Introdução: Após o surgimento e disseminação das β-lactamases (BL) de amplo espectro em membros da família Enterobacteriaceae, os antibióticos carbapenêmicos (imipenem, meropenem, ertapenem) têm sido considerados a terapia de escolha pela estabilidade apresentada contra estas enzimas. Infelizmente, em 2005, o primeiro caso de infecção fatal por um isolado de Klebsiella pneumoniae resistente aos carbapenêmicos foi relatado em nosso país. A partir deste, novos casos de infecção, inclusive por outros gêneros da família Enterobacteriaceae como Enterobacter, Providencia e Escherichia, começaram a surgir. Como mecanismo de resistência aos carbapenêmicos, a expressão de enzimas carbapenemases tem sido mundialmente relatada, enquanto que, a impermeabilidade associada à produção de enzimas do tipo AmpC ou ESBL tem sido esporádica. Com relação à mobilização dos determinantes genéticos de resistência, elementos móveis como integrons e plasmídios têm sido associados. O presente trabalho teve como objetivo caracterizar os mecanismos de resistência aos carbapenêmicos, sua mobilização genética e disseminação clonal em amostras clínicas de enterobactérias isoladas em diversos hospitais brasileiros. Material e métodos: Foram estudadas 28 cepas recuperadas de oito centros hospitalares descritas como resistentes ao imipenem. A caracterização fenotípica foi realizada por: i) determinação da CIM na presença e ausência de inibidores de BL, ii) bioensaio para produção de BL e iii) SDS-PAGE para investigar a ausência de porinas. A confirmação genotípica da resistência mediada por β-lactamases foi realizada por PCR e seqüenciamento e a sua localização plasmidial foi estudada por transformação. Por último, a tipagem molecular foi realizada pela técnica de ERIC-PCR, sendo confirmada pela técnica de PFGE. Resultados: 25 cepas apresentaram resistência para carbapenêmicos (imipenem MIC 8-128 µg/mL), todas com perfil de multiresistência incluindo cefoxitina (CIM90 ≥32 µg/mL). Foram identificados três determinantes de resistência, entre eles, a produção de carbapenemases de tipo MBL (IMP-1) e a enzima KPC-2, recentemente descrita, sendo emergente no país. O mecanismo mais prevalente nas amostras estudadas foi a impermeabilidade de membrana associada à expressão de enzimas do tipo AmpC (CMY-2 plasmidial para E. coli e AmpC cromossômica no caso de Enterobacter aerogenes), as quais mostraram uma contribuição significativa para a resistência aos carbapenêmicos. Dos 28 isolados, 18 apresentaram a perda da porina de 36 kDa, responsável pela entrada de antimicrobianos na bactéria, como os carbapenêmicos. Tanto os genes blaKPC-2 e blaCMY-2 foram transferidos com êxito para E. coli DH10B, confirmando sua localização plasmidial. A co-produção de carbapenemase ou enzimas do tipo AmpC com ESBL do tipo CTX-M foi confirmada em 68% dos isolados. A tipagem molecular mostrou uma disseminação clonal para os isolados carregando determinantes IMP-1 e as enzimas do tipo AmpC cromossômica e plasmidial. Ao contrário, isolados expressando KPC não foram clonalmente relacionadas. Conclusão: A caracterização de resistência apresentada neste trabalho demonstrou uma mudança no perfil de resistência da família Enterobactériaceae devido à sua versatilidade para a aquisição de novos mecanismos de resistência, como sua adaptação aos ambientes hostis. A perda da porina foi o mecanismo mais freqüente nesta família e a co-produção de BL foi um evento associado. Finalmente, os dados obtidos na tipagem molecular denotaram uma disseminação majoritariamente clonal na cidade de São Paulo, com exceção das cepas produtoras de KPC-2, cuja presença tem sido relatada em outras cidades do país, sugerindo a participação de uma transferência horizontal. / Introduction: After emergence, and dissemination of extended spectrum β-lactamases (ESBL) in members of the Enterobacteriaceae family, carbapenem antibiotics (imipenem, meropenem, ertapenem) have been the therapy of choice, since they are stable to ESBL hydrolysis. Unfortunately, in 2005, the first fatal case of infection by carbapenem-resistant Klebsiella pneumoniae was related in our country. From this episode, new infection cases, including by other genders of Enterobacteriaceae such as Enterobacter, Providencia and Escherichia, began to appear. Regarding carbapenem resistance mechanisms, expression of carbapenem hydrolyzing enzymes has been worldwide reported, whereas interplay between impermeability and AmpC or ESBL production has been sporadic. Furthermore, integrons and plasmids have been associated with mobilization of genetic determinants. The aim of this study was to characterize the mechanisms of resistance to carbapenems, their genetic mobilization and clonal dissemination in enterobacterial isolates recovered from clinical samples in Brazilian hospitals. Material and methods: 28 imipenem-resistant isolates recovered from 8 hospital centres were studied. Phenotypic profiles were characterized by: i) MIC of carbapenems in the presence/absence of β-lactamase inhibitors; ii) bioassay for β-lactamase production; iii) SDS-PAGE to investigate absence of outer membrane porins (OMPs). Molecular characterization of β-lactamase-mediated resistance was made by PCR and DNA sequencing and their plasmid localization was evaluated by transformation. Finally, epidemiological typing was performed by ERIC-PCR, being confirmed by PFGE. Results: 25 isolates were confirmed as being resistant to imipenem (MIC 8-128 µg/mL), exhibiting a multidrug-resistant profile, including to cefoxitin (MIC90 ≥32 µg/mL). Two main mechanism of resistance were identified: i) hydrolysis of carbapenem by class B (IMP-1-like MBL) and class A (KPC-2) enzymes, (the latter being recently reported in our country), and ii) outer membrane impermeability associated to AmpC enzyme production (plasmid-mediated CMY-2 for E. coli and chromosomal AmpC for E. aerogenes), which was the most prevalent mechanism found. Eighteen of 28 isolates lacked 36kDa OMP, which is responsible for uptake of carbapenem antibiotics. The blaKPC-2 and blaCMY-2 genes were successful transferred to E. coli DH10B, confirming the plasmid location of both genes. Co-production of carbapenemases or AmpC and CTXM enzymes was confirmed in 68% of isolates, and molecular typing showed clonal dissemination of IMP-1-, plasmid AmpC- and chromosomal AmpC-producing isolates. Otherwise, KPC-2-producing isolates were not clonally related. Conclusion: The characterization of resistance mechanisms to carbapenems, in this study, reveals a change in the resistance patterns among Enterobacteriaceae family members in Brazilian hospitals, due to versatility of isolates to acquire new resistance determinants, which it has favoured the adaptation to hostile environments. Lack of 36 kDa OMP was the most frequent resistance mechanism, being associated to co-production of β-lactamases. Finally, molecular typing denote a clonal dissemination of imipenem-resistant isolates in Sao Paulo city, with exception of KPC-2-producing isolates, which have been described in other Brazilian cities, suggesting a horizontal gene transfer.

O óxido nítrico (NO) é um mensageiro químico gerado pela atividade da enzima óxido nítrico sintase (NOS) a qual foi detectada em vários órgãos incluídos os do sistema reprodutor e parece estar envolvido na maturação oocitária. No entanto, há poucos estudos sobre o papel desse sistema em oócitos da espécie bovina. Sabe-se que o NO atua pela via da guanilato ciclase (GC) estimulando a produção do nucleotídeo GMPc, que por sua vez é capaz de influenciar nos níveis de outro nucleotídeo (AMPc) via fosfodiesterases (PDE). O AMPc é um importante elemento da via de sinalização do FSH nos complexos cumulus-oócitos e no controle da maturação oocitária. O objetivo do presente projeto foi investigar a influência do FSH na via do NOS/NO e seus componentes em oócitos bovinos maturados in vitro e o envolvimento das células do cumulus (CC) na via de sinalização. Para tanto, complexos cumulus-oócito (CCO) e oócitos desnudos (OD - maturados sem células do cumulus) foram maturados in vitro por 24h na presença ou ausência de FSH. As amostras foram avaliadas quanto a: 1) taxa da maturação nuclear; 2) níveis de produção de NO; 3) níveis de AMPc e GMPc; 4) abundância relativa de RNAm de NOS2, PDE5A, PDE6C, PKG1, PKG2, ADCY6, ADCY9, PDE3A e PKA1. O FSH, na concentração de 0,05UI/mL, estimulou positivamente a maturação nuclear em CCO e OD, com 80,6 e 89% de oócitos maturados, respectivamente. Quando comparados diretamente os grupos CCO e OD, o FSH não influenciou as taxas de maturação (71 e 71,3%, p>0,05), nem os níveis de produção de NO (12,8 e 7,4 µM/mL, p>0,05). Os níveis de GMPc em CCO aumentaram após 1 e 3 h de MIV na presença de FSH (266,3 e 187,2 pmol/pool com FSH e 240,5 e 168,5 pmol/pool sem FSH, respectivamente, p<0,05). Após 6 h os níveis de GMPc declinaram de forma mais acentuada no grupo sem FSH (46,3 e 106,9 pmol/pool, com e sem FSH, respectivamente, p<0,05). Os níveis de AMPc em CCO também foram mais elevados na presença de FSH à 1 e 3 h de MIV (7,60 e 7,81 pmol/pool, respectivamente) em comparação com CCO maturados sem FSH (0.30 e 0,76 pmol/pool, respectivamente, p<0,05). Após 6h, os níveis declinaram e foram similares para ambos os grupos (0,43 pmol/pool, p>0,05). Em relação à expressão dos genes selecionados, todos foram detectados nos oócitos (CCO e OD), porém, em células do cumulus, foram detectados apenas PDE5A, ADCY6, ADCY9 e PKA1. Quando observados os resultados do grupo CCO, apenas os genes PKG1, ADCY6 e PDE3A sofreram influência do FSH (p<0,05), apresentando um aumento destes transcritos. No grupo OD, apenas o gene PKG1 sofreu influência do FSH, também apresentando um aumento destes transcritos (p<0,05). Em células do cumulus, os genes ADCY6 e ADCY9 sofreram influência do FSH, sendo que para a ADCY6 provocou um aumentos destes transcritos, e para a ADCY9 provocou uma queda dos mesmos (p<0,05). Em conclusão, o FSH pode exercer influência positiva na maturação nuclear de oócitos bovinos, agindo sobre os níveis de GMPc e AMPc, mas não sobre o NO. O FSH pode influenciar a expressão gênica em oócitos e em células do cumulus de bovinos. / Nitric oxide (NO) is a chemical messenger generated by the nitric oxide synthase (NOS) enzyme, which was detected in several organs including the reproductive system and appears too involved in oocyte maturation. However, there are few studies on the role of this system in bovine oocytes. NO is known to act via guanylate cyclase (GC) stimulating the production of the nucleotide cGMP, which in turn is capable of influencing the levels of another nucleotide, cAMP via phosphodiesterases (PDE). cAMP is an important factor in FSH signaling in cumulus-oocyte complexes (COC) for the control of maturation. The aim of the present work was to investigate the influence of FSH on the NOS/NO pathway and its components in bovine oocytes matured in vitro and the involvement of cumulus cells (CC) in the signaling pathway. COC and denuded oocytes (DO - matured without cumulus cells) were matured in vitro for 24 h with or without FSH. Samples were assessed for: 1) maturation rate; 2) levels of NO production; 3) levels of cGMP and cAMP; 4) relative abundance for mRNA of NOS2, PDE5A, PDE6C, PKG1, PKG2, ADCY6, ADCY9, PDE3A and PKA1. FSH positively stimulated oocyte maturation at 0.05UI/mL concentration for both COC and OD (80.6 and 89% maturation rates, respectively). When COC and OD were compared directly, FSH did not affect maturation rates (71 and 71.3%, p>0.05) nor NO production levels (12,8 and 7,4 µM/mL), p>0.05). cGMP levels increased after 1 and 3 h in vitro maturation (IVM) with FSH (266.3 and 187.2 pmol/pool with FSH and 240.5 and 168.5 pmol/pool without FSH, respectively, p<0.05). After 6 h IVM, cGMP levels in COC declined more in the group cultured with FSH (46.3 and 106.9 pmol/pool, with and without FSH, respectively, p<0.05). cAMP levels in COC were also increased in the presence of FSH at 1 and 3 h IVM (7.60 and 7.81 pmol/pool, respectively) in comparison to COC cultured without the hormone (0.30 and 0.76 pmol/pool, respectively, p<0.05). After 6 h, the levels declined and were similar for both groups (0.43 and 0.02 pmol/pool, p>0.05). Regarding mRNA expression for the selected genes, all of them were detected in oocytes, but only four of them were detected in cumulus cells: PDE5A, ADCY6, ADCY9 and PKA1. For COC only PKG1, ADCY6 and PDE3A were influenced by FSH (p<0.05), with an increase in transcript relative abundance, For DO, only PKG1 was influenced by FSH and also showed an increase in these transcripts (p<0.05). In cumulus cells, ADCY6 and ADCY9 were affected by FSH, with an increase for ADCY6 and a decrease in ADCY9 transcripts (p<0.05). In conclusion, FSH may positively influence nuclear maturation, acting on cGMP and cAMP levels, but not on NO. FSH may also influence gene expression in bovine oocytes and cumulus cells.

A transmissão sináptica é essencial para o funcionamento do sistema nervoso. A neuromodulação permite regular esse processo de forma precisa. Um desses mecanismos modulatórios é a regulação da liberação de neurotransmissores. A adenosina é um importante modulador da transmissão sináptica. Além disso, a ativação do subtipo A2a dos receptores para adenosina está envolvida com a facilitação da liberação de neurotransmissores no sistema nervoso central. O presente trabalho teve como objetivo avaliar os efeitos modulatórios da ativação do receptor A2a de adenosina sobre a liberação de neurotransmissores e sua via de sinalização intracelular em modelo in vitro. Além disso, a tese contempla a construção histórica dos conceitos abordados no trabalho permitindo uma visão clara de sua evolução. Esse projeto foi o pioneiro no Brasil a utilizar o sensor biossintético fluorescente de liberação de vesículas sinápticas (supereclipse sinapto-pHluorina), o qual foi gentilmente cedido pelo professor Gero Miensenboeck do Sloan-Kettering Institute for Cancer Research. Nossos resultados demonstraram que o tratamento com o agonista do receptor A A2a de adenosina aumentou a fluorescência do supereclipse sinapto-pHluorina, assim como os níveis de glutamato e noradrenalina. Além disso, foi demonstrado que o inibidor da proteína cinase dependente de AMPc aboliu o aumento nos níveis do glutamato e noradrenalina, tal como a fosforilação da proteína sináptica sinapsina I evocado pelo agonista do receptor A2a de adenosina. Desta forma, nossos dados sugerem que a ativação do receptor A2a de adenosina em cultura de células do bulbo de ratos Wistar modula a liberação de neurotransmissores e a fosforilação da sinapsina I, assim como a proteína cinase dependente do AMPc pode ser o modus operandi desse fenômeno modulatório / Synaptic transmission is a sine qua non process for nervous system physiology. Such precise process is accomplished in part due to modulation of neurotransmitter release. Adenosine is a putative synaptic transmission modulator. Moreover, adenosine A2a receptor facilitates neurotransmitter release in the Central Nervous System. The present study focuses on the modulation of neurotransmission by adenosine A2a receptor and its intracellular signaling pathway in in vitro model. Here, we provided evidence that adenosine A2a receptor agonist increases an optical biosynthetic sensor of synaptic vesicle release (supereclipct synapto-pHluorin), as well as glutamate and noradrenaline. Furthermore, it was demonstrated that cAMP-dependent protein kinase inhibitor abolished glutamate and norepinephrin increase, as well as synapsin I phosphorylation evoked by adenosine A2a receptor agonist. Therefore, our data suggest that adenosine A2a receptor activation modulates neurotransmitter release and synapsin I phosphorylation in cultured cells from medulla oblongata of Wistar rats, as well as cAMP-dependent protein kinase might be the modus operandi of this modulatory phenomenon

Estudos anteriores de nosso laboratório demonstraram que a estimulação aguda dos receptores 2-adrenérgicos (2-AR) atenua a perda de massa muscular induzida pela desnervação motora (DEN) por meio de uma via dependente de AMPc/PKA. No entanto os mecanismos moleculares envolvidos na ativação crônica destes receptores ainda são pouco conhecidos. Por outro lado, a ativação desta via de sinalização também está envolvida no controle da estabilidade dos receptores nicotínicos (AChR) na junção neuromuscular (JNM), sugerindo que a densidade dos AChR possa estar sob controle neuro-humoral. Desta forma, aventou-se a possibilidade de que além dos efeitos protetores na massa muscular, a ativação dos receptores 2-AR pudesse mediar a estabilização dos AChR na placa motora. Para testar essa hipótese, camundongos foram submetidos à DEN através da secção do nervo ciático, um protocolo clássico de indução de atrofia muscular e desestabilização dos AChR, e tratados com salina ou clembuterol (CB), um 2-agonista seletivo, por até 14 dias. Após 3 dias de DEN, observou-se redução da massa muscular e aumento do conteúdo proteico e expressão do RNAm de genes relacionados à ativação do sistema Ubiquitina-Proteassoma (atrogina-1 e MuRF1) e do sistema autofágico/lisossomal (catepsina L e LC3). A DEN também promoveu aumento no turnover dos AChR, no número de vesículas endocíticas e na expressão do RNAm para a subunidade 1 dos AChR. Após 7 dias, a DEN reduziu a expressão dos genes relacionados à atrofia e aumentou a atividade da via do AMPc/PKA independentemente do tratamento com CB. Na tentativa de elucidar os sinais extracelulares que produziam esta resposta adaptativa, foi demonstrado que neurônios catecolaminérgicos trafegam ao longo do nervo ciático e sua ablação pela DEN reduziu o conteúdo de noradrenalina muscular. Baseados nestes resultados, foi postulado a existência de uma hipersenbilidade às catecolaminas em músculos desnervados cronicamente. O tratamento com CB por 3 dias aboliu o aumento da expressão dos atrogenes induzido pela DEN e este efeito foi associado ao maior conteúdo de AMPc e de substratos fosforilados pela PKA. Além disso, o CB diminuiu a hiperexpressão do RNAm para catepsina L e LC3 induzida pela DEN de 7 dias. Embora o CB não tenha alterado a meia-vida dos AChR em músculos inervados e desnervados, houve um total bloqueio do aumento do número de vesículas endocíticas contendo o AChR em músculos desnervados e tratados com CB. Corroborando estes dados, o CB aumentou a incorporação de AChR novos nas JNM e este efeito foi também associado à maior expressão do RNAm para a subunidade 1-AChR em músculos desnervados. Esta ação do CB no turnover dos AChR parece ser direta uma vez que neuroniôs catecolaminérgicos presentes no nervo ciático ativam receptores 2-ARe a produção de AMPc especificamente na JNM. Em estudos in vitro, foi demonstrado que a estimulação colinérgica produzida pelo carbacol (10-4M) diminuiu a velocidade de síntese de proteínas, aumentou a proteólise total e a atividade do sistema proteolítico Ca2+-dependente em músculos soleus de ratos por meio da ativação dos receptores nicotínicos. Este efeito catabólico do carbacol foi completamente bloqueado pela adição de CB (10-4M) ao meio de incubação. Os dados obtidos no presente estudo permitem sugerir que a estimulação crônica dos 2-AR no músculo esquelético induz um efeito anti-catabólico pela supressão dos sistemas proteolíticos proteassomal e lisossomal, provavelmente através da via de sinalização do AMPc/PKA. A inibição destes sistemas pode estar relacionada ao aumento do turnover dos AChR, uma vez que a velocidade de incorporação destes receptores na JNM foi aumentada pelo CB. Além disso, os achados que mostram a associação entre neurônios noradrenérgicos e colinérgicos no nervo ciático, que conjuntamente inervam as JNM, e a co-localização de receptores 2-AR e AChR na sinapse permitem sugerir a existência de uma interação funcional entre o sistema colinérgico e adrenérgico na manutenção da massa muscular e da placa motora. / Previous studies from our laboratory have shown that the acute stimulation of 2-adrenergic receptor (2-AR) attenuates the muscle loss induced by motor denervation (DEN) through a cAMP/PKA dependent pathway. However, the molecular mechanisms involved in the chronic activation of these receptors are poorly understood. Furthermore, the activation of this signaling pathway is also involved in controlling the stability of nicotinic receptors (AChR) at the neuromuscular junction (NMJ), suggesting that the density of AChR may be under neurohumoral control. Thus, we postulated that besides the protective effects on muscle mass the activation of 2-AR receptors could mediate the stabilization of AChR in the motor plate. To test this hypothesis, mice were submitted to DEN through of the sciatic nerve section, a classical protocol of induction muscle atrophy and destabilization of AChR, and were treated with saline or clenbuterol (CB), a selective 2-agonist for 14 days. DEN decreased the muscle mass and increased the protein content and mRNA expression of genes related to the activation of the ubiquitin-proteasome system (atrogin-1 and MuRF1) and autophagic/lysosomal system (cathepsin L and LC3). DEN also promoted an increase in the turnover of AChR, number of endocytic vesicles and the expression of mRNA for the 1 subunit of AChR. Interestingly, chronic DEN induced down-regulation of atrophy related-genes, and increased the activity of cAMP/PKA pathway independently of CB treatment. In an attempt to elucidate the extracellular signals that produced this adaptive response, it was demonstrated that catecholaminergic neurons travels along the sciatic nerve and its ablation by DEN reduces muscle norepinephrine content. Based on these results, it was postulated the existence of a muscle adrenergic hypersensitivity to circulating catecholamines induced by chronic DEN. CB treatment for 3 days completely abolished the higher expression of atrogenes and this effect was associated with increased Camp content and PKA phosphorylated substrates. Furthermore, CB decreased the DEN-induced hyperexpression of cathepsin L and LC3 mRNA at 7 days. Although CB has not altered the half-life of AChR in innervated and denervated muscles, it produced a total blockage of the increased number of endocytic vesicles containing the AChR in denervated muscles. Consistently, CB increased the incorporation of new AChR and this effect was associated with an increased expression of the 1-subunit AChR mRNA in denervated muscles. This action of CB on AChR turnover appears to be direct, since catecholaminergic neurons are present in the sciatic nerve stimulating 2-AR and cAMP production specifically in the NMJ. Furthermore, in vitro studies demonstrated that cholinergic stimulation produced by carbachol (10-4M) decreased the rate of protein synthesis and increased the proteolytic activity of Ca2+-dependent system in rat soleus muscle through activation of nicotinic receptors. This catabolic effect of carbachol was completely blocked by the addition of CB (10-4M) to the incubation medium. These data suggest that chronic stimulation of 2-AR in skeletal muscle induces an anti-catabolic effect by suppressing proteasomal and lysosomal proteolytic systems, probably through the cAMP/PKA signaling. The inhibition of these systems seems to be related to the increased AChR incorporation into NMJ induced by CB treatment. Moreover, the association between noradrenergic and cholinergic neurons in the sciatic nerve, both of which innervate the motor endplates, and the co-localization of AChR and 2-ARat the synapse suggest the existence of a functional interaction between cholinergic and adrenergic systems in the maintenance of muscle mass and motor endplate.

L’objectif de cette thèse était de caractériser le rôle des différentes familles de phosphodiestérases (PDEs), les enzymes de dégradation du 3'-5'- adénosine monophosphate cyclique (AMPc), dans la régulation de la signalisation de l’AMPc dans deux types de cellules musculaires lisses (CMLs), l’aorte de rat (CMLAR) et la vessie de rat néonatal (CMLVRN). Dans les CMLARs en culture, nous avons déterminé le profil d’expression et d’activité des PDE-AMPc. Nous avons alors montré, à l’aide de la technique de FRET basée sur une sonde sensible à l’AMPc pour mesurer l’AMPc en temps réel dans une cellule isolée, que l’inhibition de la PDE4 démasque un effet d’hydrolyse de l’AMPc cytosolique par la PDE1 et la PDE3, alors que les PDE3 et PDE4 agissent de façon synergistique dans le compartiment sous-membranaire. Les mécanismes de cette compartimentation subcellulaire des signaux restent à caractériser.Dans les CMLVRNs, les PDE3 et PDE4 régulent les contractions phasiques, par des mécanismes différents. L’inhibition de la PDE4 limite les contractions stimulées par le carbachol par un mécanisme dépendant de la protéine kinase A, impliquant une augmentation de la fréquence des sparks calciques, qui entrainent l’activation des canaux potassiques BK, assurant en final une diminution des transitoires calciques. Au contraire, l’effet de l’inhibition de la PDE3 implique la protéine kinase G mais par un mécanisme qui reste à définir.En conclusion, ce travail montre que dans les CMLs, les différents familles de PDE-AMPc sont douées de spécificité de fonction et/ou de mécanisme d’action, et participent ainsi à une compartimentation subcellulaire des voies de signalisation. / The aim of the present thesis was to characterize the role of the different families of phosphodiesterases (PDEs), the enzymes degrading 3'-5'-cyclic adenosine monophosphate (cAMP), in controlling the cAMP signalling in two distinct smooth muscle cells (SMCs), the rat aorta SMC (RASMCs) and the rat bladder SMC (RBSMCs).In cultured RASMCs, we firstly characterized the pattern of cAMP-PDE expression and activity. We then showed, by using a FRET-based cAMP sensor to explore cAMP signals in living cells, that PDE4 inhibition unmasks an effect of PDE1 and PDE3 on cytosolic cAMP hydrolyzis, whereas PDE3 and PDE4 act synergistically at the submembrane compartment. The mechanisms of this subcellular compartmentation need to be characterized. In neonatal RBSMCs, we showed that both PDE3 and PDE4 are involved in regulating the phasic contractions albeit through distinct mechanisms. PDE4 inhibition inhibits the carbachol-enhanced contractions through a protein kinase A-dependent pathway involving an increase in Ca2+ sparks frequency which activates BK channels to ultimately decrease Ca2+ transients, whereas PDE3 inhibition acts through a protein kinase G-dependent pathway through a still unknown mechanism.In conclusion, our work shows that in the SMC, the different cAMP-PDE families exhibit a specificity in their function and/or mechanism of action, thus participating to a subcellular signaling compartmentation.

L'AMP cyclique (AMPc) et le GMP cyclique (GMPc) sont des seconds messagers essentiels pour la régulation de la fonction cardiaque. Leurs niveaux sont régulés par l’adénylate cyclase et la guanylate cyclase, respectivement, et par les phosphodiestérases (PDEs). Cependant, une telle régulation est altérée dans l'insuffisance cardiaque (IC). En effet, la diminution de la signalisation de l’AMPc et l’augmentation de celle du GMPc est caractéristique des cœurs défaillants.Parmi la superfamille des PDEs, la PDE2 a la particularité d'être stimulée par le GMPc, conduisant ainsi à une augmentation remarquable de l'hydrolyse de l'AMPc. Ceci semble induire une interaction entre les voies de signalisation de l’AMPc et du GMPc. Cependant, le rôle de la PDE2 dans le cœur défaillant est très peu connu.Dans ce contexte, nous avons examiné si la PDE2 cardiaque est modifiée dans l’IC chez l’Homme et chez les modèles animaux d’IC, et déterminé le rôle de la PDE2 dans la signalisation β-adrénergique dans les cardiomyocytes. Grâce à l’utilisation de Western blot, de technique radioenzymatique, d’imagerie basée sur le FRET, de la planimétrie, de la microscopie à épifluorescence et des mesures du courant calcique de type L, réalisés sur les tissus myocardiques humains et/ou dans des cardiomyocytes isolés de cœurs des modèles animaux d’IC, respectivement, nous avons montré que l’expression et l’activité de la PDE2 sont augmentées dans les cœurs défaillants. Cette augmentation réduit l’effet d’une stimulation β-adrénergique aiguë, contribuant à la désensibilisation β-adrénergique observée dans l’IC. En accord avec ces résultats, la surexpression de la PDE2 dans des cardiomyocytes sains, réduit l’augmentation des taux d'AMPc et l’amplitude du courant ICa,L et abolit l'effet inotrope positif suite à une stimulation β-adrénergique aiguë, sans affecter la contractilité basale. Plus important, les cardiomyocytes surexprimant la PDE2, montrent une protection contre les réponses hypertrophiques induites par la noradrénaline et contre les arythmies induites par l'isoprotérénol.En conclusion, ce travail met en évidence l'altération de la PDE2 dans l’IC et nous laisse suggérer que l’augmentation de la PDE2 dans l’IC peut constituer un mécanisme de défense important dans des conditions de stress cardiaque, notamment en antagonisant la suractivation de la voie β-adrénergique. Ainsi, l'activation de PDE2 myocardique peut représenter une nouvelle stratégie thérapeutique anti-adrénergique intracellulaire dans l’IC. / Cyclic AMP (cAMP) and cyclic GMP (cGMP) are critical second messengers for the regulation of cardiac function. Their levels are regulated by adenylyl and guanylyl cyclases, respectively, and by cyclic nucleotides phosphodiesterases (PDEs). However, such regulation is altered in heart failure (HF). Indeed diminished cAMP- and augmented cGMP-signaling is characteristic of failing hearts.Among the PDE superfamily, PDE2 has the unique property to be stimulated by cGMP, thus leading to a remarkable increase in cAMP hydrolysis. This appears to mediate a negative cross-talk between cAMP- and cGMP signaling pathways. However, the role of PDE2 in the failing heart is only poorly understood.In this context, we investigated whether myocardial PDE2 is altered in human and experimental HF and determined PDE2 mediated effects on β-adrenoceptor (β-AR) signaling in cardiomyocytes. Using immunoblotting, radioenzymatic- and FRET-based assays, video-edge-detection, epifluorescent microscopy and L-type Ca2+ current measurements, performed in myocardial tissues and/or isolated cardiomyocytes from human and/or experimental HF, respectively, we showed that PDE2 is markedly upregulated in failing hearts. This reduces the effect of an acute β-adrenergic stimulation, and contributes to the β-adrenergic desensitization which is a characteristic feature in HF. Accordingly, PDE2 overexpression in healthy cardiomyocytes reduced the rise in cAMP levels and ICa,L amplitude and abolished the inotropic effect following acute β-AR stimulation, without affecting basal contractility. Importantly, PDE2-overexpressing cardiomyocytes showed marked protection from norepinephrine-induced hypertrophic responses and from isoproterenol-induced arrhythmias.In conclusion, this work highlights the alteration of PDE2 in HF and lets us assume that PDE2 upregulation in HF may constitute an important defence mechanism during cardiac stress, e.g. by antagonizing excessive β-AR drive. Thus, activating myocardial PDE2 may represent a novel intracellular anti-adrenergic therapeutic strategy in HF.

La mouche Drosophila melanogaster est un modèle de choix dans l’étude des grandes fonctions neurophysiologiques notamment en raison de la disponibilité d’une importante variété d’outils disponibles (approches génétiques, pharmacologiques et comportementales). Le cerveau de la mouche, malgré sa simplicité apparente, est capable de traiter des fonctions complexes d’intégration des différents paramètres environnementaux nécessaires à sa survie. Dans le cerveau drosophile, les corps pédonculés (CP) sont des structures impliquées dans de nombreuses fonctions neurophysiologiques de premier plan telles que l'apprentissage et la mémoire olfactive, la régulation de l’activité locomotrice, l'orientation spatiale, la régulation du sommeil ou encore la prise de décision. Il a été montré par des approches associant essentiellement observations comportementales et outils génétiques que la voie de signalisation de l'AMPc joue un rôle crucial dans la réalisation des fonctions diverses des CP. Les cellules de Kenyon (CK) qui sont les cellules intrinsèques des CP, reçoivent principalement des afférences du système olfactif par l’intermédiaire des neurones de projections (PN) en provenance des lobes antennaires et des afférences neuromodulatrices (dopaminergiques et octopaminergiques). Les synapses entres PN et CK se font sur un mode cholinergique grâce à des récepteurs canaux à l’acétylcholine de type nicotinique (nAchR). Nous avons utilisé une technique récente d’imagerie calcique par bioluminescence utilisant une protéine recombinante, la GFP-Aequorine. Cette technique nous a permis de suivre l’activité cellulaire calcique consécutive à l’application de nicotine, un agoniste des nAchR. Grâce à l’observation de ces réponses suite à une combinaison d’approches génétiques corroborée par des approches pharmacologiques, nous avons pu mettre en évidence une modulation complexe et régionalisée de la réponse calcique dans les CP par l’AMPc et d’autres différents partenaires tels que des canaux K+ et Ca2+. Dans un premier temps, nous avons démonté l’existence d’une modulation directe de l’intensité de la réponse par l’AMPc. Nous avons également montré, pour la première fois, que des réponses Ca2+ « spontanées » peuvent être directement inductibles par augmentation de l’AMPc. Nous avons mis en évidence l’existence d’un nouveau partenaire de la modulation de la réponse des CP indépendant de la PKA : les CNG (Cyclic Nucleotides Gated Channels) dont le rôle n’avait jusqu’ici jamais été démontré dans les corps pédonculés. Enfin nous avons pu observer une régionalisation de la régulation de l’activité Ca2+ des CP par l’AMPc. Dans un deuxième temps nous nous somme intéressé aux principales conductances calciques et potassiques. Nous avons mis en évidence que différents canaux calciques voltages dépendants (VGCC) sont impliqués de façon régionalisée et séquentielle dans la formation de la réponse calcique. Il a pu également être démontré que le signal est modulé de façon différentielle dans les calices et les lobes par l’AMPc à travers différents canaux potassiques. Enfin des protocoles originaux ont été développés, tels que la micro application de drogue ou l’électrostimulation permettant d’étudier la neuromodulation dans les CP, à réutiliser pour des travaux ultérieurs du laboratoire. Ce travail est une première étape dans la compréhension des voies de signalisations et des mécanismes intracellulaires impliqués dans l’apprentissage et la mémoire olfactive. / In Drosophila, the Mushroom-Bodies (MBs) are implicated in multiple functions, as olfactory learning and memory, locomotor activity, spatial orientation, sleep, decision making, and up to now but indirectly, in various addiction. Notably, the MBs, which express the nAchR, receive their main inputs from the cholinergic olfactory pathways, through the Projections Neurons (PNs). In this thesis we characterized, at the cellular and molecular levels, the nicotine effect on the Kenyon cells (KCs: the intrinsic neurons) of the Mushroom-Bodies. We used the in-Vivo brain imaging approach, based on the Ca2+-Sensitive bioluminescent probe (GFP-Aequorin), to characterize the nicotinic induced Ca2+-Response on the KCs of the MBs. More specifically we searched the role of different partners involved in the cAMP pathway, in order to understand their roles in the different components of the response and in its modulation. First using both genetics and pharmacological approaches to interfere with different components of the cAMP signaling pathway, we first show that the Ca2+-Response is proportional to the levels of cAMP. Second, we reveal that an acute change in cAMP levels is sufficient to trigger a Ca2+-Response. Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has a PKA-Independent effect through the cyclic nucleotide-Gated Ca2+-Channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects between the calyx/cell-Bodies and the lobes, suggesting a differential and regionalized regulation of ACSecond we exploited both genetic approaches to interfere with different types of Ca2+- and K+-Channels, first we show that the disruption of the VGCC, as cacophony, Dmcα1d and Dmcα1g lead to a striking decrease of the Ca2+-Response both in the CCB and the lobes. Moreover, for two of them, cacophony and Dmcα1d, the duration of the response is importantly increased. Second, the disruption of the fast inactivating K+-Currents, as shaker (sh), shaker-Like (shal) and slowpoke (slo) reveal that the knocked-Down of shal and slo lead to a striking decrease of the Ca2+-Response, while the knocked-Down of sh has only a mild effect. Interestingly, the stimulation of the adenylate cyclase (AC) by the forskolin with the various K+-Channels disruption show an antagonist effect of the cAMP in the CCB between sh (inhibitory) and slow (excitatory) while AC simulation mediate excitatory effects in the ML though both shal and sh. Finally, the knock-Down of the two slow inactivating K+-Currents as shaker w (shaw) and shaker b (shab) also yields to a strong decrease of the Ca2+-Response In conclusion, our results provide new insights into the complexity of the Ca2+-Response in the MBs and are a first step toward deciphering the roles of the VGCC and K+-Channels in the multiples roles of the MBs. Finaly we developed several original protocols to explore the role of the neuromodulation on the KC.This work constitutes an important step toward a better understanding of the pathway required in learning and memory.

L'interleukine-6 (IL-6) est une cytokine associée aux maladies auto-immunes de la glande thyroïde et qui pourrait aussi avoir un rôle dans le développement de l'hypertrophie cardiaque pathologique. Dans les cellules thyroïdiennes FRTL-5 et dans les cardiomyocytes de rats adultes, nous montrons que la sécrétion et l'expression de l'ARNm de l'IL-6, stimulées par l'interleukine-1 (IL-1), sont augmentées de manière synergique par la voie de l'AMPc/PKA. Dans les cellules FRTL-5, cet effet synergique s'exerce aussi au niveau de l'activation du promoteur de l'IL-6 et implique les sites AP-1 et CRE ainsi que les facteurs de transcription c-Fos et Fra2. L'IL-6 et son récepteur membranaire stimulent les voies de signalisation impliquant STAT3 et ERK-5 dans les cellules FRTL-5. Dans les cardiomyocytes, STAT3 est activé par l'IL-6 uniquement en présence de son récepteur soluble. Nous montrons que ce mécanisme de conduit à la stimulation de marqueurs associés au processus hypertrophique pathologique.

En France la prise en charge des grandes douleurs fait toute sa place à la morphine. Cependant la prise prolongée de morphine entraîne la tolérance et la dépendance. Une stratégie de protéomique différentielle a été entreprise afin d'identifier les adaptations cellulaires à un traitement par la drogue dans des cellules SH-SY5Y. Nous avons montré, notamment, qu'un traitement chronique entraîne la dégradation par le protéasome des protéines Gbeta et Ggamma2. Aussi, le niveau de dégradation de Gbeta est corrélé avec le niveau de sensibilisation de l'adénylyl cyclase (AC), phénomène impliqué dans la dépendance à la morphine. Globalement, l'analyse protéomique a permis de détecter environ 50 protéines et une centaine de phosphoprotéines modulées par la drogue. Mes travaux ont permis de proposer un nouveau mécanisme moléculaire responsable de la sensibilisation de l'AC et surtout suggèrent pour la première fois que le protéasome est impliqué dans les effets chroniques de la morphine.

L'AMP cyclique (AMPc) et le GMP cyclique (GMPc) sont des seconds messagers essentiels pour la régulation de la fonction cardiaque. Leurs niveaux sont régulés par l'adénylate cyclase et la guanylate cyclase, respectivement, et par les phosphodiestérases (PDEs). Cependant, une telle régulation est altérée dans l'insuffisance cardiaque (IC). En effet, la diminution de la signalisation de l'AMPc et l'augmentation de celle du GMPc est caractéristique des cœurs défaillants.Parmi la superfamille des PDEs, la PDE2 a la particularité d'être stimulée par le GMPc, conduisant ainsi à une augmentation remarquable de l'hydrolyse de l'AMPc. Ceci semble induire une interaction entre les voies de signalisation de l'AMPc et du GMPc. Cependant, le rôle de la PDE2 dans le cœur défaillant est très peu connu.Dans ce contexte, nous avons examiné si la PDE2 cardiaque est modifiée dans l'IC chez l'Homme et chez les modèles animaux d'IC, et déterminé le rôle de la PDE2 dans la signalisation β-adrénergique dans les cardiomyocytes. Grâce à l'utilisation de Western blot, de technique radioenzymatique, d'imagerie basée sur le FRET, de la planimétrie, de la microscopie à épifluorescence et des mesures du courant calcique de type L, réalisés sur les tissus myocardiques humains et/ou dans des cardiomyocytes isolés de cœurs des modèles animaux d'IC, respectivement, nous avons montré que l'expression et l'activité de la PDE2 sont augmentées dans les cœurs défaillants. Cette augmentation réduit l'effet d'une stimulation β-adrénergique aiguë, contribuant à la désensibilisation β-adrénergique observée dans l'IC. En accord avec ces résultats, la surexpression de la PDE2 dans des cardiomyocytes sains, réduit l'augmentation des taux d'AMPc et l'amplitude du courant ICa,L et abolit l'effet inotrope positif suite à une stimulation β-adrénergique aiguë, sans affecter la contractilité basale. Plus important, les cardiomyocytes surexprimant la PDE2, montrent une protection contre les réponses hypertrophiques induites par la noradrénaline et contre les arythmies induites par l'isoprotérénol.En conclusion, ce travail met en évidence l'altération de la PDE2 dans l'IC et nous laisse suggérer que l'augmentation de la PDE2 dans l'IC peut constituer un mécanisme de défense important dans des conditions de stress cardiaque, notamment en antagonisant la suractivation de la voie β-adrénergique. Ainsi, l'activation de PDE2 myocardique peut représenter une nouvelle stratégie thérapeutique anti-adrénergique intracellulaire dans l'IC.