La mortalité cardiovasculaire représente la première cause de mortalité chez les sujets diabétiques de type 2. La dyslipidémie de ces patients caractérisée par le quatuor : hypertriglycéridémie, baisse du HDL-cholestérol, augmentation du nombre de LDL petites et denses et hyperlipidémie postprandiale, constitue un facteur de risque prépondérant. Cette dyslipidémie est en grande partie expliquée par l’accumulation sanguine des lipoparticules riches en triglycérides (TRL) d’origine hépatique (VLDL) et intestinale (chylomicrons). L’hyperproduction des chylomicrons, déjà connue pour les VLDL, est une composante nouvellement reconnue d’insulinorésistance. L’action inhibitrice aigüe de l’insuline sur la production des VLDL est absente chez le sujet diabétique de type 2. Dans notre étude, nous avons montré l’absence d’effet aigu de l’insuline sur la production des chylomicrons chez les sujets diabétiques de type 2 grâce à une étude cinétique utilisant un isotope stable (D3-leucine). / The cardiovascular mortality represents the first cause of mortality in human type 2 diabetes. The typical diabetic dyslipidemia characterized by the quartet: high triglyceride levels, low HDL-cholesterol, increased number of small and dense LDL particles and postprandial hyperlipidemia, is a major cardiovascular risk factor. This dyslipidemia is mainly explained by the accumulation of triglyceride-rich lipoproteins (TRL) from liver (VLDL) and intestine (chylomicrons).The overproduction of chylomicrons, as is known for VLDL, is a newly characteristic of insulin resistant states. The acute inhibitory effect of insulin on VLDL production is absent in type 2 diabetic patients. In our study, we have shown the absence of acute inhibitory effect of insulin on chylomicron production in type 2 diabetic patients, using a kinetic study with stable isotope (D3-leucine).

Cette thèse se compose de deux parties qui peuvent être considérées comme deux aspects différents de l'électronique moléculaire avec pour point commun les moyens de nanofabrication mis en jeu pour réaliser des dispositifs de mesures électriques à bas courant. La première partie de la thèse concerne l'assemblage de nano-aimants entre électrodes à nanogap. Le besoin croissant de processeurs toujours plus performants et celui d’une densité de stockage toujours plus grande ont poussé la technologie CMOS couramment utilisée dans l'industrie à ses limites physiques vis-à-vis de sa miniaturisation. L'électronique moléculaire et la spintronique moléculaire se révèlent être des alternatives prometteuses à cette technologie pour les futurs dispositifs nanoélectroniques. Mes principaux travaux dans ce domaine ont porté sur l'assemblage entre des électrodes à nanogap, de nano-aimants moléculaires à base de bleu de Prusse ou de son analogue Cs–Co–Cr. Le but était ainsi de faire les premiers pas vers la construction de dispositifs en spintronique moléculaire. Des nanogaps de ~ 7 à 50 nm ont été fabriqués en palladium ou en or sur un substrat Si/SiO₂ par lithographie électronique et lift-off. Les nano-aimants ont été placés dans le gap par diélectrophorèse à courant alternatif (AC DEP). À température ambiante, un courant négligeable a été mesuré sur les jonctions utilisant des nanoparticules de Cs–Co–Cr alors qu’un courant de ~ 30 pA a été mesuré sur celles avec les nanoparticules en bleu de Prusse pour une tension de ~ 1 V. J’ai montré qu‘en fait, l’eau piégée dans les nanogaps altérait sérieusement les mesures de courants et nécessitait un recuit préalable. Pour optimiser la localisation des nanoparticules entre les électrodes, j’ai proposé un programme de simulation de la DEP ne tenant pas compte du mouvement brownien et de la dynamique des fluides. La deuxième partie de la thèse concerne la fabrication de dispositifs de type nanopatch-clamp planaire pour l'enregistrement de courants ioniques à travers les canaux ioniques des protéines membranaires. Les canaux de ces protéines incorporées dans les membranes cellulaires sont des composantes essentielles de toutes les cellules vivantes et sont à la base de divers processus physiologiques tels que ceux dans la communication nerveuse, la contraction musculaire, la sensation tactile, etc. Les mesures de transport d'ions sont maintenant utilisées dans diverses applications telles que le criblage de médicaments dans l'industrie pharmaceutique et les biocapteurs médicaux. La méthode classique pour effectuer des mesures de transport d'ions consiste à utiliser un système patch-clamp. Cependant, cette méthode nécessite d’importantes compétences, des équipements lourds et coûteux et présente une faible efficacité de mesure. Pour pallier ces inconvénients, une solution est de développer des patch-clamps planaires, qui sont modulables, automatisés et faciles d’utilisation. La fabrication du dispositif a consisté en la réalisation d’une piste conductrice constituée d’un empilement de couches Au/Ag sur un substrat de silicium oxydé. Cette piste a été passivée et isolée électriquement par une couche de Si₃N₄/SiO₂ dans laquelle j’ai gravé des micro-trous et j’ai ensuite converti la couche d’Ag en AgCl pour les mesures électriques. Afin de valider le fonctionnement du dispositif sans la membrane, j’ai procédé à des mesures de courant en fonction du temps pour diverses tensions, ce qui m’a ensuite permis de proposer un schéma équivalent électrique. / This thesis consists of two parts. The two parts correspond to two different subjects but with a common feature which is the fabrication of nanometer scale devices for low current measurements. The first part investigated the assembly of Prussian blue and Cs–Co–Cr Prussian blue analogue molecular nanomagnets into nano-patterned electrodes. The ever growing need for higher performance processors and higher storage densities has pushed the CMOS technology commonly used in industry to its physical limitations toward its miniaturization. Molecular electronics and molecular spintronics prove to be promising alternatives for the CMOS in future nanoelectronic devices. Pd or Au gaps with ~ 7–50 nm width were fabricated on a Si/SiO₂ substrate using standard electron beam lithography, metal deposition and lift-off. Nanomagnets were positioned between the gaps via AC dielectrophoresis (DEP). At room temperature, the Cs–Co–Cr Prussian blue analogue nanoparticles exhibited negligible current whereas junction with Prussian blue nanoparticles exhibited ~ 30 pA at ~ 1 V. Water trapped in nanogaps was found to seriously alter current measurements. This problem was solved by heating samples prior to measurements. A simplified DEP simulation program using Delphi was developed, which neglected Brownian motion and fluid dynamics but allowed us to better understand the DEP process. The second part of the thesis investigated the fabrication of devices for measuring electrical currents through membrane protein channels. Membrane-embedded protein channels are the basis of various physiological processes like nervous communication, muscular contraction, tactile sensation, and so on. Electrical measurements are used in different applications such as drug screening in pharmaceutical industry and biosensors. The standard method to perform such measurements is the use of patch-clamp. However, this method requires intense skill and heavy equipment while it exhibits low measurement efficiency. A solution to these drawbacks is the development of planar patch clamps, which are scalable, automated and easier to use. The first device fabrication step was the patterning of Au/Ag electrodes on thermally oxidized Si substrate by optical lithography, metallization and lift-off. Secondly, a passivation layer of Si₃N₄/SiO₂ was deposited on top of electrodes by PECVD. Then micro-holes were formed inside the Si₃N₄/SiO₂ passivation layer stack using Raith-150 e-beam lithography and reactive ion etching. Finally, Ag layer was converted to AgCl using bleach. The test of electrical current was done using Axopatch patch-clamp amplifier. Current versus time measurements for different voltages were recorded without membrane covering the holes, and an electrical model has been developed for the fabricated devices.

Neurônios GnRH e kisspeptina representam as populações neuronais de maior importância no controle da reprodução. Estradiol liga-se ao seu receptor expresso pelos neurônios kisspeptina para regular a libertação de GnRH. No modelo animal OVX+E a atividade do neurônio GnRH e pico de LH é depende do estradiol e hora do dia. Nesse estudo, embora a taxa de disparo dos neurônios GnRH seja similar entre os grupos, o padrão dos potenciais revelou uma mudança para maior duração do estouro em camundongos no proestrous, além do fato de uma maior resposta da hipófise. A prolactina tem grande impacto na modulação do eixo HPG e kisspeptina são mediadores dos efeitos da prolactina sobre a reprodução. Uma pequena porcentagem de neurônios de kisspeptina do AVPV foi indiretamente despolarizada pela prolactina. Este efeito requeria a via de sinalização PI3K. Camundongos portadores de inativação de Stat5a/b em células kisspeptina exibiram um início precoce de ciclicidade estro, indicando que os fatores de transcrição STAT5 exercem um efeito inibitório sobre o momento da puberdade. / GnRH and kisspeptina neurons represent the most important neuronal populations in the control of reproduction. Estradiol binds to its receptor expressed by the kisspeptina neurons to regulate the release of GnRH. In the animal model OVX+E the activity of the GnRH neuron and LH surge is dependent of estradiol and time of day. In this study, although the firing rate of GnRH neurons was similar between groups, the pattern of potentials revealed a change to longer burst duration in mice in proestrous, and the pituitary response was greater in this group. Prolactin has impact on HPG axis modulation and kisspeptin is a mediator of the effects of prolactin on reproduction. A small percentage of AVPV kisspeptin neurons were indirectly depolarized by prolactin. This effect required the PI3K signaling pathway. Mice bearing Stat5a/b inactivation on kisspeptin cells exhibited an early onset of estrus cyclicity, indicating that STAT5 transcription factors exert an inhibitory effect on the time of puberty.

Submitted by Carlos Alberto Zanutto Bassetto Júnior null (cbjunior@fc.unesp.br) on 2016-06-30T16:14:01Z No. of bitstreams: 1 DocNãoPublicação_carlosbassettojr.pdf: 427722 bytes, checksum: fe38077e0c0376645f906842534a3f88 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-05T16:31:24Z (GMT) No. of bitstreams: 1 bassettojunior_caz_dr_bauru.pdf: 201721 bytes, checksum: afaa803c4d84d32fc5d6b4fc9fe23555 (MD5) / Made available in DSpace on 2016-07-05T16:31:25Z (GMT). No. of bitstreams: 1 bassettojunior_caz_dr_bauru.pdf: 201721 bytes, checksum: afaa803c4d84d32fc5d6b4fc9fe23555 (MD5) Previous issue date: 2016-06-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Esta tese teve como objetivo estudar as moléculas orgânicas (N-alquilbenzenossulfonamidas) como inibidoras de canais para potássio do tipo KV3.1, heterologamente expressos em células L-929. Com o presente estudo constatou-se que as moléculas, N-alquilbenzenossulfonamidas, produzem efeitos inibitórios sobre KV3.1. Através da técnica de whole cell patch clamp, observou-se que os valores de IC50 para as moléculas que bloquearam o canal foram 13,5 μM, 16,9 μM, 25,9 μM, 34,2 μM, 34,9 μM e 60 μM, respectivamente, para 4-cloro-3-nitro-N-butilbenzenossulfonamida (SMD2), 4-cloro-3-nitro-N-furfutilbenzenossulfonamida (SMD3), 4-[N-(3’aminopropil)-2-pirrolidona]-3-nitro-N-butilbenzenossulfonamida (SMD2_APP), 4-[N-(3’aminopropil)-2-pirrolidona]-3-nitro-N-furfurilbenzenossulfonamida (SMD3_APP), 4-cloro-N-butilbenzenossulfonamida (SMD2_SN) e 4-cloro-N-furfurilbenzenossulfonamida (SMD3_SN). O efeito de todas as moléculas mostrou-se reversível quanto à ligação com o canal e todas atuaram como bloqueadores de canal aberto. Em SMD2, molécula que mostrou o menor valor de IC50, observou-se um deslocamento de -8 mV em relação ao controle, nas curvas de condutância versus voltagem, nas cinéticas de ativação e na recuperação a partir da inativação em relação à voltagem. O SMD2 não alterou as constantes de tempo de desativação, embora tenha mudado as constantes de ativação e inativação, além de ter induzido o fenômeno de tail crossover. Observou-se que para potenciais mais despolarizados, ocorreu o alívio do bloqueio (Block Relief). Não foi observado o efeito da dependência do pH para o bloqueio e SMD2 não mudou a seletividade do canal. Constatou-se que pulsos despolarizantes de curta duração induzem efeitos menos intensos, ao passo que pulsos despolarizantes mais longos, produzem efeitos mais intensos de SMD2 sobre o canal. Além disso, foi observado que, quanto mais o canal é usado, ou seja, aberto, mais ele é bloqueado por SMD2. Todos esses dados sugerem que SMD2 não interage com o estado fechado e nem com o estado inativado do canal, mas sim com seu estado aberto, apresentando também um efeito dependente de uso. De um ponto de vista farmacológico, isso indica que SMD2 pode ser uma molécula importante na modulação da atividade dos canais KV3.1, presentes em células com altas frequências de disparos de potencial de ação, podendo constituir uma nova classe de moduladores farmacológicos desses canais. / This thesis had the aim of studying the organic molecules (N-alkylbenzenesulfonamides) that block KV3.1 potassium channel heterologously expressed in L-929 cells. It was found that N-alkylbenzenesulfonamides have restrained effects on KV3.1. Through the whole cell patch clamp technique, it was observed that the values of IC50, for molecules that block the channel, were 13,5 μM, 16,9 μM, 25,9 μM, 34,2 μM, 34,9 μM and 60 μM, respectively 4-Chloro-3-nitro-N-butylbenzenesulfonamide (SMD2), 4-Chloro-3-nitro-N-furfurylbenzene-sulfonamide (SMD3), 4-[N-(3′-Aminopropyl)-2-pyrrolidone]-3-nitro-N-butylbenzenesulfona-mide (SMD2_APP), 4-[N-(3′-Aminopropyl)-2-pyrrolidone]-3-nitro-N-furfurylbenzene-sulfonamide (SMD3_APP), 4-Chloro-N-butyllbenzenesulfonamide (SMD2_SN) e 4-Chloro-N-furfurylbenzenesulfonamide(SMD3_SN). The effect of all molecules was reversible regards to the linking with the channel and all act as open channel blocker. In SMD2, molecule which showed the smallest value of IC50, it was observed a displacement of -8 mV compared to control, for conductance curves versus voltage, for the kinetics of activation and for the recovery from inactivation in relation to voltage. SMD2 did not change the deactivation of time constants, although it changed the activation and inactivation constants, and more, SMD2 have induced tail crossover phenomenon. It was observed that, for more depolarized potentials, there was a block relief. It was not observed the effect of pH dependence for the block and SMD2 did not change the channel selectivity. It was observed that, short duration depolarizing pulses prompt less intense effects, whereas long duration depolarizing pulses prompt more intense effects of SMD2 on the channels. Furthermore, it was observed that the more the channel is used, in an open state, the more it is blocked by SMD2. All of these data suggest that SMD2 does not interact neither with the closed state nor the inactivated state of channel, but with its open state presenting an use-dependent manner, also showing a use-dependent effect. In a pharmacological point of view, this indicates that SMD2 may be an important molecule in the modulation of the activity in the KV3.1 channels, presents in cells with high frequency of firing of action potential and may constitute a new class of pharmacological modulators.

Neurônios GnRH e kisspeptina representam as populações neuronais de maior importância no controle da reprodução. Estradiol liga-se ao seu receptor expresso pelos neurônios kisspeptina para regular a libertação de GnRH. No modelo animal OVX+E a atividade do neurônio GnRH e pico de LH é depende do estradiol e hora do dia. Nesse estudo, embora a taxa de disparo dos neurônios GnRH seja similar entre os grupos, o padrão dos potenciais revelou uma mudança para maior duração do estouro em camundongos no proestrous, além do fato de uma maior resposta da hipófise. A prolactina tem grande impacto na modulação do eixo HPG e kisspeptina são mediadores dos efeitos da prolactina sobre a reprodução. Uma pequena porcentagem de neurônios de kisspeptina do AVPV foi indiretamente despolarizada pela prolactina. Este efeito requeria a via de sinalização PI3K. Camundongos portadores de inativação de Stat5a/b em células kisspeptina exibiram um início precoce de ciclicidade estro, indicando que os fatores de transcrição STAT5 exercem um efeito inibitório sobre o momento da puberdade. / GnRH and kisspeptina neurons represent the most important neuronal populations in the control of reproduction. Estradiol binds to its receptor expressed by the kisspeptina neurons to regulate the release of GnRH. In the animal model OVX+E the activity of the GnRH neuron and LH surge is dependent of estradiol and time of day. In this study, although the firing rate of GnRH neurons was similar between groups, the pattern of potentials revealed a change to longer burst duration in mice in proestrous, and the pituitary response was greater in this group. Prolactin has impact on HPG axis modulation and kisspeptin is a mediator of the effects of prolactin on reproduction. A small percentage of AVPV kisspeptin neurons were indirectly depolarized by prolactin. This effect required the PI3K signaling pathway. Mice bearing Stat5a/b inactivation on kisspeptin cells exhibited an early onset of estrus cyclicity, indicating that STAT5 transcription factors exert an inhibitory effect on the time of puberty.

Orientadores: Mariana Porto Zambon, Bruno Geloneze Neto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T12:05:33Z (GMT). No. of bitstreams: 1 Silva_ClelianideCassiada_M.pdf: 3445763 bytes, checksum: 0e769f408904fd12add084d02186f8fd (MD5) Previous issue date: 2013 / Resumo: Objetivo: Avaliar a relação do índice TyG com componentes da síndrome metabólica e outros marcadores antropométricos e bioquímicos relacionados a risco cardiometabólico, e como indicador de resistência à insulina pelo clamp hiperglicêmico em adolescentes com diferentes níveis de adiposidade e estadios puberais. Materiais e métodos: Estudo transversal multicêntrico. Avaliou-se 835 adolescentes de 10-19 anos (489 meninas, média de 15,1 ± 2,4 anos e 346 meninos, média de 14,7 ± 2,4 anos). Realizou-se avaliação antropométrica, de composição corporal e bioquímica. O estadio puberal foi obtido por meio de autoavaliação, e a pressão arterial pelo método auscultatório. A resistência à insulina foi avaliada pelos índices TyG e HOMA2-IR, e pelo clamp hiperglicêmico (n = 42). A correlação entre duas variáveis foi avaliada pelo coeficiente de correlação parcial com ajuste para o estadio puberal. Na análise entre o índice TyG e o clamp hiperglicêmico foram analisados: qui-quadrado (X2), Kappa (k), curva ROC (Receiver Operating Characteristic), áreas abaixo das curvas (AUC), intervalos de confiança de 95% (IC95%) e determinado o ponto de corte para o índice TyG diagnosticar resistência à insulina. Resultados: Em ambos os sexos, o índice TyG correlacionou-se de forma positiva com circunferência da cintura (r = 0,30 feminino e 0,41 masculino; p<0,001), pressão arterial sistólica (r = 0,18; p<0,001 feminino e r = 0,13; p = 0,03 masculino) e diastólica (r = 0,20; p<0,001 feminino e r = 0,16; p = 0,005 masculino) e com o índice HOMA2-IR (r = 0,36 feminino e r = 0,45 masculino; p<0,001), e negativa com HDL-colesterol (r = -0,21 feminino e r = -0,41 masculino; p<0,001). Observou-se correlação inversa entre o clamp com o índice TyG (r = -0,55; p<0,001) e com o índice HOMA2-IR (r = -0,33; p = 0,03). Observou-se associação significante entre a presença de sensibilidade à insulina diminuída avaliada pelo clamp e a resistência à insulina aumentada segundo o índice TyG (X2 = 6,7; p = 0,009). O teste Kappa mostrou concordância satisfatória entre o índice TyG e a sensibilidade à insulina obtida a partir do clamp (k = 0,4; p = 0,009). O índice TyG mostrou bom desempenho para identificar resistência à insulina (AUC = 0,72; p = 0,02). Considerando os pacientes com índice TyG acima do percentil 90 como indivíduos resistentes à insulina, encontrou-se um valor limite para resistência à insulina de 4,47. Conclusão: O índice TyG é um instrumento válido na estimativa do grau da resistência à insulina e alterações nos componentes da síndrome metabólica e nos indicadores de risco cardiometabólico em adolescentes, tendo sido validado diante do clamp hiperglicêmico / Abstract: Objective: To evaluate the relationship of the TyG index with the metabolic syndrome components and other anthropometric and biochemical markers related to cardiometabolic risk, and as an indicator of insulin resistance by hyperglycemic clamp in adolescents with different levels of adiposity and pubertal stages. Materials and methods: A cross-sectional multicenter study. There were evaluated 835 adolescents aged 10-19 years (489 girls, mean 15.1 ± 2.4 years and 346 boys, mean 14.7 ± 2.4 years). There were performed anthropometric, body composition and biochemistry measurements. The pubertal stage was obtained through self-assessment, and blood pressure by auscultation method. The insulin resistance was assessed by TyG index, HOMA2-IR and hyperglycemic clamp (n = 42). The correlation between two variables was assessed by partial correlation coefficient adjusted for pubertal stage. In the analysis between the TyG index and the hyperglycemic clamp, there were analyzed: chi-square (X2), Kappa (k), ROC (Receiver Operating Characteristic) curve, areas under the curve (AUC), confidence intervals of 95% (CI95%) and determined the cut-off point for the TyG index to diagnose insulin resistance. Results: In both genders, the TyG index correlated positively with waist circumference (r = 0.30 female and r = 0.41 male; p<0.001), systolic blood pressure (r = 0.18, p<0.001 female and r = 0.13, p = 0.03 male) and diastolic (r = 0.20, p<0.001 female and r = 0.16, p = 0.005 male) and with the HOMA2-IR index (r = 0.36 female and r = 0.45 male, p<0.001), and negatively with HDL-cholesterol (r = -0.21 female and r = -0.41 male, p<0.001). There were observed inverse correlation between the clamp with TyG index (r = -0.55, p<0.001) and with the HOMA2-IR index (r = -0.33, p = 0.03). There was observed significant association between the presence of decreased insulin sensitivity measured by clamp and increased insulin resistance according to the TyG index (X2 = 6.7, p = 0.009). The Kappa test showed satisfactory agreement between the TyG index and insulin sensitivity obtained from the clamp (k = 0.4, p = 0.009). The TyG index showed good performance to identify insulin resistance (AUC = 0.72, p = 0.02). Considering patients with TyG index above the 90th percentile as insulin-resistant subjects, there was found a threshold value for insulin resistance of 4.47. Conclusion: The TyG index is a valid tool to estimate the degree of insulin resistance and changes in the metabolic syndrome components and cardiometabolic risk factors in adolescents, having been validated with the hyperglycemic clamp / Mestrado / Saude da Criança e do Adolescente / Mestra em Ciências

Les canaux de jonctions (CJ) composés des connexines (Cxs) assurent la communication intercellulaire directe qui par leur propriétés électriques régulent la propagation du potentiel d’action (PA) cardiaque. Dans les myocytes ventriculaires Cx43 et Cx45 exprimées à des niveaux et ratios physiopathologiques variables assurent cette fonction. Cette étude détermine la contribution de Cx43 et Cx45 dans la formation des CJ et leurs propriétés électriques. La lignée cellulaire épithéliale de foie de rat exprimant la Cx43 endogène et transfectée de manière stable pour exprimer des ratios Cx43:Cx45 précis a été utilisée. Les propriétés électriques des CJ ont été obtenues par double voltage clamp sur paires de cellules. L’expression de la Cx45 diminue le couplage électrique et augmente la dépendance au potentiel de jonction indépendamment du ratio. Les cinétiques de désactivation sont ralenties avec l’augmentation du niveau d’expression de Cx45 et les cinétiques de restitution sont modifiées en fonction du ratio. Les conductances unitaires suggèrent la formation de CJ composés de Cx43 et Cx45. La diminution du niveau d’expression de Cx43 par ARNi anti-Cx43 entraine une diminution du couplage électrique tandis que les autres propriétés électriques restent inchangées. Ces résultats montrent une contribution spécifique de Cx43 et Cx45 dans la régulation de la formation et des propriétés électriques des CJ caractérisées. Ces propriétés seront corrélées à la participation des CJ dans la régulation de la propagation du PA en fonction des profils d’expression des Cxs en conditions physiologiques et pathologiques. / Gap junction channels (GJCs), composed of connexins (Cxs) allow a direct intercellular communication that ensures the cardiac action potential (AP) propagation. Cx43 and Cx45 co-expressed in ventricular myocytes with changing expression levels and ratios in the healthy and the diseased heart ensure this function. The purpose of this study is to determine the contribution of Cx43 and Cx45 on the formation of GJCs and their electrical properties. Rat Liver Epithelial cells that endogenously express Cx43 and stably transfected to co-express accurate Cx43:Cx45 ratios have been used. The electrical properties of GJCs at each ratios were obtained by performing dual voltage clamp recordings on cell pairs. Expression of Cx45 decreases the electrical coupling and increases the voltage dependence independently of the ratio. The kinetics of deactivation are slowed with the increases of Cx45 level of expression and the kinetics of recovery are modified in a Cx43:Cx45 ratio dependent manner. Unitary conductances suggest a formation of GJCs composed by Cx43 and Cx45. The decreases of Cx43 level of by a SiRNA treatment induces a decrease of the electrical coupling, while other electrical properties are not affected. Our data show a specific contribution of Cx43 and Cx45 in regulation of the GJCs characterized by specific electrical properties. Such properties will be correlated to the function of GJCs in regulating the AP propagation in the specific patterns of expression of Cxs in the healthy and diseased heart.

Les récepteurs P2X (P2XR), activés par l’ATP, sont impliqués dans des rôles physiopathologiques. Leur fonctionnement est associé à différents états conformationnels. Le projet de thèse a mené à associer la synthèse organique et l’application de molécules photo-activables avec des techniques d’électrophysiologie patch-clamp, pour décortiquer les mouvements moléculaires de ces récepteurs et effectuer des relations structure-fonction, via trois stratégies : - La synthèse et application d’agrafes photo-isomérisables qui permet le photo-contrôle des P2XR et l’étude de mouvements - La synthèse et caractérisation d’un acide aminé (aa) photo-clivable pour étudier les implications de zones sur la fonction des P2XR via une photolyse - L’incorporation d’un aa non naturel dans les P2XR pour étudier des interactions et mouvements via un « photo-pontage ». Nous avons élucidé les mécanismes moléculaires responsables de la perméabilité des P2XR, récusé l’existence de l'état dilaté et identifié un cation organique physiologique pouvant les traverser. Nous avons aussi conçu un acide aminé photo-clivable pouvant mener à des études structure-fonction des P2XR. / P2X receptors are cationic ligand-gated ion channels, activated by extracellular ATP, involved in many physio-pathological roles. Their function is associated with different allosteric states. During this PhD, we have designed three new strategies, spanning photochemical organic synthesis and patch-clamp electrophysiology to elucidate the molecular mechanisms involved in these conformational states and to collect data in order to study structure-function relationships. - Synthesis and application of molecular tweezers, which allows the photo-control of P2X Rand the study of molecular motions - Synthesis and characterization of a photo-cleavable amino acid with the aim of incorporating it into P2XR and doing structure-function relationships - Incorporation of an unnatural amino acid for photo-crosslinking studies. We have been able to probe the molecular mechanism involved in large organic cations permeation of P2XR, to bring into question the dilated state and to identify a physiological cation that can flow through P2XR. We have also designed a photo-cleavable amino acid which could serve in the study of structure-function relationships.

Orientador: Arlete Maria dos Santos Fernandes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T08:29:22Z (GMT). No. of bitstreams: 1 Kimura_VaneskadeCarvalhoMelhado_M.pdf: 1746047 bytes, checksum: 4c3f59da8a72a362f8d251faab5d3939 (MD5) Previous issue date: 2014 / Resumo: A resistência insulínica (RI) é um estado no qual há diminuição da resposta dos tecidos-alvo à ação da insulina. A prevalência da RI na população adulta jovem não obesa e com resposta normal ao teste de tolerância oral à glicose (TTOG) é estimada em 25%. Estudos sugerem que vários fatores poderiam contribuir com a deterioração da tolerância à glicose em mulheres jovens, entre eles a elevação do peso corporal e o uso de métodos contraceptivos (MAC) hormonais. Objetivos: avaliar uma coorte de mulheres saudáveis em período reprodutivo e sem antecedentes que pudessem interferir no metabolismo de carboidratos, para determinar a prevalência e os fatores associados à RI. Sujeitos e Método: para o diagnóstico de RI foi utilizada a técnica do clamp euglicêmico hiperinsulinêmico (CEH). As variáveis estudadas foram sociodemográficas, uso atual de MAC combinado ou não hormonal, tempo de uso do MAC, composição corporal avaliada pela técnica de absortimetria de duplo feixe de raios-X (DXA), valores de perfis lipídico e glicêmico, medidas de pressão arterial e da ingestão calórica. Foi realizada análise das médias e desvio padrão (DP) das variáveis relacionadas à RI e ao uso de MAC e regressão linear múltipla para avaliar fatores que pudessem influenciar a tendência à RI. Participaram deste estudo 47 mulheres saudáveis, que responderam à veiculação da pesquisa, com idade de 18 a 40 anos, IMC<30kg/m2 e todas realizaram CEH com 40mU de insulina. Resultados: do total das mulheres, 16 usavam algum tipo de método contraceptivo hormonal combinado (34%) e 31 usavam método não hormonal (66%). A prevalência de RI foi de 19,1% e significativamente maior entre as que utilizavam método hormonal (6/16) combinado em relação às que utilizavam método não hormonal (3/31). Mulheres com RI tiveram valores médios significativamente maiores de peso, IMC, massa e percentual de massa gorda, razão androide/ginecoide, e dos valores de triglicérides, insulina de jejum, HOMA IR e HOMA Bc. Após análise linear múltipla, as variáveis associadas à tendência à RI foram não ter gestação anterior, tempo de uso de MAC >1 ano e maiores valores de triglicérides. Conclusões: Uma de cada 5 mulheres em idade reprodutiva e saudável apresentou RI e mostrou elevação de peso corporal e de cintura. É importante iniciar medidas de prevenção precoces, estimulando hábitos saudáveis de alimentação e exercícios físicos nas mulheres observadas com elevação de peso e medida de cintura durante o acompanhamento de planejamento familiar / Abstract: Insulin resistance (IR) is a condition in which there is decreased response of target tissues to the effects of insulin. The prevalence of IR in the young non obese adult population with normal response to the oral glucose tolerance test (OGTT) is estimated to be 25%. Studies suggest that several factors might contribute to the deterioration of glucose tolerance in young women, among them increased body weight and the use of hormonal contraceptive methods. Objectives: evaluate a cohort of healthy women of childbearing age, without antecedents that might interfere in carbohydrate metabolism, to determine the prevalence and the factors associated with IR. Subjects and Method: the hyperinsulinemic-euglycemic clamp technique was used for the IR diagnosis. The variables studied were: sociodemographic, current use of hormonal, combined or non hormonal contraceptive methods, how long hormonal contraceptive methods have been used, body composition evaluated by the dual-energy X-ray absorptiometry (DXA) technique, values of lipid and glucose profiles, blood pressure and caloric intake measures. The means and standard deviation (SD) of variables related with IR; use of hormonal contraceptive methods and multiple linear regression were analyzed to evaluate factors that might influence tendency to IR. Forty-seven healthy women who answered research recruiting advertisement participated in this study; their ages ranged between 18 and 40 years, presented BMI<30kg/m2 and all of them were examined by the hyperinsulinemic-euglycemic clamp technique with 40mU of insulin. Results: of the total of 47 women, 16 used some type of combined hormonal contraceptive method (34%) and 31 used a non hormonal contraceptive method (66%). The prevalence of IR was 19.1% and significantly higher among those who used a combined hormonal contraceptive method (6/16) compared with those who used a non hormonal contraceptive method (3/31). Women with IR presented significantly higher mean values of weight, BMI, mass and percentage of fat body mass, android/gynecoid ratio, and values of triglycerides, fasting insulin, HOMA IR and HOMA Bc. After multiple linear analysis, the variables associated with tendency to IR were not having been pregnant before, having used hormonal contraceptive method >1 year and higher triglycerides values. Conclusions: One in every 5 healthy women of childbearing age presented IR and increased body weight and waist circumference. It is important to adopt early prevention measures, stimulating healthy eating habits and physical exercises for women with weight and waist measure increase during the family planning follow-up period / Mestrado / Fisiopatologia Ginecológica / Mestra em Ciências da Saúde

The goal of diploma these is ‘Calculation of frequency-admittance characteristics from measured patchlamps dates’. Calculation is made on the basis of study of mentioned problematic measure ion current cell membrane by patch-clamp method and its using for measure frequency-admittance characteristics. After it selected method of calculation was realized in software calculator Matlab.