Este estudo descreve um novo método para a produção da Ca2+-ATPase do retículo sarcoplasmático de coelho em levedura utilizando um vetor de expressão regulado por choque térmico. Após solubilização das membranas de levedura com lisofosfatidilcolina, a introdução de um \"tag\" de 6 histidinas na extremidade amino-terminal da Ca2+-ATPase permitiu a sua purificação por cromatografia de afinidade utilizando uma resina carregada com níquel. Utilizando essa estratégia, foi possível obter frações enriquecidas em até 75% de Ca2+-ATPase recombinante, algo não descrito ainda na literatura. A 6xHis Ca2+-ATPase solubilizada em LPC e purificada em coluna de níquel se mantém estável desde que seja introduzido DOPC juntamente com o detergente nas etapas de lavagem e eluição. Nessas condições, a enzima purificada possui elevada atividade ATPásica cálcio-dependente (1.5-2.0 µmol/mg proteína.min) durante vários minutos de reação. A titulação da atividade ATPásica em função do cálcio livre demonstrou que a 6xHis Ca2+-ATPase purificada possui alta afinidade para o íon (K0.5= 0.15 µlM) e manteve uma forte cooperatividade na ativação por cálcio (nH = 2.07). A quantidade e o grau de pureza obtidos são suficientes para permitir a caracterização bioquímica e espectroscópica de mutantes pontuais da Ca2+-ATPase já construídos e expressos em levedura. A conversão da energia presente em ligações químicas em gradiente eletroquímico é um tema central da bioenergética. Espera-se que o estudo dos mutantes pontuais de triptofano da Ca2+-ATPase gerados nesse trabalho contribua para uma melhor compreensão do mecanismo de acoplamento entre a hidrólise de ATP e o transporte vetorial de íons nesse modêlo de estudo de proteínas de transporte. / We describe in this work a new method for the production of SERCA-l Ca2+-ATPase in yeast using a heat-shock regulated expression vector. Following solubilization of yeast membranes with lysophospholipids, the presence of an hexahistidine tag introduced at the Nterminal end of the Ca2+-ATPase allowed its purification by metal chelating affinity chromatography using a nickel-NTA resin. Using this procedure highly enriched ftactions (75% oftotal protein in the ftaction) of yeast-expressed rabbit Ca2+-ATPase were obtained. Detergent-solubilized 6xHis-Ca2+-ATPase retained highly active (1.5 - 2 µmol/mg protein .min) calcium-dependent, vanadate inhibitable ATPase activity as determined by 32P-γ-ATP hydrolysis. Titration of ATPase activity as a function of ftee calcium revealed high Ca2+ affinity (K0.5 =~ 0.15 µM) and the persistence of a strong cooperative pattem of calcium activation (Hill number of 2.07). The yield and purity of 6xHis Ca2+-ATPase fractions produced with this method allows the biochemical and spectroscopic characterization of Ca2+-ATPase mutants produced in the course of this work. Conversion of the energy present in chemical bonds to electrochemical gradient is a central theme of bioenergetics. It is hoped that the study of the Ca2+-ATPase tryptophan mutants generated in this work will contribute to a better understanding of the coupling mechanism between ATP hydrolysis and the vectorial transport of ions across membranes that occur in this model system.

Made available in DSpace on 2015-04-11T13:38:25Z (GMT). No. of bitstreams: 1 francianny.pdf: 1582573 bytes, checksum: 107d67128bf43813bf5591f4c2d0e26f (MD5) Previous issue date: 2012-06-25 / Quassia amara L. (Simaroubaceae) is a pan american plant known as pau amargo and quassia. It is used in the folk medicine to treat gastrointestinal disorders, hypertension, malaria, and as insecticide and vermifuge. Several compounds were isolated from the plant leaves and barks as β-carbonile alkaloids, 6-cantin, steroids and quassinoids (neoquassin and quassin). In this work, the effects of the standardized extracts from Q. amara collected in Manicoré/AM were studied on the cardiovascular system of rodents. The aqueous extract (AE) was prepared from the dried leaves infusion and the butanolic fraction (BuF) was obtained from the AE partition in butanol. The BuF standardization and purification in HPLC isolated 6 fractions (F1 to F6). The AE oral administration (1.0 g/kg) did not produce evident signals of pharmacological activity in rats; BuF caused sedation, anesthesia and cyanosis after 1 h. Death was not observed after 24 h of the treatment. However, BuF (1.0 g/kg) intraperitoneal administration produced intense sedation, anesthesia and cyanosis after 1 h of the treatment; all animals died after 2 h. In anesthetized rats, BuF produced fast and transient hypotension, without altering the hypertensive effect of noradrenalin, or the hypotension induced by acetylcholine. The hypotension caused by BuF (10.0 mg/kg, e.v.) was unchanged by previous treatment with atropine or prazosin. The fractions responsible for the hypotensive effect were: F1, F3 and F6. All fractions were tested on intact endothelium- and denuded- aorta rings contracted by noradrenalin. F1, F3, F4, F5 and F6 fractions relaxed the endothelium intact aorta tonus, indicating a mechanism dependent on NO cascade. The chronotropic and inotropic effects of AE and BuF were also evaluated in isolated right and left rat atrium. Neither AE (30 to 300 μg/mL) nor BuF (10 to 100 μg/mL) alter the chronotropism, however, the force of contraction increased in a concentration dependent manner. BuF potentiated the diaphragm muscle contraction induced by direct and indirect electrical stimulation. BuF (1 to 300 μg/mL) inhibited the activity of the Ca2+-ATPase isolated from rabbit skeletal muscle; this enzyme inhibition may be related to the potentiation of the diaphragm contraction. In rat vas deferens preparations, BuF inhibited the maximal contraction induced by noradrenalin without affecting the drug affinity to the α1-adrenergic receptors. The purified fractions responsible for this effect were F1, F3, F4 e F5. BuF did not inhibit the calcium influx in cardiomyocytes and cultured rat uterus cells. / A Quassia amara L. (Simaroubaceae) é planta pan-americana conhecida como pau-amargo e quassia. É utilizada na medicina popular para o tratamento de distúrbios gastrintestinais, hipertensão, malária, e também como inseticida e vermífuga. Das folhas e cascas foram isolados vários compostos químicos como alcaloides β-carbonila, 6-cantin, esteroides e quassinoides (neoquassina e quassina). Neste trabalho, foram estudados a farmacologia geral e os efeitos no sistema cardiovascular de roedores de extratos padronizados de Quassia amara coletada em Manicoré, AM. O extrato aquoso (EA) foi obtido por infusão de folhas secas, e a fração butanólica, por partição do EA em butanol. A padronização e purificação da FBut por cromatografia líquida de alta eficiência (CLAE) levou ao isolamento de 6 frações (F1 a F6). A administração oral do EA (1,0 g/kg) não produziu sinais evidentes de atividade farmacológica em ratos; a FBut produziu sinais de sedação, anestesia e cianose após 1 h. Não houve morte dos animais após 24 h do tratamento. No entanto, a administração da FBut (1,0 g/kg) por via intraperitoneal produziu sinais de sedação, anestesia e cianose intensos após 1 h de tratamento; todos os animais morreram após 2 h. Na pressão arterial em ratos anestesiados, a FBut produziu hipotensão rápida e passageira, sem alterar o efeito hipertensor da noradrenalina, ou a hipotensão produzida pela acetilcolina. A hipotensão provocada pela FBut (10,0 mg/kg, e.v.) também não foi alterada pelo tratamento prévio do animal com atropina ou prazosin. As frações responsáveis pela ação hipotensora são a F1, F3 e F6. As frações foram testadas em preparações de aorta de rato com e sem endotélio. As frações F1, F3, F4, F5 e F6 relaxaram o tônus muscular da aorta dependente de endotélio, indicando um provável mecanismo via cascata do NO. As atividades cronotrópica e inotrópica do EA e da FBut foram avaliadas em preparações de átrios direito e esquerdo isolados de rato. O EA (30 a 300 μg/mL) e a FBut (10 a 100 μg/mL) não alteraram o cronotropismo, no entanto, aumentaram o inotropismo de maneira concentração-dependente. A FBut potenciou a contração do músculo diafragma de rato sob estímulo direto e indireto. A FBut (1 a 300 μg/mL) inibiu a atividade da Ca2+-ATPase de músculo esquelético de coelho; essa inibição pode estar relacionada à potenciação da contração do músculo diafragma. Em ducto deferente de rato, a FBut diminuiu a contração máxima produzida pela noradrenalina sem alterar a afinidade da droga pelos receptores α1-adrenérgicos. As frações purificadas responsáveis por este efeito foram as F1, F3, F4 e F5. A FBut não inibiu o influxo de cálcio

Este estudo descreve um novo método para a produção da Ca2+-ATPase do retículo sarcoplasmático de coelho em levedura utilizando um vetor de expressão regulado por choque térmico. Após solubilização das membranas de levedura com lisofosfatidilcolina, a introdução de um \"tag\" de 6 histidinas na extremidade amino-terminal da Ca2+-ATPase permitiu a sua purificação por cromatografia de afinidade utilizando uma resina carregada com níquel. Utilizando essa estratégia, foi possível obter frações enriquecidas em até 75% de Ca2+-ATPase recombinante, algo não descrito ainda na literatura. A 6xHis Ca2+-ATPase solubilizada em LPC e purificada em coluna de níquel se mantém estável desde que seja introduzido DOPC juntamente com o detergente nas etapas de lavagem e eluição. Nessas condições, a enzima purificada possui elevada atividade ATPásica cálcio-dependente (1.5-2.0 µmol/mg proteína.min) durante vários minutos de reação. A titulação da atividade ATPásica em função do cálcio livre demonstrou que a 6xHis Ca2+-ATPase purificada possui alta afinidade para o íon (K0.5= 0.15 µlM) e manteve uma forte cooperatividade na ativação por cálcio (nH = 2.07). A quantidade e o grau de pureza obtidos são suficientes para permitir a caracterização bioquímica e espectroscópica de mutantes pontuais da Ca2+-ATPase já construídos e expressos em levedura. A conversão da energia presente em ligações químicas em gradiente eletroquímico é um tema central da bioenergética. Espera-se que o estudo dos mutantes pontuais de triptofano da Ca2+-ATPase gerados nesse trabalho contribua para uma melhor compreensão do mecanismo de acoplamento entre a hidrólise de ATP e o transporte vetorial de íons nesse modêlo de estudo de proteínas de transporte. / We describe in this work a new method for the production of SERCA-l Ca2+-ATPase in yeast using a heat-shock regulated expression vector. Following solubilization of yeast membranes with lysophospholipids, the presence of an hexahistidine tag introduced at the Nterminal end of the Ca2+-ATPase allowed its purification by metal chelating affinity chromatography using a nickel-NTA resin. Using this procedure highly enriched ftactions (75% oftotal protein in the ftaction) of yeast-expressed rabbit Ca2+-ATPase were obtained. Detergent-solubilized 6xHis-Ca2+-ATPase retained highly active (1.5 - 2 µmol/mg protein .min) calcium-dependent, vanadate inhibitable ATPase activity as determined by 32P-γ-ATP hydrolysis. Titration of ATPase activity as a function of ftee calcium revealed high Ca2+ affinity (K0.5 =~ 0.15 µM) and the persistence of a strong cooperative pattem of calcium activation (Hill number of 2.07). The yield and purity of 6xHis Ca2+-ATPase fractions produced with this method allows the biochemical and spectroscopic characterization of Ca2+-ATPase mutants produced in the course of this work. Conversion of the energy present in chemical bonds to electrochemical gradient is a central theme of bioenergetics. It is hoped that the study of the Ca2+-ATPase tryptophan mutants generated in this work will contribute to a better understanding of the coupling mechanism between ATP hydrolysis and the vectorial transport of ions across membranes that occur in this model system.

In heart, Ca2+ plays an important role in maintenance of normal cardiac functions. Regulation of Ca2+ is mainly through L-type Ca2+ channel (LTCC), Ryanodine receptor (RyR) and sarcoplasmic reticulum calcium ATPase pump (SERCA) on sarcoplasmic reticulum (SR), Na+-Ca2+ exchanger (NCX), plasma membrane Ca2+ ATPase (PMCA). It has been well-accepted that PMCA plays a minor contribution to elevation of Ca2+ compared to SERCA and NCX and in regulation of cytosolic Ca2+ homeostasis. There are four isoforms of PMCA, PMCA1-4, and PMCA1 is a house-keeping gene, and abundantly distributed in heart. However, the role of PMCA1 in the murine heart has not been fully explored. With a cardiac specific knockout mouse model, the electrophysiological characteristics of PMCA1 in murine hearts, particularly in atria under normal physiological and stress conditions ([Ca2+]o overload and pacing conditions) are investigated. Firstly the complete deletion of PMCA1 in the atria in PMCA1cko mice was confirmed by Western blotting and immunostaining, also the membrane localisation of PMCA1 in the atria in PMCA1loxP/loxP mice was demonstrated. Then the phenotypes of ex vivo whole hearts between PMCA1loxP/loxP and PMCA1cko mice under physiological conditions and [Ca2+]o overload condition and with different frequencies by programmed electrical stimulation (PES) were explored. Further more, the Ca2+ handling process in single atrial myocytes between the PMCA1 deletion mice and control mice under normal physiological conditions and [Ca2+]o overload condition and stimulation with different frequencies was investigated. Finally the Ca2+ handling process in single ventricular myocytes between the PMCA1 deletion mice and control mice under normal physiological condition was investigated. At the whole heart level, the PMCA1cko hearts became more susceptible to arrhythmias with PES under physiological conditions compared with the PMCA1loxP/loxP hearts, and such arrhythmic events occurred more often and had longer pacing durations under Ca2+ overload conditions and higher frequency of pacing. At the single cellular level, the NCX current decay was significantly prolonged in PMCA1cko atrial myocytes under physiological conditions. This was further increased under Ca2+ overload conditions. With frequency-dependent stimulation, the PMCA1cko atrial myocytes showed few EAD- or DAD-type APs under physiological conditions in contrast to PMCA1loxP/loxP atrial myocytes that showed no arrhythmic events. The occurrence increased significantly under Ca2+ overload condition and/or at higher frequency of stimulation. Similar findings were observed in isolated ventricular myocytes. To conclude, the role of PMCA1 in maintaining Ca2+ homeostasis and electrical function in atrial myocytes under physiological conditions is minor. ii) PMCA1 has a critical role in maintaining Ca2+ homeostasis and electrical function in the atrium under stress conditions. This is particularly important during fast efflux of Ca2+ which is required under stress conditions.

Made available in DSpace on 2015-04-11T13:38:51Z (GMT). No. of bitstreams: 1 Thiago Mattos de Araujo Lima.pdf: 914896 bytes, checksum: be7fe7f16d2a312578b3d1011a681a23 (MD5) Previous issue date: 2010-03-30 / Fundação de Amparo à Pesquisa do Estado do Amazonas / Virola surinamensis (Myristicaceae), popularly known as ucuuba, ucuuba branca and sucuuba, is widely distributed in flooded areas of the Amazonian Forest. The bark resin is used in the folk medicine to treat erysipela and the tea of the leaves, to treat colics, dyspepsia, inflammations and malaria. The main constituents described for V. surinamensis are arylpropanoids, neolignans, lignans, lignoids, flavonoids, diarylpropanes, -butirolactones, alkaloids and acid benzoic derivatives. Among them, lignans and neolignans showed anti-parasitic and anti-fungal activities. Previous pharmacological studies described anti-parasitic effect of the essential oils and anti-leishmania, anti-inflammatory, anti-fungal, insecticide and gastroprotective actions. The aim of this work was to study the pharmacological activity of the standardized buthanolic fraction of Virola surinamensis and the mechanisms of the hypotensive effect and the potentiation of the skeletal muscle contraction. The chemical standardization was obtained after partition of the leaves aqueous extract (AE) in n-buthanol originating the buthanolic fraction (BuF). The BuF purification by high performance liquid chromatography (HPLC) originated 10 high purity fractions (F1 to F10) that are still in identification process. The pharmacological screening of AE and BuF showed low action in the CNS. In the arterial blood pressure of anesthetized rats, the intravenous injection of both AE and BuF produced hypotension apparently associated with the α1-adrenoceptors blockade. The BuF was effective to reduce the arterial blood pressure in the chronic treatment also. The absorbed substances did not modify the animal behavior in the Irwin Test, but produced reversible hypotension initiating in 30 min, time necessary to the gastric emptiness. The treatment during 7 days did not modify the animal behavior, but there was a residual accumulated effect of the BuF reducing the basal arterial blood pressure of the rats. In the isolated aortic rings without endothelium, the BuF relaxed the tonus induced by noradrenalin in a concentration-dependent manner, showing that BuF did not interfere with the NO system. This effect was indicative of a competitive antagonism and reinforced the evidences obtained in the arterial blood pressure that the mechanism of the hypotension produced by BuF was the α1 adrenoceptors blockade. The BuF potentiated the direct elicited twitches of the rat diaphragm. In microssomes isolated from the rabbit skeletal muscle, BuF inhibited the Ca2+-ATPase activity similarly to thapsigargin, indicating that the potentiation of the diaphragm contraction may be related to the decrease in the Ca2+ uptake by the sarcoplasmic reticulum. / A Virola surinamensis, conhecida popularmente como ucuúba, ucuúba branca, ucuúba de igapó, sucuba, é uma árvore que cresce em várzeas e em bancos de areia em rios na Floresta Amazônica. A resina da casca é utilizada na medicina popular para o tratamento de erisipelas e o chá das folhas é indicado em cólicas, dispepsia, processos inflamatórios e para o tratamento da malária. Dentre os constituintes de Virola surinamensis foram descritos arilpropanoides, neolignanas, lignanas, sesquilignóides, lignóides diméricos, flavonóides, diarilpropanos, -butirolactonas, alcalóides e derivados do ácido benzóico. Desses, destacam-se as lignanas e neolignanas por suas atividades anti-parasitária e anti-fúngica. Estudos farmacológicos anteriores descrevem atividade anti-parasitária dos óleos essenciais, atividade anti-leishmania, anti-inflamatória, antifúngica, inseticida e gastroprotetora. Este trabalho teve como objetivo o estudo das ações farmacológicas da fração butanólica padronizada de Virola surinamensis e a análise dos mecanismos do efeito hipotensor e da potenciação da contração do músculo esquelético. A padronização química foi obtida após partição do extrato aquoso das folhas (EA) em butanol dando origem à fração butanólica (FBut). A purificação da Fbut por cromatografia líquida de alta eficiência (CLAE) originou 10 frações de elevado grau de pureza (F1 a F10) que se encontra em processo de identificação. A triagem farmacológica do EA e da Fbut mostrou baixa ação no SNC. Na pressão arterial de ratos anestesiados a injeção endovenosa tanto do extrato aquoso como da fração butanólica produziu hipotensão aparentemente associada à uma ação bloqueadora de receptores α1-adrenérgicos. A Fbut mostrou-se eficaz na redução da pressão arterial durante o tratamento crônico. As substâncias absorvidas não modificaram o comportamento animal no teste de Irwin, mas produziram hipotensão reversível com início em torno de 30 minutos, tempo necessário para o esvaziamento gástrico. O tratamento repetido durante 7 dias também não modificou o comportamento animal, mas houve um efeito residual cumulativo da FBut que a cada dia reduziu o valor basal da pressão arterial dos ratos. Em anéis de aorta sem endotélio, a Fbut relaxou o tônus induzido pela noradrenalina de maneira concentração-dependente, mostrando que Fbut não interfere no sistema NOS. Este efeito foi indicativo de antagonismo competitivo e reforça as evidências obtidas na pressão arterial de que o mecanismo da hipotensão produzida pela Fbut é o bloqueio de adrenoceptores do tipo α1. A Fbut potenciou a contração obtida por estímulo direto da fibra muscular do diaframa de rato. Em microssomas isolados da musculatura esquelética do coelho, a Fbut inibiu a atividade da Ca2+-ATPase à semelhança da tapsigargina, indicando que a potenciação da contração do diafragma parece estar relacionada à diminuição da recaptação de Ca2+ pelo retículo sarcoplasmático.

Le puissant anti-paludéen, l'artémisinine a été décrit comme inhibiteur de l'activité ATPasique de PfATP6 après son expression dans des ovocytes de Xénope. PfATP6 est l'unique ATPase Ca2+ du réticulum endo/sarcoplasmique de P. falciparum, le parasite responsable du paludisme. Quand un acide aminé de SERCA1a de lapin (E255) est muté en son équivalent dans PfATP6 (L), l'activité de ce mutant exprimé en ovocyte de Xénope est inhibée en présence d'artémisinine. Après expression de ce mutant et de PfATP6 dans S. cerevisiae puis leur purification, nous avons constaté qu'aucune de ces deux protéines n'était sensible à l'artémisinine. En parallèle, nous montrons que PfATP6 purifiée est sensible aux principaux inhibiteurs de SERCA mais elle est moins sensible à la thapsigargine que ne l'est SERCA1a. Les résultats présentés ici suggèrent que le méchanisme d'action de l'artémisinine est complexe et ne peut pas être du à une interaction directe entre l'artémisinine et PfATP6. D'autre part, la purification de PfATP6 laisse entrevoir l'opportunité de mieux caractériser cette protéine voire même de développer de nouveaux anti-paludéens en recherchant des inhibiteurs de cette protéine.

Few studies have quantified resting mouse muscle metabolism and even fewer studies have separated the contribution of sarcoplasmic reticulum (SR) Ca2+ pumping to resting metabolic rate. Furthermore, the studies that have attempted to quantify the contribution of Ca2+ pumping have used indirect methods to inhibit SR Ca2+ ATPase activity. The purpose of this study is to directly quantify resting muscle oxygen consumption and the contribution of SR Ca2+ pumping to resting oxygen consumption in mouse hindlimb muscles by using CPA to specifically inhibit Ca2+ pump activity in intact muscles at rest. The TIOX system was used to measure resting muscle VO2 of extensor digitorum longus (EDL) and soleus (SOL) muscles at 30oC and 20oC. C57BL mice aged 8-12 weeks were used with an average whole body mass of 23.8 g and EDL and SOL dry weights averaging 1.88 mg and 1.8 mg, respectively. All muscle VO2 measurements are expressed per gram dry weight. There were no differences (P>0.1) in resting muscle VO2 between EDL and SOL muscles at either 30oC (EDL, 2.05 µL/g/s; SOL, 2.27 µL/g/s) or 20oC (EDL, 0.62 µL/g/s; SOL, 0.71 µL/g/s). The average Q10 (3.1) was determined from EDL and SOL VO2 measures at 20oC and 30oC. The contribution of Ca2+ pumping by the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) was measured at 30oC using a range of CPA concentrations (1-15 µM) . There was a concentration-dependent effect of CPA on oxygen consumption with increasing CPA concentrations up to 10 µM resulting in progressively greater reductions in muscle oxygen consumption. Specifically, 1, 5, 10, and 15 µM CPA caused an 11, 35.4, 49.5, and 50.3% reduction in VO2. There were no differences (P>0.1) between 10 and 15 µM CPA indicating that 10 µM CPA induces maximal inhibition of SERCA in isolated muscle preparations. The results indicate that the Ca2+ pumping by SERCA is responsible for ~50% of oxygen consumption in resting mouse EDL and SOL muscle. This is the first study to use a direct inhibitor of SERCA to quantify the contribution of Ca2+ cycling to resting oxygen consumption and therefore is a more accurate reflection of the actual contribution of SERCA to resting muscle oxygen consumption compared to previous findings. These results suggest that SERCA energy consumption accounts for a large portion of resting muscle metabolism and may represent a potential therapeutic target for metabolic alterations to oppose obesity.

Few studies have quantified resting mouse muscle metabolism and even fewer studies have separated the contribution of sarcoplasmic reticulum (SR) Ca2+ pumping to resting metabolic rate. Furthermore, the studies that have attempted to quantify the contribution of Ca2+ pumping have used indirect methods to inhibit SR Ca2+ ATPase activity. The purpose of this study is to directly quantify resting muscle oxygen consumption and the contribution of SR Ca2+ pumping to resting oxygen consumption in mouse hindlimb muscles by using CPA to specifically inhibit Ca2+ pump activity in intact muscles at rest. The TIOX system was used to measure resting muscle VO2 of extensor digitorum longus (EDL) and soleus (SOL) muscles at 30oC and 20oC. C57BL mice aged 8-12 weeks were used with an average whole body mass of 23.8 g and EDL and SOL dry weights averaging 1.88 mg and 1.8 mg, respectively. All muscle VO2 measurements are expressed per gram dry weight. There were no differences (P>0.1) in resting muscle VO2 between EDL and SOL muscles at either 30oC (EDL, 2.05 µL/g/s; SOL, 2.27 µL/g/s) or 20oC (EDL, 0.62 µL/g/s; SOL, 0.71 µL/g/s). The average Q10 (3.1) was determined from EDL and SOL VO2 measures at 20oC and 30oC. The contribution of Ca2+ pumping by the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) was measured at 30oC using a range of CPA concentrations (1-15 µM) . There was a concentration-dependent effect of CPA on oxygen consumption with increasing CPA concentrations up to 10 µM resulting in progressively greater reductions in muscle oxygen consumption. Specifically, 1, 5, 10, and 15 µM CPA caused an 11, 35.4, 49.5, and 50.3% reduction in VO2. There were no differences (P>0.1) between 10 and 15 µM CPA indicating that 10 µM CPA induces maximal inhibition of SERCA in isolated muscle preparations. The results indicate that the Ca2+ pumping by SERCA is responsible for ~50% of oxygen consumption in resting mouse EDL and SOL muscle. This is the first study to use a direct inhibitor of SERCA to quantify the contribution of Ca2+ cycling to resting oxygen consumption and therefore is a more accurate reflection of the actual contribution of SERCA to resting muscle oxygen consumption compared to previous findings. These results suggest that SERCA energy consumption accounts for a large portion of resting muscle metabolism and may represent a potential therapeutic target for metabolic alterations to oppose obesity.

La découverte de nouveaux agents antipaludiques est primordiale. A travers le monde, les chercheurs se sont focalisés sur plusieurs stratégies. Les plus développées sont : soit les tests de molécules issues de bibliothèques chimiques dans une recherche phénotypique (comme le test direct d’agents sur des cultures de parasites in vitro), soit la recherche de nouvelles molécules agissant sur l’activité d’une cible ou d’une voie spécifique et essentielle. Cette thèse est centrée sur le second type d’approche. Nous nous sommes intéressés aux transporteurs membranaires de P. falciparum. Pour cela, nous exprimons les protéines d’intérêt dans la levure et nous les purifions. Nous optimisons les tests fonctionnels, dans le but de : a) déterminer l’effet des molécules sur les cibles spécifiques ; b) tester leur effet sur les cultures d’érythrocytes infectés par P. falciparum in vitro ; c) vérifier leur toxicité sur des cellules de mammifères ; et d) réaliser le test des molécules les plus efficaces in vivo dans un modèle de paludisme murin. Notre travail actuel est focalisé sur l’ATPase6 de P. falciparum (PfATP6) et l’adénylate translocase (PfAdT), deux protéines membranaires essentielles localisées respectivement sur le réticulum endoplasmique et la membrane mitochondriale. Nous exprimons de manière hétérologue PfATP6 dans les membranes de levure, nous purifions la protéine et mesurons une activité ATPase spécifique. Nous avons ainsi pu tester une bibliothèque chimique importante et identifier des inhibiteurs spécifiques. Ces derniers ont ensuite été testés pour évaluer leur effet sur les stades érythrocytaires du parasite in vitro et leur cytotoxicité sur des cellules de mammifères. Pour le transporteur PfAdT, nous procédons comme pour PfATP6, mais nous avons choisi un autre type de test fonctionnel dans lequel la protéine est directement exprimée sur la membrane plasmique d’E. coli. Cela devrait permettre de mesurer le transport d’ATP radiomarqué, et l’identification d’inhibiteurs spécifiques dont les effets pourront être évalués sur des cultures de parasites in vitro et dans des essais de cytotoxicité. / New drug discovery for malaria treatment urges, now more than ever. There is no optimal solution to the search for new antimalarials. Worldwide, researchers have focused their energies on several strategies. The most commonly employed are: either by screening molecules issued from chemical libraries in a phenotypic way (i.e., direct testing of drugs on in vitro parasite cultures), or by searching for new molecules acting upon the activity of a specific essential target or pathway. This PhD thesis centers on the second type of approach. We are interested in targeting membrane transporters of P. falciparum. For this, we plan to express proteins of interest in yeast and proceed to their isolation. With optimized functional tests, we aim to: a) Determine the effect of molecules upon specific targets; b) Test their effect on P. falciparum in vitro erythrocytes cultures; c) As well as verify their toxicity on mammalian cells; and d) Perform in vivo testing of the best molecules on a rodent model for malaria. Our actual work is focused on the P. falciparum Ca2+ - ATPase 6 (PfATP6) and adenylate translocase (PfAdT), two essential membrane proteins localized on the endoplasmic-reticulum and the mitochondrial membrane, respectively. We were able to express heterologously PfATP6 in yeast membranes, purify the protein and measure a specific ATPase activity. With this, we have tested a large chemical library and identified specific inhibitors. These were then tested for their effect on in vitro blood stages of P. falciparum and for their cytotoxicity on mammalian cells. For the ATP/ADP carrier PfAdT, we proceeded as previously done with PfATP6 but we have also chosen another type of functional test where we express directly this protein in the plasma membrane of E. coli. This will enable in the future the measurement of radiolabelled ATP uptake, and the identification of specific inhibitors that could then be tested for their effect on P. falciparum in vitro cultures and for their cytotoxicity.