Recentemente, o óxido nítrico (NO) tem sido relacionado com a depressão. A administração de inibidores da NO sintase (NOS) induz efeitos do tipo antidepressivo em modelos animais e há um aumento da expressão da NOS em estruturas do sistema límbico em indivíduos depressivos e em animais expostos a estresse. Além disso, sabe-se que o estresse causa um aumento da ativação de neurônios localizados em estruturas do sistema límbico e que o tratamento com antidepressivos bem como com inibidor da NOS, diminui essa marcação. Contudo, ainda não se sabe como o sistema nitrérgico dessas estruturas está relacionado com os comportamentos relacionados à depressão. Assim nosso objetivo é testar a hipótese de que o desenvolvimento do desamparo (comportamento relacionado à depressão) em ratos seria causado por um aumento de atividade de neurônios que contém nNOS em estruturas envolvidas com a resposta emocional ao estresse, e que os diferentes tratamentos induzem efeitos do tipo antidepressivo no modelo apresentando através de um efeito final comum de diminuir essa ativação e, portanto, diminuir os níveis de NO. Para isso, ratos foram submetidos ao modelo do desamparo aprendido e tratados com drogas antidepressivas. Após o teste, foi feita a imunohistoquímica com marcação para Fos (Fos-IR; marcador de atividade neuronal) e nNOS (nNOS-IR). O tratamento repetido com desipramina (DES, na dose de 25, mas não na de 12,5 mg/Kg), fluoxetina (FXT, na dose de 15, mas não na de 30 mg/Kg) e imipramina (IMI, 15mg/Kg) induziu efeito do tipo-antidepressivo no teste do desamparo aprendido (LH). O tratamento agudo apenas com imipramina, mas não com FXT ou IMI, induziu o mesmo tipo de efeito. O tratamento com DES, FXT ou IMI também aumentou o número de cruzamentos entre choques no LH, porém não induziu aumento de atividade locomotora no teste do campo aberto. O tratamento repetido com DES diminuiu a Fos-IR na amígdala basolateral (BlAm), amígdala lateral (LAm), córtex pré-frontal medial (mPFC), região CA1 e CA3 do hipocampo dorsal (dHPC) e região CA3 do hipocampo ventral (vHPC). O tratamento agudo com DES induziu um aumento de Fos-IR na amígdala central (CeAm), amígdala medial (MeAm) e CA1 e CA3 do dHPC. O tratamento repetido com FXT diminuiu Fos-IR na BlAm e LAm, enquanto o tratamento agudo aumentou Fos-IR na CeAm. O tratamento repetido com IMI aumentou nNOS-IR na MeAm e a dupla marcação no núcleo leito da estria terminal (BST); e diminuiu o Fos-IR na região CA1 do dHPC e na região parvocelular do núcleo paraventricular do hipotálamo (pPVN). Por fim, foram encontradas relações positivas entre o número de células Fos-IR e o número de falhas em fugir ou escapar dos choques no LH na BlAm, LAm, CA1 e CA3 do dHPC e CA3 do vHPC; i.e., quanto mais células ativadas nessas estruturas, maior o número de choques que os animais receberam sem consegui fugir. Os resultados aqui apresentados são, em parte, corroborados pela literatura, mostrando a participação das estruturas analisadas no comportamento do desamparo aprendido e no efeito das drogas antidepressivas. Nesse contexto, acredita-se que o BST funcionaria como um núcleo de processamento da informação vinda do mPFC, HPC e amígdala, enviando projeções para o PVN e regulando o funcionamento do eixo HPA. O trabalho abre caminho para a identificação de subpopulações específicas de neurônios que expressam a nNOS, buscando compreender o papel destas na modulação das respostas comportamentais numa situação de estresse, na busca pela formulação de um cenário cada vez mais completo da participação do sistema nitrérgico dentro do complexo neurocircuito que regula as emoções / Recently, nitric oxide (NO) has been related with the neurobiology of depression. The NO synthase (NOS) inhibition induces antidepressant-like effects in animal models and there is an increase in the NOS expression in limbic structures of depressed patients or in stress exposed animals. Besides, it is well known that stressful events causes an increase in limbic structures neuronal activation and that antidepressant treatment as well as NOS inhibition attenuates this effect. However, it is still unknown how the limbic nitrergic system is related with depression-related behaviors. Then, the aim of this work is to test the hypothesis that the helplessness behavior development (a depression-related behavior) in rats would be induced by an increased activity of nNOS-containing neurons in structures related with the neurobiology of stress responses. Furthermore, the antidepressant-like effect induced by antidepressants treatment in this model would share a final effect, decreasing the activation of such neurons, and decreasing the levels of NO in these structures. For this aim, male rats were submitted to the learned helplessness model and treated with antidepressants. After the test, immunohistochemistry assay were performed, with double labeling for c-Fos (Fos-IR; neuronal activity marker) and nNOS (nNOS-IR). The repeated treatment with desipramine (DES, 25 mg/kg but not 12,5mg/kg), fluoxetine (FXT, 15 mg/Kg, but not 30 mg/Kg) and imipramina (IMI, 15 mg/KG) induced antidepressant-like effects in the learned helplessness test (LH). The acute treatment with IMI, but not with DES or FXT, induced the same effect. The repeated treatment with DES, FXT or IMI also increased the number of intertrial crossings in the LH, but not the locomotor activity score on the open field score. The repeated treatment with DES decreased the number of Fos-IR into the basolateral amygdala (BlAm), lateral amygdala (Lam), medial prefrontal cortex (mPFC), CA1 and CA3 regions of the dorsal hippocampus (dHPC), and CA3 region of the ventral hippocampus (vHPC). The acute treatment with DES increased the Fos-IR into the central amygdale (CeAm), medial amygdala (MeAm), and CA1 and CA3 regions of the dHPC. The repeated treatment with FXT decreases the number of Fos-IR into the BlAm and Lam, while the acute treatment increases the Fos-IR into the CeAm. The repeated treatment with IMI increased the nNOS-IR into the MeAm and the double- labeled cells into the bed nucleus of stria terminalis (BST); and decreased the Fos-IR into the CA1 region of the dHPC and into the parvocellular region of the paraventricular nucleus of the hypothalamus. Finally, positive correlations between the number of Fos-IR and the number of failures in escaping or avoiding the foot shocks on the LH were found into the BlAm, Lam, CA1 and CA3 of the dHPC, and CA3 of the vHPC, i.e., with more activated cells into these structures mentioned, more foot shocks the rats received. These results are (partially) corroborated with previous scientific papers, showing the analyzed structures participation in the learned helplessness behavior as well as in the antidepressant effect of antidepressant administration. Within this context, the BST would work as a relay center, processing the information coming from the mPFC, HPC and amygdaloid nuclei, and sending the output to the PVN, modulating the HPA axis. This work open some questions about the identification of specific nNOS-containing neuronal subpopulations, aiming to clarify their role in the stress response, and searching for the formulation of a more complete scenario of the nitrergic system participation in this complex emotion-regulating neurocircuit

Recentemente, o óxido nítrico (NO) tem sido relacionado com a depressão. A administração de inibidores da NO sintase (NOS) induz efeitos do tipo antidepressivo em modelos animais e há um aumento da expressão da NOS em estruturas do sistema límbico em indivíduos depressivos e em animais expostos a estresse. Além disso, sabe-se que o estresse causa um aumento da ativação de neurônios localizados em estruturas do sistema límbico e que o tratamento com antidepressivos bem como com inibidor da NOS, diminui essa marcação. Contudo, ainda não se sabe como o sistema nitrérgico dessas estruturas está relacionado com os comportamentos relacionados à depressão. Assim nosso objetivo é testar a hipótese de que o desenvolvimento do desamparo (comportamento relacionado à depressão) em ratos seria causado por um aumento de atividade de neurônios que contém nNOS em estruturas envolvidas com a resposta emocional ao estresse, e que os diferentes tratamentos induzem efeitos do tipo antidepressivo no modelo apresentando através de um efeito final comum de diminuir essa ativação e, portanto, diminuir os níveis de NO. Para isso, ratos foram submetidos ao modelo do desamparo aprendido e tratados com drogas antidepressivas. Após o teste, foi feita a imunohistoquímica com marcação para Fos (Fos-IR; marcador de atividade neuronal) e nNOS (nNOS-IR). O tratamento repetido com desipramina (DES, na dose de 25, mas não na de 12,5 mg/Kg), fluoxetina (FXT, na dose de 15, mas não na de 30 mg/Kg) e imipramina (IMI, 15mg/Kg) induziu efeito do tipo-antidepressivo no teste do desamparo aprendido (LH). O tratamento agudo apenas com imipramina, mas não com FXT ou IMI, induziu o mesmo tipo de efeito. O tratamento com DES, FXT ou IMI também aumentou o número de cruzamentos entre choques no LH, porém não induziu aumento de atividade locomotora no teste do campo aberto. O tratamento repetido com DES diminuiu a Fos-IR na amígdala basolateral (BlAm), amígdala lateral (LAm), córtex pré-frontal medial (mPFC), região CA1 e CA3 do hipocampo dorsal (dHPC) e região CA3 do hipocampo ventral (vHPC). O tratamento agudo com DES induziu um aumento de Fos-IR na amígdala central (CeAm), amígdala medial (MeAm) e CA1 e CA3 do dHPC. O tratamento repetido com FXT diminuiu Fos-IR na BlAm e LAm, enquanto o tratamento agudo aumentou Fos-IR na CeAm. O tratamento repetido com IMI aumentou nNOS-IR na MeAm e a dupla marcação no núcleo leito da estria terminal (BST); e diminuiu o Fos-IR na região CA1 do dHPC e na região parvocelular do núcleo paraventricular do hipotálamo (pPVN). Por fim, foram encontradas relações positivas entre o número de células Fos-IR e o número de falhas em fugir ou escapar dos choques no LH na BlAm, LAm, CA1 e CA3 do dHPC e CA3 do vHPC; i.e., quanto mais células ativadas nessas estruturas, maior o número de choques que os animais receberam sem consegui fugir. Os resultados aqui apresentados são, em parte, corroborados pela literatura, mostrando a participação das estruturas analisadas no comportamento do desamparo aprendido e no efeito das drogas antidepressivas. Nesse contexto, acredita-se que o BST funcionaria como um núcleo de processamento da informação vinda do mPFC, HPC e amígdala, enviando projeções para o PVN e regulando o funcionamento do eixo HPA. O trabalho abre caminho para a identificação de subpopulações específicas de neurônios que expressam a nNOS, buscando compreender o papel destas na modulação das respostas comportamentais numa situação de estresse, na busca pela formulação de um cenário cada vez mais completo da participação do sistema nitrérgico dentro do complexo neurocircuito que regula as emoções / Recently, nitric oxide (NO) has been related with the neurobiology of depression. The NO synthase (NOS) inhibition induces antidepressant-like effects in animal models and there is an increase in the NOS expression in limbic structures of depressed patients or in stress exposed animals. Besides, it is well known that stressful events causes an increase in limbic structures neuronal activation and that antidepressant treatment as well as NOS inhibition attenuates this effect. However, it is still unknown how the limbic nitrergic system is related with depression-related behaviors. Then, the aim of this work is to test the hypothesis that the helplessness behavior development (a depression-related behavior) in rats would be induced by an increased activity of nNOS-containing neurons in structures related with the neurobiology of stress responses. Furthermore, the antidepressant-like effect induced by antidepressants treatment in this model would share a final effect, decreasing the activation of such neurons, and decreasing the levels of NO in these structures. For this aim, male rats were submitted to the learned helplessness model and treated with antidepressants. After the test, immunohistochemistry assay were performed, with double labeling for c-Fos (Fos-IR; neuronal activity marker) and nNOS (nNOS-IR). The repeated treatment with desipramine (DES, 25 mg/kg but not 12,5mg/kg), fluoxetine (FXT, 15 mg/Kg, but not 30 mg/Kg) and imipramina (IMI, 15 mg/KG) induced antidepressant-like effects in the learned helplessness test (LH). The acute treatment with IMI, but not with DES or FXT, induced the same effect. The repeated treatment with DES, FXT or IMI also increased the number of intertrial crossings in the LH, but not the locomotor activity score on the open field score. The repeated treatment with DES decreased the number of Fos-IR into the basolateral amygdala (BlAm), lateral amygdala (Lam), medial prefrontal cortex (mPFC), CA1 and CA3 regions of the dorsal hippocampus (dHPC), and CA3 region of the ventral hippocampus (vHPC). The acute treatment with DES increased the Fos-IR into the central amygdale (CeAm), medial amygdala (MeAm), and CA1 and CA3 regions of the dHPC. The repeated treatment with FXT decreases the number of Fos-IR into the BlAm and Lam, while the acute treatment increases the Fos-IR into the CeAm. The repeated treatment with IMI increased the nNOS-IR into the MeAm and the double- labeled cells into the bed nucleus of stria terminalis (BST); and decreased the Fos-IR into the CA1 region of the dHPC and into the parvocellular region of the paraventricular nucleus of the hypothalamus. Finally, positive correlations between the number of Fos-IR and the number of failures in escaping or avoiding the foot shocks on the LH were found into the BlAm, Lam, CA1 and CA3 of the dHPC, and CA3 of the vHPC, i.e., with more activated cells into these structures mentioned, more foot shocks the rats received. These results are (partially) corroborated with previous scientific papers, showing the analyzed structures participation in the learned helplessness behavior as well as in the antidepressant effect of antidepressant administration. Within this context, the BST would work as a relay center, processing the information coming from the mPFC, HPC and amygdaloid nuclei, and sending the output to the PVN, modulating the HPA axis. This work open some questions about the identification of specific nNOS-containing neuronal subpopulations, aiming to clarify their role in the stress response, and searching for the formulation of a more complete scenario of the nitrergic system participation in this complex emotion-regulating neurocircuit

Les objectifs étaient 1) l'évaluation des interactions existant entre le sédiment et le compartiment benthique; 2) le suivi des processus de restauration d'une communauté benthique ayant subi l'immersion de 6.10^6m3 de déblais de dragages. La texture sédimentaire, la qualité alimentaire de la matière organique et les paramètres faunistiques ont été mesurés dans 3 sites des parages de l'embouchure du Rhône (1 témoin, 1 sous l'influence du fleuve et 1 dans la zone d'immersion). Les phases de la restauration de la communauté ont été suivies et les facteurs ayant influencé la communauté benthique ont été mis en évidence. Ce travail a permis de préconiser aux gestionnaires des adaptations pour optimiser les prochaines campagnes de dragages et d'améliorer la connaissance des interactions entre le compartiment "macrofaune benthique" et les sédiments qui l'abrite, en montrant l'influence, non seulement de la quantité de la matière organique, mais aussi de sa qualité.

The central melanin-concentrating hormone (MCH) system has been intensively studied for its involvement in the regulation of feeding behaviour and body weight regulation. The importance of the neuropeptide MCH in the control of energy balance has been underlined by MCH knock out and Melanin-concentrating hormone receptor subtype 1 (MCHR-1) knock-out animals. The anorectic and anti-obesity effects of selective MCHR-1 antagonists have confirmed the notion that pharmacological blockade of MCHR-1 is a potential therapeutic approach for obesity. First aim of this work is to study the neurochemical “equipment” of MCHR-1 immunoreactive neurons by double-labelling immunohistochemistry within the rat hypothalamus. Of special interest is the neuroanatomical identification of other hypothalamic neuropeptides that are co-distributed with MCHR-1. A second part of this study deals with the examination of neuronal activation patterns after pharmacological or physiological, feeding-related stimuli and was introduced to further understand central regulatory mechanisms of the MCH system. In the first part of work, I wanted to neurochemically characterize MCHR-1 immunoreactive neurons in the rat hypothalamus for colocalisation with neuropeptides of interest. Therefore I performed an immunohistochemical colocalisation study using a specific antibody against MCHR-1 in combination with antibodies against hypothalamic neuropeptides. I showed that MCHR-1 immunoreactivity (IR) was co-localised with orexin A in the lateral hypothalamus, and with adrenocorticotropic hormone and neuropeptide Y in the arcuate nucleus. Additionally, MCHR-1 IR was co-localised with the neuropeptides vasopressin and oxytocin in magnocellular neurons of the supraoptic and paraventricular hypothalamic nucleus and corticotrophin releasing hormone in the parvocellular division of the paraventricular hypothalamic nucleus. Moreover, for the first time MCHR-1 immunoreactivity was found in both the adenohypophyseal and neurohypophyseal part of the rat pituitary. These results provide the neurochemical basis for previously described potential physiological actions of MCH at its target receptor. In particular, the MCHR-1 may be involved not only in food intake regulation, but also in other physiological actions such as fluid regulation, reproduction and stress response, possibly through here examined neuropeptides. Central activation patterns induced by pharmacological or physiological stimulation can be mapped using c-Fos immunohistochemistry. In the first experimental design, central administration (icv) of MCH in the rat brain resulted in acute and significant increase of food and water intake, but this animal treatment did not induce a specific c-Fos induction pattern in hypothalamic nuclei. In contrast, sub-chronic application of MCHR-1 antagonist promoted a significant decrease in food- and water intake during an eight day treatment period. A qualitative analysis of c-Fos immunohistochemistry of sections derived from MCHR-1 antagonist treated animals showed a specific neuronal activation in the paraventricular nucleus, the supraoptic nucleus and the dorsomedial hypothalamus. These results could be substantiated by quantitative evaluation of an automated, software-supported analysis of the c-Fos signal. Additionally, I examined the activation pattern of rats in a restricted feeding schedule (RFS) to identify pathways involved in hunger and satiety. Animals were trained for 9 days to feed during a three hour period. On the last day, food restricted animals was also allowed to feed for the three hours, while food deprived (FD) animals did not receive food. Mapping of neuronal activation showed a clear difference between stareved (FD) and satiated (FR) rats. FD animals showed significant induction of c-Fos in forebrain regions, several hypothalamic nuclei, amygdaloid thalamus and FR animals in the supraoptic nucleus and the paraventricular nucleus of the hypothalamus, and the nucleus of the solitary tract. In the lateral hypothalamus of FD rats, c-Fos IR showed strong colocalisation for Orexin A, but no co-staining for MCH immunoreactivity. However, a large number of c-Fos IR neurons within activated regions of FD and FR animals was co-localised with MCHR-1 within selected regions. To conclude, the experimental set-up of scheduled feeding can be used to induce a specific hunger or satiety activation pattern within the rat brain. My results show a differential activation by hunger signals of MCH neurons and furthermore, demonstrates that MCHR-1 expressing neurons may be essential parts of downstream processing of physiological feeding/hunger stimuli. In the final part of my work, the relevance of here presented studies is discussed with respect to possible introduction of MCHR-1 antagonists as drug candidates for the treatment of obesity. / Die Regulation des Körpergewichts in einem physiologischen Rahmen setzt ein internes Energiegleichgewicht voraus und wird langfristig durch Abgleich von Nahrungsaufnahme einerseits und Energieverbrauch andererseits gewährleistet. Dieses Gleichgewicht ist bei massivem Übergewicht (Adipositas) oder chronischem Untergewicht (Kachexie) dauerhaft gestört. Bei der Regulation des Energiegleichgewichts spielt der im Zwischenhirn gelegene Hypothalamus als Schaltstation eine wichtige Rolle. Hypothalamische Regelkreise gleichen sensorische, viszerale und humorale Signale miteinander ab und setzen sie in adäquates Verhalten (z.B. Nahrungsaufnahme) um. Innerhalb des Hypothalamus werden Hunger und Sättigung durch zentralnervöse Regulationssysteme kodiert. Dadurch stellt eine pharmakologische Inhibierung eines hunger-stimulierenden (orexigenen), hypothalamischen Regelkreises eine Möglichkeit dar, um Nahrungsaufnahme und Körpergewichts zu reduzieren. Das im lateralen Hypothalamus gebildete Neuropeptid Melanin-konzentrierendes Hormon (MCH) ist ein solches orexigenes Signal. In unterschiedlichen Tiermodellen wurde gezeigt, dass MCH seine physiologischen Effekte auf das Energiegleichgewicht durch den funktionellen MCH Rezeptor Subtyp 1 (MCHR-1) vermittelt. Die Behandlung von Labornagern mit selektiv wirksamen MCHR-1 Antagonisten hat in verschiedenen Tiermodellen zu einer Verminderung der Nahrungsaufnahme und Körpergewichtsreduktion geführt (anorexigene Wirkung). Das Ziel dieser Arbeit ist eine vertiefte Untersuchung des zentralen MCH Systems. Im ersten Teil der Arbeit werden MCHR-1 enthaltene Nervenzellen (Neurone) im Hypothalamus von Ratten immunhistochemisch identifiziert und neurochemisch charakterisiert. Dieser Teil der Arbeit soll mit Hilfe von Kolokalisationsstudien mögliche Interaktionen des MCH Systems mit anderen neuropeptidergen, hypothalamischen Systemen identifizieren. Der zweite Teil der Arbeit befasst sich mit der Untersuchung von pharmakologischen Effekten bei MCH und MCHR-1 Antagonist behandelten Ratten auf Nahrungsaufnahme, Wasseraufnahme sowie Veränderung des Körpergewichts. Zentrale Regulationsmechanismen wurden durch den immunhistochemischen Nachweis des Transkriptionsfaktors und neuronalen Aktivierungsmarkers c-Fos im Rattenhirn ermittelt. Diese neuronalen Aktivierungsmuster wurden mit solchen Mustern verglichen, die nach einem definierten physiologischen Stimulus (Fütterungsregime) mit derselben Methode aufgezeichnet wurden. Erste Ergebnisse zeigten, dass der hier etablierte Antikörper gegen MCHR-1 spezifisch ist und MCHR-1 in mehreren hypothalamischen Kernarealen mit Hilfe dieses Antikörpers nachgewiesen werden konnte. So konnte im lateralen Hypothalamus eine Kolokalisation von MCHR-1 mit Orexin A nachgewiesen werden, im arcuate Nukleus des Hypothalamus, einem Kernareal, das eine bedeutende Funktion in der Integration von Hunger- und Sättigungssignalen hat, zeigten MCHR-1 positive Neurone eine Kolokalisation mit dem orexigenen Neuropeptid Y oder mit dem Adrenocorticotrophin Hormon, einem Marker für das anorexigen wirkende, zentrale Melanokortin System. Der Paraventrikuläre Nukleus und der Supraoptische Nukleus des Hypothalamus spielen eine wichtige Rolle in neuroendokrinen Regulationen. Im paraventrikulären Hypothalamus konnte eine Kolokalisation von MCHR-1 mit den Neuropeptiden Vasopressin, Oxytocin und Corticotrophin-releasing Hormon festgestellt werden, außerdem konnte eine Kolokalisierung von MCHR-1 mit Vasopressin und Oxytocin im Supraoptischen Nukleus gezeigt werden. Zusätzlich konnte MCHR-1 immunhistochemisch auf Zellen der Adeno- und der Neurohypophyse nachgewiesen werden. Diese Ergebnisse lassen auf eine Interaktion von MCHR-1 im Hypothalamus nicht nur mit orexigenen (Orexin A und Neuropeptid Y) und anorexigenen (Adrenocorticotrophin Hormon) Signalen schließen, sondern weisen zusätzlich auf eine Rolle von MCHR-1 bei der Regulation des Wasserhaushalts (Vasopressin), der Fortpflanzung (Oxytocin) und bei Stress (Corticotrophin-releasing Hormon) hin. Im zweiten Versuchsvorhaben führte die zentraler Gabe (intrazerebroventrikular) von MCH ins Rattengehirn zu einer akuten und signifikanten Steigerung der Futter- und Wasseraufnahme, es konnte jedoch kein spezifisches Aktivierungsmuster in hypothalamischen Kernarealen (Nuklei) definiert werden. Im Gegensatz dazu führte eine sub-chronische Gabe eines oral verfügbaren MCHR-1 Antagonisten in Ratten zu einer signifikanten Verminderung der Nahrungs-, Wasseraufnahme und des Körpergewichts. Bei qualitativer Analyse des immunhistochemischen Signals für c-Fos bei MCHR-1 Antagonist behandelten Ratten konnte eine spezifische Aktivierung im Paraventrikulären Hypothalamus, im Supraoptischen Nukleus und im Dorsomedialen Hypothalamus gezeigt werden. Diese Ergebnisse ließen sich durch automatisierte, software-unterstützte Quantifizierung des c-Fos Signals bestätigen und heben diese Hirnareale als mögliche neuroanatomische Substrate von MCHR-1 Antagonisten hervor. Um eine mögliche neuronale Aktivierung des MCH Systems nach einem physiologischen Stimulus, hier Hunger oder Sättigung, zu untersuchen, wurden in einem weiteren Versuchsansatz Ratten in einem angepassten, neun Tage dauernden Fütterungsregime, täglich für nur drei Stunden Zugang zu Futter gewährt. Tiere, die am letzten Tag des Fütterungsregimes im 3 Stunden Zeitraum kein Futter bekamen und so als „Hunger-Stimulierte“ definiert wurden, zeigten eine signifikante Induktion von c-Fos in unterschiedlichen hypothalamischen (arcuate Nukleus, Dorsomedial Hypothalamischen Nuklei, Lateral Hypothalamus) und extrahypothalamischen Hirnarealen (Nukleus Accumbens, Basolaterale Amygdala, Paraventriculärer Thalamischer Nukleus). Dieses Aktivierungsmuster unterschied sich von Ratten, die am letzten Tag des Fütterungsregims Futter erhalten hatten, den „gesättigte Tieren“ (Aktivierung vor allem im supraoptischen Nukleus, im paraventrikulären Hypothalamus und Nukleus Tractus Solitarius), oder ad libitum gefütterten Kontrolltieren. Um durch das Fütterungsregime aktivierte Neurone dem MCH System zuzuordnen, wurden immunhistochemische Kolokalisationsexperimente von c-Fos mit MCH beziehungsweise MCHR-1 spezifischen Antikörpern durchgeführt. Zwar konnte keine Kolokalisation von c-Fos mit MCH im lateralen Hypothalamus nachgewiesen werden, aber eine Vielzahl von durch Hunger oder Sättigung aktivierte, c-Fos positive Neurone zeigte MCHR-1 Immunoreaktivität. Zusammenfassend lässt sich daraus schließen, dass Nahrungskarenz differenziert unterschiedliche intra-hypothalamische und extra-hypothalamische Zielstrukturen aktiviert. Die funktionelle Rolle des MCHR-1 in solch aktivierten Neuronen bedarf weiterer Klärung. Im abschließenden Teil der Arbeit wird eine mögliche Relevanz der hier beschriebenen Ergebnisse im Hinblick auf die Entwicklung von MCHR-1 Antagonisten und deren möglicher Einsatz bei Adipositas, diskutiert.

Depuis les années 20 du siècle précédent, les recherches effectuées dans le champ de la didactique des langues non maternelles ont enregistré l’apparition d’un public spécifique demandeur de formations en langue à des fins spécifiques. Cela a donné lieu à un domaine qui se distingue par la grande diversité de ses contextes ainsi que de ses publics, un domaine qui porte sur un jeu de désignations ou sur une jungle d’appellations qui se confondent pratiquement toutes. C’est dans le but de faire acquérir à ces publics des compétences langagières spécifiques utiles dans la pratique de leur travail qu’enseignants et chercheurs sont appelés à créer des démarches méthodologiques spécifiques.Cette thèse cherche, plus particulièrement, à apporter des éléments de réponse aux demandes des étudiants de la faculté des sciences de la santé, en Syrie, qui ont besoin d’apprendre le français médical et non le français général pour s’en servir plus tard dans leur profession. L’objectif principal de ce travail est, d’une part, d’identifier les besoins réels de ces apprenants en prenant en compte les spécificités de leur domaine et d’autre part, de concevoir un référentiel de formation FOS susceptible de contribuer au développement d’une compétence de communication spécifique appropriée aux exigences de leur terrain socioprofessionnel. / Research performed since the twenties in the field of didactics of foreign languages, helped the rise of a certain audience with specific requests in professional language. That gave rise to a domain, which is composed of an important diversity of contexts and publics called "French for Specific Purposes". Teachers and researchers are brought to create specific methodological approach, to help this audience gain specific linguistic skills.This thesis aims at bringing elements of answer to the students of the syrian scientific colleges, particularly to those of the Faculty of Health Sciences. Our purpose is to identify the real needs of the latter, taking into account peculiarities of their domain and to conceive teacher-training frame of reference allowing them to develop oral and written comprehension and expression skills.

O laser de baixa potência vem sendo utilizado em Odontologia com diversos objetivos, como diminuir o tempo de reparação de tecidos moles e duros e, atualmente, alguns profissionais tem utilizado esta ferramenta para o controle clínico da dor. O presente trabalho in vivo teve como objetivo avaliar quantitativamente os efeitos do laser de baixa potência (LBP) com diodo de GaAlAs (Gálio-alumínioarsenieto) no controle da dor após a expansão rápida da maxila (ERM), analisando a ativação de regiões cerebrais relacionadas à nocicepção, por meio da expressão de c-fos nos subnúcleos caudalis, interpolaris e oralis. Utilizou-se 75 ratos Wistar, machos, pesando em média 220g, que foram distribuídos em 4 grupos: Grupo Controle (n=5) animais não tratados (sem ERM e sem aplicação do LBP) e no período zero foram submetidos à eutanásia; Grupo Experimental I (n=20) animais submetidos apenas à aplicação do LBP no período zero e à eutanásia nos períodos 12, 24 48 e 72 horas após a aplicação do LBP; Grupo Experimental II (n=25) animais submetidos apenas à ERM e à eutanásia nos períodos 6, 12, 24 48 e 72 horas após a ERM; Grupo Experimental III (n=25) animais submetidos à ERM e logo em seguida o LBP no período zero; Os animais deste grupo foram submetidos à eutanásia nos mesmos períodos que o Grupo Experimental II. Após a eutanásia, os cérebros foram coletados e realizou-se secções coronais de 40 micrômetros. Os cortes foram processados para a imunohistoquímica para c-fos e analisados com o auxilio de um microscópio óptico. As células imunorreativas à proteína Fos foram contadas através do auxilio de um sistema de imagem (Image J). O teste de variância (ANOVA) foi usado seguido pelo pós-teste de Tukey, com nível de significância de 5%. O grupo que teve ERM apresentou um aumento significativo do número de neurônios Fos positivos nos subnúcleos interpolaris e caudalis 6 horas após a expansão maxilar, o que diminuiu expressivamente a partir de 12 horas, com um segundo pico no período de 24 horas (p<0,001). O grupo que teve aplicação do LBP teve redução expressiva do número de neurônios Fos positivos em todos os subnúcleos 12 horas após a aplicação da força (p<0,001). Os resultados sugerem que o LBP reduz a ativação neuronal de região nociceptiva e o mesmo seria uma possível alternativa para o alívio de dor em pacientes ortodônticos. / Low-level laser therapy (LLLT) has been used in Dentistry with many objectives, especially to decrease the time needed for bone and soft healing. Currently, some professionals have applied this tool for clinical management of pain. The aim of the present iin vivo study was to quantitatively evaluate the effects of Gallium-Aluminum- Arsenide (GaAlAs) low-level laser therapy (LLLT) on pain control after rapid maxillary expansion (RME) in young rats, by means of c-fos quantification in nociceptive related structures (caudalis, interpolaris and oralis subnuclei). A total of 75 male rats, weighting 220g, were assigned to 4 groups: Control Group (n=5) with no treatment (no RME and no LLLT); Experimental I (n=20) with LLLT without RME, evaluated at 12, 24, 48 and 72 hours; Experimental II (n=25) with RME without LLLT, evaluated at 6, 12, 24, 48 and 72 hours after RME; Experimental III (n=25) with RME and LLLT (54J/cm2), evaluated at 6, 12, 24, 48 and 72 hours after RME. The animals were euthanized, brain tissues were collected and coronal sections were cut at 40m, through the spinal trigeminal caudalis, spinal trigeminal interpolaris, and spinal trigeminal oralis subnuclei. The sections were processed for c-fos immunohistochemistry and were analyzed in light microscopy. The Image J software was used to quantify Fos immunoreactive neurons in sections of the rat brains. Statistical analysis was performed using ANOVA and Tukey tests with a significance level of 5%. In the experimental group I, Fos expression significantly increased in neurons in oralis and interpolaris subnuclei 6 hours after the maxillary expansion, then significantly decreased at 12 hours, and increased again at 24 hours (p<0.001). In experimental group III, Fos significantly decreased in neurons in all subnucleis 12 hours after force application (p<0.001). These results suggest that LLLT decrease cfos expression in neurons of nociceptive regions and it may be used as a therapeutic alternative to reduce pain in orthodontic patients.

A transição da adolescência (ADOLESC) para a idade adulta (ADULTO) é caracterizada por uma maturação de comportamentos, assim como de estruturas e sistemas do SNC. Este estudo investigou os efeitos da administração aguda e repetida de baixas doses de etanol (2,0 g/kg) em camundongos ADULTO (60 dias) e ADOLESC (28 dias), bem como a expressão de c-Fos e Egr-1 em distintas regiões cerebrais. Camundongos ADULTO e ADOLESC receberam agudamente salina (SAL) ou EtOH e sua atividade locomotora foi quantificada em campo aberto (CA). Uma hora após a injeção, a expressão de c-Fos e Egr-1 foi avaliada por imuno-histoquímica. Foram constatados: aumento na atividade locomotora e na expressão de c-Fos e Egr-1 nos animais tratados com EtOH. Outros animais ADULTO e ADOLESC foram tratados com EtOH ou SAL repetidamente (15 dias). Uma semana após o pré-tratamento, todos animais receberam etanol. A atividade locomotora foi quantificada e a expressão de c-Fos e Egr-1 foi avaliada. Os ADOLESC desenvolveram tolerância, enquanto que os ADULTO apresentaram sensibilização comportamental locomotora. A administração prolongada de etanol induziu alterações adaptativas diferenciadas, dependentes da idade, na expressão de c-Fos e Egr-1 em várias regiões encefálicas. / The transition from adolescence (ADOLESC) into adulthood (ADULT) is characterized by behavioral maturation, as well as of structures and systems of CNS. This study investigated the behavioral effects of acute and repeated administration of EtOH (2,0 g/kg) in ADULT and ADOLESC mice, as well as the c-Fos and Egr-1 expression induced by EtOH in distinctive brain structures. Locomotor activity from ADULT and ADOLESC mice acutely treated with either saline (SAL) or EtOH was measured in open field (OF). One hour after injections, c-Fos and Egr-1 expressions were evaluated by immunohistochemistry. The results demonstrated increased locomotor activity and increased c-Fos and Egr-1 expression in EtOH-treated mice. Other groups of ADULT and ADOLESC mice were treated repeatedly with SAL or EtOH during 15 days. One week after this pretreatment, all animals received an injection of EtOH. The locomotor activity was quantified and c-Fos and Egr-1 expressions were evaluated. While ADULT mice presented behavioral sensitization, ADOLESC mice developed tolerance. The repeated administration of EtOH induced an age-dependent differential expression of c-Fos and Egr-1 in several brain regions.

Hecogenin acetate (HA) is a steroidal sapogenin-acetylated, one of the most important precursor used by the pharmaceutical industry for the synthesis of steroid hormones. However, no studies were found on the possible analgesic profile of HA. Thus, we aimed to evaluate antinociceptive profile of HA in chronic and acute animal models. Acute pretreatment with HA (5 40 mg/kg) produced a dose dependent increase in the tail flick latency time when compared to vehicle-treated group (p<0.01) demonstrating central analgesic effect. The antinociceptive effect of HA (40 mg/kg) was prevented by naloxone (a non selective opioid receptor antagonist; 5 mg/kg), CTOP (μ opioid receptor antagonist; 1 mg/kg), nor- BNI (k opioid receptor antagonist; 0.5 mg/kg), naltrindole (δ opioid receptor antagonist; 3 mg/kg), or glibenclamide (ATP sensitive K+ channel blocker; 2 mg/kg). This effect no seems to be related to a possible myorelaxing profile of HA, since mice treated with HA (5 - 40 mg/kg) did not show motor performance alterations. Systemic administration of HA (5 - 40 mg/kg), increased the number of Fos positive cells in the periaqueductal gray and the acute pretreatment with HA, at all doses tested, significantly inhibited the Fos expression in the spinal cord dorsal horn. Additionally, intraperitoneal administration of HA (5, 10, or 20 mg/kg; i.p.) inhibited the development of mechanical hyperalgesia induced by carrageenan, TNF-α, dopamine and PGE2 in mice. When we investigated effects of HA (20 or 40 mg/kg, i.p.) on chronic neuropathic hypersensitivity (partial sciatic nerve ligation - PSNL), the acute treatment with HA was effective in producing a significanty (p<0.01) anti-hyperalgesic effect in PSNL model on mice. So, the present study demonstrates, for the first time, that HA produced consistent antinociception mediated by opioid receptors and endogenous analgesic mechanisms and that this compound may be useful in the study of new therapeutic approaches to pain treatment. / Acetato de Hecogenina (HA) é uma sapogenina esteroidal acetilada, utilizada como um importante precursor pela indústria farmacêutica para a síntese de hormônios esteróides, tais como progesterona e prednisona. No entanto, existem poucos estudos farmacológicos sobre o HA e não foram encontrados estudos especificamente sobre o seu possível efeito analgésico. Desta forma, o objetivo deste estudo foi avaliar o perfil antinociceptivo do HA em camundongos, em modelos de nocicepção crônica e aguda. O pré-tratamento agudo por via intraperitoneal (i.p.) com HA (5 - 40 mg/kg) produziu um aumento dose dependente no tempo de latência (p<0,01) da retirada da cauda no teste do tail-flick em relação ao grupo tratado com o veículo, sugerindo um efeito analgésico central. Quando avaliada juntamente com antagonistas farmacológicos, o HA (40 mg/kg) teve seu efeito revertido pela naloxona (um antagonista não seletivo do receptor opióide, 5 mg/kg), CTOP (antagonista do receptor μ opióide, 1 mg/kg), nor-BNI (antagonista do receptor K opióide, 0,5 mg/kg), naltrindole (antagonista do receptor δ opióide, 3 mg/kg), ou glibenclamida (bloqueador do canal para K sensível ao ATP, 2 mg/kg), sugerindo um efeito sobre o sistema opióide. O efeito antinociceptivo do HA não parece estar relacionado a um possível perfil miorrelaxante, uma vez que camundongos tratados com HA (5 - 40 mg/kg) não apresentaram alterações na função motora quando avaliados no teste da coordenação motora. A administração sistêmica de HA (5 - 40 mg/kg) aumentou o número de células positivas para proteína Fos na substância cinzenta periaquedutal e, por outro lado, o pré-tratamento agudo com HA, em todas as doses testadas, inibiu significativamente a expressão de Fos no corno dorsal da medula, sugerindo um possível efeito sobre a via descendente de controle da dor. Adicionalmente, a administração i.p. de HA. (5, 10, ou 20 mg/kg), inibiu de maneira significativa (p<0,05 ou p<0,001) a hiperalgesia mecânica induzida pela carragenina, TNF-α, dopamina e PGE2 em camundongos. Ao investigar os possíveis efeitos de HA (20 ou 40 mg/kg, i.p.) sobre a hipersensibilidade neuropática crônica (modelo de ligação parcial do nervo isquiático - PSNL), o tratamento agudo com HA foi eficaz em reduzir significativamente (p<0,01) o comportamento hiperalgésico, sem alterar os parâmetros motores dos animais. Diante desses resultados, o presente estudo sugere, pela primeira vez, que o HA possui perfil antinociceptivo consistente com mecanismos mediados pelo sistema opióide, e que este composto pode ser útil no estudo de novas abordagens terapêuticas para o tratamento farmacológico da dor.

Para compreensão da biologia dos tumores adrenocorticais são utilizados marcadores moleculares que em geral são genes reguladores do ciclo celular. AP-1 é um fator dimérico composto principalmente pelas proteínas JUN (JUN, JUNB e JUND) e FOS. Nesse projeto tivemos como hipótese que as proteínas da família JUN, se correlacionam com proteínas reguladoras do ciclo celular em tumores adrenocorticais, e poderiam ser utilizados no diagnóstico e prognóstico desse tipo de tumor. O objetivo foi analisar o padrão de expressão gênica e proteica (PCR e Immunobloting). A análise por PCR Array em culturas de células de tumores adrenais, mostrou que os genes da família JUN e o gene FOS estão pouco expressos nessas culturas, o que foi confirmado em ensaios de qPCR. Não foi possível determinar um padrão de expressão que diferenciasse os tipos de culturas celulares estudados, ou mesmo tumores adultos e pediátricos. Os tratamentos com ACTH aumentam a expressão da proteina JUN e JUNB, e podem ter certa importância em tumores responsivos à esse hormônio, que merecem análises futuras. / In order to have a better comprehension of adrenocortical tumor biology, molecular markers are utillized because they are in general cell cycle regulators. AP-1 is a dimeric factor, compound mainly by JUN (JUN, JUNB, JUND) and FOS proteins. In the present study, our hypothesis is that JUN proteins are correlated with others cell cycle regulatory proteins and could be utilized as a prognose and diagnostic predictor for adrenocortical tumors. To confirm our hypothesi the aim was genic and protein profile analyzes (PCR and Immunobloting). The PCR Array analysis showed that JUN and FOS gene expression were down regulated in these adrenocortical tumors cells which were confirmed through qPCR analysis. The genes expression analysis wasn´t able to stablish a standard of expression between the studied cell cultures or even between adults and pediatric tumors. The ACTH treatments increased JUN and JUNB proteins that may have some significance in responsive tumors to this hormone, and deserve further analysis.

Les circuits neuronaux responsables du sommeil paradoxal (SP) et de l'atonie musculaire qui le caractéristique sont l'objet de nombreuses recherches expérimentales, notamment en raison de l'existence de plusieurs pathologies invalidantes associées. Cette thèse de Neurobiologie s'inscrit plus spécifiquement dans la description anatomique et fonctionnelle du réseau neuronal responsable de l'atonie musculaire et son potentiel dysfonctionnement dans les troubles comportementaux en SP (RBD, REM sleep Behavior Disorder). Pour ce faire, nous avons combiné plusieurs techniques faisant appel à la neuroanatomie fonctionnelle, au traçage rétrograde de voies nerveuses, à l'hybridation in situ à la polysomnographie et à l'inactivation irréversible de populations neuronales ciblées moléculairement à l'aide de virus adéno-associés contenant des short hairpin RNAs (AAV-shRNA) chez le rat libre de ses mouvements. Nous avons ainsi montré que, contrairement à l'hypothèse généralement admise, le noyau sublatérodorsal pontique (SLD) n'est pas le générateur du SP. En effet, l'inactivation neurochimique de ses neurones glutamatergiques ou sa lésion totale diminuent les quantités de SP sans le supprimer, indiquant que le SLD n'est pas suffisant pour la genèse du SP. En revanche, ces expériences démontrent son implication directe dans la mise en place de l'atonie musculaire lors du SP. En effet, la déconnexion neurochimique des neurones glutamatergiques du SLD provoque pendant le SP l'apparition intermittente de tonus musculaire accompagné de comportements moteurs anormaux. En parallèle, nos travaux de thèse ont permis d'apporter des données expérimentales nouvelles sur la localisation, au sein de la formation réticulée bulbaire ventrale et non dans la moelle épinière, des interneurones GABA/glycine responsables de l'hyperpolarisation des motoneurones somatiques pendant le SP. En effet, ces neurones réticulaires sont exclusivement recrutés pendant le SP et envoient des projections monosynaptiques inhibitrices vers les motoneurones somatiques lombaires. De plus, leur déconnexion neurochimique ciblée déclenche des comportements moteurs anormaux sous-tendus par le maintien d'un tonus musculaire irrégulier pendant le SP. L'analyse actimétrique de ces comportements moteurs oniriques induits expérimentalement montre qu'ils sont très semblables à ceux observés après l'inactivation du SLD et à ceux décrits chez les patients RBD. Les données rapportées dans cette thèse permettent de mieux comprendre les mécanismes neurobiologiques générant le SP et ceux contribuant au contrôle moteur pendant le SP. Par la même occasion, nos travaux ont permis de valider deux modèles rongeurs du RBD humain, ouvrant ainsi des perspectives expérimentales pour l'élaboration de traitements ciblés de cette pathologie affectant le SP / A growing number of studies investigate the neuronal network responsible for paradoxical (PS) (or REM) sleep genesis and muscle atonia specific of this sleep state. The aim of this thesis was to characterize at the anatomical and functional levels the populations of neurons involved in generating muscle atonia during PS and their potential failure in REM sleep Behavior Disorder (RBD). For this purpose, we combined a large panel of experimental techniques such as functional neuroanatomy, retrograde tract-tracing, in situ hybridization, polysomnography and irreversible inactivation of genetically-targeted neurons with short-hairpin RNAs introduced in viral adenovectors (AAV-shRNA) in freely moving rats. We thus demonstrated for the first time that, in contrast to the currently admitted hypothesis, the pontine sublaterodorsal nucleus (SLD) is not the PS generator, since genetic inactivation of its glutamatergic neurons or its whole lesion diminish the quantities of but do not eliminate PS. This indicates that the SLD is not sufficient for PS generation. In contrast, our experiments clearly show that the SLD is responsible for muscle atonia because the specific inactivation of its glutamatergic neurons induces an irregular muscle tone concomitant to atypical motor behaviors during PS. In addition, we achieved original data about the location within the ventral medullary reticular formation, and not at spinal levels as often believed, of the glycine/GABA interneurons managing the sustained hyperpolarization of somatic motoneurons during PS. We indeed observed that these medullary neurons are selectively recruited during PS and send monosynaptic inhibitory efferents to the lumbar somatic motoneurons. Furthermore, their genetic inactivation is followed by an increase of abnormal motor behaviors underpinned by a sustained, although irregular, muscle tone. The actimetric analysis of these oneiric experimentally induced behaviors reveals that they are very similar to those observed after SLD inactivation or those reported in RBD patients. Taken together, data harvested during this Thesis help us to better understand the complex neurobiological mechanisms generating PS or specifically contributing to the control of the motor system during PS. At the same time, we validated two rodent models closely mimicking human RBD and thus opening new research fields for the development of targeted treatments for this pathology affecting REM sleep