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

Regensburg, Univ., Diss., 2004.

L'activité physique (AP) régulière se révèle essentielle en terme de santé publique et est un déterminant important, impactant l'endothélium, pour assurer la prévention primaire et secondaire de nombreuses pathologies et facteurs de risques cardiovasculaires (HTA, IC, hypercholestérolémie, diabète, sédentarité, obésité, surpoids).Tant chez l'homme que chez l'animal, il est couramment accepté que l'AP modérée permet de restaurer la vasodilatation endothélium dépendante médiée par le NO/EDRF (VED-NO) lorsque celle ci est altérée chez des sujets diabétiques (1 et 2), obèses, en surpoids ou sédentaires. De nombreuses investigations, tant expérimentales que cliniques restent à explorer pour évaluer les paramètres (fréquence, intensité, durée) de différents types (endurance, résistance) d'exercices se révélant les plusbénéfiques pour améliorer une VED-NO détériorée dans de nombreuses pathologies et facteurs de risques cardiovasculaires. Dans un premier temps, par une approche expérimentale, nous avons eu pour objectif de déterminer les effets d'un entraînement intense sur VED-NO. L'étude a été réalisée avec des aortes isolées de rats diabétiques (type1) sédentaires et entraînés. Dans un second temps, dans une étude clinique chez des adolescents (14-16 ans), nous avons cherché à savoir si un entrainement préalable modifiait la VED-NO après un exercice isolé aigu. Ce travail a été réalisé au niveau de la microcirculation cutanée de l'avant-bras, par débitmètrie « Laser Doppler ». Dans un troisième temps, toujours par une étude clinique par débitmétrie « Laser Doppler », nous avons enregistré les effets d'un entraînement musculaire de deux mois chez des femmes obèses tant au niveau de VED-NO qu'au niveau de biomarqueurs de l'inflammation (TNF alpha, leptine, Interleukine, visfatine).Nos principaux résultats montrent que: 1) L'inactivité physique et l'état diabétique de type 1 chez le rat provoquent, sur l'aorte isolée, une augmentation de la vasoconstriction à la Phényléphrine ainsi qu'une réduction importante de la VED -NO - inhibée par le L-NAME- induite par l'Ach comme par l'ADPβS via des récepteurs muscariniques M1/3 et purinergiques P2Y, 2) L'AP intense (sur tapis roulant, 60min/j, 25m/min, 5j/semaine,8semaines) chez le rat diabétique est inefficace à rétablir une VED-NO, 3) L'entraînement préalable de jeunes hommes (15+ ou – 1ans) permet, suite à un exercice isolé et aigu, d'améliorer la VED-NO induite par une iontophorèse d'ACh au niveau de la microcirculation cutanée de l'avant-bras (flux sanguin mesuré par Laser-Doppler), 4) .L'AP (bicyclette ergométrique 1h, intensité à 50% de la FC max, 3j/semaine, 2 mois) chez des femmes obèses -IMC=38.5-, comparativement à dessujets témoins, restaure la VED-NO induite par une iontophorèse d'ACh au niveau de la microcirculation cutanée, 5) De plus, dans cette dernière étude, les capacités aérobies sont améliorées et apparaît une réduction importante des biomarqueurs de l'inflammation. En conclusion, nos travaux montrent qu'il y a encore beaucoup d'investigations à mener pour comprendre les mécanismes moléculaires conduisant à une réhabilitation de la fonction endothéliale préalablement altérée et qu'il faut continuer à préciser, pour chaque type de pathologie cardiovasculaire, les paramètres d'AP (intensité, durée, fréquence) les plus adaptés pour rétablir la VED-NO chez l'animal comme chez l'homme. / Regular physical activity appears to be essential in terms of public health. It is an important determinant, concerning the endothelium, for primary and secondary prevention of many diseases and cardiovascular risk factors (Hypertension, heart failure, hypercholesterolemia, diabetes, sedentarity,obesity, overweight).Both in man and in animals, it is well know that moderate physical activity can restore endothelium-dependent vasodilatation mediated by NO/EDRF (EDV-NO) when it was altered in diabetics (type 1 and 2), obese, overweight, sedentary. Many investigations, both experimental and clinical remain to be explored to evaluate parameters (frequency, intensity, duration) of different types of exercise (endurance, resistance) being the most beneficial for improving EDV-NO deteriorated in many diseases and cardiovascular risk factors. In a first time, by an experimental approach, we aimed to determine the effects of intense training on EDV-NO. The study was performed with isolated aortas of sedentary and trained diabetics rats (type 1). In a second time, in a clinical study in youngmen (14-16 years), we investigated whether prior training modifies EDV-NO after an acute isolated exercise. This work was performed in the cutaneous microcirculation of the forearm by « Laser Doppler » flowmetry. In a third time, in a clinical study by « Laser- Doppler »flowmetry, we recorded the effects of two months resistance training in obese women both in EDV-NO and on inflammatory biomarkers ( TNF alpha, leptin, interleukin, visfatin). Our main results show that: 1) physical inactivity and type 1 diabetic state in rats cause on the isolated aorta, increased vasoconstriction induced by phenylephrine and a significant reduction in EDV-NO(inhibited by L-NAME) induced by Ach as ADPβS via M1/3 muscarinic and P2Y purinergic receptors. 2) Intense training (treadmill, 60min/day, 25m/min, 5d/week, , 8 weeks) in diabetic rats is ineffective in restoring EDV-NO. 3) Prior training of young men, allows, after an isolated acute exercise, to improve EDV-NO induced by ACh iontophoresis in the cutaneous microcirculation of the forearm. 4) Physical activity (treadmill, walking 1 h, intensity 50% Heart Rate max, 3d/week, 2 months) in obese women (BMI=38.5) compared to control subjects, restores EDV-NO induced by Ach iontophoresis in the cutaneous microcirculation of the forearm. 5) In addition, the previous study, aerobic capacities are improved and appears a significant reduction in inflammatory biomarkers concentration. In conclusion, our researchesshow that there is still many studies to understand the molecular mechanisms leading to a restoration of impaired endothelial function. It is necessary to continue to specify, for each type of cardiovascular disease, the most appropriate parameters of physical activity adapted to restore EDV-NO in animal and man.

Bakgrund. De senaste årtiondena har betydelsen av nitrat, nitrit och kväveoxid (NO) ikroppen blivit känd. Ett dietärt intag av nitrat har i flera kliniska studier visat sig haflera positiva hälsoeffekter, speciellt relaterat till att förbättra den kardiovaskulärafunktionen men även i idrottsammanhang har det påvisats positiva effekter, så somminskad syrekostnad och ökad muskulär verkningsgrad. De flesta studier undersökerdock akut intag av nitrater och prestationer <60 minuter.Syfte. Att undersöka om tre dagars uppladdning med nitrat i form av 500 mlrödbetsjuice om dagen samt en fjärde dos á 250 ml rödbetsjuice på testdagen kan ökaprestationen hos motionärer genom att mäta hur lång distans de kan cykla under ettlångdistanspass på 2 timmar.  Metod. En randomiserad dubbelblind placebo-kontrollerad crossover studie utfördes påtotalt 7 st fullt friska försökspersoner, tre män och fyra kvinnor. Testdeltagarnagenomgick vid två tillfällen tre dagars uppladdning med två olika typer av kosttillskott,rödbetsjuice, vilket innehåller nitrat och placebo (blåbärsjuice). En sex dagar lång washout period separerade de två testtillfällena. Totalt genomfördes sju olika mätningar;längd och vikt, blodtryck, puls, cykeltest, laktatvärden och upplevd ansträngningregistrerades. Ett parat t-test och ett icke-parametriskt test användes i den statisktiskaanalysen för att jämföra placebo och nitrat vid första och andra testtillfället.  Resultat. En signifikant skillnad (p<0.02) i prestationsförmåga kunde uppvisas efterintag av nitratprov. Puls-, laktat- och blodtrycksvärden uppvisade ingen signifikantskillnad.  Konklusion. Tre dagars uppladdning samt en fjärde dos nitrat på testdagen förbättradeprestationsförmågan med 4,1 % utan att påverka puls eller upplevd ansträngning. Dennastudie visar på att nitrat kan användas som kosttillskott för att öka sinprestationsförmåga, dock visar studien ingen skillnad i blodtryck varav man kan draslutsatsen att det främst är idrottare som drar nytta av dietärt intag av nitrat. / Aim: To investigate whether three days of dietary with nitrate intake in the form of 500 ml of beetroot juice a day and a fourth dose of 250 ml of beetroot juice on the test day could increase the performance of athletes by measuring how much distance they can bike over a long distance workouts in 2 hours. Method. A randomized double-blind placebo-controlled crossover study was conductedin a total of 7 fully healthy volunteers, three men and four women. The test participantsunderwent twice three days charging with two different types of supplements, beetroot juice, which contains nitrate and placebo (blueberry juice). A 6-day washout period separated the two test sessions. The primary outcome was the distance each cyclist covered at each occation. In addition; height and weight, blood pressure, pulse, lactate and perceived exertion were recorded. A paired t-test and a non-parametric test were used in the statistical analysis to compare placebo and nitrate in first and second testing. Results. A significant difference (p <0,02) in performance was present after intake of nitrate. Heart rate, lactate and blood pressure values showed no significant difference. Conclusion. Three days of nitrate intake and a fourth dose of nitrate on the test day improved performance by 4% without affecting heart rate or perceived exertion. This study shows that nitrate can be used as supplements to boost their performance, however, the study shows no difference in blood pressure which it can be concluded that it is mainly the athletes who benefit from dietary intake of nitrate.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A Alpinia zerumbet à uma planta da famÃlia Zingiberaceae, denominada popularmente âcolÃniaâ no Nordeste do Brasil. à utilizada com fins medicinais no tratamento de hipertensÃo e tem sido estudada em relaÃÃo as suas propriedades farmacolÃgicas. Esta pesquisa teve por objetivo analisar o efeito farmacolÃgico das fraÃÃes hexÃnica (FHOEAz), clorofÃrmica (FCOEAz) e metanÃlica (FMOEAz) do Ãleo essencial da Alpinia zerumbet (OEAz) na reatividade vascular in vitro e nos parÃmetros cardiovasculares in vivo. O projeto de pesquisa foi aprovado pela ComissÃo de Ãtica em Pesquisa Animal, da Universidade Federal do CearÃ, sob os protocolos n 55/10 e n 18/2011, de acordo com as normas de boas prÃticas que envolvem o uso de animais experimentais. Para todos os experimentos foram utilizados ratos Wistar machos. Realizaram-se experimentos de reatividade vascular no banho de ÃrgÃos, com preparaÃÃes de aorta isolada de rato, com endotÃlio Ãntegro e desnudo, mantidos em carbogÃnio e soluÃÃo de Krebs-Henseleit com concentraÃÃo em mmol/L: NaCl: 118,0; KCl: 4,7; KH2PO4: 1,2; MgSO4.7H2O; 1,2; NaHCO3: 15,0; CaCl2: 2,5 e Glicose: 5,5. Foi observada a variaÃÃo da tensÃo isomÃtrica e utilizados diferentes fÃrmacos inibidores especÃficos para anÃlise do mecanismo de aÃÃo do efeito vasodilatador. Analisou-se a pressÃo arterial indireta por pletismografia de cauda em ratos submetidos ao modelo de hipertensÃo por inibiÃÃo crÃnica do Ãxido nÃtrico, atravÃs da administraÃÃo do L-NAME, obtendo-se a pressÃo arterial sistÃlica, diastÃlica e mÃdia, frequÃncia cardÃaca, alÃm da variaÃÃo do peso corporal. Constatou-se que o OEAz, FHOEAz, FCOEAz e FMOEAz induzem relaxamento de anÃis aÃrticos prÃ-contraÃdos com Fenilefrina (1 mol/L), de forma dependente da dose, sendo a menor CE50 a da FMOEAz (150,45 g/mL). A administraÃÃo por gavagem de 100 mg/kg de OEAz, FHOEAz, FCOEAz e FMOEAz reduziu a pressÃo arterial em ratos hipertensos pelo modelo de inibiÃÃo crÃnica do Ãxido nÃtrico, um efeito que foi superior ao controle negativo com Ãgua destilada e inferior aos controles positivos com Captopril e Nifedipina. A FMOEAz (0,1 - 3000 g/mL) induz relaxamento dependente da dose em anÃis aÃrticos prÃ-contraÃdos com Fenilefrina (1 mol/L) ou KCl (80 mmol/L), com endotÃlio intacto ou desnudo. Os estudos de mobilizaÃÃo de cÃlcio mostraram que a FMOEAz inibe o influxo de Ca2+ do meio extracelular, bem como interfere na contraÃÃo induzida pela liberaÃÃo de Ca2+ dos estoques intracelulares pela Fenilefrina (1 mol/L) ou CafeÃna (30 mmol/L). A 4-aminopiridina (1 mmol/L) e a Iberiotoxina (30 nmol/L) aumentam a CE50 da FMOEAz, mas nÃo interferem no seu efeito vasodilatador final. A prÃ-incubaÃÃo com L-NAME (100 mol/L), ODQ (10 mol/L); Indometacina (10 mol/L), Atropina (1 mol/L), Catalase (500 U/ml), SOD (300 U/mL); Wortmannina (0,5 mol/L), TetraetilamÃnio (10 mmol/L), Apamina (1 mol/L); Caribdotoxina (15 nmol/L) e Glibenclamida (10 mol/L) nÃo interferiram no relaxamento induzido pela FMOEAz. Concluiu-se que o OEAz, FHOEAz, FCOEAz e FMOEAz possuem efeito hipotensor in vivo e vasodilatador in vitro, e que o mecanismo de aÃÃo da FMOEAz, provavelmente, envolve o antagonismo aos canais de cÃlcio dependentes de voltagem, aos canais de cÃlcio operados por receptor, interferindo tambÃm na liberaÃÃo de cÃlcio dos estoques intracelulares. / The Alpinia zerumbet is a plant of the family Zingiberaceae, popularly called "colÃnia" in Northeastern Brazil. It is used for medicinal purposes to treat hypertension and has been studied in relation to its pharmacological properties. This study aimed to analyze the pharmacological effect of hexanic (HFEOAz), chloroformic (CFEOAz), and methanolic (MFEOAz) fractions of the essential oil of Alpinia zerumbet (EOAz) in the vascular reactivity in vitro and cardiovascular parameters in vivo. The research project was approved by the Ethics Committee on Animal Research, of the Federal University of CearÃ, under protocol numbers 55/10 and 18/2011, according to the standards of good laboratory practice involving the use of experimental animals. For all experiments male Wistar rats were used. Experiments of vascular reactivity were conducted in organ bath, with preparations of isolated rat aorta with intact endothelium and desnuded, kept in carbogen and Krebs-Henseleit solution with a concentration in mmol/L: NaCl: 118,0; KCl: 4,7; KH2PO4: 1,2; MgSO4.7H2O; 1,2; NaHCO3: 15,0; CaCl2: 2,5 e Glicose: 5,5. Was observed variation in isometric tension and used different specific inhibitors to analyze the mechanism of action of the vasodilator effect. We analyzed blood pressure indirectly by tail plethysmography in rats submitted to chronic hypertension by inhibition of nitric oxide by the administration of L-NAME, obtaining the systolic, diastolic and mean blood pressure, heart rate, beyond variation in body weight. It was found that the EOAz, HFEOAz, CFEOAz MFEOAz induced relaxation of aortic rings pre-contracted with Phenylephrine (1 mol/L), of dose-dependent manner, with the smallest of the EC50 MFEOAz (150.45 mg/mL). The administration by gavage of 100 mg/kg EOAz, HFEOAz, CFEOAz MFEOAz reduced blood pressure in hypertensive rats by the model of chronic inhibition of nitric oxide, an effect that was greater than the negative control with distilled water and less than the positive controls with Captopril and Nifedipine. The MFEOAz (0.1 - 3000 Âg/mL) concentration dependently relaxed Phenylephrine (1 mol/L) and KCl (80 mmol/L) contracted rings with intact or denuded endothelium. Studies of calcium mobilization showed that FMOEAz inhibits the influx of Ca2+ from the extracellular environment and interferes with the contraction-induced Ca2+ release from intracellular stocks by Phenylephrine (1 mol/L) or Caffeine (30 mmol/L). The 4-aminopyridine (1 mmol/L) and Iberiotoxina (30 nmol/L) increased the EC50 of MFEOAz but do not interfere in its final vasodilator effect. The pre-incubation with L-NAME (100 mol/L), ODQ (10 mol/L), Indomethacin (10 mol/L), Atropine (1 mol/L), Catalase (500 U/mL), SOD (300 U/mL), Wortmannin (0.5 mol/L), Tetraethylammonium (10 mmol/L), Apamin (1 mol/L); Caribdotoxin (15 nmol/L) and Glibenclamide (10 mol/L) did not interfere with the relaxation induced by MFEOAz. It was concluded that the EOAz, HFEOAz, CFEOAz and MFEOAz have hypotensive effect in vivo and vasodilator effect in vitro, and that the mechanism of action of MFEOAz probably involves the antagonism of calcium channels voltage-dependent, the calcium channel operated by receptor, also interfering in the release of calcium from intracellular stores.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-01-21T12:29:23Z No. of bitstreams: 1 fabianadefariaghetti.pdf: 2678682 bytes, checksum: 4f41f688b7803865f93dd37611975999 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-01-25T18:44:48Z (GMT) No. of bitstreams: 1 fabianadefariaghetti.pdf: 2678682 bytes, checksum: 4f41f688b7803865f93dd37611975999 (MD5) / Made available in DSpace on 2016-01-25T18:44:48Z (GMT). No. of bitstreams: 1 fabianadefariaghetti.pdf: 2678682 bytes, checksum: 4f41f688b7803865f93dd37611975999 (MD5) Previous issue date: 2014-03-27 / Pacientes com hipotireoidismo subclínico (HSC) possuem maior risco para mortalidade cardiovascular. Uma das possíveis explicações para esse desfecho seria a piora da função vascular. Porém, não são conhecidos os efeitos do HSC no comportamento hemodinâmico durante manobra fisiológica de estresse mental. O objetivo desse estudo foi investigar o comportamento hemodinâmico de mulheres com hipotireoidismo subclínico durante estresse mental. Foram avaliadas 20 mulheres com HSC (Grupo HSC) e 21 eutireoidianas (Grupo Controle), pareadas por idade (37±11 vs. 38±10 anos, p=0,699, respectivamente) e índice de massa corporal (26±5 vs. 25±5 kg/m2, p=0,462, respectivamente). O fluxo sanguíneo muscular (FSM), avaliado pela pletismografia de oclusão venosa, a frequência cardíaca (FC) e a pressão arterial (PA), medidas pelo Dixtal2023, foram registrados simultaneamente durante 3 minutos de basal, seguidos de 3 minutos de estresse mental (Stroop Color Word Test). A condutância vascular do antebraço (CVA) foi calculada pela divisão do FSM pela pressão arterial média multiplicada por 100. Foram utilizados os testes t de Student e ANOVA, adotando significativo p<0,05. O grupo HSC apresentou níveis significativamente maiores do hormônio estimulador da tireoide (7,57±3,17 vs. 2,10±0,88 mU/L, p<0,001). No basal os grupos HSC e Controle foram semelhantes para FC (67±8 vs. 67±10, p=0,126, respectivamente), pressão arterial sistólica (130±11 vs. 128±10 mmHg, p=0,851, respectivamente), pressão arterial diastólica (70±7 vs. 68±6, p=0,527, respectivamente), pressão arterial média (90±8 vs. 88±7, p=0,664, respectivamente), FSM (2,50±0,79 vs. 2,55±0,71 ml/min/100ml, p=0,905, respectivamente) e CVA (2,80±0,90 vs. 2,92±0,88 unidades, p=0,952, respectivamente). Durante todo o teste de estresse mental os grupos HSC e Controle aumentaram significativamente a FC (efeito tempo, p<0,001), a PA (efeito tempo, p<0,001), o FSM (efeito tempo, p<0,001) e CVA (efeito tempo, p<0,001) em relação ao basal. Porém, o FSM e a CVA foram significativamente menores no grupo HSC durante o primeiro (FSM: 3,66±0,96 vs. 4,66±1,61 ml/min/100ml, p=0,018, tamanho do efeito=0,78; CVA: 3,95±1,08 vs. 5,19±1,96 unidades, p=0,010, tamanho do efeito=0,81;) e segundo (FSM: 3,55±1,01 vs. 4,62±2,27 ml/min/100ml, p=0,018, tamanho do efeito=0,65; CVA: 3,75±1,07 vs. 4,92±2,37 unidades, p=0,020, tamanho do efeito=0,68) minutos do teste de estresse mental. Desta forma concluímos que mulheres com HSC possuem, durante estresse mental, FC e PA preservadas, porém FSM e CVA prejudicados. / Patients with subclinical hypothyroidism (SCH) have a higher risk of cardiovascular mortality. One possible explanation for this outcome may be a worsening of vascular function. However, the effects of SCH in hemodynamic response during mental stress physiological maneuver are not known. We hypothesized that women with HSC have impaired peripheral blood flow and peripheral vascular conductance during mental stress. We evaluated 20 women with SCH (SCH Group) and 21 euthyroid (Control Group) women, matched for age (37±11 vs. 38±10 years, p=0.699) and body mass index (26±5 vs. 25±5kg/m2, p=0.462). The muscle blood flow (MBF) (venous occlusion plethysmography), heart rate (HR) and blood pressure (BP) (Dixtal2023) were evaluated simultaneously during 3 min-baseline followed by 3-min of mental stress test (Stroop Color Word Test). Forearm vascular conductance (FVC) was calculated by dividing MBF by the blood pressure mean. Student t test and ANOVA were used, adopting significant p<0.05. The SCH group had greater TSH levels (7.57±3.17 vs. 2.10±0.88 mU/L, p<0.001). In baseline the groups HSC and Control were similar to HR (67±8 vs. 67±10, p=0.126), systolic blood pressure (130±11 vs. 128±10 mmHg, p=0.851), diastolic blood pressure (70±7 vs. 68±6, p=0.527), mean blood pressure (90±8 vs. 88±7, p=0.664), MBF (2.50±0.79 vs. 2.55±0.71ml/min/100ml, p=0.905) and FVC (2.80±0.90 vs. 2.92±0.88 units, p=0.952). During all mental stress test the groups SCH and Control increased significantly HR (time effect, p<0.001), BP (time effect, p<0.001), MBF (time effect, p<0.001) and FVC (time effect, p<0.001) compared with baseline. However, MBF and FVC were significantly lower in SCH group during first (MBF: 3.66±0.96 vs. 4.66±1.61 ml/min/100ml, p=0.018, effect size=0.78; FVC: 3.95±1.08 vs. 5.19±1.96 unidades, p=0.010, effect size=0.81) and second (MBF: 3.55±1.01 vs. 4.62±2.27 ml/min/100ml, p=0.018, effect sixe=0.65; FVC: 3.75±1.07 vs. 4.92±2.37 unidades, p=0.020, effect size=0.68) minuts of mental stress test. In conclusion women with SCH compared to euthyroid women have preserved HR and BP, however impaired MBF and FVC during mental stress.

Au sein du laboratoire, deux mécanismes neurovasculaires indispensables à l'intégrité fonctionnelle de la peau sont étudiés. La PIV (Pressure-Induced vasodilation) est un mécanisme protecteur qui permet de retarder l'ischémie tissulaire. Des études antérieures ont suggéré l'implication du système nerveux central dans ce mécanisme. La première partie de ma thèse était de préciser le contrôle spinal dans la PIV et de regarder si la perte de ce mécanisme protecteur augmente le risque d'escarres. Nous avons montré l'implication de la voie sensorielle spinale dans la PIV ainsi que la corrélation directe entre la perte de ce mécanisme et l'augmentation du risque d'escarres chez des animaux sains. La CIV (current-induced vasodilation) a été décrite comme étant un mécanisme neurovasculaire de type " réflexe d'axone ". Exclusivement étudiée chez l'homme, les mécanismes impliqués restent limités. La seconde partie de ma thèse était de développer un modèle animal et approfondir la compréhension des mécanismes sous-jacents de la CIV cathodale. Après avoir développé un modèle animal, nous avons prouvé l'implication de la voie COX1/PGIS/PGI2/IP, des TRPV1 et des ASIC cutanés présents sur les fibres capsaïcino-sensibles ainsi que celles des récepteurs aux CGRP et à la substance P dans la CIV cathodale chez le rat sain. Pour conclure, la PIV est un outil diagnostique intéressant pour évaluer les capacités protectrices de la peau contre les lésions ischémiques de pression en conditions physiologiques. La CIV reflète de la libération endogène de PGI2 dans la peau, offrant un outil complémentaire à la réponse à l'acétylcholine afin d'évaluer les capacités globales de l'endothélium

Os nitrocomplexos de rutênio são estudados como agentes doadores de óxido nítrico (NO). Estes são atraentes como potenciais agentes terapêuticos porque apresentam baixa citotoxicidade, que pode ser decorrente da semelhança entre rutênio e ferro. Vários compostos de rutênio foram sintetizados em nosso laboratório, mas o complexo cis-[Ru(bpy)2(py)NO2] (PF6), (RuBPY) parece ser o mais promissor. Muitos doadores de NO tem como principal limitação clínica o desenvolvimento de tolerância, caracterizada pela perda rápida de seus efeitos anti-isquêmicos e hemodinâmicos. Os mecanismos e causas que levam à tolerância ainda são pouco conhecidos. Porém, acredita-se que a tolerância seja um processo multifatorial que envolva aumento da produção de espécies reativas de oxigênio (ERO), diminuição da atividade da enzima guanilil ciclase solúvel (GCs) e aumento da expressão e atividade das enzimas fosfodiesterases. O presente trabalho teve por objetivos avaliar se os compostos RuBPY e NaNO2, em diferentes concentrações e tempos de incubação, desencadeiam auto tolerância e tolerância cruzada à nitroglicerina (NTG). Além disso, avaliar se os receptores TRPV1 participam do relaxamento induzido pelo RuBPY, se o RuBPY libera NO ou nitrito e se é capaz de inibir a agregação plaquetária. O RuBPY e o NaNO2 são capazes de promover relaxamento total, de maneira dependente de concentração, em aortas com e sem endotélio, contraídas com fenilefrina. Estes resultados demonstram que o efeito vasodilatador destes compostos é independente do endotélio. Porém, o RuBPY é mais potente que o NaNO2. O RuBPY é capaz de induzir auto tolerância em aortas sem endotélio pré-expostas por 5 ou 10 min ao RuBPY e em aortas com endotélio, pré-expostas por 45 min ao RuBPY. A pré-exposição por 30 min ou 45 min ao RuBPY potencializa o seu efeito vasodilatador em aortas sem endotélio e com endotélio, respectivamente. A potencialização da resposta relaxante independe da ativação da GCs, mas pode estar relacionada ao aumento da liberação de NO em células do músculo liso vascular e aumento na fosforilação do resíduo de Thr495 da eNOS. O NaNO2 também induz o processo de auto tolerância em aortas sem endotélio, pela pré-exposição ao NaNO2 por 5 min ou 30 min. Não há indução de tolerância cruzada entre nitroglicerina e RuBPY. Neste trabalho demonstramos também que o novo complexo de rutênio RuBPY é um gerador de NO, e não de nitrito, capaz de inibir a agregação plaquetária. Os receptores TRPV1 não participam do relaxamento desencadeado pelo RuBPY. Um achado importante deste trabalho foi o efeito potencializador da vasodilatação induzida pelo RuBPY promovida pela sua pré-exposição. Este efeito poderia ser benéfico, considerando um potencial uso terapêutico deste composto gerador de NO / The ruthenium nitro-complexes are studied as nitric oxide (NO) donors. These complexes are attractive as potential therapeutic agents because they have low cytotoxicity, which can be due to the similarity between ruthenium and iron. Several ruthenium compounds were synthesized in our laboratory, but the complex cis-[Ru(bpy)2(py)NO2](PF6) (RuBPY) is the most promising. The main clinical limitation of NO donors is the development of tolerance that is characterized by the loss of its vasodilator and hemodynamic effects. The mechanisms and causes that lead to tolerance are still poorly understood. However, it is believed that tolerance is a multifactorial process that involves increased production of reactive oxygen species (ROS), decrease activity of thr enzyme soluble guanylyl cyclase (sGC) and increased expression and activity of phosphodiesterases enzyme. This study aimed to evaluate if the compounds RuBPY and nitrite (NaNO2) cause tolerance in different concentrations and exposure times and and cross-tolerance to nitroglycerin (NTG). In addition, to evaluate the involvement of the TRPV1 receptors in the relaxation induced by RuBPY, if RuBPY releases NO or nitrite and if RuBPY inhibits platelet aggregation. Both RuBPY and NaNO2 are able to induce complete relaxation in a concentration-dependent manner in intact endothelium or denuded contracted with phenylephrine. These results demonstrate that the vasodilation induced by these compounds is endothelium-independent. However, the RuBPY is more potent than NaNO2. The RuBPY is able to induce self-tolerance in the denuded aorta that was pre-exposed for 5 min or 10 min to RuBPY and in aorta with intact endothelium pre-exposed for 45 min to RuBPY. Pre-exposure for 30 min or 45 min to RuBPY potentiates its vasodilator effect in aorta without and with endothelium, respectively. The potentiated relaxation is independent of the GCs activation, but it is due to increased NO release in vascular smooth muscle cells and increased eNOS phosphorylation at the inhibitory residue Thr495. The NaNO2 also induces self-tolerance in denuded aortas by the pre-exposure to NaNO2 for 5 min or 30 min. It was not observed cross-tolerance between nitroglycerin and RuBPY. In this work, we have also shown that the new ruthenium complex RuBPY is a NO generator that inhibits platelet aggregation. The TRPV1 receptors do not participate in the relaxation induced by RuBPY. An important finding of this study was the potentiating effect of vasodilation induced by RuBPY promoted by its pre-exposure. This effect could be beneficial considering the potential therapeutic use of this compound NO generator.

Au sein du laboratoire, deux mécanismes neurovasculaires indispensables à l’intégrité fonctionnelle de la peau sont étudiés. La PIV (Pressure-Induced vasodilation) est un mécanisme protecteur qui permet de retarder l'ischémie tissulaire. Des études antérieures ont suggéré l’implication du système nerveux central dans ce mécanisme. La première partie de ma thèse était de préciser le contrôle spinal dans la PIV et de regarder si la perte de ce mécanisme protecteur augmente le risque d’escarres. Nous avons montré l’implication de la voie sensorielle spinale dans la PIV ainsi que la corrélation directe entre la perte de ce mécanisme et l’augmentation du risque d’escarres chez des animaux sains. La CIV (current-induced vasodilation) a été décrite comme étant un mécanisme neurovasculaire de type « réflexe d'axone ». Exclusivement étudiée chez l’homme, les mécanismes impliqués restent limités. La seconde partie de ma thèse était de développer un modèle animal et approfondir la compréhension des mécanismes sous-jacents de la CIV cathodale. Après avoir développé un modèle animal, nous avons prouvé l’implication de la voie COX1/PGIS/PGI2/IP, des TRPV1 et des ASIC cutanés présents sur les fibres capsaïcino-sensibles ainsi que celles des récepteurs aux CGRP et à la substance P dans la CIV cathodale chez le rat sain. Pour conclure, la PIV est un outil diagnostique intéressant pour évaluer les capacités protectrices de la peau contre les lésions ischémiques de pression en conditions physiologiques. La CIV reflète de la libération endogène de PGI2 dans la peau, offrant un outil complémentaire à la réponse à l’acétylcholine afin d’évaluer les capacités globales de l’endothélium / In our lab, two neurovascular mechanisms required for functional integrity of the skin are studied. The pressure-Induced vasodilation (PIV) is a protective mechanism, which delays the occurrence of tissue ischemia likely protecting the skin against pressure. Previous studies suggested the contribution of the central nervous system in this mechanism. In the first part of my PhD, we studied the spinal sensory transduction involvement in PIV and verify whether the lack of PIV increased incidence of pressure-induced ischemic lesions in healthy rats. We showed that spinal sensory disruption led to the loss of cutaneous PIV directly associated with increased incidence and severity of cutaneous lesions in response to prolonged ischemic compression in healthy animals. The current-induced vasodilation (CIV) is a neurovascular mechanism decribed as axon reflex mechanism. Exclusively studied in humans, the understanding of involved mechanisms remains limited. In the second part of my PhD, we aimed to develop an animal model and precise the underlying mechanisms involved in cathodal CIV. That we developed, we proved the involvement of the COX1/PGIS/PGI2/IP pathway, the cutaneous TRPV1 and ASIC channels present on capsaicin-sensitive fibres and the cutaneous CGRP and NK1 receptors in cathodal CIV in healthy rats. To conclude, PIV is an interesting tool to evaluate the protective capacities of the skin to withstand pressure in healthy conditions. CIV by reflecting the endogenous release of PGI2 in healthy skin offers a novel tool in complement to acetylcholine response in order to assess global capacities of the endothelium

O óxido nítrico (NO) é o principal agente vasodilatador endógeno que regula o tônus e a homeostase vascular. Dentre os compostos doadores de NO, estão os complexos nitrosilos de rutênio. No presente estudo, o doador de NO estudado, RuBPY, não apresenta citotoxicidade para células do músculo liso vascular (MLV) ao contrário do NPS. O RuBPY apresenta eficácia semelhante ao NPS em relaxar o MLV de aorta de ratos, porém o NPS é mais potente. Ambos compostos liberam NO do tipo radicalar (NO) no meio intracelular, mas o NPS libera também íon nitroxil (NO-). O sequestrador da espécie NO (hidroxocobalamina) reduziu mais a resposta relaxante estimulada com RuBPY do que com o NPS. Nenhum dos dois compostos precisa ser reduzido quimicamente para liberar NO, uma vez que houve relaxamento quando utilizamos alta concentração de KCl como agente contrátil. Porém, este relaxamento foi inibido, o que mostra a importância dos canais para K+ no relaxamento induzido pelos doadores de NO. O bloqueador não seletivo de canais para K+ (TEA), inibiu somente o relaxamento ao RuBPY. A via NO-GCs-GK é ativada por ambos doadores de NO, para induzir relaxamento. A inibição da degradação do GMPc potencializou o relaxamento estimulado com RuBPY e NPS. O armazenamento de Ca+2 no retículo sarcoplasmático (RS) via ativação da SERCA é importante somente para o relaxamento induzido com RuBPY. O composto RuBPY inibiu a resposta contrátil estimulada com fenilefrina devido ao armazenamento de Ca+2 no RS e também por inibir o influxo capacitivo de Ca+2. A presença do endotélio vascular não alterou o relaxamento induzido pelo RuBPY, porém potencializou o relaxamento induzido pelo NPS. A análise da liberação de NO por amperometria demonstrou que o RuBPY libera NO somente em presença do tecido aórtico de ratos. Portanto, não houve liberação espontânea de NO, por fotólise pela luz visível ou por redução química. É necessária a presença de heme-proteínas como a guanilil-ciclase solúvel (GCs) inibida pelo ODQ, para haver a conversão do nitrito presente no RuBPY, a NO. Pela quantificação da fluorescência emitida pela sonda DAF-2DA, RuBPY liberou cerca de 3,5 vezes mais NO do que o NPS. Pela medida do potencial de membrana, demonstramos que o RuBPY induz hiperpolarização de membrana de células isoladas do MLV da aorta de rato. RuBPY tem efeito hipotensor dose-dependente, em ratos hipertensos renais, o que não ocorre em animais normotensos. A redução da pressão arterial em ratos hipertensos é maior do que nos normotensos. Em estudos iniciais de farmacocinética, verificamos que o composto RuBPY é absorvido por via oral e é distribuído entre alguns tecidos após ser administrado aos ratos, por gavagem. / Nitric oxide (NO) is the main endogenous vasodilator agent that regulates vascular tone. Among the compounds which are able of releasing NO, are the nitrosyl ruthenium complexes. The NO donor studied, RuBPY, does not present cytotoxicity in smooth muscle cells (SMC), in contrast to SNP. RuBPY has similar efficacy to SNP in inducing rat aorta relaxation, although SNP is more potent. Both compounds release intracellular radicalar NO (NO), and SNP also release ion nitroxyl (NO-). The NO scavenger (hydroxocobalamine) had greater effect on the relaxation induced by RuBPY than by SNP. Both compounds do not need to be chemically reduced to release NO, as demonstrated in aorta relaxation after pre-contraction with high concentrations of KCl. However, this relaxation was impaired, showing the importance of K+ channels to induce relaxation by NO released from these compounds. By using non-selective blocker for K+ channels (TEA), only the relaxation induced by RuBPY was inhibited. The NO-sGC-GK pathway is activated by NO donors to induce relaxation. Inhibition of cGMP degradation, potentiated the effect of RuBPY and SNP. Storage of Ca+2 in the sarcoplasmic reticulum (SR) via activation of SERCA is important only for the relaxation induced by RuBPY. The contractile response induced by phenylephrine was inhibited by RuBPY due to the storage of Ca+2 in RS and also by inhibiting the capacitive influx of Ca+2. The presence of endothelium had no effect on the relaxation induced by RuBPY, but it potentiated the relaxation induced by SNP. RuBPY released NO only in the presence of the rat aorta. The complex RuBPY did not spontaneously release NO, by photolysis by visible light, or by chemical reduction. RuBPY requires the presence of heme-protein such as guanylyl-cyclase, inhibited by ODQ, to convert nitrite to NO. The amount of NO released from RuBPY was about 3.5 times greater than that released from SNP. RuBPY induced membrane hyperpolarization of SMC. RuBPY has hypotensive effect in renal hypertensive rats in a dose-dependent way, which does not occur in normotensive rats. The decreased of blood pressure in hypertensive rats was greater than in normotensive rats. Initial studies of pharmacokinetics demonstrated that RuBPY is orally absorbed and it is also distributed in some tissues after being administered by gavage to rats.