moderadas, ela proporciona efeitos benéficos sobre as funções cognitivas na vida adulta e no decorrer do envelhecimento. No entanto, a ingestão crescente de bebidas contendo cafeína por adolescentes tem causado preocupação, pois os efeitos desta substância sobre as funções cognitivas e a maturação do encéfalo durante a adolescência são pouco conhecidos. A cafeína atravessa a placenta e a barreira hemato-encefálica e o seu consumo tem sido associado ao maior risco de aborto espontâneo e baixo peso ao nascer. Portanto, nos estágios iniciais do desenvolvimento encefálico o consumo de cafeína também carece de maiores eslcarecimentos. Nesta tese, o impacto do consumo de cafeína durante diferentes fases de desenvolvimento do encéfalo foi investigado sobre o comportamento e proteínas sinápticas em ratos. No primeiro capítulo, ratos adolescentes machos consumiram cafeína na água de beber nas doses de 0,1; 0,3 e 1,0 g/L (correspondendo ao consumo baixo, moderado e elevado, respectivamente) somente durante o seu período ativo (das 19 às 7 horas). Nenhuma das doses testadas teve efeito sobre a atividade locomotora, porém todas desencadearam efeitos ansiogênicos. A cafeína (0,3 e 1,0 g/L) melhorou o desempenho na tarefa de reconhecimento ao objeto, enquanto na dose mais elevada (1,0 g/L) os animais não habituaram ao campo aberto, uma forma de avaliar o aprendizado não-associativo. Todas as doses testadas reduziram a densidade de proteína glial fibrilar ácida (GFAP) e proteína associada ao sinaptossoma (SNAP-25) sem causar alterações na imunorreatividade da proteína nuclear específica para neurônios (NeuN) no hipocampo e no córtex cerebral No hipocampo, a cafeína (em todas as doses testadas) aumentou a densidade de receptor de adenosina A1 e reduziu a do factor neurotrófico derivado do encéfalo (BDNF) e sua forma precursora (proBDNF) (1,0 g/L). No córtex cerebral, a cafeína (1,0 g/L) reduziu a densidade do receptor A1 e aumentou a do BDNF e do proBDNF (0,3 e 1,0 g/L). Estes resultados revelam que o consumo de cafeína por ratos adolescentes exacerba a ansiedade, mas provoca diferentes efeitos sobre a memória, melhorando a de reconhecimento e prejudicando o aprendizado não associativo. Parte destes efeitos foi associada às mudanças nos níveis de BDNF, GFAP e SNAP-25, porém sem perda da viabilidade neuronal aparente no hipocampo e no córtex cerebral. No segundo capítulo, o impacto do consumo de cafeína (0,1; 0,3 e 1,0 g/L na água de beber, das 19 às 7 horas) foi investigado sobre o comportamento e proteínas sinápticas na vida adulta dos animais que consumiram cafeína no decorrer do desenvolvimento encefálico. O consumo de três diferentes doses de cafeína iniciou 15 dias antes do acasalamento e permaneceu durante a prenhez e lactação. A partir do desmame os animais foram divididos em dois grupos: os que consumiram cafeína até a vida adulta (ao longo da vida) e os que interromperam o consumo (desenvolvimento). Esses dois grupos também foram subdivididos e analisados de acordo com o sexo. Foram comparados os efeitos destes protocolos sobre o comportamento e a densidade de proteínas sinápticas do hipocampo e córtex de fêmeas e machos adultos. A memória de reconhecimento foi prejudicada nas fêmeas que receberam cafeína (0,3 e 1,0 g/L) durante o desenvolvimento, o que coincidiu com o aumento do proBDNF e níveis inalterados de BDNF no hipocampo Ambos os protocolos de exposição causaram hiperlocomoção nos machos, enquanto que nas fêmeas somente a exposição ao longo da vida aumentou a atividade locomotora de forma significativa. Já no comportamento relacionado à ansiedade, ambos os sexos apresentaram um perfil ansiolítico ao consumir cafeína (1,0 g/L) ao longo da vida. Ambos os regimes de administração diminuíram os níveis de GFAP e SNAP-25 no hipocampo dos ratos machos. A densidade do receptor de TrkB foi reduzida no hipocampo em ambos os sexos e protocolos de exposição. No córtex cerebral BDNF e proBDNF aumentaram com o consumo de cafeína ao longo da vida nos machos. Nas fêmeas houve aumento no BDNF, mas não no proBDNF, em ambos regimes de administração. O receptor TrkB diminuiu no córtex dos ratos machos que receberam cafeína somente durante o desenvolvimento. Ambas proteínas – GFAP e SNAP-25 – aumentaram suas densidades nos machos que receberam ambos regimes de administração. Estes resultados revelaram que o consumo de cafeína ao longo da vida pode recuperar o prejuízo na memória de reconhecimento das fêmeas que consumiram a substância durante o desenvolvimento e indicam que a exposição durante um período específico do desenvolvimento do encéfalo promove alterações comportamentais dependentes do sexo, as quais nós relacionamos com modificações na sinalização BDNF. Os resultados desta tese destacam a importância de controlar o consumo de cafeína em períodos críticos para o desenvolvimento encefálico de ratos, e aponta para um efeito dependente do sexo. No entanto, mais estudos são necessários para ampliar nosso conhecimento sobre as possíveis vias de sinalização envolvidas nestes processos. / Caffeine is the most consumed psychostimulant substance worldwide, with benefits for cognitive functioning. Caffeine intake at moderate doses also prevents age-related cognitive decline. However, health experts have raised concerns about the growing intake of caffeine-containing drinks by adolescent population. In fact, the effects of caffeine on cognitive functions and neurochemical aspects of late brain maturation during adolescence are poorly understood. In addition, caffeine consumption in the early stages of fetal development has been associated with miscarriage and low birth weight, since it penetrates placenta and blood-brain barrier during pregnancy. Therefore, the impact of caffeine intake was investigated during different stages of brain development. In the first chapter of this thesis, adolescent male rats consumed caffeine in the drinking water (0.1; 0.3 and 1.0 g/L corresponding to low, moderate and high doses, respectively) only during their active period (from 7:00 p.m. to 7:00 a.m.). None of the doses tested had effect on locomotor activity, whereas all triggered anxiogenic effects. Caffeine (0.3 and 1.0 g/L) improved the performance in the object recognition task, but the higher dose of caffeine (1.0 g/L) decreased habituation in open field arena, suggesting a non-associative learning impariment. All tested doses reduced glial fibrillary acidic protein density (GFAP) and synaptosome-associated protein (SNAP-25) without causing any changes in immunoreactivity for neuronspecific nuclear protein (NeuN) in the hippocampus and cerebral cortex. In the hippocampus, caffeine (all doses tested) increased adenosine A1 receptor density and reduced brain-derived neurotrophic factor (BDNF) and proBDNF (1.0 g/L). In the cerebral cortex, caffeine (1.0 g/L) reduced adenosine A1 receptor and increased BDNF and proBDNF density (0.3 and 1.0 g/L) These findings document the effects of caffeine consumption in adolescent rats with a dual impact on anxiety and recognition memory, associated with changes in BDNF, GFAP and SNAP-25 levels without apparent neuronal loss in hippocampus and cerebral cortex. In the second chapter, it was tested whether caffeine consumption (0.1; 0.3 and 1.0 g/L in drinking water, from 7:00 p.m. to 7:00 a.m) throughout life may reverse the negative effects caused by the consumption of caffeine in the early stages of development. For this, we used exposure protocols with the end in postnatal days (PND) 21 (development) or 90 (throughout life); both protocols starting 15 days before mating. The effects of these protocols on the behavior and hippocampal synaptic proteins density of adult female and male rats were compared. Recognition memory was impaired in females receiving caffeine (0.3 and 1.0 g/L) during development, which coincided with increased proBDNF levels and unchanged BDNF in the hippocampus. Both exposure protocols caused hyperlocomotion in males, whereas in females only the exposure throughout life significantly increased locomotor activity. Considering the anxiety related behavior, both sexes presented an anxiolytic profile when consuming caffeine (1.0 g/L) throughout life. Both exposure regimens decreased hippocampal GFAP and SNAP-25 of male rats. The hipocampal TrkB receptor was reduced in both sexes and protocols of exposure In the cortex, both proBDNF and BDNF increased in males receiving caffeine throughout life as well as GFAP and SNAP-25 increased in both treatments regimen. The results revealed that caffeine consumption throughout life can recover the impairment in recognition memory of females that consumed caffeine during development and indicate that exposure for a specific period of brain development promotes sex-dependent behavioral changes, which we relate to alterations in BDNF signaling. The results of this thesis emphasize the importance of controlling caffeine intake during critical periods of brain development of rats and points to a sex dependent effect. However, more studies are needed to expand our knowledge about the possible signaling pathways involved in these processes.

La rupture de la barrière hémato-rétinienne interne (iBRB) se produit dans de nombreux troubles de la rétine et peut provoquer un œdème rétinien souvent responsable de la perte de vision. Le but de cette étude était de caractériser l'impact d'une rupture de l’iBRB sur les changements homéostatiques rétiniens de la dystrophine Dp71, AQP4 et Kir4.1 provoqués par les altérations les cellules gliales de Müller CGM. L'effet protecteur de la Dex a été étudié dans ce modèle. Par ailleurs, les explants rétiniens ont été utilisé pour étudier la formation et la résolution de l'œdème de CGM sans l'influence de l'inflammation du cristallin ainsi que l’effet de différentes doses de glucocorticoïdes (Dex, triamcinolone et fluocinolone) et des inhibiteurs de la voie de l'acide arachidonique. Nous avons observé que la chirurgie partielle du cristallin induit une rupture de l'iBRB et des changements moléculaires dans le CGM, une diminution de l’expression de la Dp71 et d’AQP4 et la délocalisation de Kir4.1. La Dex semble protéger la rétine par l’augmentation de l’expression du HSF1. Nous avons également observé que même si les glucocorticoides étudié ont des effets différents sur l’expression de la Dp71, AQP4 et Kir4.1 les trois sont capables de prévenir la formation de l’œdème de CGM. Nos résultats suggèrent que la formations d'œdème semblent être régulée par la voie des leucotriènes. Nous avons étudié le rôle des isoformes de la dystrophine Dp71 dans les processus d'adhésion intercellulaire des cellules PC12. Nos résultats suggèrent l’existence d’au moins deux mécanismes différents seraient impliqués dans l'adhésion intercellulaire associée à la Dp71, l'une impliquant Dp71dΔ71 et Cx43. / The breakdown of the internal blood-retinal barrier (iBRB) occurs in many retinal disorders and may cause retinal edema, often responsible for vision loss. The aim of this study was to characterize the impact of iBRB disruption on retinal homeostatic changes in Dp71 dystrophin, AQP4 and Kir4.1 caused by Müller glial cells (MGC) alterations. The protective effect of Dex has been studied in this model. In addition, retinal explants were used to study the formation and resolution of CGM edema without the influence of lens inflammation and the effect of different doses of glucocorticoids (Dex, triamcinolone and fluocinolone) and inhibitors of the arachidonic acid pathway. We observed that partial lens surgery induced iBRB breakdown and molecular changes in MGC, decreased expression of Dp71 and AQP4, and miss localization of Kir4.1. Dex seems to protect the retina by increasing the expression of HSF1. We also observed that although the glucocorticoids studied have different effects on the expression of Dp71, AQP4 and Kir4.1 all three can prevent the formation of MGC edema. Our results suggest that edema formation appears to be regulated by leukotrienes. We have studied the role of isoforms of dystrophin Dp71 in intercellular adhesion processes of PC12 cells. Our results suggest the existence of at least two different mechanisms involved in intercellular adhesion associated with Dp71, one involving Dp71dΔ71 and Cx43.

O complexo da esclerose tuberosa (TSC) é um transtorno clínico, com expressividade variável, caracterizado por hamartomas que podem ocorrer em diferentes órgãos. Tem herança autossômica dominante e é devido a mutações em um de dois genes supressores de tumor, TSC1 ou TSC2. Estes codificam para as proteínas hamartina e tuberina, respectivamente, que se associam formando um complexo macromolecular que regula funções como proliferação, diferenciação, crescimento e migração celular. As lesões cerebrais podem ser muito graves em pacientes com TSC e caracterizam-se por nódulos subependimários (SEN), astrocitomas subependimários de células gigantes (SEGA), tuberosidades corticais e heterotopias neuronais, podendo relacionar-se clinicamente à epilepsia refratária à terapia medicamentosa, deficiência intelectual, desordens do comportamento e hidrocefalia. O potencial de crescimento de SEGA até os 21 anos de idade dos pacientes exige acompanhamento periódico por exame de imagem e condutas clínicas ou cirúrgicas, conforme indicação médica. As lesões subependimárias têm sido explicadas por déficits de controle da proliferação, crescimento e diferenciação de precursores neuro-gliais na zona subventricular telencefálica. Embora a capacidade da tuberina em inibir a proliferação celular pela repressão do alvo da rapamicina em mamíferos (mTOR) esteja bem documentada, outros aspectos celulares do desenvolvimento de SEGA ainda não foram examinados. Assim, é importante estabelecer um sistema in vitro para o estudo de células da zona subventricular e testá-lo na análise das proteínas hamartina e tuberina. Neste sentido, o cultivo de neuroesferas em suspensão é muito apropriado. Neste estudo, buscamos relacionar a expressão e distribuição subcelular da hamartina e tuberina à capacidade proliferativa e de diferenciação das células de neuroesferas cultivadas in vitro a partir da dissociação da vesícula telencefálica de embriões de ratos normais. Analisamos a expressão e distribuição subcelular da hamartina e tuberina por imunofluorescência indireta em células entre a primeira e a quarta passagens das neuroesferas, sincronizadas nas fases G1 ou S do ciclo celular e após a reentrada no ciclo celular, através da incorporação de 5-bromo-2\'-desoxiuridina (BrdU) e imunofluorescência com anticorpo anti-BrdU. Em geral, células de neuroesferas apresentaram baixa colocalização entre hamartina e tuberina in vitro. A expressão da tuberina foi elevada em basicamente todas as células das esferas e fases do ciclo celular; ao contrário, a hamartina apresentou-se principalmente nas células da periferia das esferas. A colocalização entre hamartina e tuberina foi observada em células mais periféricas das esferas, sobretudo no citoplasma e, em G1, no núcleo celular. A proteína rheb, que conhecidamente interage diretamente com a tuberina, apresentou distribuição subcelular muito semelhante à desta. Ao carenciamento das células visando à parada do ciclo celular na transição G1/S, tuberina distribuiu-se ao núcleo celular em quase todas as células avaliadas e, de forma menos frequente, a hamartina também. À reentrada no ciclo celular pelo reacréscimo dos fatores de crescimento, avaliaram-se células com incorporação de BrdU ao seu núcleo celular, após 72 e 96 horas. Nestas, tuberina mostrou-se novamente no citoplasma de forma preponderante e hamartina manteve-se citoplasmática, em geral subjacente à membrana plasmática, em níveis mais baixos. Os grupos cujas células reciclaram por 72 ou 96 horas diferiram quanto ao aumento significativo da expressão da hamartina em células proliferativas no último. À diferenciação neuronal, aumentaram-se os níveis de expressão de hamartina observáveis à imunofluorescência indireta, tornando-se equivalentes àqueles da tuberina. Concluímos que as células de neuroesferas cultivadas em suspensão apresentam-se como um sistema apropriado ao estudo da distribuição das proteínas hamartina e tuberina e sua relação com o ciclo celular / The tuberous sclerosis complex (TSC) is a clinical disorder with variable expressivity, characterized by hamartomas that can occur in different organs. It has autosomal dominant inheritance and is due to mutations in one of two tumor suppressor genes, TSC1 or TSC2. These encode for the proteins hamartin and tuberin, respectively, which are associated in a macromolecular complex which functions as a regulator of cell proliferation, differentiation, growth and migration. TSC brain lesions may be severe and are characterized by subependymal nodules (SEN), subependymal giant cell astrocytomas (SEGA), neuronal heterotopias and cortical tubers, and may be clinically related to refractory epilepsy, intellectual disability, behavioral disorders and hydrocephaly. The growth potential of SEGA up to 21 years of age in TSC patients requires regular monitoring by imaging. Clinical and surgical interventions may be medically indicated. Subependymal lesions have been explained by deficient control of proliferation, growth and differentiation of neuro-glial progenitors from the telencephalic subventricular zone. While tuberin ability to inhibit cell proliferation by repressing the mammalian target of rapamycin (mTOR) has been well documented, other cell aspects of SEGA development have not been thoroughly examined. Therefore, it is important to establish conditions for an in vitro system to study the cells from the subventricular zone and to test its suitability for the study of the TSC proteins. In this regard, the neurosphere suspension culture is very appropriate. We evaluated the expression and subcellular distribution of hamartin and tuberin in relation to the proliferation and differentiation capability of neurosphere cells derived in vitro from the dissociation of the telencephalic vesicle of normal E14 rat embryos. These analyses were performed by indirect immunofluorescence in cells from first through fourth passages of neurospheres, synchronized in G1 or S phases of the cell cycle, and after reentry into the cell cycle by the addition of 5-brome-2\'-desoxyuridine (BrdU) and immunolabeling with anti-BrdU antibody. In general, neurosphere cells presented low colocalization between hamartin and tuberin in vitro. Tuberin expression was relatively high in basically all neurosphere cells and cell cycle phases, whereas hamartin distributed mainly to cells from the periphery of the spheres. In these cells, hamartin and tuberin colocalization was evident mostly in the cytoplasm and, in G1, also in the cell nucleus. Rheb, which is known to interact directly with tuberin, had subcellular distribution very similar to tuberin. Cell starvation indicating cell cycle arrest at G1/S redistributed tuberin to the cell nucleus in virtually all cells examined, what was accompanied by nuclear location of hamartin in a small subset of cells. When cells were allowed to reenter cell cycle by adding growth factors, we evaluated BrdU-labeled nuclei 72 and 96 hours later. In the two groups, tuberin was shown to move back to the cytoplasm as well as hamartin, which apparently maintained its lower expression levels distribution underneath the plasma membrane. Group of cells that recycled for 96 hours had significantly more expression of hamartin than those cells that cycled for only 72 hours. After neuronal differentiation, hamartin expression levels observed by immunofluorescence were similar to those of tuberin. We conclude that neurosphere cells cultured in suspension showed to be an appropriate cell system to study hamartin and tuberin distribution in respect to the cell cycle

A resposta do sistema nervoso para diversas lesões acarreta a ativação das células gliais entéricas. Este trabalho tem como objetivo analisar o efeito da isquemia e reperfusão intestinal (I/R-i) sobre as células gliais entéricas, neurônios e receptores P2X2 e P2X7. Foram analisados o íleo de ratos Controle, Sham e I/R-i com 0 hora, 24 horas e 14 dias de reperfusão. Foram realizadas dupla marcação dos receptores P2X2 e P2X7 com Hu e S100, densidade, área do perfil e marcação de proliferação celular. Os resultados mostraram dupla marcação de células gliais entéricas e neurônios com os receptores P2X2 e P2X7; a densidade apresentou um aumento de células gliais e diminuição de neurônios imunorreativos ao Hu. A área do perfil de células gliais entericas S100-IR apresentaram diminuição nos grupos I/R-i e foi detectada proliferação de células gliais entéricas nos grupos I/R-i 0 hora e 24 horas. Conclui-se que a isquemia levou a alterações diferenciadas nos receptores P2X2 e P2X7, células gliais entéricas e neurônios, que podem causar disfunções gastrointestinais. / The nervous system response to various injuries involves the activation of enteric glial cells. The aim of the work was to analyze the effect of ischemia and reperfusion (I/R-i) on enteric glial cells, neurons and receptors P2X2 and P2X7. We analyzed the ileum of Control, Sham and I/R-i with 0 hour, 24 hours and 14 days of reperfusion. Double staining were performed P2X2 and P2X7 receptors with Hu and S100, density, area profile and marking of cellular proliferation. The results show double staining of neurons and enteric glial cell with the P2X2 and P2X7; density increased by glial cells and decrease of neurons immunoreactive to Hu. The area profile of enteric glial cell S100-IR showed decreased in Groups I/R-I and enteric glial cell proliferation was observed in groups I/R-i 0 hours and 24 hours. It is concluded that ischemia has led to changes in differential P2X2 and P2X7 receptors, neurons and enteric glial cells, which can cause gastrointestinal dysfunction.

Nos últimos anos, inúmeros avanços têm sido alcançados no que diz respeito aos mecanismos moleculares que participam na indução e manutenção da dor crônica, incluindo ativação glial. Já foi demonstrado que alguns receptores de reconhecimento padrão (PRRs), como os receptores do tipo Toll (TLRs) participam desse processo e, que em modelos de inflamação/infecção do Sistema Nervoso Central, os TLRs e os receptores do tipo NOD (NLRs) cooperam na ativação das células da glia, o que nos levou a hipotetizar que os receptores NOD1 e NOD2 também possam desempenhar um papel importante no processo de dor crônica. O NOD2 é responsável pela detecção intracelular do muramil dipeptídeo (MDP) enquanto que NOD1 reconhece o ácido diaminopimélico (iE-DAP), ambos padrões moleculares associados a patógenos (PAMPs) encontrados no peptideoglicano de bactérias. Após o reconhecimento, NLRs recrutam diretamente RIPK2 (proteína 2 de interação com o receptor RICK), uma serina-treonina quinase importante na ativação do fator nuclear kB (NF-kB). Assim, o objetivo do presente estudo foi avaliar a participação de NOD1 e NOD2, via RIPK2, no desenvolvimento da hipersensibilidade mecânica neuropática focando principalmente nos mecanismos espinais envolvidos. Dessa maneira, foi observado que os animais NOD1-/-, NOD2-/- e RIPK2-/- apresentaram redução significativa da hipersensibilidade nociceptiva mecânica quando comparado aos animais selvagens após indução de neuropatia periférica pelo modelo experimental de lesão limitada do nervo isquiático (SNI, Spared Nerve Injury). Ao contrário, a hipersensibilidade inflamatória induzida pelo adjuvante completo de Freud (CFA) não foi reduzida nesses animais. A redução da dor neuropática em NOD1-/-, NOD2-/- e RIPK2-/- foi associada a uma diminuição da expressão de IBA-1, GFAP, IL-1, TNF- e P2X4 na medula espinal quando comparado ao grupo WT. In vitro, foi observado que culturas primárias de micróglia não induziram liberação de IL-1, TNF-, IL-6 em resposta ao MDP (10g/mL) e iE-DAP (100ng/mL). No entanto, quando o MDP foi administrado juntamente com uma baixa concentração de lipopolissacarídeo (LPS) (0,1ng/mL), apresentou uma forte produção destas citocinas. Além disso, também foi demonstrado que células periféricas que infiltram na medula espinal podem expressar NOD1 e NOD2 e portanto serem capazes de induzir hipersensibilidade mecânica e ativação microglial após a indução de neuropatia. Dessa maneira, os resultados sugerem que NOD1 e NOD2, via RIPK2, contribuem para a gênese da dor neuropática, possivelmente mediando a liberação de citocinas pró-nociceptivas e a ativação de células gliais. Além disso, os resultados apontam ação potencial de NOD2 com TLR4 no intuito de estimular a ativação glial. Estes mecanismos representam uma nova abordagem para elucidar os mecanismos envolvidos na fisiopatologia da dor crônica e um possível alvo para o desenvolvimento de drogas para o tratamento da dor neuropática. / In the last years, many advances have been made related to the molecular mechanisms involved in the induction and maintenance of chronic pain, including glial activation. It has been shown that some pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) are involved in this process, and that in inflammation/infection models of the CNS, the TLRs and Nod-like receptors (NLRs) cooperate in activation of glial cells, which led us to hypothesize that NOD1 and NOD2 receptors may also play an important role in chronic pain process. NOD2 are responsible by intracellular detection of muramyl dipeptide (MDP) and NOD1 detects meso-diaminopimelic acid (iE-DAP), pathogen-associated molecular patterns (PAMPs) found in the peptidoglycan from bacteria. Upon recognition, NLRs recruit directly RIPK2, an adaptor protein, important in NLRs-mediated NFB activation. In the present study, we aimed to evaluate the participation of NOD1 and NOD2, via RIPK2, in the development of neuropathic mechanical hypersensitivity focusing mainly on spinal mechanisms involved. The results demonstrate that NOD1-/-, NOD2-/-, RIPK2-/- showed a significant reduction in mechanical hypersensitivity when compared to WT mice, after submitted to an experimental model of neuropathic pain Spared Nerve Injury (SNI). Interestingly, CFA-induced chronic inflammatory hypersensitivity was not decreased in these mice. The reduction in neuropathic pain in NOD1-/-, NOD2-/- and RIPK2-/- mice was associated with a decrease in the expression of IBA-1, GFAP, IL-1, TNF- and P2X4 in spinal cord when compared with WT. In vitro, it was observed that primary cultures of microglia did not produce IL-1, TNF-, IL-6 in response to MDP (3g/mL) or iE-DAP (100ng/mL). However, MDP, together with an ineffective concentration of LPS (0.1ng/mL), produced a robust production of these cytokines. Moreover, it was also demonstrated that peripheral cells infiltrating the spinal cord could express NOD1 and NOD2 and thus, be able to induce mechanical hypersensitivity and microglial activation after induction of peripheral neuropathy. The results suggest that NOD1 and NOD2, via RIPK2, contribute to the genesis of neuropathic pain, possibly by mediating the release of pronociceptive cytokines and increased glial cells activation. Moreover, the results indicate potential action of NOD2 with TLR4 in attempt to stimulate glial cells activation. These mechanisms represent a novel approach for elucidating the pathophysiology of chronic pain, and a target for the development of drugs for the treatment of neuropathic pain.

Gene transfer to the brain is a promising therapeutic strategy for a variety of neurodegenerative disorders including Parkinson‟s disease (PD). PD is the second most common neurodegenerative disease. Although many drugs have been developed and introduced into the market to provide symptomatic treatment, there is still no cure for PD. Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for injured nigrostriatal dopamine neurons and is currently being evaluated as a potential treatment for PD. Gene therapy allows localized, long-term and stable transgene expression after a single intervention to obtain a therapeutic effect. Regulatable promoters for transgene expression furthermore allow optimizing GDNF concentration to avoid undesirable biological activity and clinical side effects. In the first part of the study, an autoregulatory tetracycline-inducible recombinant adeno-associated viral vector (rAAV-pTetbidiON) utilizing the rtTAM2 reverse tetracycline transactivator (rAAV-rtTAM2) was used to conditionally express the human GDNF cDNA. Eight weeks after a single intrastriatal injection of the rAAV-rtTAM2-GDNF vector encapsidated into AAV serotype 1 capsids (rAAV2/1), the GDNF protein level was respectively 15 fold higherand undistinguishable from the endogenous level in doxycycline(Dox) treated and untreated animals. However, a residual GDNF expression in the uninduced animals was evidenced by a sensitive immunohistochemical staining. As compared to rAAV2/1-rtTAM2-GDNF, the rAAV2/1-rtTAM2-WPRE-GDNF vector harboring a woodchuck hepatitis post-transcriptional regulatory element, which increases and stabilizes the transgene transcript, expressed a similar concentration of GDNF in the induced state but a basal level ~2.5-fold higher than the endogenous striatal level. However, the distribution of GDNF in the striatum in induced state was more widespread using the rAAV2/1-rtTAM2-WPRE-GDNF vector as compared to rAAV2/1-rtTAM2- GDNF. As a proof for biological activity, for both vectors, downregulation of tyrosine hydroxylase (TH) was evidenced in dopaminergic terminals of Dox-treated but not untreated animals. In the second part of my study, functional (behavioural) and neurochemical changes mediated by delayed intrastriatal GDNF gene delivery in the partial Parkinson‟s disease rat model were investigated. The rAAV2/1-rtTAM2-WPRE-GDNF vector (3.5 108 viral genomes) was administered unilaterally in the rat striatum 5 weeks after intrastriatal injection of 6-hydroxydopamine (6-OHDA) which produces a partial and progressive lesion of the nigro-striatal dopaminergic pathway. Rats were treated with Dox or untreated from the day of vector injection until sacrifice at 4 or 14 weeks (continuous treatment). A sub-group was Dox-treated for 7 weeks (temporary treatment) then untreated until 14 weeks. In the absence of Dox, the GDNF tissue concentration was found to be equivalent to the endogenous level in 6-OHDA-lesioned rats. In the presence of Dox, it was ~10-fold higher. Dox-dependent behavioral improvements were demonstrated 4 weeks post-vector injection. At later time points, spontaneous partial recovery was observed in all rats, but no further improvement was found in Dox-treated animals. Moreover GDNF gene delivery only transiently improved dopaminergic function. Over the long term, TH was more abundant, but not functional, and the increase was lost when GDNF gene expression was switched off. The third part of my study consisted in the evaluation of the respective dose-range of therapeutical and undesirable effects of GDNF. Functional effects appeared after delivery of 3.5 108 viral particles which produced 200-300 pg/mg protein of GDNF in the lesioned rat striatum (see above). In order to evaluate the viral dose producing undesirable effects, we compared two different doses of vector: 3.5x108 and 4.4x109 viral genome. In the low dose group, the GDNF concentration in the striatum was ~300 pg/mg protein in the Dox-treated animals and equivalent to the endogenous level in untreated animals (~20 pg/mg protein). In contrast, in the high dose group, GDNF levels reached ~1200 pg/mg protein in induced animals but up to ~300 pg/mg protein in uniduced animals. In the low dose group, Dox-dependent downregulation of TH but no asymetrical behaviour was evidenced. In the high dose group, TH downregulation was observed in both Dox+ and Dox-rats. In addition, amphetamine-induced rotational behaviour was evidenced in Dox+ but not in Dox-rats. These data suggest that low doses of virus are sufficient to induce therapeutically-relevant but not undesirable functional effects of GDNF. Nevertheless,a neurochemical effect of GDNF (TH down-regulation) did appear at low dose. In order to understand the GDNF-induced motor asymmetry, we investigated the anatomical pattern of TH down regulation in striatum. Strikingly, there was a greater loss of TH labeling in striosomes than in the surrounding matrix. Receptors which are known to be differentially expressed in the striosomes i.e. µ-opioid receptor(MOR-1) and N-methyl-D-aspartic acid (NMDA) receptor 1 (NR1) as compared to the matrix were analyzed in the high-dose group of animals. MOR-1 was not affected by GDNF gene delivery. In contrast, NR1 was down regulated. The potential relationship between TH and NR1 down-regulation as well as other previously described neurochemical effects of GDNF (as enhancement of DA release and metabolism, of DA neurons excitability or of TH phosphorylation) and behavioural asymmetry remains to be clarified. As summary, our data suggest that behavioural and neurochemical effects of striatal delivery of GDNF can be controlled by Dox by using the autoregulatory rAAV2/1-TetON- GDNF vector, provided the dose range of gene delivery is carefully adjusted. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

The social brain hypothesis (SBH) posits that complex social environments exert a major selection pressure driving the evolution of large brains and intelligence. The hypothesis was first proposed to explain the remarkable cognitive abilities of primates and has since been extended to other vertebrate groups and gained a substantial popularity. Nevertheless, the empirical support is equivocal in virtually every group where the hypothesis has been tested. In this thesis, the SBH is tested in the African mole-rats (Bathyergidae). Mole-rats share a subterranean mode of life and similar ecologies while covering the whole social spectrum, from solitary to "eusocial". The number of brain neurons is considered a better proxy for intelligence than relative or absolute brain size. Therefore, a novel approach, the isotropic fractionator, was used to estimate the total number of neurons and other cells in five brain parts (olfactory bulbs, cerebral cortex, cerebellum, diencephalon and basal ganglia, brain stem) of eleven bathyergid species. This simultaneously allows for examining if and how mole-rats differ from other rodents with respect to brain cellular scaling rules. We found that, contrary to expectations, mole-rats generally conform to these rules, with a few exceptions. They tend to have higher...

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.

Les cellules épendymaires sont des cellules multiciliées qui tapissent les parois de toutes les cavités du cerveau. Une fois différenciées, ces cellules ne se divisent plus au cours de la vie. Le battement de ces multiples cils motiles joue un rôle important pour maintenir un flux constant de liquide cérébrospinal à travers toutes les cavités cérébrales. Les cellules épendymaires assurent également des fonctions critiques d’échanges moléculaires avec le liquide cérébrospinal. Dans son ensemble, l’implication des cellules épendymaires et de leurs cils motiles s’avère d’une importance majeure dans le maintien des circuits neuraux ainsi que dans le fonctionnement plus global du cerveau. Récemment, une nouvelle caractéristique des cellules épendymaires a été identifiée ; elles font partie d’un microenvironnement appelé une « niche » centrée autour de cellules souches neurales dans le cerveau du rongeur adulte. Ces cellules souches neurales adultes sont capables de produire de nouveaux neurones qui migreront vers le bulbe olfactif des rongeurs adultes. Concernant leur origine, il a été montré que les cellules épendymaires multiciliées dérivent des cellules souches neurales durant les stades tardifs embryonnaires. Ces mêmes cellules souches peuvent d’ailleurs donner naissance à la plupart des différents types de cellules du cerveau. Cependant, les mécanismes par lesquels les cellules souches décident de leur destin cellulaire restent largement méconnus. Dans ce projet, nous étudions quel type de division donne naissance à des cellules épendymaires et nous nous intéressons également au lignage épendymaire. Nos données suggèrent que les cellules épendymaires ne migrent pas après leur dernière division et qu’elles restent à proximité de l’endroit où elles ont été produites. Chose particulièrement intéressante, nous montrons que les cellules épendymaires peuvent être générées par division symétrique ou asymétrique. Nos résultats révèlent aussi que les cellules souches neurales embryonnaires se divisent de manière asymétrique pour donner naissance à la fois à une celluleépendymaire et à une cellule souche neurale adulte. Ces données viennent s’ajouter à la connaissance actuelle que nous avons du développement du cerveau. De plus, elles pourraient contribuer à ouvrir de nouvelles perspectives et stratégies thérapeutiques pour soigner les maladies neurodégénératives à beaucoup plus long terme. / Ependymal cells are multiciliated cells lining the walls of all brain cavities. Once they are mature, they do not divide during life. Their motile ciliary beating endorses a crucial role in maintaining a proper flow of cerebrospinal fluid throughout all brain cavities. Ependymal cells also ensure critical molecular exchanges of the cerebrospinal fluid. On the whole, the involvement of ependymal cells and their multiple motile cilia in the maintenance of the neural circuits and more globally in the well-functioning of the entire brain have proven paramount. More recently, a new characteristic of ependymal cells has been brought to light. Namely, they are part of a microenvironment so called a “niche” surrounding adult neural stem cells in the adult rodent brain. Noteworthy, these adult neuralstem cells are capable of producing new neurons that will migrate to the olfactory bulb of rodents. In terms of their origin, it was shown that multiciliated ependymal cells derive from neural stem cells during late embryonic stages. Besides, the same stem cells can give rise to most cell types of the brain. However, little is known about how fate-decision is made in neural stem cells. In this project, we tackle more particularly how multiciliated ependymal cells arise from the neural stem cells. Most specifically, we address the type of celldivision and the ependymal cell lineage. We find that ependymal cells are not migrating subsequent to their last division, but rather stay where they were first produced. Most interestingly, they can be generated through both symmetric and asymmetric cell division. We also show that embryonic neural stem cells divide asymmetrically to give rise to both an ependymal cell and an adult stem cell. We are confident that these data bring major new insights in the current understanding of neural development. Additionally, these findingscould contribute in opening new therapeutic perspectives and strategies to cure neurodegenerative diseases in a much longer term.

Recent comparative studies have shown that bird brains, although small, have a high processing capacity. The brains of parrots and songbirds have higher neuronal densities than brains of mammals; especially large parrots and corvids compete with or even outnumber primates by the number of telencephalic neurons. However, the processing capacity of the avian brain appears to differ significantly between various phylogenetic lineages. Basal groups such as galliform birds have much lower absolute numbers of neurons and lower neuronal densities than songbirds and parrots. In this Master thesis, I used the isotropic fractionator to determine numbers of neurons and non-neural cells in specific brain regions in 19 species of hornbills (Bucerotiformes), woodpeckers (Piciformes) and coraciiform birds (Coraciiformes). The brains of hornbills and woodpeckers (but not coraciiform birds) have numbers of neurons comparable to that of songbirds and parrots and significantly more neurons than equivalently sized brains of pigeons (Columbiformes) and galliform birds (Galliformes). In the crown groups, we can observe similar trends such as a higher degree of encephalization, a proportionally larger telencephalon and increasing percentage of telencephalic neurons. On the contrary, in pigeons and galliform birds, we can...