Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-17T15:11:56Z No. of bitstreams: 1 viniciusdealmeidavieira.pdf: 1001088 bytes, checksum: e1032b4562d8eb7301c929f396f1eff7 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-06-28T14:38:21Z (GMT) No. of bitstreams: 1 viniciusdealmeidavieira.pdf: 1001088 bytes, checksum: e1032b4562d8eb7301c929f396f1eff7 (MD5) / Made available in DSpace on 2016-06-28T14:38:21Z (GMT). No. of bitstreams: 1 viniciusdealmeidavieira.pdf: 1001088 bytes, checksum: e1032b4562d8eb7301c929f396f1eff7 (MD5) Previous issue date: 2012-11-21 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A depressão gestacional é um assunto de grande preocupação, considerando-se os riscos de reincidência de sintomas futuros e os efeitos deletérios à saúde materna e de seus descendentes. Os antidepressivos sintéticos não diminuem a reincidência de sintomas no pós-parto, possuem muitos efeitos colaterais e não são isentos de riscos ao feto. O Hypericum perforatum L. (HP) é um fitofármaco muito utilizado no tratamento da depressão, que apresenta menos efeitos colaterais, parece não apresentar efeitos teratogênicos, e o seu uso demonstrou diminuição da recorrência futura de sintomas. Por outro lado, ainda não se sabe seus reais efeitos, quando utilizado na gravidez, no comportamento futuro da mãe e no desenvolvimento comportamental de sua prole. Portanto, este trabalho avaliou o comportamento de ratas tratadas durante a gravidez com HP no período pós-natal e de seus descendentes na fase adulta. Ratas prenhas foram divididas em três grupos tratados (T1, T2 e T3) e um grupo controle (C), que receberam, por via intragástrica e uma vez ao dia durante toda a gestação, o extrato de HP nas doses de 36, 72 e 144 mg/kg e água destilada, respectivamente. As ratas foram submetidas, com 10 e 60 dias de pós-natal, ao teste do labirinto em cruz elevado para avaliação da ansiedade e aos testes do nado forçado e suspensão da cauda para avaliação da depressão. Os seus filhotes machos foram divididos em quatro grupos, de acordo com o tratamento recebido pelas mães, e avaliados aos 90 dias de vida. Eles foram submetidos aos testes da barra giratória e do tempo de sono induzido por barbitúricos para avaliação de uma possível ação no sistema nervoso central (SNC), aos testes do labirinto em cruz elevado e da placa perfurada para avaliação da ansiedade, e aos testes do nado forçado e de suspensão da cauda para avaliação da depressão. As ratas pertencentes aos grupos T2 e T3, apresentaram um menor comportamento ansioso no teste do labirinto em cruz elevado, e um menor comportamento depressivo nos testes de suspensão da cauda e nado forçado, quando avaliadas 10 e 60 dias após o nascimento dos filhotes e suspensão do tratamento. Nos testes da barra 8 giratória e do tempo de sono induzido por barbitúricos, os descendentes das ratas dos grupos T2 e T3 apresentaram um desempenho sugestivo de ação do HP no SNC, quando comparados aos descendentes das ratas do grupo controle. Nos testes da placa perfurada e do labirinto em cruz elevado e nos testes de suspensão da cauda e do nado forçado, os animais destes mesmos grupos apresentaram um comportamento menos ansioso e depressivo, respectivamente. Portanto, o uso de HP na gestação diminuiu o comportamento depressivo e de ansiedade de ratas Wistar no período pós-natal e também de seus descendentes (F1) quando alcançam a fase adulta. / Gestational depression is a subject of great concern, considering the risks for the recurrence of symptoms and its harmful effects to the mother's and offspring's health. Synthetic antidepressants do not decrease the recurrence of symptoms in the postpartum period, present many side effects, and are not exempt of risks to the fetus. Hypericum perforatum (HP) is a phytopharmacon largely used in the treatment of depression, presents less side effects, does not seem to present teratogenic effects, and its use showed a decrease in the future recurrence of symptoms. On the other hand, its real effects in the future behavior of the mother and in the behavioral development of her offspring when used during pregnancy is not yet known. Therefore, this study evaluated the behavior of rats treated with HP during pregnancy in the postnatal period and of their offspring in their adult phase. Pregnant rats were divided into three treated groups (T1, T2 and T3) and a control group (C) that received, intragastrically, once a day, during the gestational period, HP extract in doses of 36, 72 and 144 mg/kg respectively and distilled water. At ten and 60 days of postnatal life, the rats were submitted to the elevated cross maze test for evaluation of anxiety and to the forced swim and tail suspension tests for evaluation of depression. Their male offspring were divided into four groups, according to the treatment received by the mothers, and evaluated at 90 days of age. They were submitted to the rotarod and barbiturate sleep-induced tests for evaluation of a possible action on the central nervous system (CNS), to the elevated cross maze and hole-board tests for evaluation of anxiety, and to the forced swim and tail suspension tests for evaluation of depression. The rats from groups T2 and T3 displayed less anxious behavior in the elevated cross maze test, and less depressive behavior in the tail suspension and forced swim tests when evaluated 10 and 60 days after the parturition of their offspring and discontinuation of treatment. In the rotarod and barbiturate sleep-induced tests, the offspring of the rats from groups T2 and T3 presented a suggestive performance of the HP action in the CNS, when compared with the offspring of 10 the rats from the control group. In the hole-board and elevated cross maze tests, as well as in the tail suspension and forced swim tests, the animals of these groups displayed less anxious and depressive behavior, respectively. Therefore, the use of HP during pregnancy reduced the depressive and anxiety behavior in Wistar rats in the postnatal period and also in their offspring (F1) when they reached their adult phase.

Orientador: Simone Appenzeller / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T02:14:54Z (GMT). No. of bitstreams: 1 Lapa_AlineTamires_M.pdf: 2736549 bytes, checksum: fab0371aaab88fe1695c8b704d1ee484 (MD5) Previous issue date: 2013 / Resumo: O Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune e multissistêmica. Cerca de 15-20% dos pacientes com LES desenvolve a doença ainda na infância e adolescência. O comprometimento do sistema nervoso central (SNC) é freqüente. No entanto, muitas vezes, se observa uma dissociação entre a clínica e os achados em neuroimagem. Alguns biomarcadores associados à lesão neuronal têm sido relacionados ao LES neuropsiquiátrico, mas seus papéis na patogênese e sua validade e aplicabilidade clínica não tem sido muito estudado em pacientes LESj. Objetivo: Determinar a prevalência de manifestações neuropsiquiátricas (NP) no LESj, analisarem a prevalência de alterações estruturais e lesões de substância branca em imagens de ressonância magnética (RM); além de determinar se S100B e NF-H podem estar associados a alterações estruturais e lesão de substância branca, em pacientes com LESj e controles. Método: Foram incluídos pacientes com LESj e controloes pareados. Manifestações clínicas, laboratoriais e medicação em uso foram avaliadas. A atividade da doença foi analisada através do SLEDAI (SLE Disease Activity Index) e o dano cumulativo foi analisado através do SDI (Lupus International Collaborating Clinics/American College of Rheumatology Damage Index). Os transtornos de humor foram determinados através dos inventários de Depressão (BDI) e Ansiedade (BAI) de Beck e os distúrbios cognitivos foram avaliados pelo teste de inteligência Wechsler de acordo com a idade. As manifestações NP foram analisadas através da revisão de prontuários médicos. Foram consideradas manifestações neuropsiquiátricas presentes no inicio da doença quando ocorreram nos primeiros seis meses de doença e na evolução, quando ocorreram após este período. Pacientes e controles foram submetidos ao exame de 9 RM. A dosagem dos marcadores foi realizada por ELISA (Enzyme Linked Immuno Sorbent Assay. Resultados: No estudo retrospectivo foram incluídos 71 pacientes, no estudo transversal foram incluídos 51 pacientes. Nos dois estudos foram incluídos pacientes com LESj. Observamos que no início da doença as manifestações NP estavam presentes em 49 (69,01%) pacientes e na evolução em 56 (78,87%) pacientes. As lesões da substância branca foram identificadas em 44 (86%) pacientes e em 4 (8%) controles (p<0,001). Tanto o número (número = 1029 vs número = 44) quanto o volume das lesões (volume = 35796,458 vs volume = 1870,559 mm³) foi significativamente maior em pacientes com LESj do que nos controles (p <0,001). A atrofia do corpo caloso foi identificada em seis (11,76%) (p=0,005) e atrofia do volume cerebral foi identificada em 7 (13,72%) pacientes e nenhum controle. Dez (19,61%) pacientes tinham volume ventricular aumentado. Os níveis séricos de S100? e NF-H estavam aumentados em pacientes com LESj (148,98pg/mL±102,73; 101,80pg/mL±89,40 respectivamente) quando comparados aos controles (48,10pg/mL±38,52; 57,12pg/mL±13,28 respectivamente) (p<0,001; p=0,038 respectivamente). Conclusão: As manifestações NP e as lesões de substância branca são frequentes em pacientes com LESj. Volume do corpo caloso e volume cerebral em pacientes são significativamente menores do que os controles e o volume dos ventrículos foi significativamente maior nos pacientes comparados aos controles, isso mostra que os pacientes apresentam atrofia cerebral. Os níveis séricos de S100? e NF-H estão aumentados em pacientes LESj, indicando lesão neuronal / Abstract: Systemic lupus erythematosus (SLE) is an autoimmune disease and multisystem. About 15-20% of SLE patients develop the disease in childhood and adolescence. The involvement of the central nervous system (CNS) is frequent. However, often it is observed a dissociation between clinical and neuroimaging findings. Some biomarkers associated with neuronal injury have been associated with neuropsychiatric SLE, but their roles in the pathogenesis and its validity and clinical applicability has not been studied in patients cSLE. Objective: To determine the prevalence of neuropsychiatric (NP) manifestations in SLE analyze the prevalence of structural changes and white matter lesions on magnetic resonance imaging (MRI), in addition to determining whether S100B and NF- H may be associated with structural changes and white matter lesion in JSLE patients and controls. Methods: Clinical, laboratory and medication use were assessed. Disease activity was assessed by SLEDAI (SLE Disease Activity Index) and the cumulative damage was analyzed by SDI (Lupus International Collaborating Clinics / American College of Rheumatology Damage Index). Mood disorders were determined through the Depression inventory (BDI) and anxiety (BAI) and Beck's cognitive disturbances were assessed by the Wechsler intelligence test according to age. The NP manifestations were analyzed by reviewing medical records. Neuropsychiatric symptoms were considered present early in the illness when they occurred in the first six months of disease and evolution, as occurred after this period. Patients and controls underwent MRI. The dosage of the markers was performed by ELISA (Enzyme Linked Immuno Sorbent Assay. Results: 71 patients were included in the retrospective study and 51 patients were included in cross-sectional study. 11 Both studies included patients with cSLE. Observed that early in the disease manifestations were NP present in 49 (69.01%) patients and progress in 56 (78.87%) patients.'s white matter lesions were identified in 44 (86%) patients and in 4 (8%) controls (p <0.001 ). Both the number (1029 vs number = 44) and volume of lesions (volume = volume = 1870.559 35796.458 vs mm ³) was significantly higher in patients with cSLE than in controls (p <0.001). atrophy of corpus callosum was identified in six cases (11.76%) (p = 0.005) and atrophy of brain volume was found in 7 (13.72%) and no control. Ten (19.61%) patients had a volume ventricular increased. Serum S100B and NF-H were increased in JSLE (148.98 ± 102.73pg/mL; 101.80 ± 89.40 pg/mL respectively) when compared to controls (48.10 ± 38.52 pg/mL; 57.12 ± 13.28 pg/mL respectively) (p<0.001; p=0.038 respectively). Conclusion: According to our results, the NP manifestations and white matter lesions are frequent in patients with JSLE. Corps callosum and brain volume in patients are significantly lower than the controls and the ventricle volume was significantly higher in patients compared to controls, indicating cerebral atrophy. Serum S100? and NF-H are increased in patients cSLE, indicating injury neuronal / Mestrado / Pediatria / Mestra em Ciências

La mise en place du système nerveux central (SNC) repose sur l’équilibre entre prolifération et différenciation des cellules souches neurales. Chez les mammifères, le SNC suit un développement biphasique avec une phase proliférative pendant laquelle les cellules souches neuroépithéliales s’amplifient par division symétrique puis une phase neurogénique pendant laquelle elles sont converties en progéniteurs neuraux se divisant de manière asymétrique pour générer leurs neurones. De nombreux mécanismes sont impliqués dans la régulation des phases du développement du SNC, mais leur coordination au cours du développement demeure mal comprise. Dans ce contexte, la famille des gènes oncofoetaux s’avère très pertinente. Ces gènes sont exprimés au cours du développement précoce où ils coordonnent la synchronie des évènements développementaux. Ils sont éteints en fin de développement mais leur réexpression dans de nombreux cancers témoigne de leur caractère oncogénique. Les mécanismes contrôlant leur extinction au cours du développement ou leur réactivation lors de la tumorigenèse restent obscurs. Durant ma thèse de doctorat, j’ai utilisé la mouche drosophile pour mieux comprendre comment des gènes aux propriétés oncofoetales sont contrôlés et coordonnent les évènements développementaux du SNC. Mon projet de thèse a permis d’identifier le gène aux propriétés oncofoetales, Chinmo, comme impliqué dans le développement du lobe optique de la drosophile pendant sa phase proliférative et dans le processus cancéreux. Ainsi, ces travaux pourront contribuer à une meilleure compréhension du rôle des gènes oncofoetaux dans le contexte développemental et tumoral chez les mammifères. / The development of the central nervous system (CNS) relies on the tight balance between proliferation and differentiation of the neural stem cells. The mammal CNS develops in two phases: the proliferative phase during which the neuroepithelial stem cells amplify through symmetric divisions and a neurogenic phase during which they are converted into neural progenitors that divide asymmetrically to generate their neuronal progeny. Several mechanisms are involved in the regulation of both phases of the CNS development however their coordination in the course of the development remains unclear. In this context, the oncofetal gene family seems particularly relevant. These genes are expressed during the early development where they coordinate the synchrony of the developmental events. They are switched off during the late development but their reexpression in numerous cancers brings evidence of their oncegenicity. The mechanisms governing their repression along the development and their reactivation in cancers are not well understood. During my thesis, I used the Drosophila model to better understand how oncofetal genes are controlled and regulate the developmental events in the CNS. My thesis project allowed to identify an oncofetal-like gene, Chinmo, involved in the development of the Drosophila CNS during its proliferative and in the cancerization process. This work may contribute to a better understanding of the role of oncofetal genes both in the developmental and the tumoral context in mammals.

La dystrophie myotonique de type I (DM1) constitue la plus fréquente des pathologies musculaires héréditaires chez l’adulte. Bien qu’initialement considérée comme une maladie musculaire, la DM1 présente une atteinte neurologique très handicapante. Cette maladie autosomique dominante résulte de l’expansion anormale d’un triplet CTG dans la partie 3’UTR du gène DMPK. Un effet trans du transcrit DMPK muté entraine une dérégulation de l‘épissage alternatif dans de nombreux tissus. Cependant, les mécanismes pathologiques de la DM1 dans le cerveau restent encore peu compris. Afin de disséquer ce mécanisme, notre laboratoire a créé des souris transgéniques exprimant le transcrit DMPK avec de larges expansions CUG dans de nombreux tissus. Ces souris nommées DMSXL, recréent d’importants aspects pathologiques de la DM1, comme des anomalies du comportement et électrophysiologiques du cerveau. Elles représentent donc un excellent outil pour explorer l’effet pathologique de la mutation dans le SNC. En m’appuyant sur ce modèle, j’ai exploré dans un premier temps l’effet trans des ARNs toxiques et l’ampleur de la splicéopathie dans le SNC. De façon intéressante, certains défauts d’épissage sont régions spécifiques, et ne montrent pas d’aggravation avec l’âge des souris DMSXL. Mes résultats démontrent que les ARNs mutés sont capables de déréguler l’épissage alternatif dans l’ensemble du SNC. La région du cervelet a aussi montré des anomalies de l’épissage dans les souris DMSXL, qui, en plus, présentent des perturbations cognitives dépendantes de cette région cérébrale. Le cervelet des souris DMSXL présente aussi des déficits électrophysiologiques suggérant une dysfonction cérébelleuse et plus précisément une dysfonction des cellules de Purkinje. Dans la recherche des populations cellulaires les plus affectées dans le cervelet, j’ai démontré la présence de signes de la toxicité de l’ARN plus marqués dans la glie de Bergman, entourant les cellules de Purkinje. Pour trouver les voies moléculaires perturbées dans le cervelet, et disséquer le mécanisme derrière les anomalies observées, j’ai réalisé une approche protéomique globale et trouvé une sévère baisse de l’expression du transporteur glial de glutamate GLT1/EAAT2, suggérant une dysfonction du cervelet, en conséquence d’un possible métabolisme anormal du glutamate. L’analyse protéomique globale du cerveau des souris DM1 a aussi identifié des différences d’expression et des modifications post-traductionnelles de protéines impliquées dans la signalisation du calcium. L’étude du métabolisme des ARNm dans la DM1 a mis en évidence la dérégulation de l’épissage de gènes impliqués dans le métabolisme du calcium, soutenant l’hypothèse d’une dysfonction calcique dans le SNC. Pour étudier les conséquences de la mutation sur les variations calciques cellulaires, j’ai caractérisé un modèle cellulaire astrocytaire de la DM1. Ce modèle m’a permis de démontrer une localisation anormale du récepteur GRIN1/NMDAR1, ainsi qu’une réponse calcique anormale dans les astrocytes primaires porteurs des amplifications CTG. Malgré les avancés thérapeutiques dans le muscle, on ne sait pas à quel point les stratégies en cours de développement sont efficaces dans le SNC. Pour étudier ce problème, j’ai utilisé le modèle astrocytaire de la DM1 afin de valider in cellulo une stratégie thérapeutique qui vise à rétablir une activité normale du facteur d’épissage MBNL1 endogène. Mes travaux de thèse ont permis d’avancer dans la compréhension de la neuropathologie de la DM1. Ils ont mis en évidence pour la première fois une dysfonction du cervelet, ainsi que la possible dérégulation de la voix calcique dans le SNC. Mes résultats ont donc contribué à mieux comprendre le mécanisme de la DM1 dans le SNC, pour, à long terme, développer des approches thérapeutiques ciblant des évènements moléculaires précis. / Myotonic dystrophy type 1 (DM1) is the most frequent inherited muscular disorder in adults. Although traditionally regarded as a muscle disease, DM1 presents debilitating neurological manifestations. DM1 is an autosomic dominant disease caused by the abnormal expansion of a CTG triplet within the 3’UTR of the DMPK gene. Many molecular aspects of the DM1 are mediated by a trans effect of the expanded DMPK transcripts, whose accumulation leads to splicing deregulation in many tissues. Despite recent progress in the understanding of DM1 pathogenesis in muscle and central nervous system (CNS), the detailed molecular disease mechanism operating in the brain is still poorly understood. In order to investigate the pathophysiology, our laboratory has generated DMSXL transgenic mice expressing DMPK transcripts containing large CUG expansions in many tissues. DMSXL mice mimic important features of the DM1, notably in the CNS, showing behaviour as well as electrophysiological abnormalities. Therefore, this mouse line represents an excellent tool to investigate the toxic effects of the mutation in the CNS. Taking advantages of this transgenic model, I have first explored the trans effect of the toxic RNA and the extent of DM1-associated spliceopathy in the CNS. Interestingly, some splicing defects were region-specific, and their severity did not increase with the age of the DMSXL mice. My data demonstrate that CUG-containing RNAs have a wide deleterious effect and deregulate alternative splicing in many areas of the CNS. In addition to splicing abnormalities in cerebellum, DMSXL mice also displayed deficits in cerebellum-dependant motor coordination. Plus, DMSXL cerebellum showed electrophysiological abnormalities, suggesting cerebellar dysfunction and more precisely Purkinje cell dysfunction. In the search for the cellular populations showing the greatest susceptibility to RNA toxicity in the cerebellum, I have found extensive foci accumulation as well as pronounced splicing defects in the Bergman glia, surrounding Purkinje cells, in DMSXL and DM1 patients cerebellum. In order to identify molecular pathways and mechanisms behind the behaviour and electrophysiological abnormalities detected, I have performed a global proteomics approach and found a severe decrease in the expression of a glial glutamate transporter GLT1/EAAT2, suggesting that DM1 causes cerebellum dysfunction, through abnormal glutamate metabolism. Global proteomic analysis of DMSXL cerebellum also identified expression and post-translational changes of several proteins involved in calcium signalling. Missplicing of different transcripts involved in calcium metabolism reinforces the idea of calcium dysfunction in the neuropathogenesis of the DM1. To study the effects of toxic RNA on calcium homeostasis and flux, I have established and characterised a brain cell model of DM1. DMSXL primary astrocyte cultures allowed me to show the mislocalisation of the glutamate receptor GRIN1/NMDAR1, as well as abnormal calcium responses to stimulation. Despite recent therapeutic advances in muscle, we do not know the CNS efficiency of the therapeutic strategies currently being developed. To address this problem, I have used the DM1 astrocyte cell model to validate in cellulo a therapeutic strategy aiming to restore the activity of the endogenous splicing factor MBNL1. My thesis work provided a significant step in the understanding of the DM1 pathology in the CNS. My results revealed for the first time signs of cerebellum dysfunction in DM1, as well as signs of calcium homeostasis deregulation in the SNC. My work contributed to better understand the pathological mechanisms of DM1, the brain pathways and cell types most susceptible to toxic RNA. In the long term, my data will contribute to the rational development of therapeutic strategies targeting precise and deleterious molecular events.

Les récepteurs purinergiques P2X sont des canaux ioniques activés par l’ATP. Ils sont exprimés très largement dans l’organisme, et possèdent de nombreux rôles physiologiques et pathologiques. Les récepteurs P2X4 en particulier ont été impliqués dans les processus de douleur chronique. Suite à une lésion nerveuse, l’expression des récepteurs P2X4 est induite de novo dans la microglie spinale activée, où ils sont responsables de l’hypersensibilité mécanique caractéristique des douleurs neuropathiques.Notre étude montre que les récepteurs P2X4 neuronaux sont également des acteurs centraux dans plusieurs processus pathologiques, et notamment dans la douleur inflammatoire périphérique chronique. Les récepteurs P2X4 sont exprimés par les neurones sensoriels des ganglions rachidiens et semblent impliqués dans la libération du BDNF dans la corne dorsale de la moëlle épinière. Cette libération conduit à l’activation des voies de signalisation de la voie BDNF/TrkB, et en particulier à la diminution de l’expression de KCC2. Ce processus est en partie responsable de l’allodynie tactile et de l’hyperalgésie mécanique observée en cas de douleur inflammatoire chronique. Notre étude a également permis d’étendre cette hypothèse à un modèle d’excitotoxicité in vitro dans l’hippocampe, mimant une activité épileptiforme. Nos résultats indiquent que les récepteurs P2X4 neuronaux pourraient être des acteurs importants de la libération de BDNF dans l’hippocampe lors d’un évènement excitotoxique. / Purinergic receptors P2X are ATP-gated ion channels widely expressed in the organism and involved in many physiological and pathological states. Particularly, P2X4 receptors have been involved in chronic pain. Following nerve injury, their expression is induced de novo in activated spinal cord microglia where they are responsible for the BDNF release leading to tactile allodynia, a characteristic of neuropathic pain. Our study shows that neuronal P2X4 receptors are crucial actors of other pathological processes, including inflammatory pain. We show that P2X4R are expressed in sensory neurons in dorsal root ganglions and seem involved in BDNF release in the spinal cord. This release leads to activation of BDNF/TrkB signalization pathways, and particularly to the downregulation of KCC2. This process underlies the spinal hyperexcitability in chronic inflammatory pain states. These results have been extended to model of excitotoxicity in the hippocampus mimicking the lesions caused by an epileptic activity. Our preliminary results suggest that neuronal P2X4 receptors are likely major actors in the BDNF release in the hippocampus following an excitotoxicitic insult.

L’infection par le cytomégalovirus (CMV) au cours de la grossesse est fréquente et représente la première cause de pathologie neurodéveloppementale. En dépit de cette importance médicale, il n’existe à ce jour aucun traitement préventif ou curatif satisfaisant, et les mécanismes physiopathologiques mis en jeu, en particulier au niveau du cerveau foetal, restent mal connus. Des découvertes récentes sur les modèles murins d’infection cérébrale par le CMV, principalement réalisées pendant la période néonatale, ont apporté des données convergentes sur la physiopathologie de ces infections cérébrales ; notamment, le rôle joué par les cellules immunitaires périphériques dans la résolution de l’infection, et l’implication du système immunitaire cérébral (SIC) au cours du processus infectieux. Afin de compléter et préciser les résultats précédemment obtenus dans différents modèles murins, et de comprendre le rôle joué par le SIC, le premier objectif de ma thèse a consisté à mettre au point et à caractériser un nouveau modèle d’infection cérébrale par le CMV au cours du développement in utero chez le rat. Dans l'ensemble, nos résultats confirment l'altération du SIC au cours de l'infection par le CMV du cerveau en développement, et suggèrent fortement, dans ce modèle, un rôle majeur joué par le système microglie/macrophage dans l'émergence de troubles neurologiques semblables à ceux observés dans la pathologie humaine correspondante. / Cytomegalovirus (CMV) infection during pregnancy is the leading cause of neurodevelopmental disorders (polymicrogyria, microcephaly) and may lead to severe sensorineural consequences (deafness, epilepsy, cerebral palsy and hearing loss). Despite this medical importance, no preventive or curative treatment is satisfactory to date, and the pathophysiological mechanisms, notably in the fetal brain, remain poorly understood. Recent findings in murine brain CMV infection, mostly in neonatal models, have brought converging insights into the pathogenesis of these infections; the possible role played by peripheral immune cells against infection and the involvement of the brain immune system (BIS) have been proposed. The actual roles of BIS during in utero infection, and more specifically that of microglial cells and macrophages, remain unclear. In order to expand and precise the data previously obtained in the murine models, and to clarify the role of BIS, the first objective of my thesis was to design and to characterize a novel model of CMV infection during the fetal development of the rat brain. Overall, our datas confirm the altered state of BIS as a consequence of CMV infection of the developing brain, and strongly suggest, in the rat model studied here, that the microglia/macrophages system plays critical role in the pathogenesis of neurological manifestations similar to those classically seen after human congenital CMV infection.

La distribution de principes actifs dans le système nerveux central (SNC) est entravée par la présence d'une barrière physiologique, la barrière hémato-encéphalique (BHE). L'endothélium cérébral est pourvu d'un large éventail de systèmes de transport, parmi lesquels la trancytose récepteur-dépendante, qui peut être mise à profit pour vectoriser toute une gamme d'agents thérapeutiques vers le SNC de manière non invasive. Dans le cadre de cette approche, le LDLR (Low Density Lipoprotein Receptor), exprimé à la surface de la BHE, est une cible particulièrement intéressante. L'objectif de ce travail est le développement de nouveaux ligands du LDLR en tant que vecteurs potentiels de la BHE. Le criblage d'une librairie de peptides aléatoires dirigée contre le LDLR a permis l'identification d'un peptide 15-mer cyclique ayant une haute affinité in vitro. Une étude des relations structure/activité a ensuite été menée afin d'améliorer l'affinité pour le LDLR et d'augmenter la stabilité plasmatique de ce peptide. Cette étude a abouti à l'identification d'un nouveau peptide « lead » qui a été conjugué à des molécules actives afin d'évaluer la capacité du peptide à vectoriser un principe actif à travers la BHE après administration in vivo chez la souris. / Drug delivery to the central nervous system (CNS) is hindered by the presence of a physiological barrier, the blood-brain barrier (BBB). The brain endothelium is endowed with a series of transport systems, including receptor-mediated transcytosis. This system can also be used to transport therapeutics into the brain as a non-invasive manner. Among receptors expressed on the BBB, the low density lipoprotein receptor (LDLR) is relevant as a drug delivery system. This project is dedicated to the development of new peptide-based ligands of LDLR as potential BBB-vectors. The screening of a random peptide library directed to the LDLR led to the identification of hits such as a cyclic 15-mer peptide with high in vitro affinity. A structure/activity relationship study was then carried out in order to improve its affinity towards the LDLR and to increase its plasmatic stability. This study led to the identification of a new lead peptide which was conjugated to bioactive compounds in order to assess the ability of our peptide to shuttle a drug across the BBB following in vivo administration in mice.

Ce travail avait pour objectif la synthèse d’hydrogels d’alginates stables dans le temps et leur évaluation comme support de régénération des voies nerveuses lésées du Système Nerveux Central. Différents dérivés amphiphiles de l’alginate de sodium ont été préparés en fixant chimiquement des chaînes alkyles en C12 à différents taux sur le squelette polymère par l’intermédiaire, soit de liaisons ester (alginates esters), soit de liaisons amide (alginamides) moins sensibles à l’hydrolyse. Le comportement en solution des alginamides a été étudié en termes de stabilité dans le temps, de solubilité, de comportement rhéologique, et de taux de gonflement, puis comparé à celui des alginates esters. Les alginamides ont montré des propriétés en solution différentes de celles des alginates esters, en particulier des contraintes critiques faibles, qui sont les conséquences de la formation d’agrégats liée à l’existence d’une réaction secondaire de réticulation chimique lors de la synthèse. Néanmoins, à des concentrations et taux de greffage appropriés, il est possible d’obtenir un réseau polymère tridimensionnel stabilisé par des associations hydrophobes et pouvant servir de pont de régénération pour l’application visée. L’étude in vivo conclut à l’absence de signe de régénération des voies nerveuses chez les animaux lésées sur une durée d’un an. Toutefois, cette étude a permis de valider la stratégie d’implantation d’un gel physique rhéofluidifiant et thixotrope, de définir des méthodes d’analyse des tissus post mortem en présence d’hydrogel, d’examiner la possibilité d’encapsuler un traitement pharmacologique et de définir un cahier des charges élargi de ce biomatériau (pH, stabilité, gonflement …) / The aim of this work was to synthesise alginate hydrogels, stable in time, and to evaluate their potential use as scaffolds for the damaged nerve regeneration in central nervous system. Various amphiphilic derivatives of sodium alginate were prepared by covalent attachment of alkyl chains (12 carbons) onto the polysaccharide at different substitution ratio, either via ester (alginate ester) or amide (alginamide) linkages, these last ones being more stable toward hydrolysis. The properties in solution of the alginamide derivatives were studied in terms of solubility, stability as function of time, rheological behaviour and swelling ratio. Results were compared to those obtained with the alginate ester family and highlighted a different behaviour for the alginamide series in semi-dilute regime. In particular, alginamide hydrogels exhibited a low critical strain which has been attributed to the presence of aggregates in the solution. The formation of these aggregates was due to the occurrence of a secondary cross-linking reaction during the synthesis of polymers. Nevertheless, it was possible, by appropriate tuning of the substitution yield and of the solution concentration, to obtain a three-dimensional network stabilized by intermolecular hydrophobic interactions, which has been evaluated as regenerative support for the considered application. In vivo studies demonstrated the absence of nerve regeneration for the tested injured animals after one year. However, these studies allowed us to evaluate both the strategy for the implantation of a physical gel exhibiting a shear-thinning and thixotrope behavior and the possibility to encapsulate a pharmacological treatment. An enlarged project specification has also been defined for this biomaterial (pH, stability, swelling ratio…)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Citronellol (CT) is an alcoholic monoterpene present in the essential oil of some medicinal plants such as Cymbopogon citratus. Some pharmacological effects such as the antispasmodic and anticonvulsant activities have been described, however it s possible antinociceptive and anti-inflammatory effect is unknown. Thus, the objective of this study was to evaluate the possible antinociceptive and anti-inflammatory action of CT in rodents. Therefore, 362 male Swiss mice (25-35 g) with 2 to 3 months were used. The animals were divided into groups and were treated with CT (25, 50 and 100 mg/kg, i.p.), vehicle (saline solution 0.9% + Tween 80 0.2%, i.p.) or standard drug (i.p.). To evaluate the antinociceptive activity, the animals were submitted to the test of abdomnal constrictions induced by acetic acid (0.85%), the formalin test (1%) and hot plate test. In order to evaluate the effect of CT on orofacial nociception, it was conducted the orofacial test induced by formalin (2%), capsaicin and glutamate. Motor coordination was assessed using the motor coordination test (rota rod) and the spontaneous movement test. The anti-inflammatory activity was evaluated based on the model of carrageenan-induced pleurisy, making up the total leucocyte count. It was also quantified, by using ELISA, TNF-α and nitric oxide generation by macrophages. To determine the central action, the animals were treated with CT, in three doses, or vehicle and, after ninety minutes, were anesthetized, perfused, the brains removed and cut in a cryostat. The brain sections were subjected to immunofluorescence protocol for Fos protein. Results are expressed as mean ± SEM Differences between groups were analyzed by using one way ANOVA test, and followed by Tukey test. Values of p < 0.05 were considered statistically significant. Intraperitoneal administration of CT produced a significant decrease (p < 0.001) of the abdominal constrictions induced by acetic acid. In nociception induced by formalin, the pretreatment with CT caused a significant antinociceptive effect (p <0.01) in both phases of the test. In the hot plate test, the reaction time increased significantly at all doses of CT (p < 0.05 or p < 0.001), while its effect was antagonized by naloxone. In the three orofacial nociception tests, the CT produced a significant decrease (p <0.001) in the face-rubbing time of the orofacial region. No changes were observed in the motor coordination and in the spontaneous movement test. In the evaluation of anti-inflammatory activity, treatment with CT gave rise to a significant decrease (p < 0.01) in total number of leukocytes, decreasing (p < 0.05) the levels of TNF-α and nitric oxide in macrophages (p < 0.05). By immunofluorescence, it was found that CT is able to activate signicantly (p < 0.05) neurons of the olfactory bulb, the piriform cortex, the restrosplenial cortex and the periaqueductal gray. So, it can be concluded that CT has antinociceptive and anti-inflammatory activity and its action is mediated by central and peripheral mechanisms. / O Citronelol (CT) é um monoterpeno alcóolico presente no óleo essencial de algumas plantas medicinais, como o Cymbopogon citratus. Alguns efeitos farmacológicos tais como anti-espasmódico e atividade anticonvulsivante já foram descritos, sendo desconhecido seu possível efeito antinociceptivo e anti-inflamatório. Desta forma, o objetivo do presente estudo foi avaliar a possível ação antinociceptiva e anti-inflamatória do CT em roedores. Para tanto, foram utilizados 362 camundongos Swiss machos (25 a 35 g) com 2 a 3 meses. Os animais foram divididos em grupos e foram tratados com CT (25, 50 e 100 mg/kg; i.p.), veículo (solução salina 0,9% + tween 80 0,2%; i.p.) ou droga padrão (i.p.). Para avaliação da atividade antinociceptiva, os animais foram submetidos ao teste de contorções abdominais induzidas por ácido acético (0,85%), ao teste da formalina (1%) e ao teste da placa quente. Com o intuito de avaliar a ação do CT na nocicepção orofacial, foram realizados os testes de dor orofacial induzida por formalina (2%), capsaicina e glutamato. A coordenação motora dos animais foi avaliada através do teste da coordenação motora (rota rod) e do teste da movimentação espontânea. A atividade anti-inflamatória foi avaliada a partir do modelo de pleurisia induzido por carragenina, realizando-se a contagem de leucócitos totais. Foi quantificado, ainda, através do ensaio imunoenzimático ELISA, o TNF-α e a geração de óxido nítrico por macrófagos. Para determinar a ação central, os animais foram tratados com CT, nas três doses, ou veículo e, noventa minutos após, foram anestesiados, perfundidos, os cérebros extraídos e cortados em criostato. As secções cerebrais foram submetidas ao protocolo de imunofluorescência para proteína Fos. Os resultados foram expressos como média ± erro padrão da média. As diferenças entre os grupos foram analisadas por meio do teste de variância ANOVA, uma via, seguido pelo teste de Tukey. Valores de p < 0,05 foram considerados estatisticamente significantes. A administração intraperitoneal de CT produziu uma redução significativa (p < 0,001) das contorções abdominais. Na nocicepção induzida por formalina, o pré-tratamento com CT causou um efeito antinociceptivo significativo (p <0,01) em ambas as fases do teste. No teste da placa quente, o tempo de reação aumentou significativamente em todas as doses de CT (p < 0,05 ou p < 0,001), tendo seu efeito antagonizado pela naloxona. Nos três testes de nocicepção orofacial, o CT produziu uma redução significativa (p < 0,001) no tempo de fricção da região orofacial. Não foram observadas alterações no teste da coordenação motora e no teste da movimentação espontânea. Na avaliação da atividade anti-inflamatória, o tratamento com CT causou uma diminuição significativa (p < 0,01) no número total de leucócitos, diminuindo os níveis de TNF-α (p < 0,05) e de óxido nítrico em macrófagos (p < 0,05). Através da imunofluorescência, observou-se que o CT é capaz de ativar signicativamente (p < 0,05) neurônios do bulbo olfatório, do córtex piriforme, do córtex retroesplenial e da substância cinzenta periaquedutal. Conclui-se, assim, que o CT apresenta ação antinociceptiva e anti-inflamatória, tendo sua ação mediada por mecanismos centrais e periféricos.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is a global public health problem and is considered one of the deadliest infectious diseases of the world. It is estimated that about one third of the world population is infected. TB in Central Nervous System (CNS) is one of the most severe forms of the disease, more common in children aged six months to five years old and among individuals with immunosuppression, particularly those with Acquired Immunodeficiency Syndrome (AIDS). Neurotuberculosis (NTB) is associated with a high rate of mortality in children and permanent neurological sequelae in many survivors. Currently, the way to prevent TB is BCG (Bacilo Calmette-Guérin) vaccination, however, it has limitations because it only protects children and only prevents severe forms of TB, besides presenting variable protection of 0-75%. This shows the need to develop a more effective vaccine to fight TB. One of the most promising alternatives for TB is the DNA-hsp65 vaccine, made of a plasmid DNA containing the gene encoding the heat shock protein 65 kDa of mycobacteria (M. leprae). Although, it has not studied the protective effect of DNA-hsp65 vaccine against TB in the CNS, which is the most severe form of the disease and compromises the neural tissue. Thus, we evaluated the prophylactic effect of DNA-hsp65 vaccine in experimental neurotuberculosis. C57BL/6 mice were used by three experimental groups, where one of them was immunized only with plasmideal pVAX vector (vector group) by intramuscular administration of three doses every two weeks, another group received only PBS (PBS group) and the third group DNA was immunized with hsp65-pVAX plasmideal (vaccinated group). Thirty days after the last dose, the animals of each group were challenged with H37Rv laboratorial strain of Mycobacterium tuberculosis intracerebroventricularly by stereotaxy. Thirty days after the challenge with the bacilli, the mice were sacrificed and their brains and lungs removed. These organs were analyzed using histological sections stained with hematoxylin and eosin. It was also determined amounts of colony forming units (CFU) present in the brains of animals in all groups. The results show that animals of the vaccinated group exhibited brain and lung with discrete lesions of the vector and PBS groups. Furthermore, we noticed a reduced number of bacilli in the brains of animals in the vaccinated group. Therefore, our results suggest that DNA-hsp65 vaccine (pVAX-hsp65) is promising in the prevention of injuries caused by bacillus in the CNS. / A tuberculose (TB) é causada pelo Mycobacterium tuberculosis e representa um problema de saúde pública global, sendo considerada uma das doenças transmissíveis mais mortais do mundo. Estima-se que cerca de um terço da população mundial esteja infectada. A TB no Sistema Nervoso Central (SNC) é uma das formas mais graves da doença, sendo mais incidente em crianças de seis meses a cinco anos de idade e entre os indivíduos com imunodepressão, particularmente os portadores da Síndrome da Imunodeficiência Adquirida (AIDS). A Neurotuberculose (NTB) está associada a uma alta taxa de mortalidade em crianças e permanentes sequelas neurológicas em muitos sobreviventes. Atualmente, a forma de prevenção à TB é a vacina BCG (Bacilo Calmette-Guérin), porém, a mesma apresenta limitações, pois protege apenas as crianças e previne somente as formas graves de TB, além de apresentar proteção variável de 0 a 75%. Isso aponta a necessidade do desenvolvimento de uma vacina mais efetiva para o combate da TB. Uma das alternativas mais promissoras para o combate da TB é a vacina de DNA-hsp65, constituída por um plasmídeo de DNA contendo o gene que codifica a proteína de choque térmico de 65 kDa de micobactéria (M. leprae). Ainda não foi estudado o efeito protetor da vacina de DNA-hsp65 contra a TB no SNC, que é a forma mais grave da doença e compromete o tecido neural. Dessa forma, avaliamos o efeito profilático da vacina de DNA-hsp65 na neurotuberculose experimental. Foram utilizados camundongos C57BL/6 através de três grupos experimentais, onde um deles foi imunizado apenas com o vetor plasmideal pVAX (Grupo Vetor) através da administração de três doses quinzenais intramusculares, outro grupo recebeu apenas PBS (Grupo PBS) e o terceiro grupo foi imunizado com o DNA plasmideal pVAX-hsp65 (Grupo Vacinado). Trinta dias após a última dose, os animais de cada grupo foram desafiados com a cepa laboratorial H37Rv de Mycobacterium tuberculosis via intracerebroventricular através de estereotaxia. Passados trinta dias do desafio com o bacilo, os camundongos foram sacrificados e foram removidos cérebro e pulmão. Esses órgãos foram analisados através de cortes histológicos corados com hematoxilina e eosina. Também foram determinadas as quantidades de unidades formadoras de colônias (UFC) presentes nos cérebros dos animais de todos os grupos. Os resultados demonstram que os animais do grupo vacinado apresentaram cérebro e pulmão com lesões mais discretas do que os grupos vetor e PBS. Além disso, percebemos um número reduzido de bacilos nos cérebros dos animais do grupo vacinado. Portanto, nossos resultados sugerem que a vacina de DNA-hsp65 (pVAX-hsp65) é promissora na prevenção das lesões causadas pelo bacilo no SNC. / São Cristóvão, SE