<p> Copious blood loss is the leading cause of death in military combat. Extreme exsanguination following traumatic injury causes hypotension which may culminate in hemorrhagic shock, multiple organ failure, and death. Currently, the only available strategy to treat hemorrhage is to apply tourniquets and administer resuscitative fluids. Although necessary to limit blood loss, protracted tourniquet application imposes ischemia on distal tissues. Revascularization of the injured limb reintroduces oxygenated blood into the ischemic zone, forming toxic reactive oxygen species. These highly reactive compounds can inactivate key metabolic enzymes, hamper ATP production, and cause end organ dysfunction. Fluid resuscitation provides crucial hemodynamic support, and affords an opportunity to treat the deleterious effects of hemorrhagic shock and ischemia-reperfusion. In order to mitigate the harmful effects of hemorrhagic shock and ischemia-reperfusion of tourniqueted extremities, a fluid resuscitant should contain agents capable of suppressing the formation of reactive oxygen and nitrogen species, thus, protecting cellular metabolic function; stabilizing tissue energetics; and safeguarding end organ function. Pyruvate, an endogenous energy substrate, possesses strong antioxidative properties. </p><p> This study tested whether substituting pyruvate for lactate in a Ringer's solution would be effective at mitigating reactive oxygen species formation, protect key ATP-generating and ATP-shuttling enzymes from inactivation, bolster skeletal and cardiac muscle phosphorylation potentials, and stabilize cardiac electrical function in goats subjected to hemorrhagic shock and hindlimb ischemia-reperfusion. Isoflurane-anesthetize goats were subjected to a controlled hemorrhaged to reduce the mean arterial pressure to c. 50 mmHg. After reaching this target pressure, hindlimb ischemia (HLI) was imposed for a total of 90 min by femoral artery crossclamp and tourniquet application around the hindlimb. After 30 min of hindlimb ischemia, pyruvate- (PR) or lactate- enriched (LR) Ringer's solution was infused intravenously (10mL/min) for 90 min. Time control (TC) goats were neither hemorrhaged nor subjected to hindlimb ischemia. At the conclusion of- and 3.5 h after- fluid resuscitation, the left ventricle and the right gastrocnemius were biopsied and flash-frozen for biochemical analysis of metabolites, enzymes, and markers of oxidative stress. In addition, custom-written software was developed to analyze QT interval variability- a marker of electrical instability- from the lead II electrocardiogram. </p><p> The first phase of this project tested the hypothesis that resuscitation with PR <i>vs.</i> LR effectively protects cardiac metabolism and preserves cardiac electrical performance during hemorrhagic shock and hindlimb ischemia. Resuscitation with PR effectively suppressed the formation of myocardial tissue 8-isoprostane <i>vs.</i> goats resuscitated with LR during the acute and subacute phases of the protocol. In addition, myocardial creatine kinase (CK) activity fell after LR administration <i>vs.</i> TC; however, PR preserved CK activity better than LR during fluid resuscitation and 4 h after hindlimb ischemia reperfusion. PR administration augmented myocardial phosphocreatine phosphorylation potential during fluid administration and 3.5 h later to values significantly higher than those in LR-resuscitated goats. Pro-arrhythmic QTc variability was markedly increased in LR <i> vs.</i> PR and TC during both phases of the protocol. </p><p> The second phase of this project tested the hypothesis that resuscitation with PR preserves tissue energetics in the reperfused gastrocnemius during hemorrhagic shock and hindlimb ischemia. Resuscitation with PR <i>vs. </i> LR effectively protected the gastrocnemius from oxidative stress in both protocols, as evidenced by the suppression of 8-isoprostane formation. PR prevented CK and aconitase inactivation <i>vs.</i> LR during the acute phase of reperfusion, and this enzyme protection persisted at least 3.5 h after completing fluid resuscitation. Additionally, PR augmented muscle phosphocreatine phosphorylation potential <i>vs.</i> TC and LR during the acute phase of reperfusion, and, like CK and aconitase activities, this augmented energy state persisted 3.5 h after the end of fluid resuscitation. </p><p> We conclude that 1) Pyruvate Ringer's resuscitation during hemorrhagic shock and hindlimb ischemia provides antioxidative protection in skeletal and cardiac muscle during fluid resuscitation and that this beneficial effect persists 3.5 h after the completion of fluid resuscitation; 2) Pyruvate-fortified fluid resuscitation prevents inactivation of enzymes involved in production and shuttling of ATP; 3) PR augments cardiac and muscle phosphorylation potentials during fluid resuscitation and persists 4 h after hindlimb reperfusion, <i> i.e.</i> 3.5 h after completing fluid resuscitation; and 4) Resuscitation with PR effectively protects cardiac electrical rhythm in the face of hemorrhagic shock and hindlimb ischemia. These investigations demonstrate the powerful antioxidative protection imposed by pyruvate, its positive effects on muscle and cardiac metabolism and energy state and its role in stabilizing cardiac electrical function during hemorrhagic shock and hindlimb ischemia.</p>

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Endothelin-1 (ET-1), an endothelium-derived vasoconstrictor peptide, plays a role in the pathophysiology of cardiovascular disease. Transgenic mice that overexpress human preproET-1 selectively in the endothelium exhibit endothelial dysfunction and hypertrophic remodeling of resistance-size arteries in the absence of blood pressure elevation. In order to understand the mechanisms whereby ET-1 directly induces vascular damage, we determined the changes in gene expression in mesenteric arteries of these mice using genome-wide expression profiling. This study revealed a set of genes potentially regulated by ET-1, which might be implicated in ET-1 induced-vascular damage. Our findings suggest that increased endothelium-restricted ET-1 expression results in early changes in gene expression, which increase lipid biosynthesis and decrease bone morphogenetic protein (BMP)-4 expression, and may contribute, at least in part, to ET-1-induced vascular damage. These results have the potential of defining ET-1 gene targets, which may facilitate the discovery of new therapeutic approaches against detrimental effects of ET-1. / Keywords: endothelin-1, gene expression, lipid biosynthesis, BMP4 / L'endothéline-1 (ET-1), un puissant peptide vasoconstricteur produit par l'endothélium, joue un rôle dans la pathophysiologie des maladies cardiovasculaire. Des souris transgéniques surexprimant la prépro-ET-1 humaine dans l'endothélium présentent une dysfonction endothéliale et un remodelage hypertrophique des artères de résistance en l'absence d'hypertension. Pour comprendre les mécanismes expliquant les modifications vasculaires de ces animaux, nous avons étudié les changements transcriptomiques dans les artères mésentériques. Cette étude a révélé une série de gènes régulés par l'ET-1 qui pourrait être impliquée dans les dommages vasculaires induits par l'ET-1. Ces résultats suggèrent qu'une augmentation d'expression d'ET-1 augmente la biosynthèse de lipides et diminue l'expression de la « bone morphogenic protein » (BMP)-4, ce qui pourrait contribuer, du moins en partie, aux développements des dommages vasculaires induits par l'ET-1. Ces résultats ont le potentiel de définir des cibles de l'ET-1 qui pourraient faciliter la découverte de nouvelles approches thérapeutiques contre les effets nuisibles de l'ET-1. / Mots clés: endothéline -1, changements transcriptomiques, biosynthèse de lipides, BMP4

The effect of neonatal hypoxemia on systemic arterial blood pressure at maturity was assessed in male (12 experimental, 11 control) and female (10 experimental, 9 control) adult Sprague Dawley rats. Experimental rats were exposed to hypoxia (FiO2 = 0.12) for the first ten days of life, then subsequently raised in normoxia along with age-matched controls. At 2 months of age arterial blood pressure was recorded intravascularly using telemetry. Systolic, mean, and pulse pressures were significantly greater in hypoxic pre-treated groups of both sexes compared to controls (p < 0.05). Neonatal hypoxia did not affect diastolic pressure or heart rate. Neonatally hypoxic males displayed significantly increased daytime blood pressure variability. Aortic pulse wave velocity was assessed in males and found to be significantly elevated in 6 additional neonatally hypoxic rats when compared to 5 controls. Neonatal hypoxia in the rat is associated with increased systolic arterial pressure likely due to decreased arterial compliance. / Les effets de l'hypoxémie néonatale sur la pression artérielle furent évalués sur des rats mâles adultes Sprague Dawley (12 expérimental, 11 contrôlé). Les rats expérimentaux furent exposés à l'hypoxie (FiO2 = 0.12) durant les 10 premières journées de vie pour ensuite être élevée normalement avec des rats du même âge contrôlés. À 2 mois, un prélèvement intravasculaire fut réalisé avec télémétrie. La pression systolique, la moyenne et le pouls étaient considérablement plus élevés pour les deux sexes prétraités à l'hypoxie (p < 0.05). L'hypoxie néonatale n'a pas d'effet sur la pression diastolique ou le battement du cœur. La pression sanguine au repos des mâles traités à l'hypoxie néonatale était considérablement plus variable. La vélocité des impulsions aortiques de 6 mâles expérimentaux était considérablement plus élevée que sur 5 mâles en conditions contrôlées. L'hypoxie néonatale sur les rats s'associe à une augmentation de la pression artérielle systolique résultant d'une diminution des conformités artérielles.

ABSTRACTTRPM7 (transient receptor potential melastatin), a member of the large TRP ion channel family, is ubiquitously expressed in cells and is constitutively active. It is comprised of six transmembrane domains that assemble into tetramers to form a central Mg2+ and Ca2+ permeable pore. TRPM7 and its homologue TRPM6 are some of the only channels known to carry Mg2+. Hypertension, a cardiovascular condition linked to low levels of intracellular Mg2+ is also associated with high levels of aldosterone. Previous results have demonstrated that aldosterone increases mRNA levels of TRPM7 whereas protein levels decreased in VSMCs. To understand if TRPM7 may be implicated in hypertension, we questioned whether aldosterone could regulate TRPM7 currents, and inquired for a possible underlying mechanism. Whole-cell patch clamp studies were conducted in inducible HEK-293 cells, stably expressing wild type TRPM7. We found that TRPM7 currents are enhanced after overnight stimulation with aldosterone compared to non-stimulated cells. When the mineralocorticoid receptor (hMR) is transfected two days prior aldosterone stimulation, this response is further increased. The introduction of 10mM BAPTA, a Ca2+ chelator, to the intracellular medium doubled the TRPM7 current in WT cells and also increased the response to aldosterone in cells transfected with the hMR receptor. Surprisingly, protein levels of TRPM7 do not vary, suggesting a redistribution of already existing channels to the membrane. SGK-1, a serine threonine kinase was suggested as a possible mediator of the response. In fact, when a specific blocker to SGK-1 is applied onto the cells, both current and total protein levels of TRPM7 are significantly decreased. Overall, these results demonstrate that aldosterone can regulate TRPM7 through an increase in total current. This response appears to be mediated by SGK-1 in a calcium sensitive manner. / RÉSUMÉTRPM7 (transient receptor potential melastatin), membre de la large famille des canaux ioniques des TRP, est exprimée de façon omniprésente dans toutes les cellules, et est active de façon constitutive. TRPM7 est composée de six domaines transmembranaires qui s'assemblent en tétramères pour former un pore central, perméable aux ions Mg2+ et Ca2+. TRPM7, et son homologue TRPM6, sont les seuls canaux ioniques connus pour le transport du Mg2+. L'hypertension, une maladie cardiovasculaire associée à de faibles niveaux en Mg2+ intracellulaire est aussi liée à de niveaux élevés d'aldosterone. Des recherches antérieures ont démontré que l'aldosterone augmente les niveaux d'ARNm de TRPM7 tandis que la quantité de protéines diminue dans les cellules vasculaires lisses du muscle. Afin de comprendre si TRPM7 peut être impliquée dans l'hypertension, nous nous sommes demandés si l'aldosterone pouvait réguler les courants associés à TRPM7, et si nous pouvions définir un mécanisme d'action qui pourrait expliquer une telle régulation. La technique du patch clamp a été utilisée sur des cellules HEK-293 inductibles exprimant de façon stable le phénotype humain de TRPM7. Nous avons trouvé que les courants de TRPM7 sont augmentés après une stimulation de nuit avec de l'aldosterone, comparé à des cellules non stimulées. Lorsque le récepteur humain mineralocorticoid (hMR) est transfecté deux jours avant la stimulation par l'aldosterone, la réponse en courant est rehaussée. L'ajout de 10mM de BAPTA, un chélateur du Ca2+, dans la solution intracellulaire permet de doubler la réponse en courant dans ces cellules, ainsi que d'augmenter la réponse à l'aldosterone dans les cellules transfectées avec le récepteur hMR. Etonnamment, les niveaux de protéines de TRPM7 ne sont pas affectés, suggérant une redistribution des canaux ioniques déjà existants à la membrane. SGK-1, une kinase membre de la famille des serine-threonines a été proposée comme un possible médiateur de la réponse a l'aldosterone. En effet, après l'application d'un bloquer spécifique pour le SGK-1, une diminution des courants ainsi que de la quantité de protéines associées à TRPM7 a été observée. De façon générale, ces résultats démontrent que l'aldosterone est capable de réguler TRPM7 à travers une augmentation des courants. Cette réponse, qui semble être sous l'influence de SGK-1, utilise un mécanisme sensible aux niveaux de calcium intracellulaire..

Little is known about the interaction and channeling of motor and sensory information in the control of balance with aging. The aim of this thesis was to study the mechanisms of equilibrium control and its effects on aging when large head and gaze shifts are executed under varying postural conditions. In a first study, the postural strategies used by young and elderly subjects were compared during standing and walking tasks while subjects executed a large and rapid rotation of the head. Elderly subjects restricted their body motions and velocities, likely to minimize the ensuing perturbation. An important aspect of the head turning tasks is the redirection of gaze, which must be coordinated with postural responses. Thus, in a second study we aimed to determine the mechanisms of large horizontal gaze shifts (saccades) when submitted to a support surface perturbation, in young subjects. The findings from this study revealed a saccadic facilitation mechanism, when the visual target shift stimuli was closely followed by a motion of the supporting surface, providing evidence for the need of a stable reference when all available sensors are perturbed. In a third study, the control of gaze shifts (pursuit and saccades) during surface perturbations (sequence of pseudo-random motions) and with aging was examined. As hypothesized, elderly subjects showed less precision in target tracking. As well, the surface perturbations resulted in increased tracking errors for both groups. However, gaze time lag in response to target was not affected by surface motions. This study provided evidence that gaze accuracy could be attributable for the segmental movement excursion restriction observed in elderly subjects of the first study. Moreover, the saccadic facilitatory mechanism observed in the second study is specific to the surface perturbation condition (discrete vs. sequence) and its timing relative to target shift. Therefore, the trainability of elderly subjects in executing complex tasks, as described in the present thesis, should be explored. Improved abilities in executing large head and gaze shifts under challenging postural conditions would be an important step in reducing the incidence of falls in the elderly and could guide rehabilitation interventions.

Glutathione (GSH) is the most abundant antioxidant in the lung and plays an important protective role. The level of GSH in the airway surface liquid (ASL) is significantly reduced in cystic fibrosis (CF) patients, which may exacerbate the oxidative stress in the airway lumen induced by inflammation. GSH release may also be harmful to surface cells lining the airways if it reduces intracellular levels. The mechanisms of glutathione transport by airway epithelia are not known.

Lamin A/C, an important component of the nuclear envelope, has been associated with cell differentiation and tissue development. In bone, recent studies have described that altered function of lamin A/C, due to mutations or incorrect processing, is associated with accelerated and severe bone loss. This could be due to alterations in the differentiation of mesenchymal stem cells (MSC) induced by lack of lamin A/C activity. Therefore, considering that decreased osteoblastogenesis and increased adipogenesis are the characteristic changes observed within bone marrow microenvironment that leads to age-related bone loss, we have studied the effect of inhibition of lamin A/C on MSC into either osteoblasts or adipocytes both in vitro and in vivo. For in vitro studies, we identified the effect of pharmacological inhibition of lamin A/C on adipogenesis. Subsequently, we inhibited lamin A/C by using different doses of lamin A/C siRNA in osteogenic and adipogenic differentiating MSCs as well as normal human osteoblasts. To further verify our results, we have used Zmpste24-/- null progeroid mice that lacks mature lamin A/C and studied the bone changes in absence of mature lamin A/C in in vivo. We have found that partial inhibition of lamin A/C decreased osteoblast differention and function without affecting their survival. Furthermore, our animal studies showed that absence of mature lamin A/C induced a reduction in bone mass and bone quality associated with low bone turnover. In contrast, accumulation of prelamin A induced significant levels of adipogenesis within the bone marrow cavity. In summary, our results provide evidence in support of a pivotal role of lamin A/C in the comm / Lamin A/C, un important composant de l'enveloppe nucléaire, a été associé avec la différentiation cellulaire et le développement tissulaire. Dans l'os, des études récentes ont demontrée que l'alteration de lamin A/C, due a des mutations du gène LMNA, est associée avec une perte accélérée et sevère de l'os. Ceci pourrait être dû à une altération de la différentiation des cellules souches mésenchymateuses (CSM) induit par une perte de l'activité de lamin A/C. Par conséquent, en considerant que la diminution de l'ostéoblastogènese et l'augmentation de l'adipogènese sont les changements caractéristiques observés à l'intérieur du micro-environnement de la moelle osseuse qui mènent à une perte osseuse relié à l'âge, nous avons étudié l'effet de l'inhibition de lamin A/C sur la différentiation des cellules souches soit en adipocyte ou en ostéoblast in vitro et in vivo. Pour les études in vitro, nous avons identifiés l'effet de l'inhibition pharmacologique de lamin A/C sur l'adipogènese. Par la suite, nous avons inhibé lamin A/C en utilisant différentes doses de lamin A/C siRNA sur les cellules souches en différentiatiation en ostéoblast et en adipocytes et aussi sur des ostéoblast humaine. Pour confirmer nos résultats précedents, nous avons utilisés un modèle progerique murin Zmste24-/-. Ces souris ne possedent pas la forme mature de lamin A/C. Nous avons etudiés les changements osseux en absence de la forme mature de lamin A/C in vivo. Nous avons trouvé qu'une inhibition partielle de lamin A/C diminue la différentiation et la fonction des ostéoblastes sans affecter leur survie. En outre, nos études sur les souris ont d

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