Submitted by (lucia.rodrigues@ufrpe.br) on 2016-05-31T15:10:49Z No. of bitstreams: 1 Bruno Mendes Tenorio.pdf: 4101398 bytes, checksum: d81bf09fb306302eb9abbcd6d926a6a7 (MD5) / Made available in DSpace on 2016-05-31T15:10:49Z (GMT). No. of bitstreams: 1 Bruno Mendes Tenorio.pdf: 4101398 bytes, checksum: d81bf09fb306302eb9abbcd6d926a6a7 (MD5) Previous issue date: 2011-12-12 / In recent decades, humans have been exposed to various exogenous physical agents, such as high temperatures emitted by industrial devices and electromagnetic fields emitted by the electricity distribution networks and electronic devices. Researches demonstrated that these agents can cause reproductive disorders. The chapter 1 of this study aimed to investigate the possible effects of low frequency EMF exposure of 60 Hz and 1 mT from day 13 of gestation up to puberty at 21 postnatal days. The results showed that exposure to EMF reduced parameters related to the lumen, epithelium and seminiferous tubules. It was also observed an increase in the volume of blood vessels and connective tissue cells in animals exposed to EMF. The plasma testosterone did not change due to EMF exposure. The exposure to EMF of 60 Hz and 1 mT from gestation to puberty can induce a delay in testicular development. In chapter 2, the aim was to evaluate the effects of exposure to low frequency EMF (60 Hz and 1 mT) from the 13th day of gestation up to 90 postnatal days (adult). The plasma testosterone concentration was not changed by EMF exposure. However, the histopathological and histomorphometrical analysis showed testicular degeneration in a subset of animals exposed to EMF. The degenerative process severity varied among affected animals, indicating an individual sensitivity to EMF. The electron microscopy analysis also showed testicular degeneration, the main change was high electrodense mitochondria with loss of normal organization and cristae. The exposure to EMF of 60 Hz and 1 mT seems to alter spermatogenesis and reduce the fertility. Chapter 3 aimed to analyze the effects of heat shock (43 °C / 15 min.) and Hsp90 inhibition on CaV3 T-type calcium channels and calciumactivated potassium channels (BKCa) in Leydig cells. The results showed that exposure to heat shock induced a reduction in BKCa channels steady-state current (49.8%), maximum conductance (68.9%) and activation time constant (31.9%). Heat shock slowed the BKCa channels activation and reduced its voltage dependence. Hsp90 inhibition did not change BKCa channels. The CaV3 channels current was not affected by heat shock or Hsp90 inhibition. The heat shock can inhibit BKCa channels, the Hsp90 does not seem to be involved in this process. / Nas últimas décadas, os seres humanos vêm sendo expostos a vários agentes físicos exógenos, como temperaturas elevadas induzidas por equipamentos industriais e a exposição aos campos eletromagnéticos (CEM) emitidos por aparelhos eletrônicos. Pesquisas vêm demonstrando que estes agentes podem gerar distúrbios reprodutivos. O objetivo do capítulo 1 do presente trabalho foi investigar o possível efeito da exposição ao CEM de baixa frequência de 60 Hz e 1 mT desde o 13º dia de gestação até a puberdade aos 21 dias pós-natal. Os resultados demonstraram que a exposição ao CEM reduziu os parâmetros relacionados ao lúmen, epitélio e túbulos seminíferos. Também foi observado o aumento no volume dos vasos sanguíneos e das células do tecido conjuntivo nos animais expostos ao CEM. A testosterona plasmática não se alterou devido à exposição ao CEM. A exposição ao CEM de 60 Hz e 1 mT desde a gestação até a puberdade pode induzir o retardo no desenvolvimento testicular. No capítulo 2, o objetivo foi avaliar o efeito da exposição ao CEM de baixa frequência (60 Hz e 1 mT) desde o 13º dia de gestação até os 90 dias pós-natal (adulto). A concentração da testosterona plasmática não foi alterada devido à exposição ao CEM. Entretanto, as análises histopatológicas e histomorfométricas demonstraram degeneração testicular em um sub-grupo dos animais expostos ao CEM. A gravidade do processo degenerativo variou entre os indivíduos afetados, indicando uma sensibilidade individual ao CEM. A análise de microscopia eletrônica também demonstrou degeneração testicular, sendo a principal alteração observada a presença de mitocôndrias eletrodensas e com perda da sua organização e cristas. A exposição ao CEM de 60 Hz e 1 mT parece alterar a espermatogênese e reduzir a fertilidade. No capítulo 3, o presente trabalho objetivou analisar os efeitos do choque térmico (43ºC / 15 min.) e da inibição da Hsp90 nos canais de cálcio CaV3 tipo-T e potássio ativado por cálcio (BKCa) em células de Leydig. Os resultados obtidos revelaram que nos canais BKCa o choque térmico reduziu a corrente do estado estacionário em 49,8%, a condutância máxima em 68,9% e a constante de tempo de ativação em 31,9%. O choque térmico tornou mais lenta a ativação dos canais BKCa e reduziu sua dependência de voltagem. A inibição da Hsp90 não alterou os canais BKCa. A corrente dos canais CaV3 tipo-T não foi afetada pelo estresse térmico ou pela inibição da Hsp90. A exposição das células de Leydig à temperatura elevada pode inibir os canais BKCa, a Hsp90 parece não estar envolvida neste processo.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Thymol, a monoterpene phenol derivative of cymene, is found in abundance in the essential oils produced by numerous herbs and spices such as thyme (Thymus vulgaris L.), oregano (Origanum vulgaris L.) and Lippia alba (mill.). Several biological effects have been described for thymol such as antioxidant, anti-inflammatory, local anesthetics, antinoceptive, healing and antibacterial properties. It was described the application of thymol in food Science, as herbicidad and inseticides. Moreover, essential oils containing thymol have been used in folk medicine to treat various physiological disorders such as gastrites, indigestion and stomach pains. The present study investigated the gastroprotective actions of thymol (10, 30 and 100 mg/kg, p.o.) in the acute indomethacin and ethanol and chronic acetic acid-induced ulcer models in rats. Some of the mechanisms underlying to the gastroprotective effect of thymol were investigated in the ethanol-induced ulcer model. Gastric secretion parameters (volume, pH, and total acidity) were also evaluted by the pylorus ligature model, and the mucus in the gastric content was determined. The antimicrobial activity against Helicobacter pylori of thymol was performed using the agar-well diffusion method. Thymol produced a dose dependent reduction (p < 0.01) on the total lesion area in the ethanol-induced ulcer model. This gastroprotection was also evaluated microscopically showing that the thymol at all doses decreased the loss of epithelial cells. The gastroprotective response caused by thymol (30 mg/kg) was signigicantly attenuated (P < 0.001) by intraperitoneal treatment of rats with indomethacin (a non-selective inhibitior of cyclo-oxygenase, 10 mg/kg) and glibenclamide (ATP-sensitive K channel blocker), but not by DL-Propargylglycine (PAG, a cystathionine-γ-lyase inhibitor) and Nw-nitro-L-arginine methyl ester hydrochloride (L-NAME, a non-selective inhibitor of nitric oxide synthase). Thymol (30 and 100 mg/kg) also reduced the ulcer index (p < 0.05) and the total lesion area (p < 0.001) in the indomethacin and acetic-induced ulcer models, respectively. In the model of pylorus ligature, the treatment with thymol failed to significantly change the gastric secretion parameters. However, after treatment with thymol (30 and 100 mg/kg) there was a significant increase (p < 0.01) in mucus production. Thymol showed no antimicrobial activity against H. pilory in vitro. Collectively, the present results provide evidence that thymol displays gastroprotective actions on the acute and chronic ulcer models involving mechanisms as increased amount of mucus, prostaglandins, and ATP-sensitive K+ channels. / O timol, um monoterpeno fenólico derivado do cimeno encontrado abundantemente em óleos essenciais, é produzido por numerosas plantas aromáticas e especiarias tais como tomilho (Timus vulgaris L.), orégano (Origanum vulgaris L.) e Lippia alba (mill.). Diversos efeitos biológicos têm sido descritos para o timol tais como antioxidante, anti-inflamatório, anestésico local, antinoceptivo, cicatrizante e antibactericida. É descrita a aplicação do timol em ciências dos alimentos, herbicida e inseticidas. Além disso, óleos essenciais contendo timol tem sido usados pela medicina popular para tratar diversas desordens fisiológicas como gastrite, indigestão e dores estomacais. O presente estudo investigou ações gastroprotetoras do timol (10, 30 e 100 mg/kg, v.o.) em modelos de úlcera aguda induzida por indometacina e etanol, e crônica induzida por ácido acético em ratos. Alguns dos mecanismos subjacentes ao efeito gastroprotetor do timol foram investigados em modelo de úlcera induzida por etanol. Parâmetros da secreção gástrica (volume, pH; e acidez total) também foram avaliados pelo modelo de ligadura de piloro, e o conteúdo do muco gástrico foi determinado. A atividade contra Helicobacter pylori do timol foi realizada pelo método de difusão em ágar. O timol produziu efeito dose-dependente reduzindo (p < 0,01) a área total de lesão em modelo de úlcera induzida por etanol. Esta gastroproteção também foi avaliada microscopicamente mostrando que o timol, em todas as doses, diminuiu a perda das células epiteliais. A resposta gastroprotetora causada pelo timol (30 mg/kg) foi significativamente atenuada (p < 0,001) pelo tratamento intraperitoneal dos ratos com indometacina (um inibidor não-seletivo da ciclo-oxigenase, 10 mg/kg) e glibenclamida (bloqueador de canais para potássio sensíveis a ATP), mas não pela DL-Propargilglicina (PAG, inibidor da cistationina-γ-liase) ou Nw-nitro-L-arginina metil éster cloridrato (L-NAME, um inibidor não-seletivo da sintase do óxido nítrico). O timol (30 e 100 mg/kg) também reduziu o índice de úlcera (p < 0,05) e área total de lesão (p < 0,001) por indometacina e por ácido acético, respectivamente. Em modelo de ligadura de piloro, o tratamento com timol não alterou os parâmetros de secreção gástrica. No entanto, após o tratamento com o timol houve um aumento significativo (p < 0,01) na produção do muco. O timol não apresentou atividade contra H. pylori in vitro. Coletivamente, os resultados apresentados fornecem evidências de que o timol exibe ações gastroprotetoras sobre os modelos de úlceras agudas e crônicas envolvendo mecanismos como o aumento da quantidade de muco, prostaglandinas e canais para potássio sensíveis a ATP.

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-01-15T13:24:17Z No. of bitstreams: 2 Tese - Patrícia Ferreira da Silva Castro - 2015.pdf: 4272048 bytes, checksum: bb8709720dbc4fb91db81ca23004d59d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-18T08:44:08Z (GMT) No. of bitstreams: 2 Tese - Patrícia Ferreira da Silva Castro - 2015.pdf: 4272048 bytes, checksum: bb8709720dbc4fb91db81ca23004d59d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-01-18T08:44:08Z (GMT). No. of bitstreams: 2 Tese - Patrícia Ferreira da Silva Castro - 2015.pdf: 4272048 bytes, checksum: bb8709720dbc4fb91db81ca23004d59d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-26 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Nitric oxide is a potent bronchodilator and compounds capable of increasing its supply have demonstrated clinical interest in the treatment of obstructive airways diseases. This study evaluated and compared the mechanisms of relaxation of two nitric oxide donors, ruthenium complex [Ru(terpy)(bdq)NO+]3+ (TERPY) and sodium nitroprusside (SNP) in healthy isolated trachea, Sham group, or experimental model of asthma induced by ovalbumin in rats, OVA group. The isolated trachea was sectioned into rings and contracted with carbachol in an organ chamber for studying relaxation. The relaxing effect of TERPY and SNP was evaluated at increasing concentrations from 10 nM to 100 μM. Thus, we verified the contribution of the different types of K+ channels, the importance of sGC/cGMP pathway, the inhibition of PDEs (for IBMX, amrinone, rolipram and dypiridamole), the influence of the extra and intracellular Ca2+ sources (for cyclopiazonic acid) and the influence of the store and voltage operated calcium channels. Besides inhibition of COX (diclofenac), antagonism of leukotriene receptor (montelukast) and superoxide anion scavenger (TIRON). Analyses were performed under light microscopy for evidence of inflammatory infiltration and bronchoditation by TERPY in slices bronchioles of asthmatic animals. The results verified that sensitization with ovalbumin led to intense inflammatory process and hyperresponsives to carbachol in compared Sham group. TERPY and SNP led to the relaxation of tracheal smooth muscle preparations in a dependent-concentration mode in both groups. However, the maximum effect induced by TERPY was higher than the effect of SNP in Sham and OVA groups. The relaxation mechanism of TERPY in boht groups showed differences. In Sham group, TERPY relaxation by the activation of Kv, Kir, KCa and KATP channels, cGMP-independent mechanisms and by reduction of calcium influx by blocking the store and voltage operated calcium channels. In OVA group, TERPY acts through activation K+ channels, NO/GCs/GMP way and blocking the store and voltage operated calcium channels. The relaxing effect induced by SNP in OVA group was dependent of NO/GCs/GMP pathway, Kv, KCa and BKCa channels and blocking the store and voltage operated calcium channels. However, the activation of the enzyme sGC seems to be reduced in inflamed smooth muscle, as well as the role of the sarcoplasmic reticulum calcium pump. Diclofenac, montelukast and TIRON improved relaxation of the TERPY and SNP in OVA group. The TERPY is able to reverse the contraction of carbachol in asthmatic bronchioles. In conclusion, TERPY and SNP have their mechanisms of relaxation modified by the inflammatory process. However, this modification was not able to alter the pharmacological parameters potency and efficacy to TERPY. Since SNP has less efficacy effect in asthmatic tracheas. This may result from the lower participation of the enzyme sGC and reticular calcium pump, making TERPY a promising drug to reverse the narrowing of the airways. / O óxido nítrico é um potente broncodilatador e compostos capazes de aumentar a sua oferta têm demonstrado interesse clínico no tratamento das doenças obstrutivas das vias aéreas. Estee trabalho avaliou comparativamente os mecanismos de relaxamento de dois doadores de óxido nítrico, o complexo de rutênio [Ru(terpy)(bdq)NO+]3+ (TERPY) e o nitroprussiato de sódio (SNP) em traqueias isoladas de ratos saudáveis, grupo Sham, e com asma experimental induzida por ovalbumina, grupo OVA. As traqueias isoladas foram cortadas em anéis, montadas em banho para órgãos isolados e contraída com carbacol para estudo do relaxamento. O efeito relaxante do TERPY e do SNP foi avaliado em concentrações crescentes e cumulativas (10 nM a 100 μM). Foi verificada a participação dos diferentes tipos de canais de K+, a participação da via GCs/GMPc, inibição das PDEs (pelo IBMX, amrinona, rolipram e dipiridamol), a participação dos estoques internos de Ca2+ (pelo ácido ciclopiazônico), assim como a participação do influxo deste íon pelos canais de cálcio controlados por estoque e por voltagem, além da inibição da COX (pelo diclofenaco), do antagonismo dos receptores de leucotrienos (pelo montelucaste) e do sequestro dos íons superóxidos (pelo TIRON). Foram realizadas análises em microscopia de luz para comprovação da presença de infiltrado inflamatório e da broncodilatação exercida pelo TERPY em cortes de bronquíolos de animais asmáticos. Como resultados, verificou-se que a sensibilização com ovalbumina levou a um intenso processo inflamatório com migração celular e hiperreatividade ao carbacol. Evidenciou-se que o TERPY e o SNP relaxaram o músculo liso traqueal de forma concentração-dependente em ambos os grupos. Entretanto, o efeito máximo induzido pelo TERPY foi maior do que o do SNP tanto no grupo Sham quanto no OVA. O mecanismo de relaxamento do TERPY mostrou-se diferente entre os grupos. No grupo Sham, o TERPY exerce relaxamento por ativação dos canais Kv, Kir, KCa e KATP independentemente de GMPc e por redução do influxo de cálcio através do bloqueio dos canais de cálcio operados por voltagem e por estoque. No grupo OVA, o TERPY exerce seu efeito através da por ativação dos canais K+, via NO/GCs/GMP e redução do influxo de cálcio por bloqueio dos canais de cálcio operados por voltagem e operados por estoque. O efeito relaxante do SNP no grupo OVA ocorre através da ativação da via NO/GCs/GMPc, dos canais Kv, KCa e SKCa e por redução do influxo de cálcio pelos canais de cálcio operados por voltagem e operados por estoque. Entretanto, a ativação da enzima GCs parece estar reduzida em músculo liso inflamado, assim como o papel da bomba de cálcio do retículo sarcoplasmático. O diclofenaco, o montelucaste e o TIRON melhoraram o perfil de relaxamento tanto do TERPY quanto do SNP no grupo OVA. O TERPY é capaz de reverter a contração do carbacol em bronquíolos asmáticos. Em conclusão, tanto o SNP quanto o TERPY têm seus mecanismos de relaxamento modificados pelo processo inflamatório. Entretanto, esta modificação não foi capaz de alterar os parâmetros farmacológicos de potência e eficácia do TERPY. Já o SNP, tem menor eficácia em traqueias de ratos do grupo OVA e isso pode decorrer da menor participação da enzima GCs e da bomba de cálcio reticular, fazendo do TERPY uma droga promissora para reversão do estreitamento das vias aéreas.

No Brasil, a espécie Tityus serrulatus (Ts) é a responsável pela maioria dos acidentes de envenenamento com escorpiões, bem como pela maior incidência de acidentes ocasionados por animais peçonhentos. Isso ocorre devido ao fato desta espécie possuir somente fêmeas, realizando uma reprodução assexuada (partenogênese), facilitando assim sua proliferação. Atualmente, existem 16 diferentes toxinas descritas provenientes da peçonha de Ts, sendo a Ts1 a mais abundante na peçonha solúvel. Dentre estas toxinas, as neurotoxinas com ação em canais para sódio e potássio são as que despertam maior interesse da comunidade científica, devido aos seus efeitos no envenenamento e especificidades por canais iônicos. As neurotoxinas com ação em canais para potássio são compostas por uma alfa-hélice e três fitas-beta antiparalelas, e são constituídas por 23-43 resíduos de aminoácidos. Essas estão classificadas em quatro famílias (?-, ?-, g- e k-KTx). A família ?-KTx é a de maior relevância e está dividida em 21 subfamílias. Estas toxinas com alta especificidade para diferentes subtipos de canais para sódio e potássio são de extrema importância, pois podem ser utilizadas como ferramentas terapêuticas específicas para células-alvo. Até o momento, nosso grupo realizava o isolamento dessas toxinas utilizando como primeira etapa de purificação a cromatografia da peçonha em coluna de CM-Celulose-52, segundo protocolo descrito por Arantes e colaboradores (1989). O presente trabalho padronizou um novo método de fracionamento inicial da peçonha utilizando a mesma coluna, porém incorporando o uso de CLAE (Cromatografia Liquida de Alta Eficiência), onde foi possível observar um perfil cromatográfico semelhante ao anterior (XIII Frações), porém com maior reprodutibilidade e praticidade. Algumas frações do método anteriormente descrito foram subdivididas em duas (VIA-VIB, VIIIA-VIIIB, IXA-IXB e XIA-XIB), demonstrando que o novo procedimento também apresenta melhor resolução dos componentes da peçonha. Utilizando o novo método, foram purificadas as toxinas Ts6 e Ts7. Os efeitos dessas toxinas foram avaliados em 14 diferentes tipos de canais para potássio (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.1, Kv4.3, Kv7.1, Kv7.2, Kv7.4, hERG e Shaker), expressos em células de oócitos de Xenopus laevis, através da técnica de voltage-clamp com dois microeletrodos. A toxina Ts6 (1?M) demonstrou ter ação em 11 tipos de canais (Kv1.1, Kv1.2, Kv1.3, Kv1.5, Kv1.6, Kv4.3, Kv7.1, Kv7.2, Kv7.4, hERG e Shaker), porém nos canais Kv1.2, Kv1.3 e Shaker sua ação bloqueadora foi mais intensa. Através de experimentos de dose-resposta foi possível comprovar tal seletividade, demonstrando que a toxina Ts6 atua em quantidades extremamente baixas em ambos os canais (IC50 Kv1.2 = 6,19 ± 0,35 nM /IC50 Kv1.3 = 0,55 ± 0,20 nM). A toxina Ts7 (1?M) demonstrou ter ação em 11 tipos de canais para K+ (Kv1.1, Kv1.2, Kv1.3, Kv1.4 Kv1.5, Kv1.6, Kv2.1, Kv3.1, Kv7.1, hERG e Shaker), porém a ação de bloqueio em vários subtipos de canais não apresentou diferenças significativas, mostrando baixa seletividade entre os canais analisados. Este trabalho foi de Resumo ii extrema importância por melhorar a reprodutibilidade, praticidade e resolução do método de fracionamento da peçonha de Ts, bem como por fornecer uma avaliação eletrofisiológica criteriosa das toxinas Ts6 e Ts7 em diferentes subtipos de canais para potássio. O presente estudo demonstra a potencial aplicação da toxina Ts6, seletiva para canais Kv 1.3, para o tratamento de doenças autoimunes. Além disso, indica o uso das toxinas Ts6 e Ts7 como ferramentas para o estudo de características estruturais e funcionais de canais para potássio. / In Brazil, Tityus serrulatus (Ts) species is the responsible for the most scorpion accidents and also for the major incidence of accidents caused by venomous animals. About 16 different toxins of Ts venom have been listed so far, being Ts1 the major one. Among these toxins, the neurotoxins with action on sodium and potassium channels are the most interest in the scientific community, due to their effect in the envenomation and ion channel specificity. The neurotoxins with action on potassium channels are composed of an ?-helix and three ?-strands formed by 23-43 amino acid residues. They are classified into four families (?-, ?-, g- and ?- KTx). The ?-KTx family is the most relevant and is divided into 21 subfamilies. These toxins with high specificity for different subtypes of sodium and potassium channels are very important, because they can be used as therapeutic tools to specific target cells. Until now, the fractionation of these toxins was done using a CM-Cellulose-52 column, according to the protocol of Arantes and co-workers (1989). The present work standardized a new method of isolation using the same column, but incorporated the use of HPLC (High Performance Liquid Chromatography), in which was observed a chromatographic profile such as the previous one (XIII Fractions), however with high resolution and more practical. Some fraction of the previous method were divided in two subfractions (VIA-VIB, VIIIA-VIIIB, IXA-IXB e XIA-XIB), showing that the new method also present high resolution. Using the new method, it was isolated the Ts6 and Ts7 toxin. The effects of these toxins were evaluated in 14 different types of potassium channels (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.1, Kv4.3, Kv7.1, Kv7.2, Kv7.4, hERG and Shaker), which were expressed in Xenopus laevis oocytes using the voltage-clamp technique with twomicroelectrodes. The Ts6 toxin (1?M) shows to act on 11 types of potassium channels (Kv1.1, Kv1.2, Kv1.3, Kv1.5, Kv1.6, Kv4.3, Kv7.1, Kv7.2, Kv7.4, hERG and Shaker), but the blocking of Kv1.2 and Kv1.3 was significantly more intense. Using dose-response experiments, it was possible to confirm this selectivity, in which Ts6 demonstrates to act in both channels in extremely low quantities (IC50 Kv1.2 = 6,19 ± 0,35 nM /IC50 Kv1.3 = 0,55 ± 0,20 nM). The Ts7 toxin (1?M) shows to act on 11 types of potassium channels (Kv1.1, Kv1.2, Kv1.3, Kv1.5, Kv1.6, Kv4.3, Kv7.1, Kv7.2, Kv7.4, hERG and Shaker), but the blocking action on multiple subtypes channels showed no significant differences, showing low selectivity among the channels analyzed. This work was important to improve and facilitate the method of fractionation of Ts venom, as well as evaluate the electrophysiology properties of the toxins Ts6 and Ts7 of interacting with different types of potassium channels. These studies will be essential for future applications of these toxins as drugs to treat channelpathies or as tools to study potassium channels structurally and functionally.

Parmi les différents types de canaux ioniques, les canaux potassium (K+) sont très largement exprimés au niveau des cellules eucaryotes. Ils se répartissent en plusieurs familles et sous-familles. Parmi celles-ci, les canaux K+ ATP-dépendants (KATP) représentent une classe tout à fait particulière. En effet, ils ont la particularité d’être sensibles à la concentration cytosolique d’ATP et permettent ainsi de coupler le potentiel membranaire de la cellule à son statut métabolique.<p>Le canal KATP est un complexe hétéro-octamérique constitué de 2 sous-unités :une sous-unité Kir6.x (Kir6.1 ou Kir6.2) formant le pore du canal et appartenant à la famille des canaux potassiques de type « inward rectifier », et une sous-unité régulatrice SURx (SUR 1 ou SUR2A/B) faisant partie des protéines ABC (ATP-binding cassette). L’expression hétérologue des sous-unités Kir6.x et SURx suivant différentes combinaisons conduit à la formation de plusieurs types de canaux KATP possédant des propriétés électro-physiologiques et des sensibilités aux nucléotides et aux agents pharmacologiques distinctes. \ / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

Les canaux potassiques (Kv) dépendants du voltage sont des protéines transmembranaires qui permettent le flux passif d’ions potassium à travers une membrane plasmique lorsque celle-ci est dépolarisée. Ils sont constitués de quatre domaines périphériques sensibles au voltage et un domaine central, un pore, qui délimite un chemin hydrophile pour le passage d’ions. Les domaines sensibles à la tension (VSD) et le pore sont couplés, ce qui signifie que l’activation des VSD déclenche l’ouverture du pore, et qu’un pore ouvert favorise l’activation des VSD. Le phosphatidylinositol-4,5-bisphosphate (PIP2) est un lipide mineur du feuillet interne de la membrane plasmique. Ce lipide fortement chargé négativement module le fonctionnement de plusieurs canaux ioniques, y compris les membres de la famille Kv. En particulier, l’application de ce lipide à Kv1.2 et Kv7.1, deux canaux homologues, augmente leur courant ionique. Cependant, alors que Kv1.2 est capable de s’ouvrir en l’absence de PIP2, dans le cas de Kv7.1, ce lipide est absolument nécessaire pour l’ouverture du canal. En outre, dans Kv1.2, PIP2 induit une perte de fonction, qui est manifesté par un mouvement retardé des VSD. Jusqu’à présent, les mécanismes sous-jacents à de telles modulations des canaux Kv par PIP2 restent inconnus. Dans ce travail, nous tentons de faire la lumière sur ces mécanismes en utilisant des simulations de dynamique moléculaire (DM) combinées avec une approche expérimentale, entreprise par nos collaborateurs. En utilisant des simulations de DM sans contrainte, nous avons identifié les sites potentiels de liaison du PIP2 au Kv1.2. Dans l’un de ces sites, PIP2 interagit avec le canal de sorte à former des ponts salins dépendants de l’état du canal, soit avec le VSD soit avec le pore. Sur la base de ces résultats, nous proposons un modèle pour rationaliser les données expérimentales connues. En outre, nous avons cherché à évaluer quantitativement la perte de fonction induite par la présence de PIP2 au voisinage du VSD du Kv1.2. En particulier, nous avons calculé l’énergie libre des deux premières transitions le long de l’activation du VSD en présence et en l’absence de ce lipide. Nous avons constaté que PIP2 affecte à la fois la stabilité relative des états du VSD et les barrières d’énergie libre qui les séparent. Enfin, nous avons étudié les interactions entre PIP2 et un autre membre de la famille Kv, le canal Kv7.1 cardiaque. Dans le site de liaison de PIP2 que nous avons identifié pour ce canal, l’interaction entre les résidus positifs de Kv7.1 et le lipide sont dépendants de l’état du VSD, comme dans le cas de Kv1.2. On montre que cette interaction est importante pour le couplage entre les VSD et le pore, couplage qui est par ailleurs affaibli à cause de la répulsion électrostatique entre quelques résidus positifs. Ces résultats et prédictions ont été vérifiés par les données expérimentales obtenues par nos collaborateurs / Voltage-gated potassium (Kv) channels are transmembrane proteins that enable the passive flow of potassium ions across a plasma membrane when the latter is depolarized. They consist of four peripheral voltage sensor domains, responding to the applied voltage, and a central pore domain that encompasses a hydrophilic path for passing ions. The voltage sensors and the pore are coupled, meaning that the activation of the voltage sensors triggers the pore opening, and the open pore promotes the activation of the voltage sensors. Phosphatidylinositol-4,5-bisphosphate (PIP2) is a minor lipid of the inner plasma membrane leaflet. This highly negatively charged lipid was shown to modulate the functioning of several ion channels including members of the Kv family. In particular, application of this lipid to Kv1.2 and Kv7.1, two homologous channels, enhances their ionic current. However, while Kv1.2 is able to open without PIP2, in the case of Kv7.1, this lipid is absolutely required for opening. Additionally, in Kv1.2, PIP2 induces a loss of functioning, which is manifested by delayed motions of the voltage sensors. So far, the mechanism underlying the Kv channels modulation by PIP2 remains unknown. In the present manuscript, we attempt to shed light on this mechanism using molecular dynamics (MD) simulations combined with experiments, which was undertaken by our collaborators. Using unconstrained MD simulations, we have identified potential PIP2 binding sites in Kv1.2. In one of these sites, PIP2 interacts with the channel in a state-dependent manner forming salt bridges either with the voltage sensor or with the pore. Based on these findings, we propose a model rationalizing the known experimental data. Further, we aimed to estimate the loss of functioning effect induced by PIP2 on the Kv1.2 voltage sensors. In particular, we have calculated the free energy of the first two transitions along the activation path in the presence and absence of this lipid. We found that PIP2 affects both the relative stability of the voltage sensor states and the free energy barriers separating them. Finally, we studied the interactions between PIP2 and another member of the Kv family, the cardiac channel Kv7.1. In the PIP2 binding site that we have identified for this channel, the interaction between positive residues of Kv7.1 and the lipid was state-dependent, as in the case of Kv1.2. This state-dependent interaction, however, is prominent for coupling between the voltage sensors and the pore, which is otherwise weakened due to electrostatic repulsion of some positive residues. These findings are in a good agreement with the experimental data obtained by our collaborators

KCNE peptides are a class of type I transmembrane ß-subunits that assemble with and modulate the gating and ion conducting properties of a variety of voltage-gated K+ channels. Accordingly, mutations that affect the assembly and trafficking of K+ channel/KCNE complexes give rise to disease. The cellular mechanisms that oversee KCNE peptide assembly with voltage-gated K+ channels have yet to be elucidated. In Chapter II, we show that KCNE1 peptides are retained in the early stages of the secretory pathway until they co-assemble with KCNQ1 K+ channel subunits. Co-assembly with KCNQ1 channel subunits mediates efficient forward trafficking of KCNE1 peptides through the biosynthetic pathway and results in cell surface expression. KCNE1 peptides possess two N-linked glycosylation sites on their extracellular N-termini. Progression of KCNE1 peptides through the secretory pathway can be visualized through maturation of N-glycans attached to KCNE1. In Chapter III, we examine the kinetics and efficiency of N-linked glycan addition to KCNE1 peptides. Mutations that prevent glycosylation of KCNE1 give rise to the disorders of arrhythmia and deafness. We show that KCNE1 acquires N-glycans co- and post-translationally. Mutations that prevent N-glycosylation at the co-translational site have a long range effect on the disruption of post-translational glycosylation and suggest a novel biogenic mechanism for disease. In Chapter IV, we determine the presence of an additional post-translational modification on KCNE1 peptides. We define specific residues as sites of attachment of this modification identified as sialylated O-glycans and show that it occurs in native cardiac tissues where KCNE1 plays a role in the maintenance of cardiac rhythm. Taken together, these observations demonstrate the importance of having correctly assembled K+ channel/KCNE complexes at the cell surface for their proper physiological function and define a role for the posttranslational modifications of KCNE peptides in the proper assembly and trafficking of K+ channel/KCNE complexes.

Alcoholism is responsible for more than 6% of deaths internationally per annum. The development of acute tolerance to ethanol (EtOH) is a critical component of alcoholism. Previous studies identified large conductance calcium-activated potassium (BK) channels as potential EtOH targets in a variety of species and cells. In order to elucidate mechanisms underlying tolerance development, I used inside-out patch clamp techniques to measure EtOH induced changes in channel activity (measured as open probability) of hSlo, hSlo+β1, and hSlo+β4 channels exogenously expressed in HEK 293 cells. I show that the human BK channels have subunit dependent responses to acute application of EtOH, and the magnitude of potentiation was dependent on the concentration of ethanol used and the type of β-subunit expressed. In addition the subunit dependent effects on the channels were a function of cytosolic calcium concentration. Furthermore, to determine if BK channels in ripped-off patches can become tolerant to EtOH, I monitored changes in channel activity in response to a second application of the drug, 10-minutes after washing-out the first exposure. I found that channels were less responsive to the second exposure, indicative of tolerance. I examined long-term consequences of EtOH exposure by repeating these experiments on cells cultured in 25 mM EtOH in the culture medium for 24-hours. Under these conditions, all three channel types show chronic tolerance has developed as revealed by the response to acute EtOH applications. Subunit-dependent differences to the development of acute tolerance were apparent, however. In response to a second application to EtOH, hSlo+β4 channels were now inhibited. Overall, these results indicate that BK channels respond to and develop tolerance to EtOH in the absence of cellular context, suggesting the possibility that alcohol tolerance within organisms may be in part mediated by changes imparted by EtOH on BK channels directly.

Calcium tolerant pig and rabbit cardiomyocytes were isolated using retrograde aortic perfusion of nominally calcium-free collagenase. Preconditioning protocols used 1 or 3 x l0-min episodes of ischemic pelleting or pre-incubation with 100 μM adenosine, followed by a 15-min post-incubation and 180-240-min ischemic pelleting. Control cells were incubated and washed in parallel with the experimental groups. Injury was assessed by determination of cell morphology, trypan blue permeability following osmotic swelling, lactate and HPLC analysis of adenine nucleotides. Preconditioned pig cardiomyocytes had a reduced rate of ischemic contracture, but protection occurred without conservation of ATP. Preconditioned rabbit cardiomyocytes were protected without significant changes in rates of ischemic contracture or ATP depletion. Incubation of ischemic cells with the protein phosphatase inhibitor, fostriecin, at PP2A-selective concentrations (0.1-10 μM), mimicked preconditioning in both rabbit and pig cardiomyocytes. In rabbits, the K(ATP) channel blocker, 5-hydroxydecanoate (5-HD), did not block preconditioning or fostriecin protection. In the pig, 5-HD blocked both preconditioning and fostriecin protection, with return of the rates of ischemic contracture to control. However, 5-HD was an effective blocker of protection only in early ischemia. Fostriecin mimicked preconditioning in the rabbit and the early responses of the preconditioned pig. Preconditioning appears associated with protein phosphorylation in both the rabbit and the pig, but major pathways leading to protection may differ in the two species.

Les canaux de potassium voltage-dépendant (Kv) sont des tétramères séparés en 12 familles. Chaque sous-unité est composée de six segments transmembranaires (S1-S6). Les quatre premiers (S1-S4) forment le senseur de voltage dont le rôle est de détecter des variations en potentiel membranaire grâce à des acides aminés chargés. Ces acides aminés vont bouger et ce mouvement va être transmis au second domaine, celui du pore (S5-S6). Les domaines du pore des quatre sous-unités vont se combiner pour créer le pore. Ces sous-unités peuvent former des canaux homomériques où chaque sous-unité est identique ou des canaux hétéromériques avec des membres de la même famille. Kv6.4 (KCNG4) est un membre de la famille de sous-unité silencieuse Kv6. Les familles de sous-unités silencieuses incluent également Kv5, Kv8 et Kv9. Ils ne peuvent pas former d’homomères. À la place, il doit former des hétéromères avec Kv2. Les canaux Kv2.1/Kv6.4 ont des propriétés différentes, lorsque comparées aux homomères de Kv2.1, particulièrement avec un décalage de l’inactivation vers les négatifs. Avec la technique du « cut-open voltage clamp fluorometry » (COVCF), nous avons pu déterminer que l’absence d’une charge positive à la position Kv6.4-Y345 est responsable pour une partie du décalage tout en étant capable de réduire ce décalage avec la mutation Kv6.4-Y345R. Nous avons également pu produire l’effet inverse dans Kv2.1 avec Kv2.1-R306Y. Également, nous avons déterminé que la mutation Kv6.4-L360P trouvée chez des patients souffrant de migraines mène à cette pathologie à cause d’un problème de trafic où les sous-unités mutées ne peuvent pas atteindre la surface et produire des canaux fonctionnels. Ce problème est causé par un bris dans l’hélice alpha du segment S4-S5. Uniquement des homomères de Kv2.1 se rendent à la surface ce qui réduit l’excitabilité membranaire. Nous proposons que lorsqu’exprimée dans le ganglion trigéminal, cette mutation mène à des migraines. / Voltage-gated potassium channels (Kv) are tetramers split into 12 families. Each subunit is composed of six transmembrane helices (S1-S6). The first four of those (S1-S4) form the voltage sensor domain whose role it is to detect variations in the membrane potential through charged amino acids. The movement of those amino acids will be transmitted to the second domain, the pore domain (S5-S6). The pore domain of all four subunits will combine to form the ion conducting pore. These subunits can form homomers where all four subunits are identical or heteromers with members of the same family. Kv6.4 (KCNG4) is a member of the silent subunit family Kv6, which also includes Kv5, Kv8 and Kv9. They cannot form functioning homomers. Instead, they form heteromers with Kv2. Kv2.1/Kv6.4 channels have different properties when compared to Kv2.1 homomers, particularly a negative shift of the voltage dependence of inactivation. With the cut-open voltage clamp fluorometry (COVC) technique, we were able to determine that the absence of a gating charge at position Kv6.4-Y345 is responsible for part of this shift. We were able to recover part of this shift with the mutation Kv6.4-Y345R. We were also able to produce the inverse effect in Kv2.1 with the mutation Kv2.1-R306Y. Also, we determined that the mutation Kv6.4-L360P. which is found in patients suffering from migraines, leads to this condition because of a trafficking defect caused by the mutation stopping the subunits from reaching the membrane and making functional channels. The defect is caused by a kink in the alpha helix of the S4-S5 linker. Only Kv2.1 homomers reach the membrane which reduces membrane excitability. We propose that when expressed in the trigeminal ganglion, this mutation leads to migraines because of this trafficking defect.