Conselho Nacional de Desenvolvimento Científico e Tecnológico / In recent years, the isolation of bacteria producing β-lactamases has caused concern around the world, due to the fact these enzymes hydrolysis the ring β-lactam antimicrobials used in the main clinic. This aim of this study was asses the prevalence metallo-β-lactamases (MbL) in isolates of Pseudomonas aeruginosa and Acinetobacter baumannii obtained from patients admitted at the University Hospital of Santa Maria (HUSM). The profile of susceptibility for all isolates was evaluated by the disk diffusion method standardized by CLSI. The antimicrobial disks were distributed in a way that allows the identification of strains producers of AmpC and ESBL. For the identification of the producers of MbL the test of disk approximation with EDTA 0.1 M, EDTA 0,5M and acid 2-mercaptopropionic were performed. Isolates that did not have any of the mechanisms of resistance search were classified as multiresistant (MDR). The minimum inhibitory concentration (MIC) for ceftazidima, imipenem and polymyxin B was assessed by broth method microdilution for all isolated, according to CLSI. From January to June 2006, were obtained 32 isolates the P.aeruginosa and 41 the A. baumannii, the those 17 (23.29%) were β-lactamase AmpC-type producers, 11 (15.07%) were MbL producers, and 45 (61,64%) were classified as MDR. All strains producing MbL were Pseudomonas aeruginosa. The sensitivity of the isolates according to the CIM for antimicrobial evaluated were: 90,28% for polymyxin B, 36,11% for imipenem and 18% for ceftazidima. There was a high prevalence of MDR isolates and producers of β-lactamase-type AmpC and MbL in HUSM, this is extremely worrying once there is limiting therapy available. This situation becomes even more worrying with the find of isolates resistant the polymyxin B, witch is one of the last options of treatment for MDR isolates and producers of MbL. The detection of microorganisms is extremely important for the committees of infection hospital with the goal of preventing outbreaks, as well as guide the medical team on the conduct therapy, since there are few effective antimicrobial clinically for these pathogens and no prospects for development the new antimicrobial in the near future. / Nos últimos anos, o isolamento de bactérias produtoras de β-lactamases tem causado preocupação em todo o mundo, devido ao fato dessas enzimas hidrolisarem o anel β- lactâmico dos principais antimicrobianos utilizados na clínica. Este trabalho teve por objetivo avaliar a prevalência de metalo-β-lactamases (MbL) em isolados de Pseudomonas aeruginosa e Acinetobacter baumannii obtidos de pacientes atendidos no Hospital Universitário de Santa Maria (HUSM). O perfil de sensibilidade para todos os isolados foi avaliado pelo método de disco difusão padronizado pelo CLSI. Os discos de antimicrobianos utilizados foram distribuídos de forma que permitisse a identificação dos isolados produtores de AmpC e ESBL. Para a identificação dos produtores de MbL utilizou-se o teste de disco aproximação com os seguintes agentes quelantes: EDTA 0,1M, EDTA 0,5 M e ácido 2-mercaptopropiônico. Os isolados que não possuíam nenhum dos mecanismos de resistência pesquisados foram classificados como multirresistentes (MDR). A concentração inibitória mínima (CIM) para ceftazidima, imipenem e polimixina B foi avaliada pelo método de microdiluição em caldo para todos os isolados, de acordo com o CLSI. Durante o período de janeiro a junho de 2006 foram obtidos 32 isolados de P.aeruginosa e 41 de A. baumannii, destes 17 (23,29%) foram produtores de β-lactamase do tipo AmpC, 11 (15,07%) foram produtores de MbL e 45 (61,64%) foram classificados como MDR. Todas as cepas produtoras de MbL foram de Pseudomonas aeruginosa. A sensibilidade dos isolados de acordo com a CIM para os antimicrobianos avaliados foram as seguintes: 90,28% para polimixina B, 36,11% imipenem e 18% ceftazidima. Observou-se uma alta prevalência de isolados MDR no HUSM, além de isolados produtores de β-lactamase do tipo AmpC e MbL, o que é extremamente preocupante devido limitar a terapia a poucos antimicrobianos. Esta situação torna-se ainda mais preocupante com a detecção de isolados resistentes a polimixina B, a qual é uma das últimas opções de tratamento para infecções causadas por isolados de P. aeruginosa e Acinetobacter baumannii MDR e produtores de MbL. A detecção desses microrganismos é de grande importância para as comissões de controle de infecção hospitalar com o objetivo de prevenir surtos, bem como orientar a equipe médica sobre a conduta terapêutica, uma vez que há poucos antimicrobianos efetivos clinicamente para esses patógenos e as perspectivas para o desenvolvimento de novos antimicrobianos em um futuro próximo são mínimas.

Made available in DSpace on 2014-07-29T16:11:50Z (GMT). No. of bitstreams: 1 Dissertacao DAniella Ramos Martins.pdf: 914272 bytes, checksum: 038f97a7fcd95721aab346f4a3bd0587 (MD5) Previous issue date: 2012-02-28 / The inhibition of phosphodiesterases (PDEs) increases intracellular levels of cyclic nucleotides 3 ': 5'-cyclic adenosine monophosphate (cAMP) and 3 ': 5'-cyclic guanosine monophosphate (cGMP), which has many physiological and biochemical effects, especially in cardiovascular system. The objective of this study was to analyze the pharmacological effects of a new compound derived from pyrazole, LQFM 021, which was indicated by molecular modeling studies as a possible inhibitor of PDE-3. For this purpose, aortas were isolated of rats and mounted in organ baths for isometric tension recording of the relaxing effect of LQFM 021, in preparations pre-contracted with phenylephrine. We analyzed the involvement of the vascular endothelium, soluble guanylate cyclase (sGC) and adenylate cyclase (AC), the role of K+ channels and Ca2+, besides the contribution of Ca2+ uptake by the sarcoplasmic reticulum. As a result, was demonstrated that the LQFM 021 induces vascular relaxation (Emax: 54.9 ± 6.0%), being this relaxation potentiated by endothelium (Emax: 88.1 ± 2.1%). The inhibition of AC with MDL-12.330A (10 μM) or of the sGC with ODQ (1 μM) reduced the relaxation of 88.1 ± 2.1%, to 48.35 ± 3.01% and 19.95 ± 2.32%, respectively. The pre-contraction with KCl 45 mM or treatment of preparations with TEA (5 mM), reduced almost completely the relaxing effect of the compound. Inhibition of Ca2+ / ATPase reticular with CPA (10 mM) reduced the relaxation stimulated by 021 LQFM approximately 66.5%. Concentration-response curve contractile induced by phenylephrine (0.1 nM to 1 μM) or by CaCl2 (0-3 mM, zero-calcium + phenylephrine) were reduced by pretreatment of preparations with LQFM 021 (EC50). In conclusion, this study showed that the new synthetic derivative of pyrazole LQFM 021 is a potential inhibitor of PDE-3 and has vasorelaxant activity. The endothelium potentiates the relaxation stimulated by the compound. The route of sGC and AC are involved in the mechanism of action of LQFM 021. Was also evidenced by participation from sarcoplasmatic reticulum, well as the flow of K+ and Ca2+ through the cell membrane. / A inibição das fosfodiesterases (PDEs) aumenta os níveis intracelulares de nucleotídeos cíclicos 3' : 5'-monofosfato cíclico de adenosina (AMPc) e 3' : 5'-monofosfato cíclico de guanosina (GMPc), os quais tem muitos efeitos fisiológicos e bioquímicos, sobretudo no sistema cardiovascular. O objetivo deste estudo foi analisar os efeitos farmacológicos de um novo composto derivado do pirazol, LQFM 021, o qual foi apontado por estudos de modelagem molecular como possível inibidor de PDE-3. Para tanto, artérias aortas de ratos foram isoladas e montadas em banhos de órgão para registro da tensão isométrica do efeito relaxante do LQFM 021, em preparações pré-contraídas com fenilefrina. Foi analisada a participação do endotélio vascular, da guanilato ciclase solúvel (GCs) e da adenilato ciclase (AC), o papel dos canais de K+ e de Ca2+, além da contribuição da captação de Ca2+ pelo retículo sarcoplasmático. Como resultado, foi demonstrado que o LQFM 021 induz relaxamento vascular (Emax: 54.9 ± 6.0%), sendo este relaxamento potencializado pelo endotélio (Emax:88.1 ± 2.1%). A inibição da AC com MDL-12.330A (10 μM) ou da GCs com ODQ (1 μM), reduziram o relaxamento de 88,1 ± 2,1%, para 48,35 ± 3,01% e 19,95 ± 2,32%, respectivamente. A pré-contração com KCl 45 mM ou o tratamento das preparações com TEA (5 mM), reduziram quase que por completo o efeito relaxante do composto. A inibição da Ca2+/ATPase reticular com CPA (10 μM), reduziu o relaxamento estimulado pelo LQFM 021 em aproximadamente 66,5%. Curva concentração-resposta contrátil induzida pela fenilefrina (0,1 nM a 1 μM) ou pelo CaCl2 (0 a 3 mM, em meio zero-cálcio + fenilefrina) foram reduzidas pelo pré-tratamento das preparações com LQFM 021 (EC50). Em conclusão, este estudo mostrou que o novo derivado sintético de pirazol LQFM 021 é um possível inibidor de PDE-3 e possui atividade vasorelaxante. O endotélio participa e potencializa o relaxamento estimulado pelo composto. A via da GCs e AC estão envolvidas no mecanismo de ação do LQFM 021. Também foi evidenciada a participação do retículo sarcoplasmático, bem como o fluxo de K+ e de Ca2+ através da membrana celular.

A maturação in vitro do oócito é um dos fatores limitantes na produção in vitro de embriões. In vivo, esta maturação é um processo altamente orquestrado no qual a meiose é retomada pela onda de gonadotrofina que antecede a ovulação e que induz à queda dos níveis de AMPc no oócito. No entanto, os oócitos aspirados ao serem retirados dos folículos ovarianos retomam espontaneamente a maturação comprometendo a competência de seu desenvolvimento. O AMPc é sintetizado pela adenilato ciclase (AC) e degradado pelas fosfodiesterases (PDE), existindo algumas relacionadas à degradação do AMPc e outras do GMPc. Sendo assim, a proposição deste trabalho foi averiguar a contribuição de diferentes isoformas de fosfodiesterases na retomada da meiose e nos níveis de GMPc, AMPc e ainda, determinar quando há manutenção de AMPc em níveis elevados observando sua influência na competência oocitária e ativação da MAPK. Para isso, os complexos cumulus-oócito (CCOs) foram maturados in vitro na ausência, presença ou associação de inibidores de PDEs-AMPc e GMPc específicas e FSHr. As amostras foram avaliadas em relação a: 1) taxa de maturação; 2) níveis intracelulares de AMPc e GMPc nos CCOs; 3) taxa de desenvolvimento de blastocistos ; 4) ativação da MAPK em oócitos e células do cumulus. Os resultados obtidos no primeiro experimento indiaram que o inibidor da PDE3 foi o mais eficaz (p<0,05) em atrasar a retomada da meiose, às nove horas de maturação, porém, isolado ou em associação com o inibidor da PDE8, não foi capaz de alterar (p>0,05) os níveis de AMPc. No experimento dois, o inibidor da PDE5 isolado não influenciou a retomada da meiose (p>0,05), porém, quando associado aos inibidores da PDE3 e 8 houve atraso na retomada (p<0,05) e ainda alteraram os níveis de GMPc e AMPc (p<0,05) nas primeiras horas de maturação. O experimento três mostrou a influencia do FSHr durante a MIV, o qual estimulou a retomada da meiose, mas em associação com inibidores da PDE5 e 8 atrasa a retomada (p<0,05). Além disso, o FSHr provoca aumento do nível de AMPc e sua associação com inibidores de PDE5 e PDE8 ocasionou elevação adicional (p<0,05). As condições de cultivo estudadas no experimento quatro mostraram que a maturação induzida (pré-MIV de duas horas com agentes para elevar AMPc seguindo de 22 horas de MIV com FSH associado a inibidores de PDEs) atrasaram a retomada da meiose às nove horas de maturação, mas não afetaram progressão da meiose às 24, 28 e 30 horas. Os tratamentos, porém, não melhoraram a competência oocitária após a fertilização in vitro e ocasionaram pequenas variações na ativação da MAPK em oócitos e células do cumulus. / In vitro maturation of oocytes is a limiting factor in the in vitro production of bovine embryos. In vivo, this maturation is a highly orchestrated process in which meiosis resumption by the gonadotropin surge that precedes ovulation induces the decrease in cAMP levels in the oocyte. However, when oocytes are removed from follicles, they spontaneously resume maturation compromising the competence for its development. cAMP is synthesized by adenylyl cyclase (AC) and degraded by phosphodiesterases (PDE), and there are some PDEs related to degradation of cAMP and/or cGMP. Thus, the purpose of this work was to investigate the contribution of different isoforms of phosphodiesterases in the resumption of meiosis and levels of cAMP and, also, to determine differences in signaling pathways when maintaining high levels of cAMP and its influence on oocyte competence. For this purpose, cumulus-oocyte complexes (COC) were matured in vitro in the presence, absence or combination of inhibitors of cAMP- and cGMP-specific PDEs and FSH. Samples be were evaluated in relation to: 1) maturation rate, 2) intracellular levels of cAMP and cGMP in COCs, 3) rate of blastocyst development and 4) activation of MAPK in oocytes and cumulus cells. The results of the first experiment showed that the PDE3 inhibitor is more effective (p <0.05) in delaying meiosis resumption, at nine hours of maturation, but was not capable of altering cAMP levels (p> 0.05) either alone or in combination with the PDE8 inhibitor. In experiment two, the PDE5 inhibitor alone did not affect the meiosis resumption (p> 0.05), however, when associated with PDE3 and PDE8 inhibitors it delayed their resumption (p <0.05) and also altered cGMP and cAMP levels of (p <0.05) in the early hours of maturation. The third experiments showed the influence of FSHr during IVM, which stimulated the resumption of meiosis, but in combination with PDE5 and PDE8 inhibitors meiosis was delayed (p <0.05). Furthermore, FSHr causes increased levels of cAMP and its association with PDE5 and PDE8 inhibitors caused an additional increase (p <0.05). Culture conditions studied in experiment four showed that induced maturation (pre-IVM for two hours with agents to elevate cAMP followed by 22 hours IVM with FSH associated with PDE inhibitors) delayed the resumption of meiotic maturation at nine hours, but has no effect on meiosis progression at 24, 28 and 30 hours. The treatments, however, did not improve oocyte competence after in vitro fertilization and caused minor variations in the activation of MAPK in oocytes and cumulus cells.

La glucotoxicité, ou exposition prolongée à de hautes teneurs en glucose, altère la fonction des cellules ß-pancréatiques et participe au développement du diabète. Il a été démontré que dans les cellules ß, la glucotoxicité engendre des modifications de l’expression génique, des altérations des voies de signalisation Ca2+-dépendantes et de l’exocytose ainsi qu’une augmentation de l’apoptose. Les mécanismes moléculaires responsables de ces altérations sont encore peu connus, mais ces observations suggèrent que des changements du profil d’expression génique des cellules ß-pancréatiques en sont à l’origine. Afin de mieux comprendre les conséquences de la glucotoxicité, une étude génomique a été menée dans la lignée de cellules ß-pancréatiques INS-1E. Cette étude a révélé l’existence de variations significatives des taux d’expression de plusieurs gènes importants pour la fonction des cellules ß, codant pour des protéines impliquées notamment dans le métabolisme glucidique et les différentes étapes de la voie de sécrétion d’insuline. D’autre part, cette approche a également révélé de profonds changements dans les voies de signalisation dépendantes de l’AMPc. Si le rôle prédominant du Ca2+ dans la régulation de la voie de sécrétion de l’insuline a été mis en évidence et bien caractérisé, l’implication et l’importance de l’AMPc dans ce processus restent mal définies. L’AMPc, au même titre que le Ca2+, module l’activité de nombreuses protéines de signalisation, régule l’expression génique et intervient également dans le trafic vésiculaire et la sécrétion d’insuline. De manière intéressante, l’expression de l’adénylate cyclase 8 (ADCY8) est fortement diminuée en condition de glucotoxicité. Ceci suggère qu’un défaut de synthèse d’AMPc pourrait être à l’origine du remaniement des voies de signalisation impliquées dans la régulation de la sécrétion d’insuline. Nous avons donc décidé d’étudier, plus en profondeur, les conséquences fonctionnelles de la diminution de l’expression de l’ADCY8 sur ces voies. Nos résultats suggèrent que l’ADCY8, une isoforme peu exprimée dans les cellules ß- pancréatiques? et stimulée par le Ca2+, se trouve au carrefour des voies de signalisation activées par le glucose et le GLP-1. La diminution de son expression est ainsi partiellement responsable des effets induits par la glucotoxicité sur la régulation de la sécrétion d’insuline. D’autre part, plusieurs résultats récents suggèrent l’implication des voies de signalisation AMPc-dépendantes dans la protection des cellules ß contre l’apoptose et dans ce contexte, le rôle de l’ADCY8 dans ces cellules a été abordé. / Glucotoxicity, or prolonged exposure to elevated levels of glucose, alters the function of pancreatic??-cells and is involved in diabetes pathogenesis. It has been demonstrated that glucotoxicity modifies gene expression and induces considerable changes in [Ca2+]i and in cAMP-dependent signalling (Dubois et al, Endocrinology, 148(4):1605-14 ; 2007) as well as a it decreases insulin exocytosis in response to glucose and increases apoptosis. The molecular mechanisms of these effects are not known but several observations suggest that changes in gene expression profiles are involved. To address that, a genomic study has been done in the clonal b-cell line INS-1E and revealed important modifications in the expression rates of many genes involved in glucose metabolism and vesicular traffic. This approach also revealed the alteration of cAMP-mediated signalling pathways and as the role of calcium and the importance of the correlation between cAMP and Ca2+-mediated signalling pathways had been shown, it was interesting to address the role of this second messenger in this process. Actually, cAMP regulates the activity of a large number of signalling proteins, it is also an important messenger involved in vesicular traffic, insulin secretion and gene expression. Interestingly, we also found that the expression of the adenylyl cyclase VIII (ADCY8) was largely diminished by glucotoxicity and this suggests that an alteration of cAMP synthesis could be involved in the decrease of insulin secretion in this condition. For this reason, we decided to address the functional consequences of altered ADCY8 expression on cAMP-mediated signalling pathways and on its correlation with the decrease of insulin secretion in glucotoxicity. Our results demonstrate a requirement for ADCY8 in glucose as well as in GLP-1 activated signalling pathways and strongly suggest a central role for ADCY8 in glucotoxicity. Moreover, recent publications suggest the implication of cAMP-mediated signalling pathways in the protection of b-cells against apoptosis induced by glucotoxicity, and the role of ADCY8 in this process was investigated.

L’AMP cyclique (AMPc) et le GMP cyclique (GMPc) sont des seconds messagers essentiels pour la régulation de la fonction cardiaque. La concentration de l’AMPc intracellulaire est régulée par les activités d'au moins deux familles d'enzymes: les cyclases et guanylyl cyclases, responsable de la synthèse de l'AMPc et du GMPc, et les phosphodiestérases (PDE) qui interviennent dans l’hydrolyse de l'AMPc et du GMPc.Parmi la superfamille des PDEs, la PDE2 est une enzyme à double substrat qui hydrolyse à la fois l'AMPc et le GMPc et a la propriété unique d'être stimulée par le GMPc. Il a été récemment montré que la PDE2 du myocarde est augmentée dans l'insuffisance cardiaque humaine et expérimentale (IC), tandis que d'autres (par exemple PDE3 et PDE4) sont réduites. Cependant, les conséquences physiopathologiques de l'activité PDE2 renforcée dans le cœur sont inconnues.Dans ce contexte, nous avons généré des souris transgénique (TG) avec une surexpression spécifique cardiaque de l’isoforme PDE2A3 (souris PDE2 TG).Grace à l’utilisation de Western blot et de dosage radioenzymatique nous avons montré que l'AMPc cardiaque et l'activité PDE cGMP et l'activité spécifique de PDE2 sont fortement augmentées dans les PDE2 TG par rapport à des souris de type sauvage (WT).Le raccourcissement cellulaire, les transitoires calciques et le courant calcique de type L (ICa, L) ont été enregistrés dans les myocytes ventriculaires adultes de souris WT et PDE2 TG et l'isoprénaline (ISO) a été utilisée pour examiner et comparer la réponse β-adrénergique (β-AR) de ces paramètres. Nous avons montré que lors de la stimulation β-AR, la contractilité cellulaire, la transitoire Ca2+ et l’amplitude du courant ICa,L sont fortement diminués. En conséquence, la surexpression de la PDE2 dans les cardiomyocytes a réduit les taux d'AMPc et abolit l'effet inotrope après une stimulation β-AR aiguë. L'ECG mesuré par télémétrie chez la souris PDE2 TG a montré une réduction marquée de la fréquence cardiaque au repos ainsi que de la fréquence cardiaque maximale, tandis que le débit cardiaque a est entièrement préservé en raison d'une contractilité plus forte. Fait important, les souris TG PDE2 sont résistantes à des arythmies ventriculaires déclenchées et à des arythmies induites par isoprénaline.En conclusion, ce travail montre que PDE2 joue un rôle essentiel dans la régulation du couplage excitation-contraction cardiaque. La surexpression de PDE2 semble protéger les cardiomyocytes contre une stimulation excessive β-AR et réduit le risque d'arythmie lors de l'activation sympathique.L’activation de la PDE2 peut donc représenter une nouvelle stratégie thérapeutique anti-adrénergique et anti-arythmique subcellulaire dans l’insuffisance cardiaque. / Cyclic AMP (cAMP) and cyclic GMP (cGMP) are critical second messengers for the regulation of cardiac function. Intracellular cAMP concentration is regulated by the activities of at least two families of enzymes: adenylyl and guanylyl cyclases, responsible for cAMP and cGMP synthesis and cyclic nucleotide phosphodiesterases (PDEs) that mediate cAMP and cGMP hydrolysis.Among the PDE superfamily, PDE2 is a dual substrate enzyme that hydrolyzes both cAMP and cGMP and has the unique property to be stimulated by cGMP. It was recently showed that myocardial PDE2 is increased in human and experimental heart failure (HF), while other PDEs (e.g. PDE3 and PDE4) are reduced. However, the pathophysiological consequences of enhanced PDE2 activity in the heart are unknown.In this context, we generated a transgenic (TG) mouse with a heart specific overexpression of the PDE2A3 isoform (PDE2 TG mouse). Using immunoblotting and radioenzymatic assay we showed that total cardiac cAMP and cGMP PDE activity and specific PDE2 activity was strongly increased in PDE2 TG compared to wild type (WT) mice. Sarcomere shortening, Ca2+ transients and the whole L-type Ca2+ current (ICa,L) were recorded in adult ventricular myocytes from WT and PDE2 TG mice and isoprenaline (ISO) was used to examine and compare the β-adrenergic (β-AR) response of these parameters. We showed that upon β-AR stimulation, cell contractility, Ca2+ transient and ICa,L were severely blunted. Accordingly, PDE2 overexpression in cardiomyocytes reduced the cAMP levels and abolished the inotropic effect following acute β-AR stimulation. ECG telemetry in PDE2 TG mice showed a marked reduction in resting as well as in maximal heart rate, while cardiac output was completely preserved due to greater contractility. Importantly, PDE2 TG mice were resistant to triggered ventricular arrhythmias and to isoprenaline-induced arrhythmias.In conclusion, this work demonstrates that PDE2 plays a critical role in the regulation of cardiac excitation-contraction coupling. PDE2 overexpression appears to protect the cardiomyocytes against excessive β-AR drive and reduces the risk of arrhythmias during sympathetic activation. PDE2 activation may thus represent a new subcellular anti-adrenergic and anti-arrhythmic therapeutic strategy in HF.

Dans le cœur, l’activation aiguë de la voie AMPc/PKA via la stimulation des récepteurs β-adrénergiques (β-ARs) permet de réguler la contraction cardiaque alors que l’activation chronique de cette voie est délétère, car elle est source de survenue d’arythmies cardiaques et de remodelage hypertrophique du cœur. Au niveau des cardiomyocytes, Il existe principalement deux sous-types de récepteurs β-ARs ; β1- et β2-ARs, qui exercent des effets différents sur la fonction cardiaque.Dans une première partie de ma thèse, je me suis intéressé à l’étude du rôle des récepteurs β1- et β2-ARs dans la régulation différentielle de l’activité PKA cytoplasmique et nucléaire. J’ai ainsi pu montrer que contrairement aux récepteurs β1-ARs qui ont la capacité d’activer la PKA au niveau du cytoplasme et aux noyaux, les récepteurs β2-ARs activent la PKA uniquement au niveau du cytoplasme, et ce indépendamment de la capacité des récepteurs β2-ARs à induire une augmentation des niveaux d’AMPc dans les noyaux. En accord avec ces résultats, les récepteurs β1- mais pas β2-ARs activent le facteur pro-apoptotique régulé par la PKA, ICER.Dans une seconde partie de ma thèse, je me suis intéressé aux différents mécanismes responsables de l’incapacité des récepteurs β2-ARs à activer la PKA au niveau des noyaux. Mes résultats soulignent le rôle de la localisation des récepteurs β2-ARs au niveau des cavéoles, leurs couplage aux protéines Gi, leurs désensibilisation par la GRK2 ainsi que la dégradation de l’AMPc généré par ces récepteurs par la PDE3 et 4 dans la régulation de la signalisation PKA cytoplasmique et pointent vers la PDE4 comme un régulateur central permettant de limiter l’activation de la PKA holoenzyme responsable des réponses PKA nucléaires. Mes résultats montrent également que la mAKAP est un élément clé dans la transduction de la signalisation PKA nucléaire induite par les récepteurs β2-ARs et à un moindre degré, les récepteurs β1-ARs. Dans la dernière partie de ma thèse, j’ai étudié le remodelage de la signalisation PKA nucléaire induite par les récepteurs β1- et β2-ARs au cours de l’insuffisance cardiaque. J’ai ainsi pu montrer qu’en plus de la diminution de la signalisation PKA nucléaire induite par les récepteurs β1-ARs, il existe une signalisation PKA nucléaire de novo induite par les récepteurs β2-ARs dans les cardiomyocytes de rat adulte insuffisants.En conclusion, ce travail a mis à jour une nouvelle différence entre les récepteurs β1- et β2-ARs dans la signalisation PKA au niveau des noyaux des cardiomyocytes de rat adultes, et souligne le rôle important de la PDE4 et de la mAKAP dans la régulation de la signalisation PKA nucléaire induite par les récepteurs β2-ARs. / In the heart, acute activation of the cAMP/PKA pathway upon stimulation of β-adrenoceptors (β-ARs), plays a fundamental role in the regulation of cardiac function, whereas chronic activation of this pathway is deleterious, as it is responsible for cardiac arrhythmias and hypertrophic remodeling of the heart. In cardiac myocytes, there are mainly two subtypes of β-ARs: β1- and β2-ARs, which exert different effects on cardiac function.In the first part of my thesis, my work was focused on understanding the role of β1- and β2-ARs in the differential regulation of cytoplasmic and nuclear PKA activity. Hence, I have showed that unlike β1-ARs which have the capacity to induce the activation of PKA in the cytoplasm and the nucleus, β2-ARs induce the activation of PKA only in the cytoplasmic compartment, regardless of their ability to induce an increase in cAMP in the nuclei. Consistently, β1- but not β2-ARs were able to induce the activation of the pro-apoptotic factor regulated by PKA, ICER.The second aim of my thesis was to decipher the different mechanisms involved in the inability of β2-ARs to activate PKA in the nucleus. I concentrated my efforts on investigating the role of the localization of β2-ARs in caveolae, their coupling to Gi proteins, their desensitization by GRK2 as well as the hydrolysis of cAMP by PDE3 and 4 in the regulation of β2-AR-induced cytoplasmic PKA activity. My results point to PDE4 as a central regulator which limits the activation of the PKA holoenzyme pool involved in the nuclear PKA responses. My results also show that mAKAP is a key component of nuclear PKA signaling induced by β2-ARs and to a lesser extent by β1-ARs. In the last part of my thesis, I have studied the remodeling of nuclear PKA signaling induced by β1- and β2-ARs that occurs during heart failure. I showed that, besides a decrease in β1-AR-induced nuclear PKA signaling, there is a de novo β2-AR-induced nuclear PKA signaling in cardiomyocytes from rat with heart failure.In conclusion, this work uncovers a new difference in PKA signaling between β1- and β2-ARs at the nuclear compartment of adult rat cardiomyocytes and underlines the importance of PDE4 and mAKAP in the regulation of β2-AR-induced nuclear PKA signaling.

Nous avons souhaité, dans ce travail de Thèse, préciser l'impact de l'invalidation d'AIP sur la fonction sécrétoire et la prolifération des cellules somatotropes in vivo et explorer in vitro les voies de signalisation potentiellement impliquées dans la tumorigenèse hypophysaire AIP-dépendante. L'analyse du phénotype des souris Aip+/-, en particulier de la sécrétion pulsatile de GH, montre que, contrairement à l'Homme, les animaux mutés ne développent pas de gigantisme ni d'hypersécrétion de GH et que la pénétrance de la pathologie tumorale hypophysaire est beaucoup plus faible qu'initialement décrit. Les études réalisées sur les fibroblastes de patients mutés pour AIP et porteurs d'un adénome hypophysaire ainsi que sur les cellules somatolactotropes de rat GH3 révèlent que les mutations d'AIP altèrent l'activité transcriptionnelle d'AhR (Aryl hydrocarbon Receptor), mais affectent également la voie de signalisation de l'AMPc. Enfin, des mutations germinales du gène GPR101, récemment identifiées dans des cas d'acromégalie sporadique, ont aussi été trouvées chez des patients porteurs d'un adénome hypophysaire sporadique non somatotrope, sans association avec les mutations d'AIP. Ce travail a ainsi permis de préciser les conséquences des mutations d'AIP sur la fonction somatotrope et la signalisation d'AhR. Le rôle de l'AMPc dans la tumorigenèse hypophysaire AIP-dépendante sera évalué dans un nouveau modèle de souris transgénique. / In this work, we investigated the effects of AIP deficiency in vivo on somatotroph cells, both at the secretory and proliferative levels and explored in vitro the signaling pathways potentially involved in AIP-dependent pituitary tumorigenesis. Phenotype analyzes of Aip+/- mice, especially of GH pulsatility, show that, unlike humans, mutant mice do not develop gigantism nor GH hypersecretion and present with a much lower penetrance of pituitary adenomas than initially described. In vitro studies in fibroblasts of AIP-mutation positive patients with pituitary adenomas and in somatolactotroph GH3 cells demonstrate that AIP mutations alter AhR (Aryl hydrocarbon Receptor) transcriptional activity and modify the cAMP pathway. Finally, GPR101 mutations, recently reported in patients with sporadic acromegaly, have also been identified in a small portion of patients with sporadic non-somatotroph pituitary adenomas, without any association with AIP mutations. This research work defines the consequences of AIP mutations on somatotroph cell function and AhR transcriptional activity. The role of cAMP signaling in AIP-related pituitary tumorigenesis will be further evaluated in a new transgenic mouse model.

Gene expression of porin and beta-lactamases genes during the beta-lactam antibiotic treatment and effect of inoculum size on Klebsiella pneumoniae clinical isolates ABSTRACT In recent years, Klebsiella pneumoniae has been increasingly reported to be one of the most important nosocomial pathogens, and it is usually resistant to many antibiotics. In this work, we focused on the expression of the AmpC group β- lactamase DHA-1 and its negative regulator AmpR, as well as the porins OmpK35 and OmpK36 and on effect of inoculum. We used well-characterized Klebsiella pneumoniae strains in this study. Plasmids obtained from these strains were also transformed into different wild-type Klebsiella pneumoniae strains, which were typed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Gene expression analysis was performed by RT-PCR using specific primers and TaqMan probes. In most strains, expression was dependent on the presence of an inducer. The highly resistant strain showed a different expression pattern, but the expression of blaDHA-1 remained inducible by cefoxitin. Different regulation was also observed in the transformants. Based on our data, we suggest that the previously described regulatory pathway for AmpC is not generally suitable, and we propose that there are more...

Une stimulation β-adrénergique (β-AR) aigue, par exemple au cours d’un exercice physique, accroît le second messager AMPc dans les cardiomyocytes aboutissant à une cascade d’évènements permettant d’augmenter la fonction cardiaque. Une élévation chronique des taux de catécholamines est délétère puisqu’elle participe au remodelage pathologique du cœur et à la progression vers l’insuffisance cardiaque (IC). L'IC correspond à l'incapacité du cœur à répondre aux besoins hémodynamiques de l'organisme. Si la majorité des patients meurt de défaillance cardiaque, une part importante décède d'arythmies.Les phosphodiestérases (PDEs) sont des enzymes essentielles puisqu’elles permettent non seulement la terminaison des signaux AMPc en dégradant ce nucléotide cyclique en 5’AMP inactif mais aussi l’organisation spatiale de ces signaux dans des compartiments subcellulaires spécifiques. L'IC s'accompagne de profonds remaniements de la voie β-AR et l'expression des PDEs est modifiée en conditions pathologiques, perturbant ainsi la compartimentation intracellulaire de l’AMPc. Il a été notamment démontré que l’expression d’une isoforme de PDE particulière, la PDE4B, diminue dans l'hypertrophie cardiaque et que l’invalidation du gène codant pour celle-ci favorise les arythmies ventriculaires chez la souris lors d’une stimulation β-AR. À l’inverse, l'expression d'une autre enzyme, la PDE2A, est augmentée dans l’IC, chez l’homme et différents modèles animaux. Ceci constituerait un mécanisme de défense lors d'un stress cardiaque puisqu’il a été montré que sa surexpression atténue l’hypertrophie induite par la noradrénaline ou la phényléphrine et limite les arythmies chez la souris.L’objectif de mon travail était de tester l’hypothèse qu’une augmentation de l’activité des PDEs pourrait constituer une alternative aux traitements classiques de l’IC, pour limiter le remodelage hypertrophique, la progression vers l’IC et les arythmies associées. Pour cela, j’ai réalisé une thérapie génique dans des modèles murins d'IC grâce à des virus adéno-associé de type 9 (AAV9) codant pour la PDE4B ou la PDE2A. Mes résultats suggèrent que cette approche pourrait constituer une nouvelle stratégie thérapeutique prometteuse de l'IC en limitant le dysfonctionnement cardiaque, l’hypertrophie du ventricule gauche, et la survenue des arythmies ventriculaires mais seulement lorsque la PDE2A est surexprimée. / Acute stimulation of β-adrenergic receptors (β-ARs), for example during physical activity, leads to the synthesis of the second messenger cAMP in cardiomyocytes, which triggers a cascade of events leading to the increase of cardiac function. While acute β-AR stimulation is beneficial to the heart, chronic β-AR activation is detrimental because it promotes cardiac remodeling and ultimately leads to heart failure (HF). HF is defined by the heart's inability to overcome hemodynamic needs of the body. While the majority of patients die of worsening heart function, a significant proportion dies suddenly of cardiac arrhythmias.Phosphodiesterases (PDEs) are crucial enzymes since they allow not only to terminate cAMP signals by degrading this second messenger into inactive 5’AMP but permit their spatial organization in subcellular compartments. HF is accompanied by profound rearrangements of the β-AR pathway and the expression of PDEs is modified under pathological conditions, thus disrupting cAMP intracellular compartmentation. The expression of one of these enzymes, PDE4B, is decreased in cardiac hypertrophy and the invalidation of the gene encoding PDE4B promotes ventricular arrhythmias under β-AR stimulation in mice. Conversely, the expression of another enzyme, PDE2A, is up-regulated in human and animal models of HF which may constitute an important defense mechanism during cardiac stress since its overexpression attenuates hypertrophy induced by norepinephrine or phenylephrine and limits cardiac arrhythmias.The purpose of my work was to test the hypothesis that an increase of PDE activity could constitute an alternative to conventional HF treatments to limit cardiac remodeling, HF progression and associated arrhythmias. To do so, I performed a cardiac gene therapy in mouse models of HF using serotype 9 adeno-associated viruses (AAV9) encoding for PDE4B or PDE2A. My results suggest that this approach may be a promising new therapeutic strategy during HF by limiting cardiac dysfunction, left ventricular hypertrophy, and could protect ventricular arrhythmias only when PDE2A is overexpressed.

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.