L’arrêt cardio circulatoire et le traumatisme crânien sont responsables de lésions cérébrales dont les conséquences médico économiques sont un réel enjeu de santé publique. Malgré des espoirs importants lors des travaux expérimentaux, la majorité des traitements neuroprotecteurs se sont révélés être des échecs lors du passage à la clinique humaine. Riche d’une expérience clinique vieille de plusieurs millénaires, la Médecine Chinoise Traditionnelle a démontré son efficacité en clinique sur des patients victimes d’accidents vasculaires cérébraux. Le MLC 601, et sa formule simplifiée le MLC901, produits issus de celle-ci, ont déjà été étudiés dans un travail expérimental réalisé sur un modèle d'ischémie focale dans le laboratoire d’accueil. L’effet pléiotrope du produit avait alors été souligné. L’objet de notre travail a été d’étudier les effets neuroprotecteurs et neurorégénérateurs du MLC901 sur 2 autres modèles expérimentaux de lésions cérébrales : l’ischémie globale, mimant les conséquences cérébrales d’un arrêt cardiaque et le traumatisme crânien par percussion liquidienne latérale. Nous insistons, dans ce travail, sur l’effet neuroprotecteur du produit agissant sur les mécanismes de nécrose, d’apoptose et de stress oxydant se mettant en place après la lésion initiale. Nous retrouvons également une action neurorégénérative avec une stimulation de la neurogenèse induite par la lésion. L’ensemble de ces mécanismes cellulaires mis en place est associé à une amélioration de la récupération des fonctions neurologiques des animaux mis en évidence par l'utilisation de tests comportementaux moteurs et cognitifs. Nous démontrons donc dans ce travail, l’effet neuroprotecteur et neurorégénérateur du MLC901 sur deux modèles expérimentaux de « cérébro lésion », l’un ischémique et l’autre traumatique. / Cardiac arrest and traumatic brain injury are a socio economic health problem. Despite lot of hopes on neuroprotective therapies, few confirmed promising experimental results in clinical studies. Traditional Chinese Medicine has been used for several centuries. Despite lot of clinical investigations, few data are available on mechanisms involved in their effects. Interesting results have been published in stroke patients, and experimental studies using MLC601 and MLC901 have been conducted in mouse focal ischemia models. The multiple mechanisms of action, neuroprotective and neuroregenerative, of these treatments have been highlighted. The purpose of our study was to analyse the neuroprotective and neuroregenerative actions of MLC901 on rat global ischemia and traumatic brain injury models. In these models, we confirmed the neuroprotective action on necrosis, apoptosis and oxidative stress and the neuroregenerative action by the way of neurogenesis activation. These cellular actions are associated with functional recovery in the two models. We confirmed in these two experimental models, the neuroprotective and neuroregenerative effects of MLC901 on post ischemic or post traumatic brain injuries. This approach is essential for Traditional Chinese Medicine to be accepted by occidental one.

La maladie d’Alzheimer est une maladie très complexe, aujourd’hui encore mal comprise et cette démence est devenue un réel problème de santé publique. La protéine précurseur de l’amyloïde (APP) et la protéine Tau sont deux acteurs majeurs impliqués dans la maladie. De nombreuses recherches se sont investies dans la compréhension du métabolisme, de l’action et de l’implication de ces deux protéines dans les mécanismes pathologiques de la maladie et d’autres maladies neurodégénératives. Elles sont notamment l’objet de la plupart des approches thérapeutiques passées et actuelles, et étudiées pour le diagnostic biologique de la maladie. Dans ce projet de thèse, notre objectif fut d’explorer le métabolisme des protéines APP et Tau au cours de la différenciation neuronale à l’aide d’outils biochimiques et de systèmes innovants d’immunodétection multiplex très sensibles (MSD®) dans plusieurs modèles de culture cellulaire de la maladie. L’objectif était d’obtenir une vision globale des processus physiopathologiques au travers d’analyses d’échantillons générés au cours de la différenciation neuronale de cellules souches pluripotentes induites (iPSC) de patients Alzheimer comparées aux cellules souches embryonnaires humaines (hESC). Nous avons ainsi généré et caractérisé plusieurs lignées cellulaires d’IPSC d’une personne saine contrôle et de patients atteints des formes sporadiques et familiales de la maladie. Ce projet offre l’opportunité unique de combiner des approches innovantes pour tenter de comprendre comment les fragments et les peptides Ab sont générés, ainsi que les modifications de Tau en conditions normales et pathologiques. / Amyloid precursor protein (APP) and Tau protein are two main molecular actors of the Alzheimer’s disease (AD), which is of prime importance in Human Health. Intensive research is ongoing to understand these proteins’ metabolism, action and implication in the pathological mechanism of these affections. They are the target of most therapeutic approaches and are used for biological diagnosis. In the present PhD project, our objective was to investigate neuronal APP and Tau protein processing and metabolism using biochemical tools and innovative multiplex immunodetection system (MSD®) in diverse cell culture models of AD. The goal was to get a comprehensive view oh physiopathological processes based on the analysis of samples generated in neuronal differentiated human embryonic stem cell and induced pluripotent stem cells derived from AD-patients. We generated several cell lines from an healthy control individual, and AD patients showing sporadic and familial forms of the disease. This project offer the unique opportunity to combine state-to-the-art approaches to understand how the APP fragments and peptides are generated as well as the modifications of the Tau protein in normal and pathological situation.

Malgré sa complexité, le cerveau intègre en permanence de nouvelles cellules – à la fois neuronales et gliales – au-delà du développement embryonnaire et ce, tout le long de la vie. La période postnatale est caractérisée par une gliogenèse intense. A l’âge adulte, de nouveaux neurones et cellules gliales sont produits dans des régions restreintes à partir de cellules souches/progénitrices (CSP) localisées dans des niches. Les deux niches de CSP adultes les mieux décrites sont la zone sous-ventriculaire des ventricules latéraux, qui produit de nouveaux interneurones olfactifs, et la zone sous-granulaire du gyrus denté de l’hippocampe, où de nouveaux neurones en grain sont produits localement. Des travaux menés ces dernières années ont montré qu’une neuro- et une gliogenèse avaient aussi lieu dans l’hypothalamus postnatal, une petite région du diencéphale ventral qui régule des processus physiologiques vitaux tels que le métabolisme, la reproduction, le sommeil et la thermorégulation. Si l’identité des CSP hypothalamiques reste débattue, de nombreux travaux s’accordent sur l’importance de la neurogenèse hypothalamique postnatale dans le contrôle du métabolisme. Cependant, la possibilité que la genèse postnatale de cellules contribue aussi au contrôle de la fonction de reproduction, une autre fonction clé de l’hypothalamus, restait à explorer. L’objectif premier de mon travail de thèse était de rechercher si la genèse de cellules dans l’hypothalamus postnatal est impliquée dans le contrôle de la reproduction, une fonction physiologique qui requière un haut degré de plasticité. La fonction de reproduction est orchestrée par une petite population de neurones produisant la neurohormone Gonadotrophin-Releasing Hormone (GnRH). Ces neurones, qui naissent en dehors du cerveau, sont en place dans la région préoptique (RPO) de l’hypothalamus à la naissance. Cependant, ils doivent subir une maturation postnatale pour acquérir le profil de sécrétion qui leur permettra d’initier la puberté et d’assurer la fertilité de l’individu. Dans une première étude, grâce à une combinaison d’approches in vitro et in vivo, nous avons mis en évidence une vague d’astrogenèse dans l’environnement des neurones à GnRH au sein de la RPO au cours des deux premières semaines de vie postnatale chez la ratte. Nos résultats suggèrent que les neurones à GnRH utilisent la prostaglandine D2 pour attirer les progéniteurs environnants et que ce recrutement est important pour la maturation sexuelle. Dans une deuxième étude, nous avons recherché si de nouvelles cellules naissent à l’âge adulte dans des régions hypothalamiques qui contrôlent la fonction de reproduction. Nous montrons que des cellules sont produites dans la RPO chez la ratte adulte et que leur taux varie au cours du cycle oestral, suggérant une régulation par les stéroïdes sexuels. De plus, nous montrons que la survenue d’une gestation stimule la néogenèse cellulaire dans une zone de la RPO qui contrôle le comportement maternel. Si la néogenèse hypothalamique adulte a surtout été étudiée chez les rongeurs de laboratoire, il reste à déterminer si ce phénomène existe aussi chez l’homme. Pour aborder cette question, nous avons évalué dans une troisième étude l’expression de marqueurs de CSP dans l’hypothalamus humain adulte, comparativement au rongeur (souris, rat) et à un primate lémurien, le microcèbe. Nous montrons que l’hypothalamus humain adulte contient des populations de cellules au profil antigénique de CSP, dont certaines semblent propres à l’homme. Au total, ces travaux montrent que de nouvelles cellules naissent dans des régions hypothalamiques qui contrôlent la fonction de reproduction au cours de la vie postnatale et à l’âge adulte chez la ratte, et que ce phénomène est important pour la maturation sexuelle. L’observation de CSP putatives dans l’hypothalamus humain adulte suggère que la capacité de l’hypothalamus à produire de nouvelles cellules à l’âge adulte existe aussi dans notre espèce. / Despite its complexity, the brain keeps adding new cells – both neuronal and glial – beyond embryonic development and throughout life. The postnatal period is characterized by intense and widespread gliogenesis. During adulthood, both glio- and neurogenesis occur in restricted locations from stem/progenitor cells (NPC) residing in niches. The two best-described niches of adult NPC are the subventricular zone of the lateral ventricles, which provides new interneurons to the olfactory bulb, and the subgranular zone of the hippocampal dentate gyrus that locally produces new granule cells. The last decade has seen an accumulation of studies showing that neuro- and gliogenesis also occur in the postnatal hypothalamus, a small portion of the ventral forebrain surrounding the third ventricle that regulates essential physiological processes such as metabolism, reproduction, sleep and thermoregulation. Even though the identity of hypothalamic NPC remains a matter of debate, a growing body of evidence points to postnatal hypothalamic neurogenesis relevance for the control of metabolism. However, a possible contribution of postnatal hypothalamic cell generation to the central control of reproduction, another key function of the hypothalamus, remained to be explored.The main aim of my doctoral researches was to evaluate whether the generation of new cells in the postnatal hypothalamus contributes to the central control of reproduction, a physiological function known to require a high degree of plasticity. The reproductive function is controlled by a small population of neurons producing the neurohormone Gonadotrophin-Releasing Hormone (GnRH). These neurons, which are born in the nasal placodes, are in place at birth in the preoptic area (POA) of the hypothalamus. However, they need a postnatal maturation to reach a mature secretory pattern that will trigger puberty and subsequent fertility.In a first study, using a combination of in vitro and in vivo experiments, we showed that a wave of astrogenesis occurs in the POA from local progenitors in the environment of GnRH neurons during the first weeks of postnatal life in the female rat. We identified prostaglandin D2 as a factor used by GnRH neurons to attract progenitors in their vicinity and showed that impaired progenitor recruitment alters sexual maturation.In a second study, we evaluated whether cell neogenesis still occurs during adulthood in hypothalamic regions relevant for the reproductive function. Our results showed that new cells are born in the POA of adult female rats. The rate of cell neogenesis varies across the estrus cycle, suggesting a regulatory influence of gonadal steroids. Moreover, we showed that gestation impacts the rate of cell neogenesis in a POA region implicated in the control of maternal behavior.While cell neogenesis in the adult hypothalamus has been mainly studied in laboratory rodents, it remains to be known whether this phenomenon also occurs in humans. To start addressing this question, we evaluated in a third study the expression of a panel of NPC markers in the adult human hypothalamus and compared it to that found in rodents (mouse, rat) and a lemur primate, the grey mouse lemur. Our results showed that the adult human hypothalamus contains populations of cells with an antigenic profile of NPC, some of which appear specific to humans.Altogether, this work shows that new cells are born in hypothalamic regions controlling reproduction throughout postnatal and adult life in female rats, and that this process is required for sexual maturation. The identification of NPC marker-expressing cells in the adult human hypothalamus suggests that the capacity for cell neogenesis also exists in the hypothalamus of our species.

xi, 88 p. A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Neuroblasts are the neural stem cells of the Drosophlia central nervous system. They are large cells that divide asymmetrically to renew another neuroblast and generate a smaller ganglion mother cell (gmc) that will divide once to produce two neurons. Combining genetic lineage tracing experiments with cell fate markers I isolated two separate neural stem cell populations with distinct locations and cellular behaviors in the larval brain. In my first chapter I introduce the central nervous system of Drosophila and in the next two sections of chapter I, I introduce the development of the optic lobe and central brain, two separate structures of the central nervous system. In my second chapter I characterize the lineage relationship of cells within the developing larval optic lobe and use cell fate markers to determine the identity of these cells. Next I examine the effect of spindle orientation on cell fate within epithelial cells of the optic lobe. In my third chapter I characterize another novel neural stem cell lineage in the larval brain containing GMCs with greater proliferation potential than a "canonical" GMC, and I term these, transit amplifying gmcs (TA-GMCs). Further I show that the parent neuroblast of these novel TA-GMCs does not asymmetrically segregate the fate determinant Prospero (Pros) thereby producing a GMC with greater proliferation potential. Finally I show that TA-GMCs do asymmetrically segregate the fate determinant Pros, divide slowly and give rise to up to 10 neurons which normal gmcs never do. In my fourth chapter I show preliminary work on the characterization of a mutation that causes excessive production of neuroblasts specifically in novel TA-GMC lineages. These findings reveal novel neural stem cell lineages, patterns of asymmetric cell division and patterns of neurogenesis that could aid in our understanding of neural stem cell biology and tumorogenesis. This dissertation includes both my previously published and my co-authored materials. / Adviser: Chris Doe

It was long believed within the scientific community that the adult brain was unable to generate new neurons. In the end of the 1990s the consensus changed and it is since believed that the adult brain can and does generate new neurons after birth, a process referred to as adult neurogenesis. Adult neurogenesis takes place in two places in the adult brain: the subventricular zone (SVZ) in close proximity to the olfactory bulb and the subgranular zone (SGZ) in the hippocampus. The level of adult hippocampal neurogenesis (AHN) can be upregulated and one part of the aim was to examine the effect of voluntary chronic aerobic exercise (VCAE) on AHN. It is clear that voluntary chronic aerobic exercise reliably increases AHN. Still, the function of these new brain cells is under debate. Spatial learning and memory are among the main abilities that have been focused on. The other part of the aim was to examine the effect of VCAE and AHN on spatial learning and memory. The reviewed literature suggests that both AHN and spatial learning and memory increase together from VCAE, although it does not show causation, that an increase of AHN from VCAE causally effects spatial learning and memory. More studies are needed to investigate if a causal relationship exists.

Les microfaisceaux (MBs) de rayons X générés par un synchrotron permettent de délivrer des doses de radiation très élevées, jusqu'à plusieurs centaines de gray (Gy), sans pour autant induire de dommages tissulaires irréversibles dans les zones avoisinant la lésion. La réactivité du réseau vasculaire à se régénérer grâce aux cellules endothéliales, est probablement un mécanisme clef dans la radio-tolérance des tissus sains, puisqu'il permet une recolonisation rapide des zones tissulaires lésées. Cette méthode permet ainsi de reproduire de façon non invasive une incision chirurgicale précise du cortex tout en préservant son architecture. Cette caractéristique présente un intérêt certain et permet d'envisager le développement de nouveaux modèles neurobiologiques expérimentaux ouvrant la voie à des traitements innovants pour de nombreux troubles cérébraux. Durant cette thèse, les microfaisceaux ont été utilisés sur la structure corticale et l'hippocampe de rats. Concernant le cortex, les MBs ont été appliqués au niveau du cortex sensori-moteur (taille 100 µm/600 µm, of 400 µm/1200 µm crête à crête, dose pics - vallèe de 360-240 Gy/150-100 Gy) chez des rats males Wistar sains ainsi que chez des rats développant des attaques cérébrales consécutivement à l'injection corticale d'Acide Kaïnique (KA). Suite aux traitements par MBs, les performances motrices étaient évaluées par le test du Rotarod et les structures corticales étudiées par immunohistochimie grâce au marquage par NeuN et GFAP des neurones et astrocytes matures. Aucun déficit neurologique n'a été observé chez les rats sains soumis au protocole de traitement par MBs et une diminution significative (normalement là il faut mettre des statistiques ou au moins les avoir sinon tu peux dire diminution importante) de la durée des crises de convulsions a pu être observée chez les rats KA. L'incidence des MBs (9 microfaisceaux de 75 μm de largeur séparés de 400 μm crête à crête, dose d'entrée: 1000 Gy) délivrés au niveau de l'hippocampe de rats Wistar sains a été comparée avec un traitement par rayons X de 10 Gy délivré uniformément. Le but de cette expérience était d'évaluer l'impact d'un traitement par microfaisceaux par rapport aux techniques d'irradiation conventionnelles. Pour cela, une évaluation quantitative (i) comparative a été faite sur la neurogénèse grâce à l'utilisation de marqueurs cellulaires (BrdU et Ki-67). Une étude (ii) a aussi été menée sur le long terme (1 an) pour mettre en évidence la corrélation entre la neurogénèse, les troubles de l'apprentissage et de la mémoire. Une préservation des cellules prolifératives a été observée au sein du groupe traité par MBs. Les transsections de l'hippocampe de rats par MBs n'induisent pas de troubles significatifs (là aussi, il faut avoir des stat ou alors utiliser troubles marquants) du comportement de plus, l'observation histologique et immunohistochimique des tissus a permis de mettre en évidence la conservation de sa structure.Le travail effectué au cours de cette thèse confirme que le traitement spécifique de zones radiosensibles du cerveau par les microfaisceaux est sûr et permet d'améliorer le pronostique des animaux traités par rapport aux techniques conventionnelles. Ainsi, le développement d'appareils permettant de délivrer des faisceaux de rayons submillimétriques capables de générer une destruction précise et limitée au niveau du cortex ou de l'hippocampe pourra permettre une prise en charge innovante plus sûre de nombreuses pathologies fonctionnelles cérébrales comme l'épilepsie ou certaines douleurs. / Synchrotron-generated X-ray microplanar beams (microbeams, MBs) are characterized by the ability to avoid widespread tissue damage following delivery of doses ranging from hundreds to over a thousand of Grays. The resistance of normal tissues to high doses of MBs is likely related to the fast repair of the microvessels and to the wide interface between normal and irradiated tissue, allowing fast recolonization of the microbeam ablated columns of tissue. The preservation of the cortical architecture following high-dose microbeam irradiation and the ability to induce non-invasively the equivalent of a surgical cut over the cortex is of great interest for the development of novel experimental models in neurobiology and new treatment avenues for a variety of brain disorders. In this Thesis microbeams transections were delivered to the rat cortex and hippocampus. MBs cortical transections were delivered to the sensory motor cortex (size 100 µm/600 µm, center-to-center distance of 400 µm/1200 µm, peak-valley doses of 360-240 Gy/150-100 Gy) of Wistar male healthy rats and rats developing seizures following cortical injections of Kainic Acid (KA)-. The motor performances following sensorimotor cortex transections was assessed by rotarod test. The effect of microbeam transections on cortical architecture was assessed by immunohistology with NeuN and GFAP, markers of mature neurons and astrocytes. No neurological deficit was observed in healthy animals undergoing sensorimotor cortex microbeam transections. Convulsive seizure duration was markedly reduced following transections in KA rats. MBs hippocampal transections (75 µm thickness, 400 c-t-c distance and 600 Gy as peak entrance dose) were performed in adult healthy Wistar rats. MBs transections were compared with an uniformly delivered X-ray dose (broad beam, BB) at 10 Gy. Our aims were to quantify (i) the impact of microbeams versus conventional irradiation on neurogenesis (using proliferative cells markers such as BrdU and Ki-67), and (ii) to investigate on a long term (1 year) the correlation between neurogenesis and impairments in learning and memory. Preservation of proliferative cells in the microbeam treated group were observed. Microbeam transections delivered to the hippocampus did not induce significant behavioral impairment histological and immunohistochemical findings showed a preserved hippocampal structure with evidence of higly precise transections diving the hippocampus in columns. This work confirms the safe delivery of high doses of radiation to specific and radiosensitive parts of the brain, if performed using microbeams Additionally, the development of clinical devices delivering submillimetric beams able to generate cortical or hippocampal transections might become a new powerful new tool for the clinical treatment of epilepsy, pain and other functional brain disorders.

A neurogênese hipocampal é modulada por muitos fatores que incluem envelhecimento, estresse, enriquecimento ambiental, atividade física e aprendizado. Atividade física voluntária (espontânea) estimula a proliferação celular no giro denteado e facilita a aquisição e/ou retenção de tarefas dependentes do hipocampo, incluindo o Labirinto Aquático de Morris. Embora seja bem estabelecido que o exercício físico regular melhore o desempenho em tarefas de memória e aprendizado, não está claro qual a duração desses benefícios após o final da atividade física. Neste estudo investigamos a relação temporal entre os efeitos benéficos da atividade física associado ao aprendizado de tarefa dependente da função hipocampal, e sua relação com a neurogênese, levando em consideração também o tempo decorrido desde o término da atividade física. Grupos independentes de ratos tiveram acesso a roda de atividade ao longo de 7 dias (Grupo EXE) ou roda bloqueada (Grupo Ñ-EXE) e receberam injeções de BrdU nos últimos 3 dias de exposição roda. Após um INTERVALO de 1, 3 ou 6 semanas após o final da exposição a roda de atividade após o final da exposição a roda de atividade, os animais foram testados no labirinto aquático de Morris, sendo uma parte deles expostos a tarefa de memória operacional espacial, dependente da função hipocampal (H), e outra parte a uma tarefa de busca por uma plataforma visível, independente da função hipocampal (ÑH). Em ambos os casos, o intervalo entre as tentativas (ITI) foi de 10 minutos durante as sessões 1-6 e (virtualmente) zero minutos durante as sessões 7-10. Concluída a tarefa os cérebros foram processados para imuno-histoquímica. Foram feitas imunoistoquímicas para a detecção de Ki-67 (proliferação celular), BrdU, NeuN (para identificar neurónios maduros), e DCX (para identificar imaturo neurônios). Nossos dados suportam a ideia que atividade física voluntária induz um aumento na proliferação celular e na diferenciação neuronal (neurogênese) no giro denteado. A introdução de um período de intervalo entre o final do exercício e a execução da tarefa comportamental causa uma redução significativa na sobrevivência dos novos neurônios, como observado com 1 semana de intervalo em comparação com os animais testados com 6 semanas de intervalo. Em contraste, entretanto, o presente resultado não confirma que esse aumento da neurogênese é acompanhado por melhora na memória espacial, como avaliado por meio da versão que envolve memória operacional no labirinto aquático de Morris. O aprendizado da tarefa do labirinto aquático de Morris, na versão de memória operacional que é dependente do hipocampo, leva a um aumento da sobrevivência dos novos neurônios que foram produzidos no período de exercício, ao passo que o aprendizado da versão independente da tarefa leva a uma redução do número absoluto de novos neurônios / Hippocampal adult neurogenesis is modulated by many factors including age, stress, environmental enrichment, physical exercise and learning. Spontaneous exercise in a running wheel stimulates cell proliferation in the adult dentate gyrus and facilitates acquisition and/or retention of hippocampal-dependent tasks including the Morris water maze. While it is well established that regular physical exercise improves cognitive performance, it is unclear for how long these benefits last after its interruption. In this study, we investigate the temporal relation between exercise-induced benefits associated with learning of a hippocampal-dependent task, this relationship with neurogenesis, considering the time after exercise has ended. Independent groups of rats were given free access to either unlocked (EXE Group) or locked (No-EXE Group) running wheels for 7 days, having received daily injections of BrdU for the last 3 days. The animals were then transferred to standard home cages. After a time period of either 1, 3 or 6 weeks, the animals were tested in the Morris water maze, one of them being exposed to the spatial working memory task dependent on hippocampal function (H) and partly to a task search for a visible platform, independent of hippocampal function (NH). In both cases, the interval between trials (ITI) was 10 minutes during sessions and 1-6 and (virtually) zero minute during the sessions 7-10. After the task brains were processed for immunohistochemistry. Cell proliferation and net neurogenesis were assessed in hippocampal sections using antibodies against BrdU, NeuN (to identify mature neurons), and DCX (to identify immature neurons). Data of the present study confirm that exposure of rats to 7 days of spontaneous wheel running increases cell proliferation and neurogenesis. In contrast, however, the present results did not confirm that this neurogenesis is accompanied by a significant improvement in spatial learning, as evaluated using the working memory version of the Morris’ water maze task. The introduction of a delay period between the end of exercise and cognitive training on the Morris water maze reduces cell survival; the number of new neurons was higher in the EXE1 week delay group as compared to the EXE6 week delay. We showed that learning the Morris water maze in the working memory task dependent on hippocampal function (H) increases the new neurons survival, in contrast, learning hippocampal-independent version of the task decreases number of new neurons

Made available in DSpace on 2015-03-26T13:35:15Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2208057 bytes, checksum: 45a242fadca486c484bfa0df0f61ec59 (MD5) Previous issue date: 2011-07-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The characterization of fractals and the search for general rules and mechanisms for description of non-linear phenomena brought the attention of the scientific community to the interdisciplinary area of complex systems. Complex phenomena can be found in almost every branch of natural science. One example is the simulation using cellular automata of self-organization in life. This work focuses on cellular automata models to simulate the growth of branched structures in animal body guided by the characteristics of interactions in cell migration. Our models are guided by the dynamics of angiogenesis and neurogenesis, apparently two different mechanisms that can be classified under the same rules regarding local interactions and emergent behaviors. The proposed models can be very useful in studies of neurological diseases and cancer therapies. / A caracterização de estruturas fractais e a busca por mecanismos ou regras universais para descrição de diversos fenômenos não-linares fez com que a comunidade científica se voltasse para uma área que pode ser considerada uma das pioneiras na interdiciplinaridade: os Sistemas Complexos. Praticamente toda disciplina possui em seu domínio fenômenos complexos. Uma das linha de destaque dessa área são as simulações em autômatos celulares, principalmente quando voltada para estudos de auto-organização em seres vivos. O presente trabalho propõe uma modelagem em autômatos celulares para simulação de crescimento de estruturas ramificadas no organismo animal guiadas pelas interações características da migração celular. Nosso modelo foi baseado na angiogênese e na neurôgenese, estruturas que são aparentemente distintas, mas que na verdade pertencem a um mesmo grupo se classificados quanto às regras de interações locais e seus comportamentos emergentes. Esse modelo pode ser de grande utilidade no estudo de doenças relacionadas ao sistema nervoso e de terapias de combate ao câncer.

Les macrophages primitifs envahissent la moelle épinière dès E11.5-12.5 chez l'embryon de souris. Ces cellules interagissent avec les neurites spinaux en croissance des neurones sensoriels des ganglions de la racine dorsale dès E12.5. La microglie est connue pour réguler plusieurs processus développementaux dans le système nerveux central, mais leur rôle dans le développement du système nerveux périphérique reste inconnu. Nous montrons que les macrophages primitifs régulent le développement des neurones sensoriels dans l'embryon de souris. Nous avons d'abord découvert que les macrophages primitifs interagissent avec les neurites périphériques des neurones sensoriels dès E11.5. Nous avons ensuite démontré que l'absence de macrophage chez les souris PU.1KO ou leur ablation immunopharmacologique, entraînait une réduction initiale des neuronaux TrkB+ et TrkC+ à E11.5, suivie d'une augmentation transitoire de leur nombre à E12.5. Cette augmentation est associée à une augmentation transitoire de leur mort développementale ce qui expliquerait pourquoi leur nombre redevient normal à partir de E13.5. Chez les embryons dépourvus de macrophage la mort augmente à nouveau à E15.5 pour les neurones TrkC+ et dès E14.5 pour les neurones TrkB+. Concernant les neurones TrkA+, leur nombre reste déficitaire entre E12.5 et E15.5, bien que leur mort développementale ne soit pas affectée. Nous avons enfin montré que la prolifération cellulaire était diminuée à E12.5 dans les ganglions des souris PU.1KO. Ces résultats sont la première démonstration que les macrophages primitifs et/ou les microglies immatures peuvent aussi réguler le développement embryonnaire du système nerveux périphérique. / The primitive macrophages invade the spinal cord as early as E11.5-12.5 in the mouse embryo. These cells interact with growing spinal neurites of the dorsal root ganglia sensory neurons as early as E12.5. Microglia is known to regulate several developmental processes in the central nervous system, but their role in the development of the peripheral nervous system remains unknown. We show that primitive macrophages regulate the development of sensory neurons in the mouse embryo. We first discovered that primitive macrophages interact with peripheral neurites of sensory neurons as early as E11.5. We then demonstrated that the absence of macrophage in PU.1KO mice or their immunopharmacological ablation resulted in an initial reduction of TrkB+ and TrkC+ neurons at E11.5, followed by a transient increase in their number at E12.5. This increase is associated with a transient increase in their developmental death which would explain why their numbers become normal again from E13.5. In macrophage-free embryos, death increases again to E15.5 for TrkC+ neurons and as early as E14.5 for TrkB+ neurons. Concerning TrkA+ neurons, their number remains deficient between E12.5 and E15.5, although their developmental death is not affected. We have finally shown that cell proliferation was decreased at E12.5 in ganglions of PU.1KO mice. These results are the first demonstration that primitive macrophages and / or immature microglia can also regulate the embryonic development of the peripheral nervous system.

A esquizofrenia (EZ) é uma das doenças neuropsiquiátricas mais prevalentes, afetando cerca de 1% da população ao redor do mundo, sendo caracterizada pela presença de delírios, alucinações, disfunção cognitiva e apatia, entre outros. Dentre as hipóteses que procuram explicar as bases biológicas da EZ, a que tem tido um maior embasamento e credibilidade é a Hipótese do Neurodesenvolvimento, que afirma que a EZ é uma desordem de desenvolvimento do sistema nervoso central, originada nos estágios intermediários da vida intra-uterina. Ao mesmo tempo o envolvimento de componentes genéticos em EZ é fortemente sugerido, principalmente por estudos envolvendo gêmeos e famílias. Uma série de estudos de ligação, realizados com famílias contendo indivíduos afetados, têm apontado para diferentes locos cromossômicos que devem estar associados à EZ. Enquanto tais estudos sugerem locos candidatos, a descoberta dos genes envolvidos com a doença, mapeados nestes locos, trará grande progresso ao estudo da genética da EZ. Dessa forma, o objetivo deste estudo foi analisar a possível associação entre polimorfismos em genes envolvidos com neurodesenvolvimento, mapeados em locos importantes sugeridos por estudos de ligação, e a EZ. Após uma série de análises in silico e validações in vitro, um total de 41 polimorfismos, mapeados em 39 genes candidatos, foram selecionados neste estudo. Estes polimorfismos foram genotipados em amostras de DNA, obtidas de 200 pacientes esquizofrênicos e 200 controles da população Brasileira. Uma análise individual das freqüências genotípicas e alélicas de cada polimorfismo identificou quatro polimorfismos como associados à EZ, mapeados nos genes AKT1, MAP1B, FZD3, e NUMBL. Em seguida, esses quatro polimorfismos foram analisados em um segundo set amostral, composto por amostras de DNA de casos e controles Dinamarqueses, de forma a tentar replicar os achados com a amostra Brasileira. Para dois destes polimorfismos (NUMBL e FZD3) observamos uma tendência de associação na amostra dinamarquesa, o que reforça o possível envolvimento destes polimorfismos com a EZ. Avançamos na caracterização haplotípica do SNP de FZD3 em amostras do Brasil e da Dinamarca e também investigamos as conseqüências funcionais deste SNP, utilizando ensaios de gene-repórter em duas diferentes linhagens celulares. Por fim, uma análise multilocus preliminar, utilizando o método set association, foi realizada para os marcadores bialélicos por nós avaliados (SNPs e indels), a fim de identificar a existência de um set de genes que estivessem contribuindo em conjunto para a etiologia da EZ. Os resultados dessa análise apontaram para o efeito aditivo de 5 genes para a etiologia da EZ: FZD3, AKT1, MAP1B, SEMA6C e ADAM28. Os genes encontrados em nosso estudo como associados à EZ, tanto pela análise individual quanto pela análise multilocus, se encontram envolvidos direta ou indiretamente com as vias sinalizadoras de WNT e NOTCH, sugerindo o envolvimento destas duas vias sinalizadoras para a etiologia da doença. / Schizophrenia (SZ) is one of the most prevalent neuropsychiatry disorders, affecting about 1% of the world\'s population. SZ is characterized by the presence of delirium, hallucinations, cognitive dysfunction, apathy, etc. Among the hypotheses that try to explain the biological basis of SZ, the Neurodevelopment Hypothesis is one of the most accepted. This hypothesis states that SZ is a neurodevelopment disorder, originated during intermediate stages of the intrauterine life. On the other hand, the involvement of genetic components in the etiology of SZ has been suggested by twin and family studies. A series of linkage studies pointed different genomic loci associated with the disease. While these studies suggest candidate loci, the determination of which genes/genetic alterations inside these loci are involved with the disease will bring great progress to the study of SZ genetics. The objective of this study was to analyze the possible associations between DNA polymorphisms in genes related to neurodevelopment and mapped to genomic loci previously associated with SZ. After a series of in silico and experimental analysis, a total of 41 polymorphisms, mapped to 39 candidate genes, were selected for analysis. These polymorphisms were genotyped in DNA samples obtained for 200 SZ patients and paired 200 controls from the Brazilian population. An individual analysis of the genotypic and allelic frequencies of each polymorphism identified 4 polymorphisms as associated with SZ, mapped in the AKT1, MAP1B, FZD3, and NUMBL genes. These four polymorphisms were then analyzed in a second set of samples, derived from Danish cases and controls. A trend for association was observed for two of these polymorphisms (FZD3 and NUMBL), reinforcing their putative association with EZ. Haplotipic analysis was performed for the FZD3 SNP and its functional consequences were confirmed in two different cell lines by using gene reporter assays. Finally, multilocus analysis, performed by the use of the set association method, pointed to an additive effect of 5 genes contributing to the etiology of SZ: FZD3, AKT1, MAP1B, SEMA6C e ADAM28. All the genes pointed by our study as associated with SZ, by both the individual and the multilocus analysis, are directly or indirectly involved in the WNT and NOTCH pathways, which strongly suggests the involvement of these pathways in the etiology of SZ.