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L’ocytocine (OT) est un neuropeptide synthétisé au sein de l’hypothalamus. On sait aujourd’hui que l’OT est fortement impliquée dans la modulation de nombreux comportements et émotions. Pourtant, il reste encore difficile d’expliquer comment s’organise le système ocytocinergique, par exemple en sous-ensembles spécifiques. De même, les circuits neuronaux impliqués dans leur recrutement restent obscures, tout comme leur potentielle plasticité. C’est pourquoi, au cours de ma thèse, je me suis attachée à mieux comprendre ces différents points. Les résultats obtenus ont montré que i) un sous-ensemble spécifique de neurones OT est recruté par la peur ; ii) le système OT fait preuve d’une grande plasticité après une exposition à un contexte effrayant ; iii) le neuropeptide S est capable de recruter une sous-population de neurones OT afin d’exercer une action anxiolytique ; iv) les neurotransmetteurs monoaminergiques sont eux-mêmes capables de recruter différents sous-ensembles de neurones OT. En conclusion, mon travail a mis en évidence la plasticité de ce système peptidergique et différentes manières de recruter de manière spécifiques certains sous-ensembles existants de neurones OT. / Oxytocin (OT) is a peptide synthesized within the hypothalamus. We now know that OT is strongly involved in the modulation of many behaviors and emotions. However, it is still difficult to explain how the oxytocinergic system is organized, for example in specific sub-populations. Similarly, the neuronal circuits involved in their recruitment remain obscure, like their potential plasticity. That is why, during my thesis, I tried to better understand these different points. The results obtained showed that i) a specific sub-population of OT neurons is recruited by fear; ii) the OT system exhibits great plasticity after exposure to a scary context; iii) the neuropeptide S is able to recruit an OT neuron sub-population in order to exert an anxiolytic effect; iv) monoaminergic neurotransmitters are themselves able to recruit different sub-populations of OT neurons. In conclusion, my work has highlighted the plasticity of this peptidergic system and different ways to recruit in a specific manner some existing sub-populations of OT neurons.

Made available in DSpace on 2014-12-17T15:37:19Z (GMT). No. of bitstreams: 1 ManaraBBCD_DISSERT.pdf: 1554643 bytes, checksum: e4b820c02db19b42acca3889a1b08851 (MD5) Previous issue date: 2014-04-25 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Neuropeptide S (NPS) is an endogenous 20-aminoacid peptide which binds a G protein-coupled receptor named NPSR. This peptidergic system is involved in the modulation of several biological functions, such as locomotion, anxiety, nociception, food intake and motivational behaviors. Studies have shown the participation of NPSR receptors in mediating the hyperlocomotor effects of NPS. A growing body of evidence suggests the participation of adenosinergic, dopaminergic and CRF systems on the hyperlocomotor effects of NPS. Considering that little is known about the role of dopaminergic system in mediating NPS-induced hyperlocomotion, the present study aims to investigate the locomotor actions of intracerebroventricular (icv) NPS in mice pretreated with α-metil-p-tirosine (AMPT, inhibitor of dopamine synthesis), reserpine (inhibitor of dopamine vesicle storage) or sulpiride (D2 receptor antagonist) in the open field test. A distinct group of animals received the same pretreatments described above (AMPT, reserpine or sulpiride) and the hyperlocomotor effects of methylphenidate (dopamine reuptake inhibitor) were investigated in the open field. NPS and methylphenidate increased the mouse locomotor activity. AMPT per se did not change the locomotion of the animals, but it partially reduced the hyperlocomotion of methylphenidate. The pretreatment with AMPT did not affect the psychostimulant effects of NPS. Both reserpine and sulpiride inhibited the stimulatory actions of NPS and methylphenidate. These findings show that the hyperlocomotor effects of methylphenidate, but not NPS, were affected by the pretreatment with AMPT. Furthermore, methylphenidate- and NPS-induced hyperlocomotion was impaired by reserpine and sulpiride pretreatments. Together, data suggests that NPS can increase locomotion even when the synthesis of catecholamines was impaired. Additionally, the hyperlocomotor effects of NPS and methylphenidate depend on monoamines vesicular storaged, mainly dopamine, and on the activation of D2 receptors. The psychostimulant effects of NPS via activation of dopaminergic system display clinical significance on the treatment of diseases which involves dopaminergic pathways, such as Parkinson s disease and drug addiction / Neuropept?deo S (NPS) ? um pept?deo end?geno formado por 20 amino?cidos e ? o ligante de um receptor acoplado ? prote?na G chamado NPSR, o qual est? envolvido na modula??o de v?rias fun??es biol?gicas centrais como locomo??o, ansiedade, nocicep??o, ingest?o de alimento e comportamentos motivacionais. J? ? conhecido que o efeito hiperlocomotor do NPS ? mediado pelos receptores NPSR e parece depender da ativa??o do sistema adenosin?rgico, dopamin?rgico e do sistema peptid?rgico do CRF. Considerando o pouco conhecimento acerca do envolvimento do sistema dopamin?rgico na media??o do aumento da atividade locomotora induzido pelo NPS, o presente estudo objetiva investigar as a??es motoras da administra??o intracerebroventricular (icv) de NPS em camundongos pr?-tratados com α-metil-p-tirosina (AMPT, inibidor da enzima de s?ntese de dopamina), reserpina (inibidor do armazenamento da dopamina em ves?culas) ou sulpiride (inibidor de receptores D2 de dopamina), em animais submetidos ao teste de atividade locomotora no campo aberto. Camundongos Swiss machos (30-35 g) foram submetidos ? cirurgia estereot?xica para a implanta??o de uma c?nula-guia no ventr?culo lateral. No 3? dia ap?s a cirurgia, os animais foram pr?-tratados com AMPT (250 mg/kg, ip, 24 h antes do teste), reserpina (2 mg/kg, SC, 24h) ou sulpiride (25 mg/kg, ip, 45 min) e depois foram tratados com NPS (1 nmol, 2 μl; icv, 5 min) e submetidos ao teste do campo aberto. Para fins de compara??o, um grupo distinto de animais recebeu os mesmos pr?-tratamentos acima descritos (AMPT, reserpina ou sulpiride) e o efeito hiperlocomotor do metilfenidato (5 mg/kg, sc, 15 min; inibidor da recapta??o de dopamina) foi investigado no campo aberto. O teste do campo aberto avalia a locomo??o espont?nea dos animais atrav?s da dist?ncia percorrida (m) e do tempo de imobilidade (m) durante 60 min. O NPS aumentou a atividade locomotora dos animais na dose de 1 nmol. O AMPT per se n?o causou altera??o na locomo??o dos animais. Por outro lado, o AMPT reduziu parcialmente o efeito hiperlocomotor do metilfenidato, mas n?o foi capaz de afetar a a??o hiperlocomotora do NPS. Tanto o pr?-tratamento com reserpina como o com sulpiride foram capazes de inibir o efeito estimulat?rio do NPS, assim como o do metilfenidato. Estes achados mostram que o efeito hiperlocomotor do metilfenidato, mas n?o do NPS, foi afetado pela administra??o de AMPT. Al?m disso, tanto o efeito do metilfenidato quanto o do NPS foram prejudicados pelos pr?-tratamentos com reserpina e sulpiride. Em conjunto, sugere-se que o NPS pode promover est?mulo excitat?rio mesmo quando a s?ntese de catecolaminas foi prejudicada. Ainda conclui-se que o efeito hiperlocomotor do NPS e do metilfenidato depende dos estoques vesiculares de monoaminas, em particular dopamina, e da ativa??o do receptor dopamin?rgico D2. O efeito psicoestimulante do NPS por meio da ativa??o do sistema dopamin?rgico pode apresentar import?ncia cl?nica no tratamento de doen?as que envolvem a via dopamin?rgica, como o Mal de Parkinson e a depend?ncia qu?mica

Made available in DSpace on 2014-12-17T15:37:11Z (GMT). No. of bitstreams: 1 JuliaJD_DISSERT.pdf: 2580317 bytes, checksum: 265bbbe6825ce7a3d27c569cde32cf8d (MD5) Previous issue date: 2012-06-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Neuropeptide S (NPS) is the endogenous ligand of a G-protein coupled receptor. Preclinical studies have shown that NPSR receptor activation can promote arousal, anxiolytic-like behavioral, decrease in food intake, besides hyperlocomotion, which is a robust but not well understood phenomenon. Previous findings suggest that dopamine transmission plays a crucial role in NPS hyperactivity. Considering the close relationship between dopamine and Parkinson Disease (PD), and also that NPSR receptors are expressed on dopaminergic nuclei in the brain, the current study attempted to investigate the effects of NPS in motor deficits induced by intracerebroventricular (icv) administration of 6-OHDA and systemic administration of haloperidol. Motor deficits induced by 6-OHDA and haloperidol were evaluated on Swiss mice in the rota-rod and catalepsy test. Time on the rotating rod and time spent immobile in the elevated bar were measured respectively in each test. L-Dopa, a classic antiparkinsonian drug, and NPS were administrated in mice submitted to one of the animal models of PD related above. 6-OHDA injection evoked severe motor impairments in rota-rod test, while the cataleptic behavior of 6-OHDA injected mice was largely variable. The administration of L-Dopa (25 mg/kg) and NPS (0,1 and 1 nmol) reversed motor impairments induced by 6-OHDA in the rota-rod. Haloperidolinduced motor deficits on rota-rod and catalepsy tests which were reversed by L-Dopa (100 e 400 mg/kg), but not by NPS (0,1 and 1 nmol) administration. The association of L-Dopa 10 mg/kg and NPS 1 nmol was also unable to counteract haloperidol-induced motor deficits. To summarize, 6-OHDA-, but not haloperidol-, induced motor deficits were reversed by the central administration of NPS. These data suggest that NPS possibly facilitates dopamine release in basal ganglia, what would explain the overcome of motor performance promoted by NPS administration in animals pretreated with 6-OHDA, but not haloperidol. Finally, the presented findings point, for the first time, to the potential of NPSR agonist as an innovative treatment for PD. / O neuropept?deo S (NPS) ? o ligante end?geno do receptor NPSR acoplado ? prote?na G. Estudos pr?-cl?nicos mostraram que a ativa??o do receptor NPSR promove aumento da vig?lia, ansi?lise, efeito anor?xico, al?m de hiperlocomo??o. Este ?ltimo efeito ? robusto e ainda pouco entendido. Evid?ncias apontam para o envolvimento do sistema dopamin?rgico no efeito estimulat?rio do NPS. Tendo em vista a modula??o exercida pelo sistema NPS-receptor NPSR na locomo??o espont?nea de animais, a express?o do receptor NPSR em n?cleos dopamin?rgicos e a rela??o intr?nseca entre a dopamina e a Doen?a de Parkinson, o presente estudo visou investigar o papel exercido pelo sistema do NPS no preju?zo motor de camundongos induzido pela administra??o intracerebroventricular (icv) de 6-OHDA e sist?mica de haloperidol. Para avalia??o dos preju?zos motores induzidos por 6-OHDA e haloperidol, camundongos Swiss foram submetidos aos testes do rota-rod e da catalepsia e o desempenho motor na barra girat?ria do rota-rod e o tempo de imobilidade na plataforma elevada foram registrados, respectivamente. O efeito do tratamento dos camundongos com L-Dopa (via oral), um antiparkinsoniano cl?ssico, e do NPS (icv) foi avaliado nos dois testes comportamentais citados acima. No teste do rota-rod, tr?s dias ap?s a administra??o de 6-OHDA, os animais apresentaram significativo preju?zo motor. A revers?o do preju?zo motor foi verificada ap?s a administra??o de L-Dopa (25 mg/kg) ou de NPS (0,1 e 1 nmol). A administra??o de 6-OHDA tamb?m elevou o tempo de perman?ncia na plataforma elevada (teste da catalepsia), mas este efeito foi muito vari?vel, n?o sendo, portanto, utilizado para investigar a a??o do NPS. O preju?zo motor induzido pelo haloperidol no teste do rota-rod e catalepsia foi revertido pela administra??o de L-Dopa (100 e 400 mg/kg), mas n?o pelo NPS (0,1 e 1 nmol) ou pela associa??o de LDopa 10 mg/kg e NPS 1 nmol. Em conclus?o, os danos motores induzidos pela administra??o de 6-OHDA, mas n?o de haloperidol, foram revertidos pelo tratamento central com NPS. Estes dados sugerem que o NPS parece facilitar a libera??o de dopamina na via nigroestriatal, o que justificaria a melhora do desempenho motor induzida pela administra??o do NPS em animais tratados com 6-OHDA, mas n?o com haloperidol (que causa bloqueio de receptores dopamin?rgicos). Por fim, os achados aqui apresentados apontam, pela primeira vez, para a possibilidade de agonistas do receptor NPSR atuarem como agentes terap?uticos ou adjuvantes no tratamento da Doen?a de Parkinson.

The duodenal epithelium is regularly exposed to HCl, digestive enzymes, bacteria and toxins, and sometimes also to ethanol and drugs. The imbalance of aggressive factors in the intestinal lumen and mucosal barrier function increases the risk of tissue injury and inflammation. The key components of the duodenal barrier function include mucosal permeability, bicarbonate transport and the secretion or absorption of fluids. This thesis aims to elucidate the role of melatonin, neuropeptide S (NPS) and short chain fatty acids (SCFAs) in the regulation of intestinal mucosal barrier function and motility in the anesthetized rat in vivo and in tissues of human origin in vitro. Melatonin was found to reduce ethanol-induced increases in paracellular permeability and motility by a neural pathway within the enteric nervous system involving nicotinic receptors. In response to luminal exposure of ethanol, signs of mild mucosal edema and beginning of desquamation were observed in a few villi only, an effect that was not influenced by melatonin. Melatonin did not modify increases in paracellular permeability in response to luminal acid. NPS decreased basal and ethanol-induced increases in duodenal motility as well as bethanechol stimulated colonic motility in a dose-dependent manner. Furthermore, NPS was shown to inhibit basal duodenal bicarbonate secretion, stimulate mucosal fluid absorption and increase mucosal paracellular permeability. In response to luminal exposure of acid, NPS increased bicarbonate secretion and mucosal paracellular permeability. All effects induced by the administration of NPS were dependent on nitrergic pathways. In rats, administration of NPS increased the tissue protein levels of the inflammatory biomarkers IL-1β and CXCL1. Immunohistochemistry showed that NPS was localized at myenteric nerve cell bodies and fibers, while NPSR1 and nNOS were only confined to the myenteric nerve cell bodies. Perfusing the duodenal segment with the SCFAs acetate or propionate reduced the duodenal mucosal paracellular permeability, decreased transepithelial net fluid secretion and increased bicarbonate secretion. An i.v. infusion of SCFAs reduces mucosal paracellular permeability without any effects on mucosal net fluid flux. However, it significantly decreased bicarbonate secretion. Luminal SCFAs changed the duodenal motility pattern from fasting to feeding motility while i.v. SCFAs was without effect on motility. The systemic administration of glucagon-like peptide-2 (GLP-2) induced increases in mucosal bicarbonate secretion and fluid absorption. An i.v. GLP-2 infusion during a luminal perfusion of SCFAs significantly reduced the duodenal motility. In conclusion, the results in the present thesis show that melatonin, NPS and SCFAs influence the neurohumoral regulation of intestinal mucosal barrier function and motility. Aberrant signaling in response to melatonin, NPS and to luminal fatty acids might be involved in the symptom or the onset of disease related to intestinal dysfunction in humans. / <p>Research funders and strategic development areas:</p><p>- Bengt Ihre Foundation (grant SLS-177521)</p><p>- Socialstyrelsen(grant SLS-176671)</p><p>- Erik, Karin, and Gösta Selanders Foundation</p><p>- Emil and Ragna Börjesson Foundation</p><p>- Uppsala University </p><p>- Ministry of Education of Malaysia</p><p>- Universiti Malaysia Sabah, Malaysia</p>

Made available in DSpace on 2014-12-17T15:37:10Z (GMT). No. of bitstreams: 1 LailaSA_DISSERT.pdf: 1680302 bytes, checksum: b5c16765c49be4a6aa33996242e86d2f (MD5) Previous issue date: 2012-04-30 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Anxiety is an emotional phenomenon, and normally it is interpreted as an adaptative behavior front to adversities. In its pathological form, anxiety can severely affect aspects related to the personal and professional life. Studies have shown a close relationship between anxiety disorders and aversive memory processing. Considering that the pharmacotherapy of anxiety disorders is still limited, innovative anxiolytic agents are needed. In this regard, neuropeptides systems are interesting therapeutic targets to the treatment of psychopathologies. Neuropeptide S (NPS), a 20-aminoacid peptide, is the endogenous ligand of a G-protein coupled receptor (NPSR), which has been reported to evoke hyperlocomotion, awakefull states, besides anxiolysis and memory improvements in rodents. This study aimed to investigate the effects of biperiden (BPR; an amnesic drug), diazepam (DZP; an anxiolytic drug) and NPS at three distinct times: pre-training, post-training, and pre-test, in order to assess anxiety and memory process in the same animal model. The elevated Tmaze (ETM) is an apparatus derived from the elevated plus-maze test, which consists of one enclosed and two open arms. The procedure is based on the avoidance of open spaces learned during training session, in which mice were exposed to the enclosed arm as many times as needed to stay 300 s. In the test session, memory is assessed by re-exposing the mouse to the enclosed arm and the latency to enter an open arm was recorded. When injected pre-training, BPR (1 mg/kg) impaired learning and memory processing; DZP (1 and 2 mg/kg) evoked anxiolysis, but only at the dose of 2 mg/kg impaired memory; and NPS 0.1 nmol induced anxiolysis without affecting memory. Post-training injection of DZP (2 mg/kg) or BPR (1 and 3 mg/kg) did not affect memory consolidation, while the post-trainning administration of NPS 1 nmol, but not 0.1 nmol, improved memory in mice. Indeed, pre-trainning administration of NPS 1 nmol did not prevent memory impairment elicited by BPR (2 mg/kg, injected before training). In the open field test, BPR 1 mg/kg and NPS 1 nmol induced hyperlocomotion in mice. In conclusion, the proposed ETM task is practical for the detection of the anxiolytic and amnesic effects of drugs. The anxiolytic and memory enhancement effects of NPS were detected in the ETM task, and reinforce the role of NPS system as an interesting therapeutic target to the treatment of anxiety disorders / A ansiedade ? um fen?meno emocional comum e ?til, que funciona com valor adaptativo frente ?s altera??es do meio. Em sua forma patol?gica pode severamente interferir com a condi??o normal de vida de um indiv?duo e estudos tem demonstrado haver uma estreita rela??o entre os transtornos de ansiedade e o processamento da mem?ria aversiva. Entretanto, ainda hoje o tratamento dos transtornos de ansiedade ? limitado, refor?ando a necessidade de novas op??es farmacol?gicas. Neste contexto, os neuropept?deos atuam como mensageiros qu?micos no sistema nervoso central e constituem-se de alvos terap?uticos inovadores para o tratamento de psicopatologias. O neuropept?deo S (NPS), composto por 20 amino?cidos, ? o ligante end?geno de um receptor acoplado ? prote?na G (NPSR) e promove estado de vig?lia, hiperlocomo??o, melhora da mem?ria e ansi?lise em roedores. Diante disso, este trabalho tem por objetivo avaliar os efeitos do biperideno (BPR) (droga amn?sica), diazepam (DZP) (droga ansiol?tica) e NPS, em tr?s momentos distintos, antes da sess?o treino, ap?s a sess?o treino e antes da sess?o teste, com o intuito de investigar os efeitos dos tratamentos sobre o aprendizado/aquisi??o, consolida??o e evoca??o da mem?ria, respectivamente, em camundongos submetidos ? tarefa do labirinto em T elevado (LTE). Os animais foram submetidos a uma sess?o treino, na qual foram re-expostos ao LTE quantas vezes foram necess?rias para atingir o ponto de corte (300 s no bra?o fechado). Ap?s 24, 48 ou 96 h, foi realizada a sess?o teste, na qual os animais foram submetidos ao LTE, a fim de avaliar a evoca??o do comportamento de evita??o aos bra?os abertos. Foi observado que a mem?ria induzida na sess?o treino do LTE ? duradoura mantendo-se intacta 15 dias ap?s a sess?o treino. Quando administrados pr?-treino, BPR (1mg/kg) induziu d?ficit de aprendizado e mem?ria; DZP (1 e 2 mg/kg) casou ansi?lise, mas somente a dose de 2 mg/kg induziu d?ficit de mem?ria; e NPS 0,1 nmol promoveu ansi?lise sem afetar a mem?ria. A administra??o p?s-treino de BPR (1 e 3 mg/kg) e DZP (2 mg/kg) n?o causou efeitos comportamentais, enquanto que NPS 1 nmol, mas n?o 0,1 nmol, induziu melhora na consolida??o da mem?ria. A administra??o pr?-teste de BPR (1 mg/kg) e DZP (1 e 2 mg/kg) n?o modificou a evoca??o da mem?ria na tarefa do LTE. Ainda, a administra??o pr?-treino de NPS 1 nmol n?o preveniu o preju?zo de mem?ria evocado pelo BPR (2 mg/kg) injetado pr?-treino. Quando avaliados no campo aberto, somente os tratamentos com BPR 1 mg/kg e NPS 1 nmol induziram hiperlocomo??o. Estes dados sugerem que o LTE ? uma tarefa capaz de avaliar aprendizado, ansiedade e mem?ria simultaneamente em camundongos. Os efeitos ansiol?ticos e facilitadores da mem?ria do NPS tamb?m foram detectados nesta tarefa, que juntamente com achados pr?vios, refor?am o papel deste sistema peptid?rgico no tratamento de transtornos de ansiedade

L'asma és una malaltia d'etiologia complexa, formada per factors genètics i ambientals, on la interrelació de ambdós factors mitjançant interaccions gen-ambient juga un paper clau. L'objectiu d'aquesta tesi ha sigut aprofundir en el coneixement del paper dels polimorfismes genètics, i la seva interacció amb factors ambientals, en la ocurrència d'asma, atòpia i hiperreactivitat bronquial. Aquest objectiu ha estat desenvolupat a través de la replicació de variants genètiques prèviament identificades, l'avaluació d'interaccions gen-ambient i la identificació de nous gens de susceptibilitat mitjançant un disseny basat en el genotipatge de variants genètiques all llarg del genoma en pools d'ADN. La tesi ha estat majoritàriament duta a terme dins l'estudi European Community Respiratory Health Survey (ECRHS) que està comprès per 5.000 individus seguits durant 9 anys, pels quals es disposa d'un qüestionari complet sobre símptomes respiratoris, avaluacions clíniques, informació sobre exposicions ambientals i mostres de ADN. Aquesta tesi a replicat l'associació del polimorfismes dels gens TNFA i NPSR1 amb asma. A més s'han establert les interaccions entre TNFA i obesitat, NQO1 i contaminació atmosfèrica, i NPSR1 i edat d'inici d'asma. L'anàlisi de pools d' ADN ha permès associar la regió on es situa el gen SGK493 amb atòpia. Aquesta tesi contribueix al coneixement de l'etiologia d'asma amb la identificació i replicació d'associacions genètiques i interaccions gen-ambient. / Asthma is a disease with a complex etiology, involving multiple genetic and environmental factors, and with an important role of the interplay of these factors through gene-environment interactions. In this thesis I aimed to advance our knowledge on the importance of genetic polymorphisms and their interaction with environmental data for the occurrence of asthma and related phenotypes (atopy and bronchial hyperreactivity). This objective was developed through the replication of genetic associations previously reported, the assessment of gene-environment interactions and the identification of new susceptibility genes using genome-wide analysis based on a pooling DNA strategy. The thesis was, mostly, performed within the European Community Respiratory Health Survey (ECRHS). This cohort has information and DNA samples from approximately 5,000 adult subjects followed-up for 9 years, with extensive questionnaires on respiratory symptoms, clinical evaluations and information on environmental exposures. This thesis replicates previous effects on asthma of polymorphisms in TNFA and NPSR1 genes. In addition, interactions have been established between TNFA and obesity, NQO1 and air-pollution, and NPSR1 and age at onset of asthma. The approach based on genome-wide analysis of DNA pools identified the SGK493 region being associated with atopy. This thesis contributes to the understanding of the etiology of asthma through the identification and replication of genetic associations and gene-environment interactions.