductase (SQR) was significantly reduced at these sites. We propose that the site-specific alterations in H2S production and inactivation during colitis promote the resolution of inflammation and injury. Chapter 4 examined whether the ability of hyperhomocysteinemia (Hhcy) to exacerbate colonic inflammation occurred through impaired H2S synthesis. Hyperhomocysteinemia is often reported in patients with inflammatory bowel disease and is a consequence of decreased vitamin B intake. In all three models tested, diet-induced Hhcy significantly exacerbated colitis. Being dependent on vitamin B6 as a co-factor, the increased H2S production normally observed during colitis was abolished during Hhcy. Administration of an H2S donor to Hhcy rats significantly decreased the severity of colitis. These results also uncovered a novel role for IL-10 in promoting H2S production and homocysteine metabolism, which may have therapeutic value in conditions characterized by Hhcy. Finally, in Chapter 5 we looked for a mechanism through which H2S can promote resolution of colitis. Using CSE-deficient mice we found that H2S production was required to maintain HIF-1α signaling in the colon. Additionally, proper HIF-1α signaling was required for H2S-donating molecules to promote the resolution of colitis. These results suggest that HIF-1α signaling is a critical event through which H2S promotes resolution of colitis. Collectively, these chapters further highlight the importance of H2S production in colon during inflammation and injury and offer insight into new therapeutic targets mediated through H2S. / Dissertation / Doctor of Philosophy (PhD)

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The purposes of this dissertation were to 1) determine the effectiveness of the neutrophillymphocyte ratio (NLR) as an athlete monitoring tool in resistance training and 2) determine if repetition maximum or relative intensity loading scheme is superior in managing fatigue through the hormonal, inflammatory, and performances response throughout a 10-week periodized resistance training program. Results from the dissertation give merit to continued research regarding the use of NLR as a monitoring tool to help determine the degree of recovery. Furthermore, results from this dissertation lead to questioning the effectiveness of using a repetition maximum (RM) loading scheme within a periodized training model. Results indicated statistical significant time x group interaction effects for training strain and T:C, statistical main effects for time for NLR, IPF, and IPFa. Under an identical programming model, RM loading subjects experienced a 48.7% increase in training strain over the course of ten weeks. This intensification in training strain likely contributed to the increased negative immune and endocrine response the RM subjects experienced when compared to the relative intensity (RISR) group. When dissecting the individual pre-post performance results, the three largest decreases in static jump height (out of four) participated in the RM loading group. Additionally, only two subjects experienced decreases in their maximal strength (based on isometric mid-thigh pull), both of which participated in the RM loading group. Lastly, it is highly likely that one subject from the RM group was at exceedingly high risk of entering a state overtraining. At a minimum, the subject entered a state of a nonfunctional overreach, based on an increase in cortisol concentrations, NLR, T:C levels, along with decreases in testosterone concentrations and maximal strength performance. When combined, results suggest that using an RM loading scheme and a periodized model may not allow for adequate recovery, especially during phases where recovery is of utmost importance (e.g. a taper).

Caffeine is one of the most widely used neurostimulants in the world and there is considerable debate on its effect in immune cells. One of its main targets is proposed to be adenosine receptors which mediate an anti-inflammatory switch in activated immune cells while another target is phosphodiesterase where it acts as an inhibitor. In macrophages, caffeine has been shown to cause both pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. If the primary effect of caffeine on macrophages were to antagonize adenosine receptors we would expect cells exposed to caffeine to have a prolonged M1 response. However, we show that caffeine suppresses phagocytosis at physiological concentrations (an indicator of M2 phenotype). This suppression was reversed when macrophages were pretreated with protein kinase A inhibitor, suggesting that at physiological concentrations caffeine's phagocytic suppression may be due to its function as a phosphodiesterase inhibitor, pushing cells towards an M2 fate. However, mRNA expression profiling suggests that caffeine can modulate A2A receptor expression and suppress MKP-1 expression, a hallmark of M1 macrophages. Caffeine is, therefore an immunomodulator that can suppress or prolong inflammatory responses in macrophages, which may account for the abundance of contradicting evidence in the literature. Additionally, these effects are complicated by regular caffeine intake and fitness level, emphasizing that tolerance and immune robustness are important factors in macrophage activation.

Periodontitis potentially contributes to many systemic diseases. Specific gram-negative bacteria such as Porphyromonas gingivalis (P.g) and Treponema denticola (T.d) contribute to the initiation and progression of periodontal disease via factors such as NF-κB, TNF-α, IL-1β, and CXCL1. Down-regulation of these factors by natural compounds or short hairpin RNAs (shRNAs) reduces periodontal bacteria-induced inflammation. Our preliminary data indicated that P.gingivalis / lipopolysaccharide (LPS) stimulates chemokine CXCL1 production in macrophages. CXCL1 stimulates LPS-induced TNF-α expression, resulting in inflammation. We hypothesize that inhibiting the expression of CXCL1 will reduce LPS-induced TNF-α production. We recently demonstrated that a CXCL1 shRNA inhibits LPS-stimulated CXCL1 production in macrophages and leads to reduced expression of LPS-stimulated pro-inflammatory cytokines TNF-α and IL-1β. This indicates that CXCL1 shRNAs have potential as inhibitors of LPS-induced inflammation. Further studies are needed to confirm these as well as to identify the signaling pathway.

Cortical injuries, such as those caused by stroke and other insults, are the leading cause of death and disability worldwide. While thrombolytics can be used to restore blood flow immediately following the onset of symptoms of an ischemic stroke, there are currently no neurorestorative therapeutics that can enhance long-term recovery of function following injury. Neuregulins are a family of growth factors involved with the survival and function of neurons and glia. Glial Growth Factor 2 (GGF2) is an isoform of neuregulin-1 that has demonstrated significant effects in the recovery of function in rodent models of stroke. Histological analyses suggest GGF2 promotes recovery by enhancing endogenous mechanisms to reduce inflammation and promote plasticity. To further explore the efficacy of GGF2, we used our rhesus monkey model of cortical injury and fine motor impairment to compare the rate and pattern of recovery in monkeys treated with GGF2. Twenty-four young adult male monkeys (ages 4-10 years old) were pre-trained on our task of fine motor function of the hand before undergoing surgery to produce a cortical lesion limited to the hand representation of the primary motor cortex on one side. Intravenous (IV) administration of GGF2 (0.5 mg/kg) began 24 hours after surgery and continued daily for 7 days. This was followed by 21 days of sub-cutaneous administration of GGF2 at two different dose levels (0.1 mg/kg or 0.3 mg/kg). Post-operative testing began two weeks after the lesion and continued for 12 weeks. All trials were video recorded and latency to retrieve a reward was quantitatively measured to assess the trajectory of post-operative response latency and grasp pattern compared to pre-operative levels. The results showed no significant differences between the groups in the recovery of fine motor function. Moreover, all vehicle control monkeys returned to their pre-operative levels of latency and grasp pattern despite no significant differences in lesion volume from the experimental groups. In addition to measures of behavioral recovery, we processed the brain tissue with immunohistochemistry to investigate the role of GGF2 treatment in reducing the pro-inflammatory response of microglia and enhancing axonal sprouting and synaptogenesis following injury. All groups had a greater density of Iba1+ microglia in the perilesional grey matter and sublesional white matter, but there were no significant differences in the numerical density or phenotypes of microglia between the groups. We also found no significant differences in axonal sprouting between the groups. However, GGF2 treatment did enhance expression of synaptophysin in the contralesional hemisphere of monkeys that received subcutaneous doses of GGF2 following the initial 7 days of intravenous GGF2 treatment. This suggests that high dose GGF2 treatment may enhance plasticity of compensatory circuits involving the intact hemisphere and that this effect is dose dependent. In addition, we followed up these analyses using a subset of monkeys from the larger GGF2 study to optimize and validate a method that labels newly synthesized myelin. This is accomplished by in vivo administration of a choline analog, propargylcholine (P-Cho) that labels newly synthesized myelin and can be visualized post-mortem. Our results demonstrate effective and stable incorporation of P-Cho with post injection survival of 1 to 6 weeks. Using this method to quantify new myelin after cortical injury to the primary motor cortex, showed significantly greater P-Cho labeling and co-localization with myelin basic protein (MBP) in the white matter underlying the ipsilesional hemisphere when compared with the contralesional hemisphere. This validates P-Cho for assessing myelin plasticity in a nonhuman primate brain and how it might be used to assess therapeutics aimed at inducing remyelination and enhancing myelin synthesis. Finally, this dissertation also includes the comparison of sex differences in recovery of motor function after cortical injury. In a cohort of aged male and female monkeys, postmortem analysis showed no differences in lesion volume between the males and females. However, behaviorally, the females returned to their pre-operative latency and grasp patterns significantly faster and more completely than the males. These findings demonstrate the need for additional studies to further investigate the role of estrogens and other sex hormones that may differentially affect recovery outcomes in the primate brain. Collectively, the results presented in this dissertation highlight the complexity of evaluating treatments and mechanisms underlying recovery of function by enhancing neuroplasticity. Specifically, we were unable to effectively evaluate GGF2 as a treatment due to the behavioral recovery of all control monkeys. Follow up studies should investigate treatment with GGF2 in aging monkeys and compare the results with our findings. Additionally, it is necessary to further explore the recovery of fine motor function in young monkeys. Finally, our study showing sex differences in recovery of function provides evidence that sex hormones may play a significant role in providing neuroprotection in the aging brain following cortical injury. Future studies should measure post-operative estrogen levels and evaluate supplementation as a potential treatment option.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.