Les canaux TREK sont des canaux mécano- et thermosensibles appartenant à la famille des canaux potassiques de fond à deux domaines pore, qui jouent un rôle majeur dans l’excitabilité cellulaire. Notre équipe a précédemment montré que les canaux TREK-1 et TRAAK sont des senseurs moléculaires essentiels dans la perception polymodale de la douleur et qu’ils interviennent dans l’excitabilité des nocicepteurs, modulant ainsi le message douloureux.Dans la première partie de ma thèse, j’ai étudié l’implication du canal TREK-1 dans l’activité antalgique de la morphine. Nous avons mis en évidence un couplage fonctionnel entre les canaux TREK-1 et TREK-2 et les récepteurs opioïdes µ et δ. Nous avons également montré que le canal TREK-1 est impliqué dans les effets périphériques et centraux de la morphine. Ces travaux ont permis d’impliquer le canal TREK-1 comme un médiateur de l’analgésie induite par la morphine agissant en aval des récepteurs aux opioïdes. La seconde partie de ma thèse a porté sur l‘étude du rôle du canal TREK-2 dans la perception de la douleur. En étudiant l’impact des canaux TREK-2 sur l’activité des nocicepteurs, j’ai pu montrer que le canal TREK-2 module la perception thermique des nocicepteurs cutanés. Toutefois, l’activité de TREK-2 dans la thermo perception est complémentaire de celle déjà rapportée pour les canaux TREK car, contrairement à TREK-1 et TRAAK, TREK-2 intervient dans la perception de températures non nocives. En conclusion, ces travaux montrent que les canaux potassiques de fond TREK jouent un rôle essentiel dans la perception et la modulation du message douloureux et pourraient être des cibles intéressantes pour le traitement de la douleur. / TREK channels are mechano- and thermo-activated channels belonging to the two-pore domains potassium channels family, which play a major role in neuronal excitability and cell firing. Our group previously demonstrated that TREK-1 and TRAAK channels are essential molecular sensors in polymodal pain perception and that they are involved in the excitability of nociceptors, thus modulating the nociceptive message. In the first part of this work, I investigated the implication of TREK-1 channel in the analgesic action of morphine, one of the most used analgesic. We reveal a functional coupling between TREK-1 and TREK-2 channels and opioid receptors µ and δ. We also show that TREK-1 channel is involved in peripheral and central effects of morphine. This work demonstrate that TREK-1 channel, downstream of the µOR, is an important mediator of morphine induced analgesia. During the second part of my work, I investigated the role of TREK-2 channel in pain perception. I assessed the impact of this channel on nociceptors and we reveal that TREK-2 channel modulates thermal perception of cutaneous nociceptors. However, we show that TREK-2 activity in thermal perception complements the one already reported for TREK-1 and TRAAK channels. Whilst TREK-1 and TRAAK are involved in noxious temperatures perception, TREK-2 intervene in non-aversive temperatures detection. Taken together, these results show that background potassium channels TREK are major contributors to pain message perception and transmission and that they might be interesting targets for the treatment of pain.

Made available in DSpace on 2014-12-17T15:28:52Z (GMT). No. of bitstreams: 1 HermanyMV_DISSERT.pdf: 1765987 bytes, checksum: dedd84315ca1c69652d27407ffd67d85 (MD5) Previous issue date: 2013-03-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Recently, genetically encoded optical indicators have emerged as noninvasive tools of high spatial and temporal resolution utilized to monitor the activity of individual neurons and specific neuronal populations. The increasing number of new optogenetic indicators, together with the absence of comparisons under identical conditions, has generated difficulty in choosing the most appropriate protein, depending on the experimental design. Therefore, the purpose of our study was to compare three recently developed reporter proteins: the calcium indicators GCaMP3 and R-GECO1, and the voltage indicator VSFP butterfly1.2. These probes were expressed in hippocampal neurons in culture, which were subjected to patchclamp recordings and optical imaging. The three groups (each one expressing a protein) exhibited similar values of membrane potential (in mV, GCaMP3: -56 ?8.0, R-GECO1: -57 ?2.5; VSFP: -60 ?3.9, p = 0.86); however, the group of neurons expressing VSFP showed a lower average of input resistance than the other groups (in Mohms, GCaMP3: 161 ?18.3; GECO1-R: 128 ?15.3; VSFP: 94 ?14.0, p = 0.02). Each neuron was submitted to current injections at different frequencies (10 Hz, 5 Hz, 3 Hz, 1.5 Hz, and 0.7 Hz) and their fluorescence responses were recorded in time. In our study, only 26.7% (4/15) of the neurons expressing VSFP showed detectable fluorescence signal in response to action potentials (APs). The average signal-to-noise ratio (SNR) obtained in response to five spikes (at 10 Hz) was small (1.3 ? 0.21), however the rapid kinetics of the VSFP allowed discrimination of APs as individual peaks, with detection of 53% of the evoked APs. Frequencies below 5 Hz and subthreshold signals were undetectable due to high noise. On the other hand, calcium indicators showed the greatest change in fluorescence following the same protocol (five APs at 10 Hz). Among the GCaMP3 expressing neurons, 80% (8/10) exhibited signal, with an average SNR value of 21 ?6.69 (soma), while for the R-GECO1 neurons, 50% (2/4) of the neurons had signal, with a mean SNR value of 52 ?19.7 (soma). For protocols at 10 Hz, 54% of the evoked APs were detected with GCaMP3 and 85% with R-GECO1. APs were detectable in all the analyzed frequencies and fluorescence signals were detected from subthreshold depolarizations as well. Because GCaMP3 is the most likely to yield fluorescence signal and with high SNR, some experiments were performed only with this probe. We demonstrate that GCaMP3 is effective in detecting synaptic inputs (involving Ca2+ influx), with high spatial and temporal resolution. Differences were also observed between the SNR values resulting from evoked APs, compared to spontaneous APs. In recordings of groups of cells, GCaMP3 showed clear discrimination between activated and silent cells, and reveals itself as a potential tool in studies of neuronal synchronization. Thus, our results indicate that the presently available calcium indicators allow detailed studies on neuronal communication, ranging from individual dendritic spines to the investigation of events of synchrony in neuronal networks genetically defined. In contrast, studies employing VSFPs represent a promising technology for monitoring neural activity and, although still to be improved, they may become more appropriate than calcium indicators, since neurons work on a time scale faster than events of calcium may foresee / Neur?nios se comunicam por meio de sinapses, trocando mensagens capazes de modificar o potencial de membrana de outros neur?nios. Demonstrar o papel desses sinais e decodificar essa linguagem el?trica representa o grande objetivo da neuroci?ncia moderna. Atualmente, a eletrofisiologia ? o ramo da neuroci?ncia capaz de investigar esses recursos el?tricos de neur?nios - que v?o desde registros de condut?ncia e comportamento cin?tico de canais i?nicos individuais at? a demonstra??o de neur?nios individuais implicados em comportamentos complexos. Nesse sentido, diferentes estados cerebrais e comportamentos implicam o recrutamento de grandes conjuntos de neur?nios se comunicando em um estado coerente, din?mico. Al?m disso, essas grandes popula??es s?o formadas por diversos subtipos neuronais cuja an?lise requer t?nicas que possibilitem uma resolu??o temporal e espacial de c?lulas individuais e, prefencialmente, de subtipos espec?ficos. Apenas recentemente, indicadores ?pticos geneticamente codificados surgiram como ferramentas n?o invasivas de alta resolu??o espacial e temporal utilizados para monitorar a atividade de neur?nios individuais e popula??es neuronais espec?ficas. O n?mero crescente de novos indicadores optogen?ticos, juntamente com a aus?ncia de compara??es em condi??es id?nticas, gerou dificuldade em escolher a mais adequada das prote?nas, dependendo do desenho experimental. Portanto, o objetivo deste estudo foi comparar tr?s prote?nas rep?rter recentemente desenvolvidas: os indicadores de c?lcio GCaMP3 e R-GECO1, e o indicador de voltagem VSFP butterfly1.2. Foram expressos em neur?nios do hipocampo em cultura, os quais foram submetidos a registros de patch-clamp e de imageamento ?ptico. Os tr?s grupos (cada um expressando uma prote?na) exibiram valores semelhantes de potencial de membrana (em mV, GCaMP3: -56 ? 8,0; R-GECO1: -57 ? 2,5; VSFP: -60 ? 3,9; p = 0,86), no entanto, o grupo de neur?nios que expressam VSFP mostrou uma m?dia mais baixa de resist?ncia de entrada do que os outros grupos (em Mohms, GCaMP3: 161 ? 18,3; GECO1-R: 128 ? 15,3; VSFP: 94 ? 14,0; p = 0,02). Cada neur?nio foi submetido a inje??es de correntes com frequ?ncias diferentes (10 Hz, 5 Hz, 3 Hz, 1,5 Hz, e 0,7 Hz) e as suas respostas de fluoresc?ncia foram registradas. Em nosso estudo, apenas 26,7% (4/15) dos neur?nios que expressam VSFP mostraram sinal de fluoresc?ncia detect?vel em resposta a potenciais de a??o. O valor m?dio de sinal-para-ru?do (SNR), obtido em resposta a cinco potenciais de aҫ?o (a 10 Hz) foi pequeno (1,3 ? 0,21), no entanto a cin?tica r?pida do VSFP permite a discrimina??o de disparos, como picos individuais, com detec??o de 53% dos APs evocados. Freq??ncias abaixo de 5 Hz, assim como variaҫ?es no potencial de membrana subliminares, foram indetect?veis devido ao alto ru?do do sinal de fluoresc?ncia. Por outro lado, os indicadores de c?lcio mostraram maior altera??o na fluoresc?ncia, seguindo o mesmo protocolo (cinco potenciais de aҫ?o a 10 Hz). Entre os neur?nios expressando GCaMP3, 80% (8/10) exibiram sinal, com um valor m?dio de SNR de 21 ? 6,69 (soma), enquanto que para os neur?nios expressando R-GECO1, 50% (2/4) dos neur?nios demonstraram sinal com um valor m?dio SNR de 52 ? 19,7 (soma). Para protocolos de 10 Hz, 54% dos disparos foram detectados com evocado GCaMP3 e 85% com o R-GECO1. Disparos foram detectados em todas as frequ?ncias e os sinais de fluoresc?ncia foram tamb?m detectados a partir de despolariza??es subliminares. Sendo GCaMP3 o indicador mais prov?vel de produzir sinal de fluoresc?ncia e com alto SNR, alguns experimentos foram realizados somente com essa prote?na. Observamos que GCaMP3 ? eficaz na detec??o de inputs sin?pticas (envolvendo influxo de Ca2+), com alta resolu??o espacial e temporal. Tamb?m foram observadas diferen?as entre os valores de SNR resultantes dos disparos evocados, em compara??o com os disparos espont?neos. Em registros de grupos de c?lulas, GCaMP3 mostrou clara discrimina??o entre c?lulas ativadas e sil?ncio, revelando-se como uma ferramenta potencial em estudos de sincroniza??o neuronal. Assim, nossos resultados sugerem que os indicadores de c?lcio dispon?veis atualmente permitem estudos detalhados sobre a comunica??o neuronal, que v?o desde dendritos individuais at? a investiga??o de eventos de sincronia em redes neuronais geneticamente definidas. Em contraste, VSFPs representam uma tecnologia promissora para monitorar a atividade neural e, apesar de ainda requererem melhoramentos, podem se tornar mais apropriados do que os indicadores de c?lcio, uma vez que os neur?nios trabalham em uma escala de tempo mais r?pida do que eventos de c?lcio podem prever

A obesidade induz resistência à insulina que é um dos passos para o diabetes do tipo 2 e as doenças cardiovasculares. Neste panorama, o papel do tecido adiposo visceral é inquestionável, mas o mesmo não é verdade para o tecido adiposo subcutâneo, especialmente da região abdominal. Isto confirma a importância da análise da composição e distribuição do tecido adiposo no corpo para definir risco metabólico. Se o papel do tecido adiposo subcutâneo é objeto de controvérsias, mais ainda é o efeito da retirada do mesmo, através da dermolipectomia na sensibilidade à insulina, aferida pelo clamp. O objetivo deste estudo foi avaliar o efeito da dermolipectomia abdominal na sensibilidade à insulina e nas adipocitocinas (adiponectina e leptina). Avaliamos 17 pacientes do sexo feminino, com idades entre 22 e 51 anos, obesas ou com sobrepeso. Todas eram ex-obesas mórbidas que haviam sido submetidas à gastrectomia parcial em Y de Roux, há no mínimo um ano antes da entrada no protocolo, com perda de pelo menos 30% do peso inicial, com peso estável (mas, ainda com grande quantidade de tecido subcutâneo abdominal). Destas, 12 concluíram o estudo, com realização do clamp euglicêmico hiperinsulinêmico, no início e três meses após a cirurgia plástica. O clamp teve duração total de três horas, com coletas seriadas de glicose e insulina a cada 10 minutos. A dose de infusão de insulina regular foi de 1 mU/kg/min, para elevar de forma aguda e mantida a insulinemia. No momento basal de cada teste foram coletadas amostras para dosagens bioquímicas, hormonais e para as adipocitocinas. A captação de glicose (valor M) foi calculada em mg de glicose por kg de peso corpóreo total por minuto, considerando os últimos trinta minutos do teste. O HOMA-IR também foi calculado. Antes de realizar o primeiro clamp, as pacientes realizaram o exame de DEXA (densitometria de corpo inteiro), todas no aparelho Hologic, o que permitiu a correlação da sensibilidade à insulina com a massa magra no antes da dermolipectomia. Considerando para análise estatística, as 12 pacientes, houve variação significativa de peso de 84 kg na fase pré para 81,8 kg na fase pós (p=0,015) e também de IMC (31,1 x 30,3 kg/m2, com p=0,017). Não houve variação de massa magra (46,4 x 44,7 kg, com p=0,119) nem de captação de glicose (p=0,742). Não houve tampouco variação do HOMA-IR (p=0,722) como era de se esperar, já que não houve variação de captação. Notamos que na fase pré a sensibilidade à insulina correlacionou-se diretamente com a massa magra (r=0,80; p=0,002) e inversamente com a idade (r=-0,71; p=0,10). Não houve diferença das adipocitocinas: leptina (p=0,739) e adiponectina (p=0,940) quando comparadas as fases antes e depois da dermolipectomia. Concluímos que a sensibilidade à insulina, aferida pelo clamp não se altera com a dermolipectomia abdominal, em mulheres ex-obesas mórbidas, embora ainda com sobrepeso ou obesidade e metabolicamente normais. A sensibilidade à insulina aumenta com a massa magra e diminui com a idade, após a dermolipectomia, nesta população estudada. / Obesity induces insulin resistance, which is one of the steps to type 2 diabetes and cardiovascular diseases. In this scenario, the role of visceral adipose tissue is undoubted, but the same is not true for subcutaneous adipose tissue, especially in abdominal region. This confirms the importance of analysis of composition and distribution of adipose tissue in the whole body to define metabolic risk. If the role of adipose subcutaneous tissue is debatable, even more is the abdominal dermolipectomy impact on insulin sensitivity, measured by clamp. The aim of the present study was to evaluate the effect of abdominal dermolipectomy on insulin sensitivity and adipocytokines (adiponectin and leptin). We evaluated 17 female patients, age range 22-51 years, obese or overweight. All patients were ex-morbid obese and had performed Roux en Y gastric bypass surgery, at least one year before the baseline, with lost of at least 30% of their initial body weight, with stable weight (but still with huge amount of abdominal subcutaneous adipose tissue). From these, 12 concluded the study, performing the clamp at the beginning of the protocol and three months after the plastic surgery. Clamp lasted three hours, with glucose and insulin samples drawn each ten minutes. Regular insulin infusion dose was 1 mU/kg/min to raise and sustain insulinaemia. At baseline from each test, samples were draw for biochemistry, hormones and adipocytokines. Glucose uptake (M-value) was calculated in mg of glucose, per kg of body weight per minute, considering the last thirty minutes. HOMA-IR was also calculated. Before underwent the first clamp, patients performed DEXA (dual energy X-ray absorptiometry), all in the same equipment (Hologic), what enabled correlation of insulin sensitivity and fat free mass (FFM) in the first phase. Considering for statistical purposes the 12 patients, there was significant weight variation; from 84 kg to 81,8 kg (p=0,015) and also BMI variation from 31,1 x 30,3 kg/m2 (p=0,017). There was no FFM variation: 46,4 x 44,7 kg (p=0,119) neither glucose uptake (p=0,742). Even not of HOMA-IR (p=0,722) as expected, since there was not glucose uptake variation. In the first phase, insulin sensitivity was directly correlated with FFM (r=0,80; p=0,002) and inversely with age (r= - 0,71; p=0,10). There were no differences on adipocytokines: leptin (p=0,739) and adiponectin (p=0,940) before and after dermolipectomy. We conclude insulin sensitivity measured by clamp, was not modified by abdominal dermolipectomy in ex-morbid obese women, but still overwight or obese and metabolically healthy. Insulin sensitivity raises with FFM and lowers with age, after dermolipectomy, in this population.

Orientador: Bruno Geloneze Neto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T04:57:20Z (GMT). No. of bitstreams: 1 Lima_MarceloMirandadeOliveira_M.pdf: 5830772 bytes, checksum: 60a5a321b7a0db75dbf4b6b163df924a (MD5) Previous issue date: 2010 / Resumo: A obesidade visceral está associada à resistência à insulina (RI), à presença de síndrome metabólica (SMet), à presença do diabetes tipo 2 (DM2) e a risco cardiovascular e mortalidade elevados. Entretanto, não está claro se existe uma relação causal. A ressecção cirúrgica da gordura visceral (lipectomia visceral) é um modelo experimental excelente para esclarecer esta questão. Em modelos animais, a lipectomia visceral melhora a sensibilidade à insulina (SI), a tolerância à glicose e o perfil de adipocinas. A omentectomia combinada com a cirurgia bariátrica em humanos tem efeitos controversos sobre a SI no longo prazo, e não há dados sobre o curto prazo, no qual o efeito da perda de peso poderia ser minimizado. Para investigar o papel da gordura visceral no metabolismo, este estudo prospectivo randomizado analisou, como objetivo primário, os efeitos aditivos da omentectomia combinada ao bypass gástrico em Y de Roux na SI corporal total (valor de M), mensurada pelo clamp euglicêmicohiperinsulinêmico (método padrão-ouro) no curto prazo (1 mês pós-cirurgia). Também foram analisados o HOMA-IR (marcador substitutivo de SI), as adipocinas, outros parâmetros bioquímicos e cardiovasculares, medidas antropométricas, composição corporal e as medidas ecográficas da gordura abdominal, subcutânea e visceral. Vinte mulheres em menacme com obesidade grau III e SMet foram randomizadas para bypass gástrico isolado (grupo-controle) ou combinado à omentectomia total e estudadas précirurgia e 1 mês pós-cirurgia. Na análise do conjunto dos dois grupos, não houve melhora na SI medida pelo clamp (valor de M) no primeiro mês pós-cirurgia apesar de haver redução de peso (pequena mas significativa). Ao contrário, observou-se melhora no HOMA-IR. A omentectomia não potencializou os efeitos do bypass gástrico na SI apesar de associar-se a maior perda de peso. Em conclusão, não se provou que a gordura visceral é um fator causal para a diminuição da SI. Um mês após a cirurgia, o metabolismo da glicose no jejum melhora independente de mudanças na SI periférica e a omentectomia não influenciou este resultado / Abstract: Visceral obesity is linked to insulin resistance, metabolic syndrome, diabetes, cardiovascular risk and mortality. Whether this relationship is causative or correlative is unclear. The surgical resection of visceral fat is an excellent experimental model to address this issue. It has been shown to improve insulin sensitivity (IS), glucose tolerance and adipokine profile in animal models. The omentectomy has been combined to bariatric surgery in humans in order to study its long-term metabolic effects with controversial results on IS. To approach the role of the visceral fat tissue in metabolism, the present prospective randomized trial assessed the additional effects of omentectomy combined to Roux-en-Y gastric bypass (RYGBP) on whole-body IS, measured by the "gold standard" method, i. e., the euglycemic-hyperinsulinemic clamp, a month post-surgery, as a primary objective. HOMA-IR (a surrogate marker of IS), adipokines, other basal blood and cardiovascular parameters, anthropometric measurements, body composition and ecographic measures of the subcutaneous and visceral fat thicknesses in the abdomen were also evaluated. Twenty grade-III obese premenopausal women with metabolic syndrome were randomized to either RYGBP alone (control group) or combined to a total greater omentectomy and were studied at baseline and shortly after surgery (first month). In the analysis of the pooled data from both groups, IS measured by the clamp (M-value) did not improve in the first post-surgery month despite of a decrease in body weight. This finding was discordant to the observation of an improvement in HOMA-IR. Omentectomy did not potentiate the effect of RYGBP on IS despite of being associated with greater weight loss. In conclusion, it has not been proven that the visceral fat is a causal factor on impaired IS. A month after RYGBP, fasting glucose metabolism improves independent of a change in peripheral insulin sensitivity and omentectomy did not influence this outcome / Mestrado / Clinica Medica / Mestre em Clinica Medica

Introdução: A acromegalia é uma doença rara, caracterizada pela produção aumentada de hormônio do crescimento, causada geralmente por um adenoma hipofisário, ocasionando uma série de comorbidades como apneia do sono e resistência insulínica que acarretam um aumento na mortalidade e redução da expectativa de vida. Objetivo: O objetivo deste estudo foi avaliar o impacto da terapêutica da apneia do sono com um dispositivo de pressão positiva contínuas nas vias aéreas (CPAP) e avaliar o impacto desta terapêutica na resistência insulínica pela realização do clamp euglicêmico hiperinsulinêmico (CEH). Pacientes: De 156 acromegálicos regularmente atendidos na unidade de Neuroendocrinologia do HC-FMUSP, foram selecionados 12 indivíduos com apneia do sono de moderada a grave em uso de análogos da somatostatina (AS). Método: Os pacientes foram randomizados em dois grupos com seis integrantes. O grupo A iniciou o tratamento com CPAP, e o grupo B, um adesivo dilatador nasal com efeito de placebo. A avaliação basal incluiu a polissonografia, determinação do GH, IGF-1, HbA1c, ácidos graxos livres, lípides, CEH, bem como os índices de resistência periférica à insulina (HOMA, HOMA2 e QUICKI). Após 3 meses de tratamento, os pacientes foram reavaliados pelos mesmos exames, sendo trocado o tratamento entre os grupos e feita nova avaliação, após mais 90 dias. Resultados: Analisando os resultados finais de todos os pacientes que fizeram uso do CPAP, foi observada uma redução significante na resistência periférica à insulina, verificada pelo índice de sensibilidade derivado do clamp (ISCLAMP, pré e pós- CPAP, 3,83 versus 6,11, p=0,032). Esta redução não foi observada no grupo que fez uso do adesivo nasal (ISCLAMP, pré e pós-adesivo, 5,53 versus 5,19, p=0,455). Não houve diferença significante nos níveis de lípides, HbA1c nem nos índices de resistência periférica à insulina. Conclusão: O tratamento da apneia do sono moderada a grave com CPAP, em pacientes acromegálicos em uso de AS, levou a uma redução da resistência periférica à insulina aferida pelo CEH, dado não observado por meio dos índices HOMA, HOMA2 e QUICKI / Introduction. Acromegaly is a rare disease, characterized by the production of high GH levels usually by pituitary adenoma leading to comorbidities as sleep apnea and insulin resistance, bringing increase of mortality and life span reduction. Objective: This study aims to assess the impact of treatment of sleep apnea with a continuous positive air pressure device (CPAP) on the insulin resistance by performing the hyperinsulinemic euglycemic clamp (HEC). Patients: From 156 acromegalic patients regularly attended on Neuroendocrine Unit of the Hospital das Clínicas, University of São Paulo Medical School, 12 subjects on somatostatin analogs (SA) harboring moderate to severe sleep apnea were selected. Methods: Patients were randomized in two groups of six subjects. Group A started treatment with CPAP while group B started treatment using a nasal dilator adhesive with placebo effect. Basal assessment included polysomnography, determination of GH, IGF-1, HbA1c, free fat acids, lipids assays, HEC as well as insulin resistance indexes (HOMA, HOMA2 and QUICKI). Patients were reevaluated after three months of treatment by the same tests and then the treatment was switched between groups with new assessment 90 days later. Results: A significant reduction on insulin resistance determined by the clamp derived sensibility index was observed after assessing the final data of all patients on CPAP (SICLAMP, pre and post CPAP, 3,83 versus 6,11, p=0,032). This reduction was not seen in the nasal dilator adhesive group (SICLAMP, pre and post adhesive, 5,53 versus 5,19, p=0,455). There was no significant difference on lipids, HbA1c or on peripheral insulin resistance indexes. Conclusion: CPAP treatment of acromegalic patients on AS with moderate to severe sleep apnea leaded to significant reduction on peripheral insulin resistance assessed by the HEC. HOMA, HOMA2 and QUICK did not detect this data

Orientador: Antônio Fernando Ribeiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T16:58:35Z (GMT). No. of bitstreams: 1 Kmit_Arthur_M.pdf: 1478515 bytes, checksum: 34c7bf6fcce0ab6ab1b63b0927edcf98 (MD5) Previous issue date: 2013 / Resumo: O receptor purinérgico P2X7 é um canal iônico permeável a cátions pertencente da família P2X (P2X1-P2X7). Ele é ativado por altas concentrações (100?M - 1000?M) de ATP (Adenosine 5?-triphosphate), apresentando duas distintas respostas: 1) uma rápida ativação do canal, 2) uma segunda ativação, lenta e continua, que gera largos poros na membrana celular, permeáveis a grande moléculas (900 Da). O receptor P2X7 está envolvido em processos como morte celular, formação de células gigantes e secreção de citocinas como IL-1? e está predominantemente presente em células imunes. Neste estudo foi examinado como as altas correntes do P2X7 são geradas e qual o mecanismo necessário para serem ativadas em oócitos de Xenopus laevis. Os oócitos foram cirurgicamente retirados de uma rã adulta de Xenopus laevis e injetamos o cRNA do P2X7 para expressa-los na membrana celular. Medimos a condutância através da técnica de Voltage Clamp (TEVC). A incubação dos Oócitos superexpressos com P2X7 em BABTA-AM demonstrou que o Ca2+ extracelular, e não intracelular, é necessário para gerar altas correntes não seletivas através do P2X7, e a reposição de íons extracelular (Cl- e Na+) demonstrou regula-las. A mutação de truncamento da cauda C-terminal na proteína P2X7 gerou uma corrente menor após a estimulação com 1mM de ATP. E ainda três bloqueadores de canais o Ácido Tânico, o AO1 e o NPPB inibiram significativamente as correntes geradas pelo P2X7. Nós concluímos que (i) Os oócitos de Xenopus que expressam P2X7 produzem altas correntes não seletivas após estimulação com ATP, (ii) A ativação do P2X7 requer tanto o influxo de Ca2+ e a cauda C-terminal, e (iii) as correntes do P2X7 são regulados por cátions e ânions extracelulares / Abstract: The purinergic P2X7 receptor is an ion channel permeable to cations which belong to the P2X family (P2X1-P2X7). It is activated by high concentrations (100?M - 1000?M) of ATP (adenosine 5'-triphosphate), presenting two distinct responses: 1) a rapid activation of the channel, 2) a second activation, slow and continuous, which generates a large pore in the cell membrane permeable to large molecules (900 Dalton). The P2X7 receptor is involved in processes such as cell death, giant cell formation and secretion of cytokines such as IL-1? and is present predominantly on immune cells. In this study we examined how the P2X7 high currents are generated and what is the mechanism required to be activated in Xenopus laevis oocytes. Oocytes were surgically removed from an adult frog Xenopus laevis and injected with cRNA to express the P2X7 in the cell membrane. We measure the conductance through the Voltage Clamp technique (TEVC). Incubation of oocytes overexpressed with P2X7 receptors in BABTA-AM demonstrated that extracellular Ca2+, and do not intracellular, it is necessary to generate nonselective high currents through P2X7, and replacing extracellular ions (Cl- and Na+) showed regulate them. The truncation mutation in C-terminal tail of the P2X7 protein generated a smaller current after stimulation with 1 mM ATP. And three channel blockers Tannic Acid, AO1 and NPPB significantly inhibited the generated currents by P2X7. We conclude that (i) Xenopus oocytes expressing P2X7 produce a nonselective high currents after stimulation with ATP (ii) Activation of the P2X7 requires both the influx of Ca2+ and C-terminal tail, and (iii) the currents of the P2X7 are regulated by extracellular cations and anions / Mestrado / Saude da Criança e do Adolescente / Mestre em Ciências

De nombreux processus physiologiques tels que les mouvements stomatiques, l’absorption des nutriments, l’élongation cellulaire et la signalisation cellulaire impliquent des flux d’anions entre les membranes plasmique et vacuolaire des cellules végétales. Ces flux ioniques sont régulés par des canaux et transporteurs membranaires. Les canaux ioniques transportent passivement les ions au travers des membranes selon le gradient électrochimique. Les transporteurs actifs permettent le transport contre le gradient électrochimique de l’ion transporté induisant son accumulation dans un compartiment cellulaire. Dans les cellules végétales, le gradient de H+ entre différents compartiments constitue la principale source d’énergie couplée par les symports et les antiports au transport de NO₃⁻ et Cl⁻. Au cours de ma thèse, j’ai analysé ces flux ioniques avec deux approches. Une première approche a consisté en l’étude fonctionnelle par électrophysiologie de deux protéines membranaires, AtCLCc et AtCLCg impliquées dans le transport d’anions. Dans une deuxième approche, un biosenseur, clopHensor a été exprimé chez A. thaliana et a permis de mesurer simultanément la [NO₃⁻] et le pH cytosoliques in vivo. Les cellules de garde ont été choisies comme modèle cellulaire pour l’étude de la dynamique in vivo de la [NO₃⁻]cyt et du pH. Nous avons mis en évidence que la [NO₃⁻]cyt est influencée par les conditions extracellulaires dans ces cellules. Enfin l’expression de clopHensor en plantes KO pour un antiport NO₃⁻/H⁺ vacuolaire, AtCLCa, et d’un canal anionique de la membrane plasmique, SLAC1, nous a permis d’étudier la contribution de deux membranes dans la régulation de [NO₃⁻] et du pH cytosolique. Les travaux menés ont permis de visualiser l’activité de canaux et de transporteurs d’anions et H⁺ in vivo et de quantifier leur impact sur l’homéostasie du cytosol. / Many physiological processes like stomata aperture, nutrient up-take, cellular elongation and cell signalling involve anion fluxes at the two main membranes, the plasma and vacuolar membranes of plant cells. Specialized membrane proteins form active and passive anion transport systems mediating and regulating anion fluxes. Ion channels are passive transport systems mediating ion fluxes across membranes along the electrochemical gradient. Whereas active transporters work against the electrochemical gradient of the transported ion allowing its accumulation into a cellular compartment. In plant cells, the H⁺ gradient is the main energy source of antiporters and symporters that couple the transport of anions like NO₃⁻ and Cl⁻ to the transport of H⁺. In the presents work, we aimed at analysing anion and H⁺ fluxes at two levels. First, we used an electrophysiological approach to study the functional properties of two anion transport systems acting at the vacuolar membrane, AtCLCc and AtCLCg. We also expressed a biosensor, clopHensor in A. thaliana to dynamically measure in vivo the [NO₃⁻] and pH of the cytosol. We chose stomata guard cells as a cellular model to study these fluxes. Our results illustrate the in vivo dynamics of cytosolic [NO₃⁻] and pH variations in the cytosol of guard cells. Our data show that in guard cells the cytosolic [NO₃⁻] is highly influenced by the extracellular [NO₃⁻]. At last, clopHensor’s expression in plants KO for the vacuolar NO₃⁻/H⁺ antiporter AtCLCa and for the plasma membrane anion channel SLAC1 allowed us to dissect the role of the two membranes in controlling the variation of cytosolic [NO₃⁻] and pH. This work enabled to visualize the activity of an anion channel (SLAC1) and of a NO₃⁻/H⁺ antiporter (AtCLCa) in vivo and to quantify the impact of anion and proton fluxes on cytosolic homeostasis of guard cells.

L’ischémie cérébrale est caractérisée par une interruption totale ou partielle de l’apport sanguine au cerveau, conduisant à une privation d’oxygène et de glucose pour les cellules du cerveau. La série de processus cellulaires qui sont déclenchées par une ischémie cérébrale sont nombreux et complexes. La réduction sévère d’oxygène et de glucose la diminution de la production d’ATP et un changement drastique de la concentration de K+, du pH intracellulaire et extracellulaire et de la production de lactate. La perturbation du métabolisme énergétique au sein des tissus ischémiés conduit rapidement à la dépolarisation membranaire et au relarguage de neurotransmetteurs dans le milieu extracellulaire. Dans le cervelet, l’impact d’un stress ischémique à largement été étudié sur les cellules de Purkinje, seule voie de sortie neuronale du cortex cérébelleux. Il a été montré que le glutamate, relargué par une surexcitation des fibres glutamatergique et par l’inversion des transporteurs du glutamate, est la cause principale de la dépolarisation anoxique des cellules de Purkinje. Cependant, la compréhension de la réponse astrocytaire et l’influence des astrocytes vis-à-vis de l’ischémie ne sont pas encore connu.La cellule de Bergmann est un astrocyte radiaire qui compose un réseau couplé électriquement, formant des interactions anatomiques et fonctionnelles complexes avec les neurones du cortex cérébelleux. En utilisant un modèle in vitro d’ischémie cérébrale, la privation d’oxygène et de glucose (OGD), plusieurs caractéristiques de base de la réaction astrocytaire à l'ischémie sont analysés. Des expériences en patch clamp et d’imagerie calcique sont réalisées sur tranche de cervelet adulte révélant la réponse de la glie de Bergmann à l’OGD par une dépolarisation progressive de la membrane, avec en parallèle une augmentation de calcium cytosolique soutenue. L’enregistrement apparié entre cellule de Purkinje et cellule de Bergmann révèle des différences importantes de réponse à l’OGD entre ces deux types cellulaires. De plus, nous avons mesuré les changements de la concentration de K+ extracellulaire durant l’OGD en utilisant des microélectrodes sensibles aux ions. Nos résultats montrent une corrélation importante entre la dynamique du K+ extracellulaire et la dépolarisation membranaire de la cellule de Bergmann au cours de l’OGD. / Cerebral ischemia is characterized by partial or total interruption of the blood supply to the brain resulting in glucose and oxygen deprivation to brain cells. The series of cellular processes that are unleashed by cerebral ischemia are complex. The severe reduction in oxygen and glucose induces decreases in ATP production and dramatic changes in extracellular K concentration, pH of intracellular and extracellular space and lactate production. The disruption of energy metabolism in the ischemic tissue rapidly lead to membrane depolarisation and neurotransmitters are released into the extracellular space. In the cerebellum, the impact of an ischemic stress has been extensively studied in Purkinje cells, the only neuronal output of the cerebellar cortex. It has been shown that glutamate released from overexcited fibers and from reversal of glutamate transporters, is the principal cause of the dramatic, anoxic depolarization in Purkinje cells. However a detailed understanding of the astrocytic response to cerebellar ischemia and the potential influence of astrocyte to ischemia outcome is still lacking.Bergmann glia (BG) are radial gial cells that form networks of electrically coupled cells underling complex anatomical and functional interactions with the neurons of the cerebellar cortex. Using an in vitro model of cerebral ischemia, the oxygen and glucose deprivation (OGD), several basic features of astrocytic reaction to ischemia are analyzed. Patch clamp and calcium imaging experiments performed in cerebellar slices from adult mice revealed that BG respond to OGD with a progressive membrane depolarisation that is paralleled with a sustained cytosolic calcium increase. Double patch-clamp recordings between Purkinje neurons and BG reveal different responses to OGD in these cell types. Furthermore, we measured extracellular potassium concentration changes during OGD by using ion-sensitive microelectrodes. Our results indicate an important correlation between the BG membrane depolarisation and the extracellular K dynamics during OGD.

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Die vorliegende Dissertation verfolgt das Ziel, die von Nervenzellen maximal erreichte Signalrate zu bestimmen. Außerdem werden die bislang weitgehend unbekannten Anpassungen einer Synapse an die Anforderungen hochfrequenter Signalübertragung untersucht. Die maximale Übertragungsrate spielt im zentralen Nervensystem eine wichtige Rolle für die Codierung und Verarbeitung von Informationen. Neben den Grundlagen der synaptischen Übertragung und der neuronalen Informationscodierung werden in der Einleitung die anatomischen Gegebenheiten der Kleinhirnrinde und der Moosfaser-Körnerzell-Synapse vorgestellt. Präsynaptische patch-clamp-Messungen von Moosfaserboutons und die erstmals durchgeführten Messungen von präsynaptischen Boutons und postsynaptischen Körnerzellen („Paarableitungen“) werden erläutert. Mit Hilfe dieser Methoden wird gezeigt, dass die Kommunikation zwischen Nervenzellen mit Raten von bis zu einem Kilohertz stattfinden kann. Hierbei ist die präsynaptische Freisetzung von Botenstoffen schneller und effizienter als bisher bekannt. Ein einzigartiges Repertoire präsynaptischer Mechanismen wird charakterisiert und bildet die Grundlage der nachgewiesenen, hochfrequenten Informationsübertragung.