This doctoral thesis includes four projects. The first project explored the role of innate HIV genetic polymorphisms in drug-selected mutations. By means of drug selection experiments, we comparatively evaluated emergent mutations in protease and reverse transcriptase in HIV-1 and HIV-2 subtypes. Genotypic and phenotypic analyses were used to determine time to appearance of resistance mutations and the levels of drug resistance, respectively. Natural polymorphisms in HIV-2 seemed to facilitate selection of protease inhibitor (PI) resistance: The acquisition of the I54M, I84V, L90M and L99F mutations resulted in multi-PI-resistant viruses. Nucleoside reverse transcriptase inhibitor (NRTI) combinations preferentially selected for the K65R mutation in HIV-1 subtype C, while in HIV-1 subtype B, and HIV-2 cultures favored the development of M184I. The K65R mutation was not detected in HIV-2 selections. These results underscore potential differences in emergent drug resistance pathways in HIV-1 and HIV-2 and the role that innate polymorphisms may have in dictating antiretroviral resistance pathways. In the second project, we studied the relationship between mutations in the RNase H domain on viral susceptibility to nucleoside analogues. Our results showed that although some mutations were observed in the connection domain, the presence of the L74V and M184V mutations was the most significant determinant of phenotypic resistance to ZDV in patients with thymidine analog-associated mutations. The third project performed a near full-length genomic analysis of a novel HIV-1 recombinant involving subtypes A1 and C in Quebec. The fourth project evaluated the genetic diversity of an / Cette thèse de doctorat comprend 4 projets. Le premier explore le rôle du polymorphisme génétique du VIH dans la sélection des mutations de résistance. Au cours des tests de sélection, nous avons évalué comparativement le délai dans lequel les mutations de résistance émergent dans la protéase et la transcriptase inverse des sous-types de VIH-1 et de VIH-2. Des analyses génotypiques et phénotypiques ont été utilisées pour déterminer le délai d'émergence et le niveau de résistance des mutations, respectivement. Le polymorphisme naturel du VIH-2 semble faciliter la sélection des mutations de résistance aux inhibiteurs de protéase (IPs). L'acquisition des mutations de résistance I54M, I84V, L90M et L99F entraine une résistance croisée à plusieurs IPs. Les combinaisons d'inhibiteurs nucléosidiques de la transcriptase inverse (INTI) sélectionnent préférentiellement la mutation K65R chez le VIH-1 de sous-type C alors que les cultures du sous-type B et le VIH-2 favorisent le développement de M184I. La mutation K65R n'a jamais été détectée dans les sélections de VIH-2. Ces résultats mettent en évidence des différences dans les mécanismes de résistance du VIH-1 et du VIH-2 et le rôle que certain polymorphisme jouent dans les voies génétiques de résistance. Dans le second projet, nous avons étudié la relation existante entre le polymorphisme dans la RNAse H et la sensibilité aux analogues nucléosidiques. Nos résultats montrent que bien que certaines mutations soient observées dans le domaine de connection, la présence des mutations L74V et M184V était le facteur déterminant de la résistance phénotypique à la ZDV chez les

Objective measurement of the subglottic lumen is still lacking in clinical practice. The objective of this study is to evaluate the accuracy of ultrasound and video-bronchoscopy in measuring the subglottic diameter in the pediatric population. This was a blind, prospective clinical study. Ten children undergoing non-life threatening bronchoscopy had their subglottic diameters measured with ultrasound, video-bronchoscopy and endotracheal tube sizing. / Ultrasound and video-bronchoscopy strongly correlated with endotracheal tube sizing in measuring the subglottic diameter. Ultrasound had measurements that were always smaller while video-bronchoscopy had measurements that were slightly larger than endotracheal tube sizing. Video-bronchoscopy may be more accurate than endotracheal tube sizing as the latter method will often underestimate the size of the lumen due to its own limitations. The smaller values obtained by ultrasound suggest it is not ideal to give absolute measurements in this area, but rather a potential tool to detect change of lumen size on follow-up.

Background. Up to 1600 Canadians/yr suffer a serious complication due to the diagnostic test ERCP (endoscopic retrograde cholangiopancreatography) used in the management of patients with suspected biliary obstruction. MRCP (magnetic resonance cholangiopancreatography) is a new highly accurate noninvasive alternative that is not associated with any significant complications. / Aims. To assess the real-life effectiveness and costs of MRCP versus ERCP in a planned interim analysis of a randomized clinical trial, in intermediate risk patients. To assist in the decision regarding study continuation versus termination. / Methods. 200 patients with a clinical and/or ultrasonographic (US) suspicion of obstruction were randomized to MRCP or ERCP, using block allocation stratified by level of obstruction. The primary endpoint was the occurrence of a negative outcome attributable to the biliopancreatic disease or procedures within 12 months of randomization. Secondary outcomes included complication-related length of stay (CRLOS), number of procedures, costs, mortality and accuracy. / Results. 102 patients were randomized to MRCP and 98 to ERCP with mean ages of 56.4 +/- 18 (SD) and 51.8 +/- 19 yrs, respectively. Median follow-up was 12 months. According to intention-to-treat analysis, 18 (17.6%) MRCP patients had a complication (CRLOS = 0.80 d/patient (excluding 1 outlier with CRLOS = 157d) versus 14 (14.3%) ERCP patients (CRLOS = 0.68d/patient); difference = 3.3% (95%CI: -7 to 13%). There were 45 fewer ERCPs but 90 extra MRCPs in the MRCP arm. ERCP was "dominant", with both marginally lower complications and lower costs by $1411/person (ITT). There was neither evidence for confounding nor effect modification. / Conclusions. In this interim analysis, it appears that a strategy that involves a screening MRCP in this group of patients is not cost-effective. The results of this primary analysis are unlikely to change with the recruitment of 200 additional patients into the trial.

Innovations in the practice of pediatric cardiology and cardiovascular surgery have resulted in significant improvements in the survival of children born with congenital heart disease. This has resulted in a growing population of adults with repaired or palliated congenital heart disease. However, these patients are at increased risk of impaired cardiovascular health, including mortality, particularly under conditions of physiological stress, such as operative reintervention, in later life. We hypothesized that neonatal hypoxia engenders lasting changes in cardiovascular structure and function which may adversely influence myocardial and vascular responses to physiological stress at maturity. To test this hypothesis, Sprague-Dawley rats reared initially in hypoxic conditions (FiO2 = 0.12) for days 1 to 10 of life were compared to rats reared only in ambient air. Neonatal hypoxia was associated with significant changes in the left ventricular expression of 1945 and 422 genes in 10- and 90-day-old animals, respectively. Functional annotation revealed several genes involved in adaptive vascular remodeling and energy metabolism, as well as the regulation of apoptosis. Trends in gene expression suggested a proapoptotic paradigm which was corroborated by data showing decreased survival, following an ischemia-reperfusion insult, of cardiomyocytes isolated from adult animals exposed to neonatal hypoxia. Based on the above observations, we next sought to characterize additional changes in cardiovascular structure and function, induced by neonatal hypoxia, which might enhance cardiomyocyte vulnerability to physiological stress in the adult animal. Hypoxic animals had significant left ventricular hypertrophy, as well as impaired cardiomyocte calcium homeostasis and sarcomere relaxation, observations which were consistent with in vivo echocardiographic evidence of severe diastolic dysfunction. Neonatal hypoxia was also associated with the development of significant remodeling of left ventricular arterioles resulting in decreased lumen area. Significant reduction in agonist-induced activation of Akt and ERK1/2 survival signalling, as well as decreased mitochondrial hexokinase 2 expression, were observed. Finally, the left ventricular protein expression of Gαi3, Gαi2, and HIF-1α was significantly increased in adult animals following neonatal hypoxia.The above observations were evaluated in the context of the clinical challenges associated with the care of adult patients with congenital heart disease, and the potential clinical implications of these data are discussed. / Les innovations dans la pratique de la cardiologie et de la chirurgie cardio-vasculaire pédiatrique ont entraîné des améliorations importantes dans la survie des enfants nés avec une cardiopathie congénitale. Cela a mené à une population croissante d'adultes ayant une maladie cardiaque congénitale réparée ou palliée. Cependant, ces patients sont à risque de santé cardio-vasculaire affaiblie, y compris la mortalité, en particulier dans des conditions de stress physiologique, tel que la réintervention opératoire. Notre hypothèse est que l'hypoxie néonatale engendre des changements durables dans la structure et la fonction cardio-vasculaire qui peuvent influencer négativement les réponses du myocarde au stress physiologique.Pour vérifier cette hypothèse, des rats Sprague-Dawley élevés initialement dans des conditions hypoxiques (FiO2 = 0,12) pour les jours 1 à 10 de vie ont été comparés à des rats élevés dans l'air ambiant. L'hypoxie néonatale a été associée à des changements significatifs dans l'expression, au niveau du ventricule gauche, de 1945 et de 422 gènes chez les animaux âgés de 10 et de 90 jours, respectivement. L'annotation fonctionnelle a révélé plusieurs gènes impliqués dans le remodelage vasculaire adaptatif et le métabolisme énergétique, ainsi que dans la régulation de l'apoptose. Les tendances dans l'expression des gènes ont suggéré un paradigme proapoptotique qui a été corroboré par des résultats montrant une diminution de la survie, suite à une insulte ischémie-reperfusion, de cardiomyocytes isolés chez les animaux adultes exposés à l'hypoxie néonatale.Sur la base des observations ci-dessus, nous avons ensuite cherché à caractériser les changements dans la structure et la fonction cardio-vasculaire, induits par l'hypoxie néonatale, qui pourraient accroître la vulnérabilité des cardiomyocytes au stress physiologique chez l'animal adulte. Les animaux hypoxiques avaient une hypertrophie ventriculaire gauche significative, ainsi que des altérations de l'homéostasie du calcium dans le cardiomyocyte et de la détente des sarcomères, des observations compatibles avec des observations échocardiographiques montrant une dysfonction diastolique sévère. L'hypoxie néonatale a également été associée au développement du remodelage des artérioles du ventricule gauche. Nous avons observé une réduction significative de l'activation d'Akt et ERK1/2 induite par l'isoproterenol, ainsi qu'une diminution de l'expression mitochondriale de hexokinase 2. Enfin, l'expression des protéines Gαi3, Gαi2, et HIF-1α a été significativement augmentée, au niveau du ventricule gauche, chez les animaux adultes suivant l'hypoxie néonatale.Les observations ci-dessus ont été évaluées dans le contexte des défis cliniques associés aux soins des patients adultes atteints de cardiopathies congénitales, et les implications cliniques potentielles sont discutées.

We hypothesize that continuous electrical stimulation (ES) of denervated muscle using implanted electrodes and a pulse generator lessens the effects of denervation and helps to preserve muscle integrity. Ten dogs underwent sciatic nerve severance, common peroneal nerve repair, and implantation of stimulating devices. The proportion of muscle weight preserved was significantly greater in the stimulated muscle than in the untreated denervated muscle (p $<$ 0.01). There was evidence of less atrophy and degeneration in DS seen in both light and electron microscopy. Dependent t test analysis using the normal contralateral muscle groups as the control showed that twitch contraction time was maintained within normal limits by stimulation whereas in the unstimulated group prolongation of twitch time occurred (p $<$ 0.004). The electrical resistance property of the stimulated muscle was also preserved when compared to the normal control (p = 0.7) but did differ between D and N (p $<$ 0.04). Although trends in the data suggested beneficial functional effects of ES, statistical analysis failed to support differences between the stimulated and unstimulated groups in functional testing. (Abstract shortened by UMI.)

Despite the best of modern microsurgical skills, the functional result that is achieved following the repair of a severed peripheral motor nerve has usually been less than complete. In an attempt to reduce or prevent denervation atrophy we have devised a totally implantable system of electrical stimulation and have examined its effect on the target muscle of a repaired peripheral nerve. In this study, twenty New Zealand white rabbits were divided into two groups: in one group the rectus femoris muscle is electrically stimulated after microsurgical repair of the femoral nerve; whereas the control group undergoes microsurgical repair of the femoral nerve only. Comparing the muscles to their contralateral counterparts at eight weeks post-op we found that electrical stimulation resulted in (a) the retention of 79+/$-$5(SEM)% of muscle bulk (compared to 42+/$-$3% in the non-stimulated group) p $<$ 0.001, (b) a maximum force of tetanic contraction averaging 41+/$-$5% of normal (compared to 19+/$-$4% in the non-stimulated group) p $<$ 0.01, (c) less myofiber atrophy, interfascicular fibrosis, and fatty infiltration, (d) significantly less myofilamentous disruption at the ultrastructural level, and (e) a preferential atrophy of type-II fibres (p $<$ 0.001) in the absence of electrical stimulation. There was no statistical difference between the two groups in the compound action potential waveform or amplitude of evoked contractions. In conclusion, these results suggest that the electrical stimulation of skeletal muscle will improve the ultimate functional results in patients undergoing peripheral motor nerve repair.

Injured adult mammalian cardiac muscle does not respond with significant regeneration. Skeletal muscle, on the other hand, has an efficient regeneration mechanism attributable to myogenic stem (satellite) cells. This study investigated a hypothesis which states that skeletal muscle satellite cells (SCs), if grafted into an injured heart, can form muscle tissue and repair the damaged myocardium. / In the first study SCs obtained from 28 dogs were isolated, purified and cultured. Percoll density gradient and preplating methods for the purification of SC culture were investigated. Beta-galactosidase ($ beta$-Gal) labelled SCs underwent autografting into acutely cryoinjured myocardium. Three control dogs received only sham implants into the cryoinjured heart. Two (n = 6), 4 (n = 16) and 6 (n = 6) weeks after SC implantation, hearts with SC graft sites were procured and processed for histrionical examination, as well as for detection of a $ beta$-Gal marker. Control specimens were obtained at 2 (n = l), 4 (n = l), and 6 (n = l) weeks after sham implantation and were processed as described above. / In the second study on rats, SCs were obtained from donor animals (n = 30). An intramuscular bupivicaine method to increase SC yield was adopted. Purified, cultured, and $ beta$-Gal labelled SCs were implanted either into acutely cryoinjured myocardium (n = 15), or into the mature cardiac scar (n = 15) of isogenic donor animals. Myocardial specimens were obtained at 1 month after SC implantation. Techniques of rodent SC labelling in vitro with human alkaline phosphorate genes were also explored. (Abstract shortened by UMI.)

Background. A growing number of patients suffering from ischemic cardiomyopathy are not eligible for conventional revascularization. This has prompted new research in the field of angiogenesis. This study hypothesized that since inflammation is probably the mechanism behind TMR induced angiogenesis; a larger inflammatory response induced by thermal injury may lead to increased angiogenesis. / Methods. The model used for this study was coronary artery ligation in the Rat. Four groups of animals were used to compare the novel experimental approach with conventional TMR and with ischemia alone. Neovascularization was determined by immunohistochemical techniques using anti-Factor VIII antibody. Evaluation of VEGF, Ang-1 and Ang-2 expression was also carried out using immunohistochemistry. / Results. The experimental "HOT" TMR technique resulted in significantly increased angiogenesis presumably due to the thermal injury induced by the novel technique. Also a significant increase in VEGF expression was observed in all ischemic groups. Ang-1 expression was decreased in the experimental group while it was similar in the other groups. Finally Ang-2 was induced by ischemia as evidenced by increased expression among all ischemic groups. However Ang-2 expression did not significantly vary among ischemic groups. / Conclusions. The addition of thermal injury by heating of the needle led to an increased angiogenic response compared to ischemia alone and compared to conventional TMR. This increased angiogenesis was associated with increased VEGF expression at one week, however there was a significant inverse correlation between VEGF expression and angiogenesis among the ischemic groups. Also angiopoietin expression was in agreement with expression characteristics described in the literature.

Amyotrophic Lateral Sclerosis (ALS) is the most common adult motor neuron disease. Currently there is only one modestly beneficial pharmacological treatment, Riluzole, approved by the FDA. It has been documented that polyunsaturated fatty acid (PUFA) concentrations can affect the progression of neurodegenerative conditions however most interventions rely on nutritional supplementation and have limited long-term effectiveness. This thesis describes experiments using fenretinide, a synthetic retinoid capable of altering membrane PUFA concentrations, in a mouse model of ALS (SOD1(G93A)mice). Our treatment resulted in delayed onset, improved motor coordination, and increased life expectancy. Fenretinide also increased plasma levels of the ω-3 PUFA docosahexaenoic acid (DHA) while decreasing ω-6 PUFA arachidonic acid (AA) and products of lipid peroxidation malonyldialdehyde (MDA) and nitrotyrosine (NT). Spinal cord immunohistochemistry revealed a significant reduction in inflammation as assessed by the quantity of activated microglia and astrocytes. These results indicate that fenretinide represents a promising treatment strategy for ALS. / La Sclérose latérale amyotrophique (SLA) est la maladie affectant les neurones moteurs adultes la plus commune. Il n'existe qu'un seul traitement pharmacologique approuvé par la FDA ayant certains effets bénéfiques, soit le Riluzole. Il est par ailleurs documenté que des concentrations d'acides gras polyinsaturés (PUFA) peuvent affecter la progression d'un état neurodégénératif. Cependant, la plupart des interventions s'appuient sur des suppléments nutritifs et ont une efficacité à long terme plutôt limitée. Cette thèse décrit une série de traitements utilisant le fenretinide, un rétinoïde synthétique capable d'altérer la concentration de PUFA dans les membranes, dans un modèle de souris de SLA (souris SOD1(G93A)). Les traitements ont entrainé un retardement du déclenchement de la maladie avec une meilleure coordination motrice ainsi qu'une espérance de vie améliorée. Le fenretinide a également accrue les niveaux plasmatiques de l'acide docosahexaenoique tout en diminuant les niveaux d'arachidonate ainsi que les produits de peroxydation lipidiques tel que malonyldialdehye et nitrotyrosine. L'analyse immunohistochimique de la moelle épinière a révélé une réduction significative de l'inflammation déterminé par la quantité d'astrocytes et de microglies activés présentes. Ces résultats indiquent que le fenretinide représente un traitement prometteur contre la SLA.

Objectives. To study marrow stromal cells (MSCs) induced angiogenesis. To examine the possible mechanisms involved in the process. To evaluate neovascularization following implantation of MSCs in ischemic hind limb model. / Methods and result. Using murine Matrigel angiogenesis model, we compared MSCs related angiogenesis to that produced by vascular endothelial growth factor (VEGF) and basic fibroblast growth factor. We found that MSCs result in an efficient and organized angiogenesis, arteriogenesis and vasculogenesis. MSC-related angiogenesis is VEGF dependent. MSCs in vivo produce VEGF that through paracrine effect induces local angiogenesis and through an autocrine loop stimulates FLK1+MSCs to differentiate into endothelial cells. MSCs implanted into ischemic hind limb resulted in marked improvement in blood flow and collateral vessels formation. / Conclusion. MSCs spontaneously induce efficient and mature angiogenesis in ischemic/hypoxic tissues with significant arteriolar component resulting in increased blood flow. They are also capable of spontaneous differentiation into endothelium. VEGF appears to be necessary for MSC-related angiogenesis and vasculogenesis.