Extended periods of oxygen deprivation cause brain death in mammals but the western painted turtle overwinters in anoxic mud for months without damage. Neural protection is achieved through decreases in the whole cell currents of N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (NMDAR and AMPAR) that are dependent on a mild increase in intracellular calcium from the mitochondria. The goal of this research was to determine if natural anoxic decreases in reactive oxidative species (ROS) serve as the signal to bring about these changes. Reductions in cellular ROS levels were demonstrated to have no effect on AMPAR currents or intracellular calcium and produced massive increases in NMDAR currents, indicating that ROS depression does not directly mediate anoxic alterations. Interestingly, mammalian neural tissue also experiences a similar increase in NMDAR whole cell current in response to reducing agents suggesting a possible conserved mechanism for normoxic receptor control.

Gout is one form of inflammatory arthritis, which is caused by the precipitation of crystals of monosodium urate (MSU) in the joints. Acute gout is associated with sudden and painful inflammatory episodes characterized by high neutrophil infiltration. In spite of years of study gout treatment remains a challenge due to its relative ineficcacy. Thus, search for new and efficient therapies is necessary. The objective of this study was to investigate the involvement of glibenclamide in a model of acute gout in rats induced by MSU. MSU crystals produced nociception and edema when injected into the ankle joint of rats. Treatment with glibenclamide (3 mg/kg, s.c.) or dexamethasone (8 mg/kg, s.c., used as a positive control) decreased spontaneous nociception (67% ± 11 and 70 ± 7% inhibition, respectively) and edema (28 ± 7% and 77 ± 7% inhibition, respectively) induced 6 hours after MSU injection. The number of leukocyte infiltrates in the synovial fluid as well as the release of interleukin 1β (IL-1β) and prostaglandin E2 (PGE2) significantly increased at 6 hours after injection of MSU joint, but these effects were not reversed by treatment with glibenclamide (3 mg/kg, s.c.). In contrast, dexamethasone reduced the leukocyte infiltration and release of IL-1β and PGE2. To confirm if the dose of glibenclamide was able to block the KATP channels, we determined the levels of glucose in the blood of animals. Glibenclamide decreased (23 ± 2%) and dexamethasone increased the blood glucose of the rats compared to vehicle-treated animals / MSU. Therefoe, the effects of glibenclamide on nociception and edema induced MSU, suggests that this sulfonylurea may be an interesting option as an adjunct therapy in pain observed in acute attacks of gout. / A gota é uma forma de artrite inflamatória, causada pela precipitação de cristais de urato monossódico (MSU) nas articulações. A forma aguda de gota está associada a episódios inflamatórios súbitos e dolorosos caracterizados por uma grande infiltração de neutrófilos. Apesar dos anos de estudo sobre a gota, o seu tratamento ainda é um desafio pela relativa ineficácia dos fármacos disponíveis no mercado. Assim, a busca por novos agentes terapêuticos mais efetivos e seguros se faz necessário. Desta forma, o objetivo deste estudo foi investigar o possível potencial farmacológico da glibenclamida em um modelo de gota aguda induzida por MSU em ratos. Os cristais de MSU produziram nocicepção e edema quando injetados na articulação do tornozelo de ratos. O tratamento com glibenclamida (3 mg/kg, s.c.) ou dexametasona (8 mg/kg, s.c., usada como controle positivo) reduziu a nocicepção espontânea (67 ± 11% e 70 ± 7% de inibição, respectivamente) e o edema (28 ± 7% e 77 ± 7% de inibição, respectivamente) induzidos pelo MSU, 6 horas após a injeção do cristal. O número de leucócitos infiltrados no líquido sinovial, assim como a liberação de interleucina 1β (IL-1β) e de prostaglandina E2 (PGE2) foram consideravelmente aumentados, 6 horas após a injeção de MSU na articulação, porém esses efeitos não foram revertidos pelo tratamento com glibenclamida (3 mg/kg, s.c.). Em contrapartida, dexametasona reduziu a infiltração de leucócitos e a liberação de IL-1β e de PGE2. Para confirmar se a dose utilizada de glibenclamida foi capaz de bloquear os canais de KATP, foi avaliado os níveis de glicose no sangue dos animais. A glibenclamida reduziu (23 ± 2%) e a dexametasona aumentou a glicemia dos ratos quando comparado aos animais tratados com veículo /MSU. Assim, frente aos efeitos desempenhados pela glibenclamida sobre a nocicepção e edema induzidos pelo MSU, sugere-se que esta sulfonilureia possa ser uma opção interessante como um tratamento adjuvante na dor observada em ataques agudos de gota.

L’infarctus du myocarde (IM) survenant après l’obstruction de l’artère coronaire est la cause principale des décès cardiovasculaires. Après l’IM, le coeur endommagé répond à l’augmentation du stress hémodynamique avec une cicatrice et une hypertrophie dans la région non-infarcie du myocarde. Dans la région infarcie, la cicatrice se forme grâce au dépôt du collagène. Pendant formation de la cicatrice, les cardiomyocytes ventriculaires résidant dans la région non-infarcie subissent une réponse hypertrophique après l’activation chronique due au système sympathique et à l’angiotensine II. La cicatrisation préserve l’intégrité structurale du coeur et l'hypertrophie des cardiomyocytes apporte un support ionotropique. Le canal CaV1.2 joue un rôle dans la réponse hypertrophique après l’IM. L’activation du CaV1.2 déclenche la signalisation dépendante de Ca2+ induisant l’hypertrophie. Cependant, il est rapporté que l’ouverture des canaux potassiques (KATP) ATP sensitifs joue un rôle sélectif dans l’expansion de la cicatrice après IM. Malgré leur expression dans les coeurs mâles, les KATP fournissent une cardioprotection sexe dépendante limitant l’expansion de la cicatrice chez les femelles. L’administration de rapamycine aux rates ayant subi un infarctus produit l’expansion de la cicatrice, soutenant la relation possible entre la cible de rapamycine, mTORC1 et les KATP dans la cardioprotection sexe spécifique. Effectivement, dans les cellules pancréatiques α, la signalisation mTORC1 était couplée à l'activation du KATP. Cependant, le lien entre mTORC1 et les canaux KATP dans le coeur reste inconnu. L'objectif de la thèse est d’examiner le rôle des canaux ioniques dans le remodelage cardiaque post-IM, surtout des canaux calciques dans l'hypertrophie et d'élucider la relation entre les KATP et mTORC1. L’hypothèse première teste que l’hypertrophie médiée par le système sympathique des cardiomyocytes ventriculaires des rats néonataux (NRCM) produit une augmentation de l’influx calcique après une augmentation des sous-unités du CaV1.2. Le traitement de norépinéphrine (NE) quadruple l’amplitude du courant calcique type L et double l’expression protéique des sous unités de CaVα2δ1 et CaVβ3. L’hypertrophie des NRCM au NE s’associe à une augmentation de la phosphorylation de la Kinase ERK 1/2. Le β1-bloqueur metoprolol et l’inhibiteur ii de ERK1/2 diminuent l’effet de NE sur CaVα2δ1. Cependant, l’augmentation de CaVβ3 et de la réponse hypertrophique persiste. Ainsi, le signal β1-adrenergique à travers ERK augmente les sous-unités CaVα2δ1 outre l’hypertrophie. L’autre hypothèse examine la spécificité du sexe sur l’expansion cicatricielle médiée par rapamycine et l’influence de mTOR sur l’expression de KATP. Rapamycin augmente la surface de la cicatrice et inhibe la phosphorylation de mTOR chez les coeurs de femelles. Dans les coeurs des deux sexes, la phosphorylation de mTOR et l’expression de KATP, Kir6.2 et SUR2A sont similaires. Cependant, une grande inactivation de la tubérine et une faible expression de raptor sont détectées chez les femelles. Le traitement à l’ester de phorbol des NRCM induit l’hypertrophie, augmente la phosphorylation de p70S6K et l’expression SUR2A. Le prétraitement par Rapamycine atténue chacune des réponses. Rapamycin démontre un patron d’expansion cicatriciel sexe spécifique et une régulation de phosphorylation de mTOR dans IM. Aussi, l’augmentation de SUR2A dans les NRCM traités par PDBu révèle une interaction entre mTOR et KATP. / Myocardial infarction (MI) secondary to the obstruction of the coronary artery is the main cause of cardiovascular death. Following MI, the damaged heart adapts to the increased hemodynamic stress via formation of a scar and a hypertrophic response of ventricular cardiomyocytes in the non-infarcted myocardium. In the infarcted region, a scar is formed via the rapid deposition of collagen. With ongoing scar formation, ventricular cardiomyocytes in the non-infarcted myocardium undergo a hypertrophic response secondary to the chronic activation by the sympathetic system and angiotensin II. Collectively, scar formation and cardiomyocyte hypertrophy preserve the structural integrity of the heart and provide inotropic support, respectively. CaV1.2 channels play a significant role in the hypertrophic response post-MI. Notably, the activation of CaV1.2 channel triggers Ca2+-dependent signaling that induces hypertrophy. By contrast, the opening of ATP-sensitive potassium (KATP) channels was shown to partake in selective scar expansion following MI. Notwithstanding its expression in male hearts, KATP channels endow a sex-dependent cardioprotection limiting scar expansion selectively in females. Moreover, administration of the macrolide rapamycin to the infarcted female rat heart led to scar expansion, supporting the possible relationship between the target of rapamycin, mTORC1 and KATP channels in providing sex-specific cardioprotection. Indeed, in pancreatic-α cells, mTORC1 signaling was coupled to KATP channel activation. However, whether mTORC1 targets KATP channels in the heart remains unknown. Thus, the AIM of the thesis was to explore the role of ion channels in cardiac remodeling post-MI by specifically addressing the role of Ca channels in cardiomyocyte hypertrophy and elucidate the potential relationship between KATP channels and mTORC1 signaling. The first study tested the hypothesis that hypertrophied neonatal rat ventricular cardiomyocytes (NRVMs) following sympathetic stimulation translated to an increase in calcium influx secondary to the augmentation of CaV1.2 channel subunits. NE treatment led to a 4-fold increase of L-type Ca2+ peak current associated with a 2-fold upregulation of CaVα2δ1 and CaVβ3 protein subunits in hypertrophied NRVMs. The hypertrophic response of NNVMs to NE was associated with the increased phosphorylation of extracellular regulated kinase (ERK1/2). The β1-blocker metoprolol and the ERK1/2 inhibitor suppressed NE-mediated protein upregulation of CaVα2δ1 whereas CaVβ3 upregulation and the hypertrophic response persisted. Therefore, sympathetic mediated β1-adrenergic signaling via ERK selectively upregulated the CaVα2δ1 subunit independent of NRVM hypertrophy. The second study tested the hypothesis that rapamycin-mediated scar expansion was sexspecific and mTOR influenced KATP channel subunit expression. Rapamycin administration translated to scar expansion and inhibited mTOR phosphorylation exclusively in females. In normal adult male and female rat hearts, mTOR phosphorylation and protein levels of KATP channel subunits Kir6.2 and SUR2A were similar. However, greater tuberin inactivation and reduced raptor protein levels were detected in females. NRVMs treated with a phorbol ester induced hypertrophy, increased p70S6K phosphorylation and SUR2A protein levels and rapamycin pretreatment attenuated each response. Thus, rapamycin administration to MI rats unmasked a sex-specific pattern of scar expansion and highlighted the disparate regulation of mTOR phosphorylation. Moreover, rapamycin-dependent upregulation of SUR2A in PDButreated NRVMs revealed a novel interaction between mTOR and KATP channel subunit expression

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L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1. També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut. / Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation. We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.