Orientadores: José Antonio Rocha Gontijo, Patricia Aline Boer / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T15:49:33Z (GMT). No. of bitstreams: 1 Cardoso_BarbaraVaccari_M.pdf: 1480102 bytes, checksum: c17066766c9415b675dc34743e6d7526 (MD5) Previous issue date: 2014 / Resumo: Estudos têm mostrado que a restrição proteica gestacional programa o fenótipo da prole a fim de torná-lo compatível com o provável ambiente encontrado na vida pós-natal. No entanto, se a oferta nutricional tornar-se abundante após o nascimento aumenta-se o risco etiológico de desenvolvimento de doenças cardiovasculares, como a hipertensão arterial, na prole adulta. Um importante fator que contribui para esse processo é a alteração do metabolismo hidrossalino, caracterizado pela alta retenção de sódio nesse modelo. Visto que o rim é o principal órgão de controle hidroeletrolítico e sua função é minuciosamente modulada pelo sistema nervoso por meio da transmissão catecolaminérgica central, alterações nesse sistema teriam um papel fundamental nas modificações da função renal e, consequentemente, na etiologia da hipertensão arterial. Diante disso, o objetivo desse trabalho foi avaliar as implicações da transmissão noradrenérgica central mediada por ?-adrenoceptores sobre o balanço hidroeletrolítico renal em ratos machos adultos submetidos à restrição proteica durante o período fetal. Ratas Wistar prenhes foram submetidas à dieta hipoproteica durante a gestação e, na prole de machos, foi realizado a caracterização antropométrica e pressórica. Na idade adulta, estes animais foram submetidos à implantação de uma cânula intracerebroventricular para microinjeção de antagonistas ?1- e ?2-adrenoceptores durante a avaliação de função renal pelo clearance de lítio e creatinina. Os animais submetidos à restrição proteica durante o desenvolvimento fetal apresentaram significativa redução do peso ao nascer e aumento da pressão arterial sistólica. O estudo da função renal revelou que o estímulo simpatomimético por epinefrina frente ao bloqueio prévio de ?2-adrenoceptores centrais promoveu antidiurese acentuada, bem como elevação significativa da natriurese nesses animais. Os resultados sugerem que a massiva retenção de sódio e água observada nesse modelo experimental se deve, pelo menos em parte, à uma possível alteração da atividade e/ou número de ?2-adrenoceptores centrais, isolada ou em conjunto com modificações na sinalização adrenérgica mediada por ?1-adrenoceptores presentes no sistema nervoso central de animais programados. O estudo também mostra que a retenção renal de sódio ocorre nos segmentos proximais e pós proximais do nefro e, não são acompanhados por alterações na filtração glomerular. Assim, o presente trabalho demonstrou que alterações na transmissão catecolaminérgica central mediada por ?-adrenoceptores possui um importante papel no estabelecimento e/ou manutenção da hipertensão arterial na prole de mães submetidas à restrição proteica gestacional / Abstract: Studies have shown that gestational protein restriction programs the offspring phenotype in order to make it compatible with the likely environment found in postnatal life. However, if the nutrient supply becomes abundant after birth, it increases the etiological risk of developing cardiovascular diseases such as hypertension in adult offspring. An important factor contributing to this process is the alteration of the salt and water metabolism characterized by high sodium retention in this model. Since the kidney is the main hydroelectrolytic control organ and its function is thoroughly modulated by the nervous system through central catecholaminergic transmission, alterations in this system may have a key role in the renal function changes and, consequently, in the etiology of hypertension. Therefore, the aim of this study was to evaluate the implications of central noradrenergic transmission mediated by ?-adrenoceptors in the renal fluid and electrolyte balance in adult male rats subjected to protein restriction during the fetal period central. Pregnant Wistar rats were subjected to a low protein diet during pregnancy and blood pressure and anthropometric characterization were performed in the male offspring. In adulthood, these animals underwent an intracerebroventricular cannula implantation towards microinjection of ?1-and ?2-adrenoceptors antagonists during the evaluation of renal function by creatinine clearance of lithium. The animals submitted to protein restriction during fetal development showed significant reduction in the birth weight and increased systolic blood pressure. The renal function study revealed that the sympathomimetic stimulation by epinephrine with prior blockade of central ?2-adrenceptores promoted marked antidiuresis, as well as a significant increase in natriuresis level in these animals. The results suggest that the massive sodium and water retention observed in this experimental model is due, at least in part, to a possible change in activity and/or number of central ?2-adrenoceptors, alone or in conjunction with changes in ?1-adrenoceptors-mediated signalling in the central nervous system of programmed animals. The study also shows that renal sodium retention occurs in the proximal and post-proximal nephron segments and are not accompanied by changes in glomerular filtration. Thus, the present study demonstrated that changes in central catecholaminergic transmission mediated by ?-adrenoceptor plays an important role in the establishment and/or maintenance of hypertension in the offspring of rats subjected to gestational protein restriction / Mestrado / Fisiopatologia Médica / Mestra em Ciências

Orientador: José Antonio Rocha Gontijo / Tese (doutorado) - Universidade Estaual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T13:09:43Z (GMT). No. of bitstreams: 1 Lima_MarceloCardosode_D.pdf: 7673135 bytes, checksum: e538dba1f2c8583b18739df7c2e1a919 (MD5) Previous issue date: 2012 / Resumo: O presente estudo avaliou a repercussão da restrição protéica gestacional sobre os receptores da Angiotensina II (AT1R e AT2R) no sistema nervoso central e seu impacto na programação da hipertensão arterial na vida adulta. Os resultados deste estudo demonstraram que o ambiente intrauterino inadequado, desencadeou nos animais do grupo hipoprotéico (LP), um baixo peso ao nascimento, uma expressão significativamente menor de receptores AT1R no extrato de tecido hipotalâmico aos 12 dias de idade e não significativa na 16a semana de vida quando comparados com os animais do grupo normoprotéico (NP). Inversamente, foi observado uma maior e significativa expressão de receptores AT2R no estrato total de hipotálamo de animais do grupo LP com 12 dias de vida, permanecendo nos animais com 16 semanas de vida, quando comparados ao grupo NP. Adicionalmente, observou-se a redução na resposta dipsogênica frente à administração Intracerebroventricular (i.c.v.) de Angiotensina II (AngII) nos animais LP em comparação com os animais do grupo NP. Estes resultados podem estar relacionados à expressão diminuída de vasopressina (AVP) hipotalâmico, demostrada aqui pela imunohistoquímica, na prole submetida à restrição protéica gestacional. A presente investigação também demonstrou uma menor expressão de receptores mineralocorticóide (MR) e glicocorticóide (GR) associada a uma elevação de propiomelanocortina (POMC) e hormônio adrenocorticotrófico (ACTH), assim sugerindo uma participação de mineralocorticoides e glicocorticóides como resposta compensatória ao longo da vida para atenuar as alterações na fisiologia renal comprovadas pela queda acentuada na excreção fracional de sódio urinário (FENa+) nos animais do grupo LP sem ocorrer alteração na filtração glomerular estimada pelo clearance de creatinina (CCr). O Clearance de lítio demonstrou que esta excreção urinária aumentada foi devida à perda da capacidade reabsortiva do néfron proximal a despeito do aumento na reabsorção pós-proximal. Estes efeitos foram associados com um significativo aumento na pressão arterial no grupo LP, mas, o mecanismo exato destes fenómenos permanecem desconhecidos / Abstract: Recent studies have shown that the central nervous system, during development, can be influenced by alterations in the intrauterine environment. This organizational phenomenon is termed 'early-life programming'. Here, in maternal protein-deprived offspring model, we focus on adult hypertension development, hypothalamic changes and renal function disorders as an outcome and confirm the hypothalamus as a structure in which there are early and permanent changes that underlie the developing hypertension. The present study shows that LP male pup body weight was significantly reduced when compared to that of NP pups. However, the body masses at 12-days-old and 16-wk-old were similar to observed in NP age-matched group. Furthermore, the immunoblotting analysis in the current study demonstrated a significantly decreased expression of type 1 AngII receptors in the entire hypothalamic tissue extract of LP rats at 12 days of age and unaltered expression in16-wk-old rats, compared to that observed in NP offspring. The unchanged AT1R expression by blotting in the whole hypothalamic extract of 16- wk LP rats may results of uneven expression of protein, revealed by immunohistochemistry, of different analyzed hypothalamic structures. Conversely, the expression of the type 2 AngII receptors in 12-days and 16-wk-old LP hypothalamus was significantly increased, when compared with the NP agematched group. The current data shows the influence of central AngII administration on spontaneous water consumption in a concentration-dependent fashion, but also demonstrated that the water intake response to graded AngII concentrations was strikingly attenuated in 16-wk-old LP, compared with agematched NP controls. These results may be related with decreased brain AVP expression showed in maternal protein-restricted offspring. The present investigation also shows an early and pronounced decrease in fractional urinary sodium excretion in maternal protein-restricted offspring. It also shows a decreased central expression of MR and GR associated, reciprocally, with enhanced immunoreactivity to POMC e ACTH. The decreased FENa was accompanied by a fall in FEPNa and occurred despite unchanged CCr and an enhanced FEPPNa. All these effects was associated with a significant enhance in arterial blood pressure in the LP group but, the precise mechanism of these phenomena remains unknown / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Ciências

Caffeine is one of the most popular psychostimulant drugs worldwide. Its effects are exerted through a variety of complex mechanisms, apparently primarily via interactions with adenosine A1 and A2A receptors. This drug has also been proven to elicit neuroprotective responses in a number of different brain disorders of the Central Nervous System (CNS), as well as provide enhancement of cognitive abilities. Moreover, a biphasic set of functional and structural neurological changes are often found in these receptors among diverse vertebrates. I investigated the effects of chronic caffeine exposure on functional recovery of the dorsal light reflex (DLR) in hemilabyrinthectomized common goldfish, Carassius auratus. In this lesion model the unilateral removal of vestibular organs results in the temporary loss of gravitational regulated postural control, which over time corrects itself by a vestibular compensation (VC) mechanism and can be quantified via the DLR. We compared the functional recovery over 24 post -surgery days in goldfish continuously held in a caffeine solution of 2.5mg/L (n=10), 5.0mg/L (n=10), 10.0mg/L (n=11) or a control 0.0mg/L (n=9). Compared to a sham surgery group (n=11), statistically significant changes in the DLR of all hemilabyrinthectomized goldfish was observed on day 1. The control group recovered over the study period by approaching but not entirely reaching sham surgery DLR. The 2.5mg/L and 5.0mg/L groups initiated postural recovery similar to the controls, but then regressed to a stronger DLR. Beginning on day 10 the caffeine groups deviated from the control and all three experimental caffeine groups were statistically different from the control group on days 15-24. Results suggest early caffeine exposure may be innocuous; however, chronic exposure inhibits the functional recovery process.

Les récepteurs nucléaires sont des facteurs de transcription régulant l’expression de gènes impliqués dans plusieurs processus physiologiques. En général, ces récepteurs sont activés par une interaction avec un ligand spécifique, mais leur activité peut également être modulée par des modifications post-traductionnelles comme la SUMOylation. Notre laboratoire a récemment trouvé que le récepteur des estrogènes ER est une cible de cette modification sur un site de SUMOylation atypique nommé pSuM. Ce site non-consensus se retrouve également sur Nor-1, un récepteur nucléaire impliqué dans une variété de processus physiologiques comme le métabolisme du glucose, la prolifération cellulaire ainsi que la survie neuronale. Pour l’instant, le rôle de ce motif atypique reste inconnu et aucune évidence n’indique que Nor-1 est SUMOylé dans la littérature. Nos objectifs étaient donc de déterminer le potentiel de SUMOylation de Nor-1 sur son pSuM dans un contexte neuronal et de caractériser l’effet de la SUMOylation sur l’activité transcriptionnelle de Nor-1 ainsi que l’expression de gènes cibles. Nos résultats ont démontré que Nor-1 peut effectivement être SUMOylé sur son pSuM, que cette SUMOylation est dépendante d’une phosphorylation de la sérine-139 et que la voie des MAPK augmente l’état global de SUMOylation de Nor-1 ainsi que son expression protéique. De plus, les résultats ont indiqués que Nor-1 peut conjuguer deux isoformes de SUMO, soit SUMO-1 et SUMO-2. Les résultats démontrent également que la SUMOylation augmente l’activité transcriptionnelle de Nor-1 et que la SUMOylation de son site pSuM est importante pour le maintien de son activité transcriptionnelle basale. Nos résultats préliminaires suggèrent que la SUMOylation du pSuM de Nor-1 affecte l’expression de gènes cibles comme l’énolase-3 ce qui propose un rôle de la SUMOylation de Nor-1 dans la régulation du métabolisme des neurones. En conclusion, les résultats de ce projet permettent d'identifier un nouveau processus de SUMOylation impliqué dans la régulation des récepteurs nucléaires et de Nor-1 suggérant un rôle de ce processus de SUMOylation atypique dans plusieurs réseaux géniques et processus physiologiques. / Nuclear hormone receptors are transcription factors that regulate the expression of many genes implicated in a wide variety of physiological functions. They are generally activated by an interaction with a specific ligand but their activity can also be modulated by post-translational modifications such as SUMOylation. Recently, our laboratory identified ER as a SUMOylation target on an atypical SUMOylation motif named pSuM. This non-consensus SUMOylation motif was also found on Nor-1, a nuclear receptor implicated in physiological processes such as glucose metabolism, cellular proliferation and neuronal survival. Until now, the function of this pSuM motif is still unknown and there are no evidence that Nor-1 is a target of SUMOylation. Therefore, the objectives of this work were to determine the SUMOylation potential of Nor-1 on his pSuM in a neuronal context and to characterize the effect of SUMOylation on Nor-1 transcriptional activity and target genes expression. Our results showed that Nor-1 can indeed be SUMOylated on his pSuM, that this SUMOylation was dependent on serine-139 phosphorylation, and that the MAPK pathway augmented the global SUMOylation state of Nor-1 and its protein expression levels. Furthermore, we demonstrated that Nor-1 can conjugate SUMO-1 and SUMO-2. Our results also indicated that SUMOylation activates the transcriptional activity of Nor-1 and that the pSuM site of Nor-1 was important in maintaining its basal transcriptional activity. Preliminary results suggests that Nor-1 SUMOylation on the pSuM has an impact on the expression of target genes such as enolase-3, suggesting a potential role for Nor-1 in the regulation of the metabolism of neurons. In conclusion, the results presented in this work identify a new SUMOylation process implicated in regulating Nor-1 and nuclear receptors activities, suggesting an extended role of this atypical SUMOylation process in many gene networks and physiological processes.

Les infections fongiques invasives, sont des infections sévères grevées d’une lourde mortalité. Elles sont actuellement un problème majeur de santé publique et leur incidence augmente. Les candidémies représentent la quatrième cause d’infection hématogène nosocomiale aux Etats-Unis, les cryptococcoses sont responsables de 600 000 décès chaque année en Afrique et l’aspergillose invasive infecte 10% des patients transplantés de cellules souches. La mortalité de ces infections reste élevée avec des taux de mortalité de 50, 15 et 40% respectivement. L’augmentation de leur incidence est due à l’accroissement des populations immunodéprimées à risque de développer des infections fongiques en raison de l’augmentation des thérapeutiques immunosuppressives et de l’allongement de la durée de vie des patients immunodéprimés. Les infections fongiques invasives surviennent chez des patients immunodéprimés, majoritairement dans un contexte d’immunodépression acquise (neutropénie, chimiothérapie, greffe de cellules souches périphériques ou transplantation d’organe solide, diabète, infection par le virus de l’immunodéficience humaine), mais également secondairement à un déficit immunitaire héréditaire (granulomatose septique chronique, déficit immunitaire combiné, neutropénie congénitale, défaut de l’axe IFNγ-IL12). Cependant certains patients développent des infections fongiques invasives sans immunodépression ou facteurs de risques identifiés. Nous avons donc émis l’hypothèse que ces infections avaient possiblement une origine génétique non identifiée. Au cours de ma thèse, j’ai étudié une cohorte de patients présentant des infections fongiques invasives sans facteur favorisant identifié afin de rechercher une étiologie génétique à ces infections. Le premier groupe de patients que j’ai étudié présentait une dermatophytose invasive ou dermatophytose profonde sans immunodépression. Contrairement à la dermatophytose superficielle, en général bénigne et fréquente dans la population générale; la dermatophytose profonde est une infection rare, invasive et sévère, dans laquelle les dermatophytes (qui sont des champignons filamenteux) envahissent les tissus dermiques et hypodermiques, les ganglions et parfois les organes profonds. Les patients que j’ai étudié étaient tous originaires d’Afrique du Nord, pour la plupart issus de familles consanguines, dont certains avec des cas multiples. Ces observations suggéraient une origine génétique de la dermatophytose profonde avec une hérédité probablement récessive. Au cours de ma thèse, j’ai étudié les caractéristiques cliniques, immunologiques et génétiques de 18 patients atteints d’une dermatophytose profonde, issus de neuf familles Marocaines, Algériennes, Tunisiennes ou Egyptiennes. En parallèle, j’ai étudié des patients ayant présenté des infections fongiques avec localisations cérébrales. L’une de ces patients a présenté des abcès cérébraux suite à une infection disséminée à Exophiala dermatitidis et trois patients ont développé des infections du système nerveux central à Candida spp.. Les infections invasives à Exophiala dermatitidis sont des infections rares, avec de fréquentes atteintes du système nerveux central, survenant majoritairement chez des patients sans déficit immunitaire identifié suggérant l’existence d’une origine génétique probable méconnue chez ces patients. Les candidoses invasives surviennent habituellement chez des patients neutropéniques, ayant récemment subi une intervention chirurgicale ou étant porteurs d’un cathéter intraveineux. Parmi les candidoses invasives, les localisations au système nerveux central sont rares, et classiquement rapportées chez des nouveau-nés prématurés ou suite à une intervention neurochirurgicale. J’ai par ailleurs étudié une patiente ayant développé des infections invasives des tissus sous-cutanés et des adénopathies dues à un champignon filamenteux. (...) / Invasive fungal diseases are a major health problem as they are severe infections complicated with high mortality rates and with rising incidence. Invasive fungal diseases occur mainly in patients with acquired immunodeficiencies, but also with primary immunodeficiencies (chronic granulomatous disease, defect in IFN-ϒ/IL-12 axis, congenital neutropenia). However, few patients develop invasive fungal disease without known risk factor. We therefore hypothesized that these infections probably have an unidentified genetic etiology. I studied a cohort of patients who developed invasive fungal diseases without risk factors and searched for a genetic etiology to their infections. The first group of patients presented with deep dermatophytosis without known immunodeficiency. Deep dermatophytosis is a rare, invasive and severe infection where dermatophytes invade dermis, hypodermis, lymph nodes and sometimes deep organs. I could study clinical, immunological and genetic characteristics of 18 patients from nine families who presented deep dermatophytosis. I also studied patients who developed central nervous system (CNS) fungal infections; one patient with CNS Exophiala dermatitidis infection and three patients with CNS Candida spp. infection. Invasive E. dermatitidis infections are rare, with frequent CNS location, mainly reported in patients without known immunodeficiencies, suggesting a potential unknown genetic etiology in these patients. CNS candidiasis are also rare infections usually occuring in preterm neonates or following neurosurgery. Based on literature data previously reporting a large consanguineous Iranian family with CARD9 deficiency that developed chronic mucocutaneous and central nervous system candidiasis; according to candidate gene approach, I sequenced CARD9 in all patients. CARD9 is an adaptor protein expressed by myeloid cells that signals downstream Dectin-1 and Dectin2 that are the main Pattern Recognotion Receptor implicated in antifungal immunity. I identified in all studied patients homozygous CARD9 mutations. Among 18 patients with deep dermatophytosis, 16 had homozygous nonsense Q289X and two homozygous missense R101C mutation in CARD9. I identified R18W, R35Q and R70W homozygous missense mutations in the patients who developed E. dermatitidis and two patients who developed CNS candidiasis, respectively. Transmission was autosomal recessive for all patients, except for the one with E. dermatitidis infection who had an uniparental disomy. In contrast with controls, CARD9 expression is abolished in Q289X, reduced in R70W and normal in R18W patients’ myeloid cells. CARD9 deficient patients whole blood and dendritic cells display a selective response defect to Candida albicans and Saccharomyces cerevisiae ; with IL-6 and TNF-α production impairment after Candida albicans and Saccharomyces cerevisiae stimulation. This defect can explain elective fungal susceptibility of CARD9 deficient patients to invasive fungal infections. This work evidenced that CARD9 deficiency was the main genetic etiology of deep dermatophytosis. It also could evidence that CARD9 deficiency is associated with Exophiala dermatitidis and Candida spp. CNS infections. This susceptibility is associated with proinflammatory cytokines defect by dendritic cells and whole blood to fungal agents. Various fungal clinical phenotypes in CARD9 deficient patients assess CARD9 central role in skin and central nervous system antifungal immunity.

This thesis examines the development of communication skills of children with developmental dysphasia, with the use of augmentative and alternative communication. The work is made up of theoretical and practical part. The research focuses on the influence of elements augmentative communication intervention in children with developmental dysphasia in preschool facilities. The research part is formed by empirical research based on a case study of a boy with developmental dysphasia, case reports describing the progress of special education intervention in preschool institutions, research part is completed by the conclusions of the investigation and recommendations for practice. The aim is to show the positive influence the course of education of children with developmental dysphasia support structured learning and noticeable advances in communication development of children with the support of the graphic expression.

Les syndromes neurologiques paranéoplasiques (SNP) sont des maladies neurologiques rares, associés à une réponse immunitaire efficace contre un cancer sous-jacent exprimant un antigène également exprimé par des cellules du système nerveux central (SNC). Le cancer déclenche alors une réponse auto-immune secondaire qui provoque la destruction des cellules du SNC. Certains travaux récents suggèrent que l'immunité à médiation cellulaire associée à des auto-anticorps reconnaissant des antigènes intracellulaires pourrait jouer un rôle majeur, bien qu'encore mal compris, dans la physiopathologie des SNP. Les exemples de SNP les plus représentatifs sont le syndrome Hu, qui conduit à la perte de diverses populations de neurones du SNC et l'ataxie cérébelleuse subaiguë (PCD en anglais, pour Paraneoplastic Cerebellar Degeneration), caractérisée par la perte sélective des cellules de Purkinje du cervelet. Alors que le syndrome Hu se développe en général chez des patients présentant des tumeurs du poumon à petites cellules qui expriment l'antigène HuD spécifique des neurones, la majorité des patients souffrant de PCD présente un cancer gynécologique qui exprime la protéine CDR2, également exprimée dans les cellules de Purkinje. Afin de mieux cerner la physiopathologie des SNP et de tester l'implication de l'immunité cellulaire, notamment des lymphocytes T, nous avons durant ma thèse développé et analysé deux modèles murins, l'un pour le syndrome Hu et l'autre pour la PCD. Ces modèles reposent sur l'utilisation de souches de souris génétiquement modifiées : la souris CamK-HA, qui exprime l'hémagglutinine (HA) du virus de la grippe dans la plupart de ses neurones du SNC et la souris L7-HA dans laquelle la protéine HA est exprimée exclusivement par les cellules de Purkinje du cervelet. Dans ces souris, une réponse anti-tumorale est provoquée par l'injection de cellules tumorales 4T1 exprimant HA (4T1-HA). Afin le faciliter le suivi des réponses cellulaires contre l'antigène HA, nous avons injecté des lymphocutes T CD4+ et/ou T CD8+ naïfs isolées à partir de souris transgéniques pour des récepteurs de lymphocytes T spécifiques de HA. Nos résultats montrent que seul le transfert in vivo des cellules tumorales 4T1-HA, et non celui des cellules 4T1 témoins, peut conduire à l'activation, la prolifération et la différentiation des deux types de lymphocytes T spécifiques pour l'antigène HA. De plus, nous avons observé que les populations de lymphocytes T CD4+ et CD8+ sont toutes deux requises, non seulement pour une réponse anti-tumorale efficace, mais aussi pour le déclenchement d'une réaction auto-immune collatérale chez la souris CamK-HA. Enfin, nous avons montré qu'il était nécessaire d'injecter en parallèle des anticorps contre le récepteur inhibiteur CTLA-4 chez la souris L7-HA, afin de permettre la migration des lymphocytes T spécifiques de HA dans le cervelet. Chez ces souris L7-HA, nous avons en outre démontré que les lymphocytes T CD8+ cytotoxiques sont les effecteurs principaux de la maladie. Ces nouveaux modèles murins représentent donc des outils précieux pour une meilleure compréhension des mécanismes moléculaires responsables du développement des SNP. De plus, ils pourraient permettre de tester et de valider de nouvelles approches thérapeutiques visant à bloquer la pénétration dans le SNC d'effecteurs immunitaires potentiellement pathogènes, tout en préservant l'efficacité de la réponse anti-tumorale en périphérie. / Paraneoplastic neurological disorders (PNDs) are rare human autoimmune diseases that mostly affect the central nervous system (CNS). They are triggered by an efficient immune response against a neural self-antigen that is ectopically expressed in neoplastic tumor cells and naturally expressed in CNS cells. Due to this shared antigenic expression, the immune system reacts not only to tumor cells but also to neural cells resulting in neurological damage. Growing data point to a major role of cell-mediated immunity in PNDs associated to autoantibodies against intracellular proteins. However, its precise contribution in the pathogenesis remains unclear. Two illustrative examples of possibly cell-mediated PNDs are the Hu-syndrome, characterized by inflammation and widespread los of neurons, and paraneoplastic cerebellar degeneration (PCD), characterized by the selective loss of Purkinje cells. PCD develops mostly in patients with gynecologic carcinomas that express the Purkinje neuron-specific CDR2 protein whereas most patients with the Hu-syndrome harbor small cell lung cancer expressing the neuron-specific protein HuD. In this context, our study aimed to investigate the impact of anti-tumor cellular immune responses in the development of these PNDs. To this end, we developed two animal models mimicking the Hu-syndrome and PCD. We used a tumor cell line expressing the hemagglutinin (HA) of influenza virus to induce an anti-tumor response in CamK-HA mice, which express HA in CNS neurons and L7-HA mice, which express HA only in cerebellar Purkinje neurons. To promote and track the T cell response against the HA antigen, naïve HA-specific CD8+ and/or CD4+ T cells, originating from TCR-transgenic animals, were transferred into these mice. We demonstrate that HA-expressing tumors, but not control tumors, induce in vivo activation, proliferation and differentiation of naïve HA-specific CD4+ and CD8+ T cells into effector cells. Moreover, the collaboration between these two T cell subsets was needed to control tumor growth and induce CNS inflammation in CamK-HA mice. In L7-HA mice the additional injection of the antibody against the inhibitory receptor CTLA-4 was necessary to allow T cells to enter the cerebellum to cause inflammation and the subsequent destruction of Purkinje neurons. Furthermore, in L7-HA mice we demonstrate that cytotoxic CD8+ T cells are the main effectors driving the disease. Thus, these two new mouse models provide further insights into the cellular mechanisms of PND whereby a potent anti-tumor immunity triggers a cancer-associated autoimmune disease, and may therefore help to develop new therapeutic strategies against PND.

The nervous system is the most complex system in an organism. Functioning of the nervous system requires proper formation of neural cells, as well as accurate connectivity and signaling among them. While the major events that occur during these processes are known, the finer details are yet to be understood. Hence, an attempt was made to look for novel genes that could be involved in them. The focus of the present study is on CG9650, a gene that was uncovered in a misexpression screen, as a possible player in neuronal development in Drosophila melanogaster. The first chapter of the thesis reviews existing knowledge about neuronal development and function. The first section of this chapter explains in brief the formation and specification of neural stem cells, and their differentiation to neurons and glia. Sections 2 and 3 describe neuronal connectivity and signaling with respect to axon growth, synapse formation, function and plasticity. A comparison of invertebrate and vertebrate neuronal development is provided in section 4 of this chapter. This part also explains the use of Drosophila as a model for neuronal development and function. Chapter 2 describes the expression pattern of CG9650, which was characterized to gain insights into the possible role it plays during Drosophila neurogenesis.CG9650 is expressed in multiple cell types in the nervous system at the embryonic stage. Some of the cell sub-types have been identified from their morphology and position. Expression was restricted to neurons in the larval stage (except in the optic lobe, where it was expressed in precursors also), and continued in the pupal stage. No expression was seen in adults (except in the optic lobe). CG9650 has a putative DNA binding region, which bears homology to the mouse proteins CTIP1 and CTIP2, implying that CG9650 is possibly a transcription factor. In order to understand the function of CG9650, the protein was knocked down panneuronally. The resultant animals showed locomotor defects at both larval and adult stages, which have been described in chapter 3. Knock down larvae showed reduced displacement and speed of movement. The number of peristaltic cycles was also reduced in these animals but the cycle period was normal. In adults, movement was uncoordinated and righting reflex was lost, resulting in inability to walk, climb or fly. These results imply a defect in neuronal signaling. Sensory perception was unaffected in these animals. Stage specific knockdown of CG9650 indicated that the requirement for this protein is primarily during the larval stage. All CG9650-expressing neurons in the ventral nerve cord were glutamatergic, implying that its role in controlling locomotor activity is likely through glutamatergic circuits. Following up on these observations, signaling at the neuromuscular junction was assessed in CG9650 knock down animals. Chapter 4 discusses the signaling defects seen on CG9650 knock down, and the possible role of this protein. Electrophysiological recordings from Dorsal Longitudinal Muscles showed reduced and irregular neuronal firing in the knock down animals. These animals also had reduced bouton and active zone numbers. Moreover, overexpression of BRP, an active zone protein, rescued the locomotor defects caused by knock down of CG9650. Chapter 5 reports the effect of over expression of CG9650. Pan-neural over expression of CG9650 resulted in embryos with severe axon scaffolding defects, as well as aberrant neuronal and glial pattern. However, the incorrectly positioned glial cells in these embryos did not express CG9650, indicating that their aberrant positioning was probably due to incorrect signaling from the neurons. In conclusion, this study reports the requirement for CG9650, a hitherto unknown protein, in locomotor activity and signaling, thus ascribing for it a role in neuronal development and function of Drosophila melanogaster.

Introduction: Little is known about the aetiology of cancer in teenagers and young adults (TYA) aged 15-24 years, although in England, cancer is the most common cause of disease-related mortality in this age group. The most common cancers at this age are lymphomas, central nervous system (CNS) tumours and germ cell tumours (GCT). The commonest carcinomas seen at older ages including lung, breast, large bowel and prostate account for only 3-4% of TYA cancers. In this thesis I describe the incidence patterns of selected cancers in TYA and the variation seen with geography, time and in population subgroups. The focus is on CNS tumours, GCT and bone tumours as they either peak in incidence in TYA and/or contribute disproportionately to cancer related mortality in TYA. This will allow formulation of hypotheses regarding aetiology of cancer in this age group which can then be tested by further research. Methods: For the majority of the analysis, anonymised national cancer registration data from England on individual patients of all ages with newly diagnosed cancer between 1979 and 2003 were used. To contrast the incidence patterns in England with that of India, data from five Indian urban population based cancer registries were used for part of the analysis. Age, sex, site and histology specific incidence rates were calculated and expressed per million person years. All rates, where appropriate, were adjusted to the world standard population using direct methods. To explore the link of growth with development of osteosarcoma and Ewing sarcoma, a random-effects meta-analysis was undertaken on studies which investigated an association of these tumours with height at diagnosis. Results: The incidence of cancer in TYA overall in England exceeded that of India. This was also true for most individual sites including epithelial cancers of lung, colon/rectum, breast, ovary and cervix, and non-epithelial cancers including melanoma, Hodgkin lymphoma and testicular cancer. Notable exceptions to this pattern were cancers of the mouth, gall bladder and stomach (females only) where incidence was higher in India. In England, CNS tumours in TYA were a composite of pilocytic astrocytomas and embryonal tumours (representing tail end of childhood CNS tumours), pituitary tumours, nerve sheath tumours, high grade astrocytomas and meningiomas (representing early-onset of CNS tumours that peak in incidence in the 6th and 7th decade of life), and of CNS GCTs, pleomorphic xanthoastrocytomas and neurocytomas which show a peak incidence in TYA. Irrespective of site or histology, GCT in England showed a peak in incidence between ages of 10 to 39 years which was more marked in males. This however varied by site and the peak incidence was seen at 10 to 14 years in the CNS, 15 to 19 years in ovary, 25 to 29 in mediastinum & thorax and abdomen & pelvis, and 30 to 34 years in testicular tumours. Osteosarcoma and Ewing sarcoma were the predominant bone tumours in TYA in England and showed a distinct peak of incidence at 10 to 14 years age in females and a larger peak at 15 to 19 years age in males. The peak incidence of osteosarcoma of long bones of the lower limb was six times more than that at any other site while the peak incidence of Ewing sarcomas located in the bones of the central axis exceeded those in long bones of the lower limb. The average height of patients with osteosarcoma at diagnosis was found to be significantly above the average height of the reference population, at the 95% level. The association of greater height at diagnosis with Ewing sarcoma was also significant at the 95% level but much weaker. Conclusion: In this thesis I have explored the epidemiology of cancer in TYA using some of the established methodologies which have previously been used in advancing our knowledge of childhood and older adult cancers. These studies provide some clues to aetiology. Variation in environmental exposures and lifestyle factors between England and India can explain the majority of the differences in incidence patterns observed. Genetic predisposition to cancer along with carcinogen exposure could lead to early onset of some cancers generally seen in older adults. Regardless of site, the similarity in age-incidence patterns of GCT, suggests a common initiation of these tumours in embryonic/foetal life with variable rates of tumour progression as a result of local factors or events during postnatal and pubertal period. The incidence patterns of osteosarcoma along with the strong and consistent association with a greater height at diagnosis indicate that bone growth is important in the development of this tumour while different biological pathways which may be unrelated to growth could also be relevant for Ewing sarcoma.

Methylenedioxymethamphetamine (MDMA) popularly known as "Ecstasy" continues to gain popularity as a recreational drug that has been shown to increase serotonin and dopamine levels. The present study has demonstrated that repeated exposure to MDMA produces long-term damage to serotonergic and dopaminergic neurons in various regions of the rat brain.