A Ouabaína (OUA), esteroide cardiotônico endógeno, ao ligar-se a enzima NKA pode sinalizar modulando a atividade da Src quinase e os níveis intracelulares de Ca2+, desencadeando a ativação de diversas vias de sinalização, dentre elas a via do NFκB, que uma vez ativada, é capaz de ativar outras vias de sinalização, como a via canônica da WNT. O objetivo dessa tese foi verificar os efeitos da administração intrahipocampal de OUA, em ratos, na sinalização das vias NFκB, BDNF/CREB e canônica da WNT, assim como os possíveis efeitos morfológicos e funcionais. Os resultados sugerem que a OUA altera o microambiente celular favorecendo a ativação das vias propostas levando ao aumento da arborização dendrítica de neurônios do CA1 e do GD, com melhora da memória de referência espacial dos animais e piora do processo de extinção de memória de longa duração, também conhecido como flexibilidade comportamental, além de proteger os neurônios da apoptose frente a um estímulo excitotóxico. / It is well established that Ouabain (OUA), an endogenous cardiotonic steroid, can bind to NKA and trigger activation of signaling pathways dependent on Src kinase activation and intracellular Ca2+ levels oscillation, what can lead to NFκB activation, and in turn, can transactivate other signaling pathways, such as the canonical WNT pathway. The aim of this thesis was to investigate the effects of intrahippocampal administration of OUA in adult rats, whether it was able to activate NFκB, BDNF/CREB and the canonical WNT signaling pathways, as well as the possible morphological and functional effects of this injection. Results suggested that OUA changed the cellular microenvironment in favor to the activation of the proposed pathways leading to increased dendritic branching in CA1 and DG neurons, with spatial reference memory improvement and worsening of long-term memory extinction. Furthermore, OUA protected neurons from apoptosis stimulus triggered by excitotoxicity.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Traumatic brain injury (TBI) is a major cause of morbidity and mortality in industrialized countries leading to the motor and cognitive deficits. Evidence demonstrated that exercise is neuroprotective in traumatic brain injury. However, the effects of exercise before of the TBI at the cognitive function are unknown. Role of excitotoxicity and oxidative damage in secondary damage of TBI, however, until this moment, were not demonstrated if exists a relationship between early phase of damage and the late cognitive deficit. In the current study, we proposed that improvement cognitive response induced by exercise prior in rats after a TBI can be associated with the neuroprotection of early phase after injury. To demonstrate this hypotheses, adult rats practice swimming exercise during 6 weeks followed for TBI operation. We assessed the motor alterations of early phase, the glutamate uptake and antioxidant defense in twenty four hours (24 h) and 15 days after TBI. Acquisition of memory was assessed by recognition object task on days 15 post TBI. Moreover, we evaluated the brain-derived neurotrophic factor (BDNF) to assessement the synaptic plastic. In the present study, we showed that TBI induced by fluid percussion injury (FPI) in adult male Wistar rats induced early motor impairment 24 h, followed by learning retention deficit (2 weeks after neuronal injury). Previous swimming training improved the memory in object recognition task per se and protected against FPI-related disabilities. Although the FPI did not alter hippocampal expression of glutamate transporters (EAAT1 / EAAT2) and brain-derived neurotrophic factor (BDNF), the alterations in the redox status, herein characterized by DCFH-DA oxidation and SOD activity inhibition, led to marked impairment of protein functionally (Na+, K+-ATPase activity inhibition) and glutamate uptake inhibition 24 h after neuronal injury in sedentary injured rats. Indeed, the early increase of nuclear factor erythroid 2-related factor (pNRF2/NRF2 ratio) followed by a repair mechanism (protein HSP70 expression), 24 h and 2 weeks after neuronal injury, suggests that FPI-induced signal transduction may exert compensatory effect on pathophysiological processes. In this report we showed that previous physical exercise induced the increase of immune content of glutamate transporters (EAAT1/ EAAT2), pNrf2/Nrf2 ratio, SOD enzyme and HSP70 per se besides preventing against FPI-induced Na+, K+ - ATPase activity, glutamate uptake inhibition DCFH-DA oxidation 24 h after neuronal injury. The enhancement of hippocampal pNrf2/Nrf2 and HSP70 immune content in trained injured when compared with sedentary rats suggest that protein expression modulation associated to antioxidant defense elicited by previous physical exercise prevent against toxicity induced by TBI. The significant increase of BDNF levels in trained injured rats 24 h and 2 weeks strongly reinforce the idea that physical activity alters neuronal functions and thus delays or prevents secondary cascades that leave the neurobehavioral disability after TBI. / O traumatismo cranioencefálico (TCE) é uma das maiores causas de morte e morbidade nos países industrializados podendo levar ao comprometimento motor e déficits cognitivos. Evidências demonstram que o exercício físico é neuroprotetor na recuperação após o TCE. Porém, os efeitos do exercício físico antes do TCE na função cognitiva não são totalmente conhecidos. Sabe-se da participação da excitotoxicidade e do estresse oxidativo na cascata do dano secundário após o TCE, entretanto até o momento não foi demonstrado qual a relação da fase inicial após o TCE com os déficits cognitivos tardios. Portanto, no presente estudo, nós propomos que a melhora cognitiva tardia induzida pelo exercício prévio em ratos após o TCE pode estar associada com a neuroproteção da fase inicial após o dano. Para demonstrar esta hipótese, ratos adultos praticaram treinamento de natação durante 6 semanas e posteriormente foram submetidos a cirurgia para o TCE. Nós avaliamos as alterações motoras iniciais, a captação de glutamato e a defesa antioxidante em 24 horas (24 h) e 15 dias após o TCE. Aquisição da memória foi avaliada pela tarefa de reconhecimento de objetos em 15 dias após o TCE. Além disso, nós avaliamos o fator neurotrófico derivado do encéfalo (BDNF) para avaliar a plasticidade sináptica. No presente estudo, nós mostramos que o TCE induzido pela lesão de percussão de fluido (LPF) em ratos Wistar machos adultos induziu déficit motor inicial 24 h, seguido por déficit de aprendizagem (15 dias após o dano neuronal). O treinamento de natação prévio melhorou a memória na tarefa de reconhecimento de objeto per se e protegeu contra desabilidades relacionadas ao LPF. Embora o LPF não tenha alterado a expressão dos transportadores de glutamato (EAAT1/EAAT2) e de BDNF, causou uma alteração no estado redox, caracterizado pela oxidação de DCFH-DA e inibição da atividade da SOD. O LPF também causou prejuízo acentuado da funcionalidade de proteínas (inibição da atividade da enzima Na+, K+-ATPase) e inibição da captação de glutamato 24 h após o dano neuronal em ratos sedentários lesionados. De fato, o aumento inicial do fator de transcrição Nrf2 (relação pNrf2/Nrf2), 24 h após o TCE, seguido por um mecanismo de reparo (expressão da proteína Hsp70), 24 h e 15 dias após o dano neuronal, sugerem que a transdução de sinal induzida pelo LPF pode exercer um efeito compensatório em processos patofisiológicos. Neste trabalho, nós mostramos que o exercício físico prévio induziu o aumento do imunoconteúdo dos transportadores de glutamato (EAAT1/EAAT2), relação pNrf2/Nrf2, enzima SOD e a proteína Hsp70 per se, além de prevenir contra inibição da atividade da Na+, K+-ATPase, inibição da captação de glutamato e oxidação de DCFH-DA induzida pelo LPF, 24 h após o dano neuronal. O aumento do imunoconteúdo hipocampal de pNrf2/Nrf2 e Hsp70 em ratos treinados e lesionados quando comparado com ratos sedentários, sugerem que a modulação da expressão das proteínas associadas às defesas antioxidantes induzidas pelo exercício físico prévio preveniu contra a excitotoxicidade induzida pelo TCE. O significante aumento nos níveis de BDNF em ratos treinados e lesionados 24 h e 15 dias, reforçam fortemente a ideia que a atividade física altera a função neuronal e assim retarda ou previne as cascatas do dano secundário que levam a desabilidade neuronal após o TCE.

La récupération fonctionnelle des patients victimes d’un accident vasculaire cérébral (AVC) ischémique est largement sous-tendue par les propriétés plastiques du cerveau et plus précisément par sa capacité à remodeler les réseaux de neurones épargnés par l’infarctus. Les études réalisées sur différents modèles animaux d’infarctus cérébral s’accordent à montrer que ces changements plastiques sont induits par le BDNF (Brain-Derived Neurotrophic Factor). Aussi, augmenter les taux cérébraux de BDNF est considéré comme une stratégie thérapeutique prometteuse de réduction des déficiences post-AVC. Dans ce contexte, notre travail avait 2 objectifs : 1) chez le rat, identifier les cellules impliquées dans la surproduction de BDNF et évaluer la pertinence de la mesure des taux circulants de BDNF pour estimer les taux de BDNF présents dans le cerveau, 2) chez le patient victime d’un infarctus cérébral, étudier l’efficacité de la fluoxétine sur la récupération motrice à 3 mois, la fluoxétine étant un inhibiteur spécifique de la recapture de la sérotonine commercialisé comme antidépresseur et capable non seulement d’augmenter la production cérébrale de BDNF mais aussi de stimuler la plasticité post-lésionnelle.Les études précliniques ont été réalisées chez le rat soumis à l’embolisation unilatérale du cerveau par un nombre variable de microsphères (en carbone et calibrées à 50 µm) afin de reproduire le large panel de souffrance cérébrale rencontré en clinique. Le BDNF a été mesuré dans le cerveau et dans le sang (plasma et sérum par technique ELISA) avant et après (4, 24h et 8j) embolisation. Nos résultats montrent :- que la production de BDNF est plus intense et plus durable dans l’hémisphère embolisé que dans l’hémisphère non embolisé et que cette production est indépendante du degré d’embolisation, marqueur indirect de la souffrance cérébrale. - que les cellules non-neuronales deviennent une source non négligeable de BDNF en cas d’ischémie, notamment les cellules endothéliales et microgliales avant 24h et les astrocytes au temps 8j.- que les taux circulants et cérébraux de BDNF ne sont pas corrélés mais qu’il existe une corrélation entre le BDNF plasmatique mesuré au temps 4h et le degré d’embolisation.L’étude clinique correspond à un essai randomisé contrôlé en double aveugle comparant la fluoxétine (20mg/j, voie orale, pendant 3 mois et débutée entre 5 et 10j après les premiers symptômes) au placebo chez des patients présentant un déficit moteur modéré à sévère sur l’échelle motrice de Fugl-Meyer (n=59 dans chaque groupe). Nos résultats montrent que l’amélioration de la fonction motrice est meilleure sous fluoxétine que placebo. En conclusion, notre travail montre l’intérêt des médicaments capables d’augmenter le BDNF et la plasticité post-lésionnelle pour améliorer le pronostic clinique de l’AVC et identifie pour la première fois les cellules endothéliales cérébrales comme une cible potentielle de ces médicaments. Il remet également en cause l’idée largement répandue selon laquelle les taux circulants de BDNF varient dans le même sens que les taux cérébraux. / Functional recovery after ischemic stroke largely involves brain plasticity and more accurately its ability to reorganize the neuronal networks spared by the infarct. Studies conducted on animals using different ischemic stroke models have demonstrated that plastic changes are induced by BDNF (Brain-Derived Neurotrophic Factor). Hence, increasing levels of BDNF in the brain is considered a promising therapeutic strategy to reduce post-stroke impairments. In this context, our work had 2 aims: 1) In a rat model, to identify cells involved in the over-production of BDNF and to evaluate the pertinence of the measurement of circulating BDNF levels to estimate brain BDNF levels; 2) In ischemic stroke patients, to study the effectiveness of fluoxetin on 3-month motor recovery. This drug is a selective serotonin-reuptake inhibitor commercialized as an antidepressant treatment that is not only able to increase brain production of BDNF, but also to stimulate post-lesion plasticity. Animal studies were performed on rats that underwent unilateral embolization of the brain with various amounts of carbonized calibrated (50 µm) micropsheres in order to mimick the large panel of brain injury observed in humans. BDNF levels were measured in the brain and the blood (plasma and serum, ELISA method) before and after (4, 24h, and 8d) embolization. Our results show that:- The production of BDNF was more intense and longer lasting in the embolized than in the non-embolized hemisphere, and this production was independent of the degree of embolization, an indirect marker of brain injury.- Several non-neuronal cells become a non-negligible source of BDNF after ischemia, particularly endothelial cells and microglia before 24h, and astrocytes at 8d.- Brain and circulating levels of BDNF did not correlate, but a correlation between plasma BDNF at 4h and the degree of embolization was noted.Our clinical study was a randomized placebo-controlled trial that evaluated the efficacy of fluoxetine (20mg/d, oral route, over 3 months, and starting between 5 and 10d after stroke onset) in patients with moderate to severe motor impairment measured by the Fugl-Meyer motor scale (n=59 in each group). Our results showed a greater improvement in motor recovery under fluoxetin than placebo.To conclude, our work underlines the fact that treatments able to increase BDNF levels and post-lesion brain plasticity are of interest to improve the prognosis after stroke. We have shown, for the first time, that endothelial cells are a potential target for these treatments. Our study also calls into question the widespread idea according to which circulating levels of BDNF vary in the same way as levels of BDNF in the brain.

L’objectif de ce travail a été de tester l’hypothèse selon laquelle l’effet bénéfique de l’administration de la forme recombinante de l’activateur tissulaire du plasminogène (rt-PA) chez le patient victime d’un accident vasculaire cérébral (AVC) ischémique ne résulte pas uniquement de son action fibrinolytique mais aussi de sa capacité à augmenter le brain-derived neurotrophic factor (BDNF) dans le cerveau. Dans ce but, nous avons conduit une première étude visant à évaluer chez l’animal sain, l’effet du rt-PA sur les taux cérébraux de BDNF. Dans une seconde approche, nous avons étudié l’effet du rt-PA sur les taux sériques de BDNF chez le patient victime d’un AVC ischémique et chez l’animal soumis à une ischémie cérébrale focale. Le sang a été prélevé chez l’Homme à l’admission (J0), J1, J7 et J90 après l’AVC, et chez le Rat avant et après (1h, 4h et 24h) l’ischémie. Le BDNF a été mesuré dans le cerveau par technique de Western blot et dans le sang par technique ELISA. Dans les deux études le rt-PA (Actilyse®) a été administré sous forme d’un bolus suivi d’une perfusion d’une heure. La première étude montre que 1) le rt-PA augmente les taux de BDNF dans l’hippocampe, 2) le traitement par MK801 (un antagoniste des récepteurs NMDA) mais pas par l’acide tranexamique (un inhibiteur de la plasmine) annule l’effet du rt-PA sur les taux de BDNF. La deuxième étude met en évidence que 1) la récupération neurologique est meilleure chez les patients recevant le rt-PA, 2) le traitement par rt-PA augmente les taux sériques de BDNF à J1 et J7 chez l’Homme, mais ne modifie pas les taux sanguins de BDNF chez l’animal, 3) les taux de BDNF ne sont pas corrélés à la récupération neurologique mais sont inversement corrélés au score cardiovasculaire du patient. En conclusion, nos résultats suggèrent que le rt-PA peut exercer un effet protecteur extra-fibrinolytique en augmentant les taux de BDNFm par une potentialisation de l’activité glutamatergique. Même si le rt-PA induit une meilleure récupération neurologique et augmente les taux circulants de BDNF chez les patients victimes d’un AVC, l’absence de corrélation entre ces deux paramètres n’est pas en faveur de l’utilisation du BDNF circulant comme un marqueur prédictif de récupération neurologique, mais pourrait être un reflet de la capacité de l’endothélium à sécréter le BDNF. / Our objective was to test the hypothesis that the beneficial effect of the administration of the recombinant form of tissue plasminogen activator (rt-PA) in ischemic stroke patient not only results from its fibrinolytic activity but also from its ability to increase brain-derived neurotrophic factor (BDNF) in the brain. To this end, we conducted an initial study to evaluate the effect of rt-PA on brain BDNF levels in healthy animals. In a second study, we investigated the effect of rt-PA on serum BDNF levels in ischemic stroke patients and in animals subjected to permanent focal cerebral ischemia. Blood samples were obtained from patient on admission (D0), D1, D7 and D90 after stroke and in rats before and after (1h, 4h and 24h) ischemia. BDNF was measured in the brain by Western blot and in the blood by ELISA. In both studies, the rt-PA (Actilyse®) was administered as a bolus followed by an infusion of one hour. The first study evidences that 1) rt-PA increases the BDNF levels in the hippocampus, 2) treatment with MK801 (a NMDA receptor antagonist) but not with tranexamic acid (a plasmin inhibitor) canceled the effect of rt-PA on BDNF levels. The second study exhibits that 1) neurological recovery was higher in the patients receiving rt-PA, 2) treatment with rt-PA increases serum BDNF at D1 and D7 in patients, but does not change the blood BDNF levels in animals, 3) BDNF levels are not correlated with neurological recovery but are inversely correlated to the patient cardiovascular score. In conclusion, our results suggest that rt-PA may have a protective extra-fibrinolytic effect by increasing in BDNF levels through a potentiation of glutamatergic pathway. Although rt-PA induces a better neurological recovery and increases circulating BDNF levels in stroke patients, the lack of correlation between these two parameters is not in favor of using circulating BDNF as a predictive marker of neurological recovery, but could be a reflect of the endothelium ability to synthesize BDNF.

Introdução - Os efeitos na saúde de dietas vegetarianas (DV) apontam principalmente para a diminuição do risco de Doenças Crônicas Não Transmissíveis (DCNT), uma vez que modula positivamente parâmetros bioquímicos, particularmente aqueles relacionados ao controle da glicemia e lipemia, além de ser uma medida para o controle de peso. Estudos recentes em adultos demonstram que a dieta possa também modular parâmetros moleculares. Nesse cenário, atenta-se para o papel do Fator Neurotrófico Derivado do Encéfalo (BDNF) o qual parece estar relacionado com a DV em relação à redução de triglicerídeos e colesterol e aumento da sensibilidade à insulina. Objetivo - Avaliar adultos que adotaram uma DV, em relação ao estado nutricional, aos parâmetros bioquímicos e moleculares comparados aos adultos com dieta onívora. Métodos - A população avaliada foi constituída por 96 indivíduos, 56 vegetarianos e 40 onívoros, adultos e de ambos os sexos, em um estudo do tipo transversal. Para o levantamento dos dados sociodemográficos e de estilo de vida foi aplicado questionário e aferidas às medidas de peso corporal (PC) e altura, para posterior cálculo de Índice de Massa Corporal (IMC) e circunferência de cintura (CC). Foi realizada também coleta de sangue para estudos bioquímicos e expressão de BDNF plasmático. Os índices de Castelli 1 e 2 (razões lipídicas) são indicadores de risco cardiovascular (RCV) com maior valor preditivo do que parâmetros isolados, por isso, também foram calculados. A resistência à insulina (IR) foi avaliada pelo índice HOMA_IR. As análises foram conduzidas pelo software SPSS (Statistical Package for Social Sciences) versão 20.0 e para todas foi considerado um nível de significância de 5 por cento .Foi realizada análise de regressão logística para identificar se a DV e outros fatores podem prever a redução da chance de ter RCV, determinado pelos índices de Castelli 1 e 2.Resultados Em relação às variáveis de estilo de vida, os VEG têm uma tendência a praticar mais atividade física (64,3 por cento vs 42,5 por cento , p = 0,056) e ingerir suplementos alimentares (48,1 por cento vs 20,5 por cento , p = 0,012), embora o número de fumantes se apresente semelhante em ambos os grupos. Não houve diferença estatisticamente significante para as variáveis: idade, sexo, prática de fumar, triglicerídeos (TG), Colesterol Total (CT) e lipoproteína de baixa densidade (LDL- c) entre os dois grupos. Já os valores dos índices de Castelli 1 (3,23 ± 0,84 vs 3,90 ± 0,99, p =0,001)e 2 (1,91 ± 0,69 vs 2,42 ± 0,79, p = 0,001) foram menores em vegetarianos (VEG) do que em onívoros (ONV). O grupo VEG tinha significativamente menor PC (63,9 ± 10,4 vs 69,4 ± 14,6 kg, p = 0,032); IMC (22,5 ± 2,6 vs 25,0 ± 3,9 kg/m2, p = 0,001); CC( 81,8 ± 8,2 vs 87,8 ± 10,9 cm, p = 0,003 ) e maior lipoproteína de alta densidade (HDL-c) (54,88 ± 14,44 vs 47,30 ± 12,27 mg / dl , p = 0,008) . Os VEG também apresentaram menor HOMA-IR (1,17 ± 0,70 vs 1,48 ± 0,8, p = 0,02) em comparação com ONV. Quanto a variável BDNF, não houve diferença entre os grupos VEG e ONV (662,8 + 276,5pg/ml vs 698,1 + 314,9 pg/ml, p=0,563). Conclusão - Sugere-se, portanto, que a DV pode ter efeitos protetores na saúde cardiovascular e no metabolismo desses indivíduos. / Introduction - The effects of vegetarian diets (VD) on health points out mainly to a decrease in the risk for noncomunnicable chronic disease (NCDs) once it positively modulates the biochemical parameters, particularly those related to the control of glicemic and lipemia being also a way of controlling weight. Recent studies have shown that diet can also modulate molecular parameters. In this scenario, one must pay attention to the role of the brain-derived neurotrophic factor (BDNF) which seems to be related to the VG in what regards the reduction of triacylglycerol and cholesterol, and the increase of insulin sensitivity. Objective - To assess adults that adopted a VD in what regards their nutritional status, biochemical and molecular parameters, in comparison to adults that adopted an omnivorous diet. Methods- A cross-sectional study assessed a population composed of 96 individuals, 56 vegetarians and 40 omnivores, adults of both genders. A questionnaire was administered in order to gather sociodemographic and life-style related data, body weight (BW), height and waist circumference (WC) were surveyed. In order to carry out the biochemical study and the expression of plasmatic BDNF, blood was collected. The Castelli indexes 1 and 2 (lipid ratios) are indicators of cardiovascular risk (CVR) with a higher predictive value than isolated parameters and therefore were calculated. Insulin resistance (IR) was calculated by the HOMA_IR index. The analyses were carried out through the SPSS (Statistical Package for Social Sciences) software, 20.0 version, taking into account a 5 per cent significance level. An analysis of logistic regression was done in order to identify if the VD and other factors are able to prevent the reduction of CVR, determined by the Castelli indexes 1 and 2. Results - There was no statistically significant difference between both groups regarding the following variables: age, gender, smoking habits, triglyceride (TG), Total Cholesterol (TC) low-density lipoprotein cholesterol (LDL- col). On the other hand, the values of the Castelli indexes 1 (3,23 ± 0,84 vs 3,90 ± 0,99, p =0,001) and 2 (1,91 ± 0,69 vs 2,42 ± 0,79, p = 0,001) were lower in vegetarians (VEG) than in omnivores (OMV). The VEG-groups showed significant lower BW (63,9 ± 10,4 vs 69,4 ± 14,6 kg, p = 0,032); BMI (22,5 ± 2,6 vs 25,0 ± 3,9 kg/m2, p = 0,001); WC ( 81,8 ± 8,2 vs 87,8 ± 10,9 cm, p = 0,003 ) and more high-density lipoprotein cholesterol (HDL col) (54,88 ± 14,44 vs 47,30 ± 12,27 mg / dl , p = 0,008). The VEG-group also presented lower HOMA-IR (1,17 ± 0,70 vs 1,48 ± 0,8, p = 0,02) in comparison to the OMV-group. Regarding life-style parameters, the individuals of the VEG-group displayed a tendency for practicing more physical activity (64,3 per cent vs 42,5 per cent , p = 0,056) and for ingesting food supplement (48,1 per cent vs 20,5 per cent , p = 0,012), although the number of smokers was quite similar between both groups. Regarding the BDNF variable, there was no difference between the VEG-group and the OMVGroup (662,8 + 276,5 pg/ml vs 698,1 + 314,9 pg/ml, p=0,563).Conclusion - In relation to these results it is to be suggested that a VD may exert protective effects on cardiovascular health and on the metabolism of the individuals that adopt it.

La consommation chronique et excessive d’éthanol provoque des modifications neurobiologiques adaptatives. Les mécanismes qui les contrôlent sont multiples et certains ont été reliés à des régulations épigénétiques conduisant à des modifications structurelles et fonctionnelles. L’éthanol induit également une neurodégénérescence de l’hippocampe responsable de déficits cognitifs. Parmi l’ensemble des modèles animaux qui sont utilisés pour étudier les effets d’une consommation chronique d’alcool, figurent les souris de la lignée C57BL/6J. Ces souris possèdent une appétence naturelle pour l’éthanol faisant d’elles un modèle de choix pour étudier les conséquences de la consommation chronique d’éthanol. Le but de ce travail de thèse a été d’étudier les relations entre les mécanismes épigénétiques et la modulation de la neuroplasticité de l’hippocampe à la suite d’une consommation chronique d’éthanol chez les souris C57BL/6J, et d’en évaluer les conséquences comportementales. Nous avons montré que la consommation chronique d’éthanol induit, au niveau de l’hippocampe, des modulations épigénétiques globales corrélées à un remodelage chromatinien au sein du gène du BDNF, impliquant à la fois les modifications post-traductionnelles des histones et la méthylation de l’ADN. Ces modifications épigénétiques sont certainement responsables de l’augmentation d’expression protéique du BDNF observée dans l’hippocampe, et plus particulièrement dans le gyrus denté, après 3 semaines de consommation chronique d’éthanol en libre choix. L’accroissement de l’expression du BDNF induit une stimulation des voies de la signalisation intracellulaire dépendantes de l’activation du récepteur TrkB du BDNF, et une augmentation de la neurogenèse du gyrus denté. Les effets de l’antagoniste spécifique du récepteur TrkB, ANA 12, démontrent que l’augmentation de la neurogenèse observée chez les souris C57BL/6J après la prise chronique d’éthanol, est sous le contrôle unique du complexe BDNF/TrkB. L’analyse comportementale des souris C57BL/6J ayant consommé de l’éthanol, montre une détérioration des capacités d’apprentissage et de mémoire sans modification de la plasticité synaptique dans l’hippocampe, suggérant ainsi que d’autres mécanismes sont impliqués dans ces déficits cognitifs. L’ensemble de ces données apporte de nouveaux éléments de compréhension concernant la stimulation de la neurogenèse hippocampique chez les souris C57BL/6J lors d’une consommation chronique en libre choix d’éthanol. Il est probable que cette apparente augmentation de plasticité soit un mécanisme adaptatif et compensatoire à la détérioration des fonctions cognitives induite par une consommation chronique d’alcool. / Chronic and excessive ethanol consumption triggers neurobiological adaptations within the central nervous system, which are responsible for the development of an addiction. Ethanol induces adaptive mechanisms linked to epigenetic regulations leading to functional and structural changes, and also provokes a neurodegeneration responsible for the cognitive deficits observed in alcohol abusers. Among the different animal models available for studying the effects of chronic ethanol consumption, C57BL/6J mice are among the most relevant. These mice display high ethanol preference, making them a good model for studying the consequences of chronic and free-choice ethanol consumption. The purpose of this work was to study the links between epigenetic mechanisms and hippocampal neuroplasticity and to evaluate the behavioural consequences induced by chronic ethanol consumption in C57BL/6J mice. We showed that, in the hippocampus, chronic ethanol consumption induced global epigenetic modulations that were correlated with chromatin remodelling at the BDNF gene level. These effects involved post-translational histone modifications and DNA methylation. Epigenetic changes at the BDNF gene level probably allowed the increase in BDNF protein expression observed within the hippocampal dentate gyrus in mice having consumed ethanol for 3 weeks. Upregulation of BDNF expression was linked to both the stimulation of intracellular cascades downstream BDNF/TrkB receptor activation, and the increase in neurogenesis within the dentate gyrus. Using a specific TrkB receptor antagonist, ANA-12, we demonstrated that the hippocampal neurogenesis induced by chronic ethanol intake was under the control of BDNF. Behavioural analysis evidenced learning and memory impairments after ethanol consumption without synaptic plasticity alteration within the hippocampus, suggesting the involvement of other mechanisms in the cognitive deficits. Altogether, these data bring new elements for understanding the hippocampal neurogenesis stimulation observed under chronic and voluntary ethanol consumption in C57BL/6J mice. Moreover, this apparent increase in plasticity might probably be considered as an adaptive and compensatory mechanism in response to the cognitive deficits induced by ethanol consumption.

Le GABA et la glycine, principaux neurotransmetteurs inhibiteurs de la moelle épinière adulte, jouent un rôle clé dans la plasticité neuronale. Ils peuvent être excitateurs selon la concentration en chlorure du neurone cible. Celle-ci est principalement régulée par les co-transporteurs, KCC2, spécifique des neurones et NKCC1, ubiquitaire. Ces protéines font respectivement sortir et entrer les ions chlorure. Au cours du développement, l’expression de KCC2 augmente alors que celle de NKCC1 diminue, au moment où l’effet des potentiels post-synaptiques inhibiteurs sur le potentiel de membrane des neurones passe d’une dépolarisation à une hyperpolarisation. Une lésion médullaire à P0 bloque cette augmentation développementale. Cela est corrélé à une perte de fonction de KCC2, laquelle est restaurée après traitement avec un agoniste des récepteurs 5-HT2, indiquant que les voies descendantes issues du tronc cérébral, en particulier sérotoninergique, sont essentielles à la maturation du système inhibiteur. Une lésion chez l’adulte, induit une diminution de l’expression KCC2, en particulier à la surface des motoneurones et de ce fait, de la force de l’inhibition post-synaptique. Le BDNF est impliqué dans cette diminution mais son effet s’inverse 15 jours après la lésion, permettant le réacheminement de KCC2 à la surface des cellules. La phosphorylation des tyrosines et sérines serait impliquée dans l’adressage et la stabilisation de KCC2 dans la membrane plasmique alors que celle des thréonines jouerait un rôle dans son activation fonctionnelle. Ces résultats indiquent que KCC2 est une cible de choix pour restaurer l’inhibition neuronale dans la moelle épinière après traumatisme. / Le GABA et la glycine, principaux neurotransmetteurs inhibiteurs de la moelle épinière adulte, jouent un rôle clé dans la plasticité neuronale. Ils peuvent être excitateurs selon la concentration en chlorure du neurone cible. Celle-ci est principalement régulée par les co-transporteurs, KCC2, spécifique des neurones et NKCC1, ubiquitaire. Ces protéines font respectivement sortir et entrer les ions chlorure. Au cours du développement, l’expression de KCC2 augmente alors que celle de NKCC1 diminue, au moment où l’effet des potentiels post-synaptiques inhibiteurs sur le potentiel de membrane des neurones passe d’une dépolarisation à une hyperpolarisation. Une lésion médullaire à P0 bloque cette augmentation développementale. Cela est corrélé à une perte de fonction de KCC2, laquelle est restaurée après traitement avec un agoniste des récepteurs 5-HT2, indiquant que les voies descendantes issues du tronc cérébral, en particulier sérotoninergique, sont essentielles à la maturation du système inhibiteur. Une lésion chez l’adulte, induit une diminution de l’expression KCC2, en particulier à la surface des motoneurones et de ce fait, de la force de l’inhibition post-synaptique. Le BDNF est impliqué dans cette diminution mais son effet s’inverse 15 jours après la lésion, permettant le réacheminement de KCC2 à la surface des cellules. La phosphorylation des tyrosines et sérines serait impliquée dans l’adressage et la stabilisation de KCC2 dans la membrane plasmique alors que celle des thréonines jouerait un rôle dans son activation fonctionnelle. Ces résultats indiquent que KCC2 est une cible de choix pour restaurer l’inhibition neuronale dans la moelle épinière après traumatisme.

De nombreuses données indiquent qu'un petit poids à la naissance, résultant en partie d'une sous-nutrition materno-fœtale, est associé à une augmentation de la morbidité et de la mortalité durant la période néonatale, et conduit également à un risque accru de développer à l'âge adulte un syndrome métabolique (diabète de type 2, obésité, hypertension artérielle et dyslipidémie). Les mécanismes de cette programmation prénatale sont encore mal connus et impliqueraient plusieurs molécules et systèmes physiologiques distincts. De nombreuses études suggèrent que le placenta serait impliqué dans la programmations de ces pathologies métaboliques. En effet, celui-ci constitue un organe de communication entre la mère et son fœtus et participe à la régulation de l'homéostasie fœtale. En raison de la proportion croissante de femmes présentant des troubles de la nutrition durant la grossesse et en lien avec leurs répercussions potentielles chez la descendance, il est nécessaire de mieux comprendre les interactions entre l'alimentation maternelle et l'unité fœto-placentaire et d'identifier les mécanismes impliqués dans les altérations de la croissance fœtale. En conséquent, le placenta constitue un organe de choix pour étudier les interactions entre l'alimentation maternelle et le fœtus au cours de la grossesse. Durant cette thèse, nous avons tenté d'identifier de nouvelles voies moléculaires placentaires impliquées dans le contrôle de la croissance fœtale chez le rat, puis d'étudié l'expression de ces facteurs dans des placentas humains provenant de grossesses impliquant des anomalies de la croissance fœtale. Comme la malnutrition maternelle constitue une part importante dans l'étiologie de la restriction de croissance intra-utérine (RCIU), nous avons utilisé un modèle expérimental effectué chez le rat, qui consiste en une réduction (de 50% à 70%) de la ration alimentaire quotidienne maternelle durant la gestation. Ces régimes conduisent à des troubles de la croissance de l'unité fœto-placentaire révélés par des réductions drastiques du poids du placenta et des poids de naissance à terme. Afin d'identifier de nouvelles voies placentaires impliquées dans RCIU, nous avons utilisé deux méthodologies différentes: une approche protéomique et une évaluation de deux protéines récemment caractérisées.Premièrement, nous avons étudié le protéome placentaire chez le rat RCIU provenant de mères dénutris par une analyse protéomique (2D-PAGE et spectrométrie de masse). Cette stratégie nous a permis de découvrir de nouvelles voies modulées par le RCIU et, étonnamment, des modulations importantes ont été observées pour plusieurs protéines mitochondriales, suggérant un effet ciblé de la dénutrition sur ces organites. Par la suite, en utilisant diverses techniques d'analyses moléculaires, protéomiques et fonctionnelles, nous avons montré que ces organites élaborent une réponse adaptative à la restriction alimentaire maternelle qui pourrait avoir des conséquences sur la régulation de la croissance fœtale. Deuxièmement, nous avons étudié deux autres protéines atypiques: le brain-derived neurotrophic factor et l'hormone apéline. Nos résultats suggèrent que ces deux facteurs pourraient être impliqués, au niveau placentaire, dans le contrôle de la croissance fœtale à la fois chez le rat et chez l'homme. En conclusion, comme les techniques cliniques actuelles ne permettent pas de diagnostiquer avec précision un RCIU, nos résultats pourraient permettre une meilleure compréhension de la physiopathologie placentaire et permettre de développer de nouveaux marqueurs de diagnostique et/ou de traitement dans le but d'améliorer la croissance placentaire et fœtale en conditions pathologiques.

Mental health has become a prominent issue in society. Yet, much remains unknown about the etiology of psychiatric disorders. The aim of the present thesis was to investigate the association between biological, psychological and social factors of risk and resilience and behavioral problems in a birth cohort of Swedish children. 1723 mothers and their children were followed from birth to the age of 12 as part of the South East Sweden Birth Cohort Study (the SESBiC study). Information was gathered through register data, standardized questionnaires and DNA samples. In study I, stability of maternal symptoms of depression and the impact on child behavior at age 12 were investigated. The prevalence of depressive symptoms was found to be 12.0 % postpartum. Symptoms of postpartum depression significantly increased the risk for subsequent depressive symptoms 12 years later in women. Children whose mothers reported concurrent symptoms of depression and anxiety had an increased risk for both internalizing and externalizing problems at age 12, but no long term effect on child behavior was seen for postpartum depressive symptoms. The greatest risk was seen for children whose mothers reported symptoms of depression on both occasions. In study II, the impact of gene-environment interaction of 5-HTTLPR and BDNF Val66Met and experience of life events together with symptoms of maternal depression and anxiety on child behavior at age 12 was studied. A main effect of 5-HTTLPR was noticed, but no geneenvironment effects were shown. Similarly to study I, concurrent symptoms of maternal depression and anxiety were an important predictor of child behavioral problems. A high degree of psychosocial stress around childbirth was found to have long lasting detrimental effects on child behavior, increasing the risk for internalizing problems at age 12. Study III investigated the impact of geneenvironment interactions of 5-HTTLPR and BDNF Val66Met and life events together with symptoms of maternal depression and birth characteristics on behavioral problems at age 3. Symptoms of postpartum depression were found to predict internalizing as well as externalizing problems in children three years later. Child experience of life events was a stable predictor of behavioral problems across the scales similar to sociodemographic factors such as parental immigration status and unemployment. No gene-environment interaction effects of 5-HTTLPR or BDNF Val66Met were shown. Study IV used the risk factors identified in studies I-III to investigate factors of resilience to behavioral problems at age 12. The l/l genotype of 5-HTTLPR was associated with a lower risk for behavioral problems at age 12, especially for children facing low adversity. Good social functioning was found to be a general resource factor, independent of the level of risk, while an easy temperament was associated with resilience for children with a high degree of adversity. However, effect sizes were small. In summary, the results from the present thesis emphasize the importance of maternal mental health and sociodemographic factors for child mental health at ages 3 and 12, which must be taken into account in clinical settings. Moreover, it adds to the null-findings of the gene-environment effect of 5-HTTLPR and BDNF Val66Met on behavioral problems in children, but indicates a main effect of 5-HTTLPR on internalizing symptoms at age 12.

Smith-Magenis Syndrome (SMS) [OMIM, #182290] is a congenital anomaly and mental retardation (MCA/MR) syndrome associated with deletion of chromosome17p11.2 [1]. The clinical phenotype has been well described and includes minor craniofacial anomalies, self-injurious behaviors as well as sleep disturbances, speech delays, and obesity [1,2,3]. The incidence of SMS is estimated to be ~ 1:15,000 - 25,000 births [2,6]. Among SMS patients, ~90% are comprised of 17p11.2 deletions, while ~10% have RAI1 mutations [8]. All 17p11.2 deletions associated with SMS include RAI1 deletion [10]. RAI1 is thought to function as a transcriptional factor although its cellular role is still unclear. First, in order to better understand the role of RAI1 as a transcriptional factor and its relation to SMS, we confirmed that RAI1 regulates BDNF within an intronic region. This sequence was further narrowed down by utilizing the luciferase reporter assay. This test confirmed what was previously found using ChIP-chip assay and microarray analysis of Rai1+/- mice hypothalami. Next, in order to evaluate the role of Bdnf, an ampakine drug was administered to the Rai1+/- mouse model. A mouse model is a powerful tool for studying a specific gene. Rai1+/- mice exhibit the SMS phenotypes of obesity, craniofacial abnormalities, reduced pain sensitivities, seizures and others. Many physical, neurological, and behavioral tests were performed on the mice to see if any of the phenotypes can be rescued. Interestingly, twice-daily injections of ampakine CX1837 restored the pain sensitivities in Rai1+/- mice. The hot plate data suggest that BDNF potentially has a role in regulating the SMS phenotype of decreased pain sensitivity. In order to evaluate other genes that are altered as a result of the CX1837 ampakine drug, the whole brain's global gene expression was evaluated via microarray analysis. Two potential pain-related genes were identified to be upregulated due to drug administration, which could account for the pain phenotypes observed. One of the genes upregulated in treated mice was Osm, which is interesting because Osm is responsible for pain sensitivity. Further analysis is needed to confirm that an ampakine drug can potentially be used to treat SMS patients.