L’objet de ce travail était d’observer les effets éventuels d’un nouveau traitement chez des enfants avec des Troubles Envahissants du Développement. Pour cela, nous avons comparé deux groupes d’enfants TED, l’un étant traité l’autre constituant un groupe témoin, à travers des domaines de la cognition, de la sensorialité et des comportements de la vie quotidienne et de la socialisation. Nous avons rassemblé différents outils pour notre méthodologie : questionnaires parentaux, évaluations psychométriques et évaluations psychophysiques.Nous avons également utilisé une approche comparative pour le domaine sensoriel en intégrant un groupe d’enfant au développement typique.Les résultats obtenus dans le cadre de notre travail ont permis de mettre en avant que l’ensemble des comportements évalués n’apparaissaient pas altérés dans notre population d’enfants TED. Nous avons mis en évidence des améliorations accrues des enfants traités concernant leurs capacités visuo-spatiales. Nous avons également constaté que ces enfants présentaient des améliorations de certains processus verbaux. Nous avons confirmé la présence de patterns particuliers de comportements sensoriels chez les enfants TED par rapport à la population neurotypique. En revanche, nous n’avons pas mis en évidence de différence d’évolution entre nos deux groupes d’enfants TED au regard de ces altérations sensorielles. Enfin, les comportements répétés et restreints apparaissaient améliorés de manière accrue chez les enfants TED traités mais nous n’avons pas confirmé les améliorations des comportements sociaux rapportées par les études précédentes. Cette étude a apporté des résultats qui nécessitent d’être scrutés plus en détail, à plus grande échelle. / The purpose of this work is to observe the potential effects of new treatment in children with Pervasive Developmental Disorders. For this, we compared two groups of children with PDD, one being treated the other constituting a control group, across domains of cognition, sensory and behaviors of daily life and socialization. We collected different tools for our methodology: parental questionnaires, psychometric assessments and psychophysical assessments. We also used a comparative approach for the sensory domain integrating a typical child development group.The results of this work highlight that whole of the evaluated behaviors did not appear altered in our population of children with PDD. We pointed out increased improvements in treated children regarding their visuospatial abilities. We also found that these children had improvements in some verbal processes. We confirmed the presence of particular patterns of sensory behavior in children with PDD compared to the typically developed population.However, we did not show any difference in evolution of these sensory alterations between our two groups of children with PDD. Finally, repeated and restricted behaviors appeared to be more alleviated in treated PDD children than control PDD children but we did not confirm the improvements in social behaviors reported by previous studies. This study has brought results that need to be scrutinized in more detail, on a larger scale.

Há evidências convincentes de que o início da doença de Alzheimer é precedido por uma redução de estímulos sensoriais, como ocorre durante a aposentadoria, catarata, surdez e degeneração macular, em um cérebro idoso que apresenta deficiência de receptores tipo GABAA. Neste trabalho foi utilizado um modelo computacional fenomenológico do koniocortex, que é a primeira camada cortical que recebe estímulos sensoriais, adaptado para simular as fases iniciais da doença de Alzheimer. A arquitetura e as propriedades dos neurônios do modelo computacional do koniocortex se assemelham as do cérebro, sendo também capaz de aprender, permitindo com isso que a memória de curto prazo seja testada em qualquer momento. Usando o modelo computacional é possível também analisar as fases iniciais da doença de Alzheimer simulando o \"envelhecimento\" do koniocortex artificial através de um conjunto de parâmetros referentes à plasticidade intrínseca, à acetilcolina, aos estímulos sensoriais, ao pruning sináptico, entre outros. O modelo computacional revela que, quando o envelhecimento afeta os neurônios que expressam receptores GABA-A ocorrendo na sequência uma redução dos estímulos sensoriais, o resultado dessa cascata de eventos leva ao hipermetabolismo e ao início da fase de deposição excessiva das placas -amiloide / There is compelling evidence that Alzheimers disease onset is preceded by a reduction of sensory stimuli like during job retirement, cataract, deafness or even macular degeneration, over an aged brain with impaired GABA-A receptor inhibitions. In this paper, was adapted a phenomenological computational model of the koniocortex which is the first cortical layer that receives sensory stimuli to simulate the initial phases of Alzheimers disease. The architecture and neurons properties of the modeled koniocortex resemble those of the brain, so that the model is also able to learn, thereby allowing the assessment of short-term memory at any moment. By using the computational model, it is possible to analyze the initial phases of Alzheimers disease by aging the artificial koniocortex through a set of parameters related to intrinsic plasticity, acetylcholine, sensory stimuli, synaptic pruning, among others. The computational model shows that when aging occurs in such way that GABA-A receptor expressing neurons are affected, and, in the sequence, a reduction of sensory stimuli takes place, the result of this cascade of events leads to hypermetabolism and to the initial phase excessive deposition of beta-amyloid plaques

A substância cinzenta periaquedutal dorsal (SCPd) e o colículo inferior (CI) são duas estruturas do teto mesencefálico que, juntamente com a amígdala, o hipotálamo dorsomedial e o colículo superior, estão envolvidas na modulação da expressão comportamental dos estados de medo. A estimulação química ou elétrica destas estruturas produz uma série de respostas comportamentais defensivas. Além disso, dados comportamentais com modelos animais de ansiedade têm fornecido evidências da existência de uma regulação inibitória tônica GABAérgica na SCPd e CI. Neste estudo investigamos o envolvimento da neurotransmissão GABAérgica na expressão do medo condicionado e do medo incondicionado. Para isso, os efeitos da administração de muscimol (agonista GABA-A) e semicarbazida (inibidor da descarboxilase do ácido glutâmico) na SCPd e CI foram analisados no teste do sobressalto potencializado pelo medo, na resposta de congelamento condicionada, nos limiares de congelamento e fuga determinados por estimulação elétrica dessas estruturas e no congelamento pós-estimulação. No modelo de medo incondicionado, microinjeções de muscimol intra-SCPd reduziram a aversividade da estimulação elétrica, mas não o congelamento pós-estimulação, ao passo que a semicarbazida produziu efeitos pró-aversivos em ambas as condições. O muscimol também causou redução significativa no sobressalto potencializado pelo medo e congelamento condicionado, enquanto que a semicarbazida não alterou essas respostas. Já a microinjeção de ambas as drogas no CI não produziu efeitos no modelo condicionado, mas no teste incondicionado, o muscimol reduziu a aversividade da estimulação elétrica. Esses dados mostram uma participação diferencial de mecanismos GABAérgicos no medo condicionado e incondicionado. Estes mecanismos na SCPd parecem estar envolvidos tanto no medo condicionado quanto no incondicionado, enquanto que no CI eles parecem participar somente do medo incondicionado. / The dorsal periqueductal gray (dPAG) and inferior colliculus (IC) are two structures of the midbrain tectum that, together with amygdala, dorsomedial hypothalamus and superior colliculus, are involved in the modulation of the expression of fear-related behaviors. The chemical or electrical stimulation of these structures produces a series of behavioral defensive responses. Moreover, behavioral data from animal models of anxiety have provided evidences of tonic inhibitory GABAergic regulation in dPAG and IC. This study investigated the involvement of GABAergic neurotransmission in the expression of unconditioned and conditioned fear. To this aim, the effects of intra-dPAG and IC administration of muscimol (GABA-A agonist) and semicarbazide (glutamic acid decarboxylase inhibitor) were examined in the fear potentiated startle test, in conditioned freezing, in the thresholds for freezing and escape determined by electrical stimulation of these structures, and in the post-stimulation freezing. In the unconditioned model, intra-dPAG injections of muscimol reduced the aversiveness of the electrical stimulation but had no effects on the post-stimulation freezing, while semicarbazide produced aversive-like effects in both conditions. Muscimol also caused significant reduction in fear potentiated startle and conditioned freezing, while semicarbazide had no effect in these responses. In contrast, intra-IC injections of both drugs were ineffective in the conditioned model. In the unconditioned model, however, muscimol reduced the aversiveness of the electrical stimulation. These data show a differential participation of GABAergic mechanisms on conditioned and unconditioned fear. These mechanisms in the dPAG seem to be involved in both conditioned and unconditioned fear, while in IC they seem to participate in unconditioned fear only.

Les cellules souches neurales quiescentes (CSN) sont le réservoir de la neurogenèse adulte, permettant de produire des nouveaux neurones tout au long de la vie. Cependant, la neurogenèse décroit au cours du vieillissement, provoquant des déclins cognitifs incurables. Afin de mieux comprendre les mécanismes qui contrôlent la prolifération des CSN, nous avons mis en place une méthode de tri par cytométrie en flux qui permet pour la première fois d’isoler les CSN quiescentes et leurs cellules filles dans la ZSV adulte murine. Cette technique nous a permis de prouver que le blocage de la voie GABAAR in vivo provoque l’entrée en cycle des CSN quiescentes. Ainsi, les signaux GABA produits par les neuroblastes dans la ZSV permettent de maintenir les CSN dans leur état de quiescence. Au cours du vieillissement, nous montrons que la production progressive de TGFβ1 par les cellules endothéliales de la niche allonge la phase G1 des CSN activées, diminuant sensiblement la production de nouveaux neurones, sans toutefois diminuer le stock de CSN. Nous mettons ainsi en évidence deux voies majeures contrôlant la prolifération des CSN in vivo, la voie du GABAAR et la voie TGF-β/Smad-3. En vue d’une application thérapeutique, nous prouvons que leur blocage pharmacologique permet de stimuler efficacement la neurogenèse in vivo. / Quiescent neural stem cells (NSCs) are considered the reservoir for adult neurogenesis, generating new neurons throughout life. However, neurogenesis decreases during aging, causing a progressive decline that is currently untreatable. To study the regulatory mechanisms of NSCs proliferation, we set up a new technique allowing the isolation of quiescent NSCs and their progeny. We show that GABAAR directly regulates NSCs quiescence in vivo as the depletion of GABA-producing neuroblasts or GABAAR pathway pharmacological blockade provoked NSCs cell cycle entry in the SVZ. During aging, the stock of NSCs is not perturbed, but we show that an over-production of TGFβ1 by brain endothelial cells directly lengthens activated NSCs G1 phase, strongly decreasing the production of new neurons. These findings highlight GABAAR and TGF-β/Smad-3 as two major pathways controlling NSCs proliferation. In line with a future therapeutic application, we also prove that their blocking stimulates endogenous neurogenesis in vivo.

Os animais são expostos a diferentes situações que podem colocar em risco sua sobrevivência. Na natureza estas situações, em geral, eliciam medo e ou ansiedade. A agressão é um conjunto de comportamentos direcionados a um indivíduo co específico, ou não, que tem como objetivo a aquisição de recursos ou proteção em situações de risco à sobrevivência. Considerando a interação entre medo/ansiedade e agressividade, este trabalho teve como objetivo estudar se essas situações podem modificar o comportamento agressivo agressividade em camundongos. O propósito deste estudo foi investigar se a pre-exposição de camundongos a estímulos estressores incondicionados (campo aberto, labirinto em cruz elevado, exposição ao rato, exposição a odor de rato) e condicionados (choque nas patas) podem modular o futuro comportamento agressivo em camundongos. Para atingir esse objetivo, os animais foram previamente expostos a diferentes situações capazes de provocar um estado de ansiedade e/ou medo e posteriormente submetidos ao encontro agonístico (teste residente intruso). As alterações na reatividade emocional induzidas pelas variáveis independentes foram medidas usando a resposta de sobressalto potencializado pelo medo e a análise de vocalizações ultrassônicas. Devido à influência relevante da neurotransmissão de GABA na agressão, as mudanças comportamentais induzidas pelas variáveis utilizadas foram associadas com o benzodiazepínico diazepam. Os dados obtidos no presente estudo após análise mostrou que a pré-exposição de camundongos a situações aversivas que provocam medo e / ou ansiedade alteram o seu comportamento. / Aggression is defined as a behavioral repertoire mainly directed to a conspecific for acquisition of resources and protection. In this context, anxiety and fear-like behaviors is commonly triggered by these survivors situations. Since aggression and fear are highly correlated in the present study we investigated whether previous exposure to environmental unconditioned (rat presence and rat odor, open field and elevated plus-maze tests, foot-shocks) and conditioned aversive stimuli (fear-potentiated startle) can modulate future aggressive behavior in mice. To achieve this goal, the animals were previously exposed to different situations able to elicit a state of anxiety and/or fear and later submitted to the agonistic encounter. Changes on the emotional reactivity induced by the independent variables used were measured using the fear-potentiated startle response and ultrasonic vocalizations analysis. Due to the relevant influence of GABA neurotransmission on aggression, behavioral changes induced by the variables used were challenged with the prototypic benzodiazepine diazepam. In addition, regarding human aggression, the most effective and enduring pharmacological intervention rely on compounds that act as dopaminergic antagonists. Therefore, in our study, in order to verify the influence of dopamine neurotransmission on the modulation of aggression pharmacological manipulation was conducted with the systemic administration of the dopamine D2 agonist apomorphine. Both drugs were administered previously to the resident-intruder test. The data obtained in the present study after analysis show that the pre-exposure to aversive situations that trigger fear and/or anxiety changes mice behavior.

O ácido gama-aminobutírico (GABA), principal neurotransmissor inibitório, está envolvido no mecanismo intrínseco do início da puberdade. Os efeitos inibitórios do GABA sobre a secreção de GnRH (hormônio hipotalâmico estimulador da secreção das gonadotrofinas) são mediados pelo receptor tipo A (GABAA) que é composto por diferentes subunidades organizadas de forma heteropentamérica. A subunidade a1, codificada pelo gene GABRA1 localizado no locus 5q34-35, é a mais implicada na atividade inibitória do GABA. A puberdade precoce dependente de gonadotrofinas (PPDG) predomina no sexo feminino, sendo na maioria dos casos idiopática. Recentemente, defeitos moleculares das subunidades dos receptores de GABA têm sido identificados em pacientes com anormalidades eletroencefalográficas específicas. Neste estudo, investigamos a presença de mutações funcionais ou polimorfismos do GABRA1 em meninas com a forma idiopática de PPDG e avaliamos as anormalidades EEG neste grupo. Trinta e uma meninas com diagnóstico clínico e hormonal da forma idiopática da PPDG, sendo 6 casos familiais (19,4%) e 25 casos esporádicos (80,6%), e 73 controles não relacionados foram selecionados. Todas as pacientes com PPDG apresentaram ressonância magnética de sistema nervoso central normal. Vinte e três meninas foram submetidas a estudo eletroencefalográfico (EEG). O DNA genômico foi extraído do sangue periférico de todas as pacientes e controles. A região codificadora do GABRA1 foi amplificada utilizando-se oligonucleotídeos intrônicos específicos, seguida por purificação enzimática e seqüenciamento automático. Dois polimorfismos conhecidos do GABRA1 foram também estudados pelo programa GeneScan e pela técnica de digestão enzimática (enzima TaiI). O seqüenciamento automático do GABRA1 não revelou mutações funcionais. Identificamos 7 polimorfismos no GABRA1: duas variantes exônicas 156T>C e 1323G>A, localizados no éxons 4 e 11, respectivamente, e 5 polimorfismos intrônicos - IVS2-712(GT)n, no íntron 2, caracterizado por número variável de repetições GT; IVS3+12A>T, no íntron 3; IVS8+45T>G no íntron 8; IVS9+76T>G no íntron 9 e IVS10+15G>A, no íntron 10. Estes polimorfismos não alteram o uso do sítio de splice original. Não houve diferença estatisticamente significante entre a distribuição genotípica e a freqüência alélica dos 2 polimorfismos exônicos e do polimorfismo IVS2-712(GT) encontrados no grupo de pacientes e no grupo controle. O EEG revelou anormalidades em 6 de 23 meninas (4 sem epilepsia). A distribuição genotípica e a freqüência alélica dos polimorfismos do GABRA1 não difereriram significativamente entre as pacientes com PPDG sem e com anormalidades eletroencefalográficas. Nós concluímos que mutações funcionais ou polimorfismos no GABRA1 não estão envolvidos na etiologia da forma idiopática da PPDG e não estão associadas às anormalidades eletroencefalográficas encontradas. Adicionalmente, a presença de alterações eletroencefalográficas em pacientes com PPDG sem epilepsia sugere que a análise eletroencefalográfica deva ser incluída na investigação da PPDG / The gamma-aminobutyric acid (GABA), a dominant inhibitory neurotransmitter, is involved in the intrinsic mechanism of the onset of the puberty. Their inhibitory effects on the GnRH (hypothalamic gonadotropin release hormone) secretion are mediated by type A receptor (GABAA), composed by different subunits which are organized in a heteropentameric form. The alpha-1 subunit, encode by GABRA1 gene located at locus 5q34- 35, is the most implicated in the inhibitory activity of GABA. The gonadotropin-dependent precocious puberty (GDPP) is predominant in females, being idiopathic in the majority of the cases. Recently, molecular defects of the GABA receptor subunits have been identified in patients with specific electroencephalographic (EEG) abnormalities. In this study, we investigated the presence of functional mutations or polymorphisms of the GABRA1 in girls with the idiopathic form of the GDPP and evaluated EEG abnormalities in this group. Thirty-one girls with clinical and hormonal diagnosis of GDPP idiopathic form, being 6 familial cases (19.4%) and 25 sporadic cases (80.6%), and 73 unrelated controls were selected. All patients with GDPP had normal magnetic resonance of central nervous system. Twenty-three girls were submitted to electroencephalographic study. Genomic DNA was extracted from peripheral blood of all patients and controls. The entire coding region of the GABRA1 was amplified using specific intronic oligonucleotides, followed by enzymatic purification and automatic sequencing. Two known polymorphisms of the GABRA1 were also studied by GeneScan software and digestion with restriction endonuclease TaiI. The automatic sequencing of the GABRA1 did not reveal any functional mutations. We identified 7 polymorphisms in the GABRA1: two silent exonic variants 156T>C e 1323G>A, located at exons 4 e 11, respectively, e 5 polymorphisms - IVS2-712(GT)n, at intron 2, characterized by a variable number of repeat GT; IVS3+12A>T, at intron 3; IVS8+45T>G at intron 8; IVS9+76T>G at intron 9 and IVS10+15G>A, at íntron 10. These polymorphisms did not alter the use of original splicing site. No significant statistical difference of the genotypic distribution and allele frequency of the exonic polymorphisms (156T>C and 1323G>A) and IVS2-712(GT)n between unrelated patients and control group was obtained. Electroencephalographic tracings were abnormal in 6 of 23 girls (4 without epilepsy). No significant statistical difference of the genotype distribution and allele frequence were found between patients without and with EEG abnormalities. We conclude that functional mutations or polymorphisms in the GABRA1 are not involved in the etiology of idiopathic GDPP in this study, and they are not associated with electroencephalografic abnormalities. In addition, EEG abnormalities present in girls with GDPP without epilepsy, suggest that EEG analysis should be included in the investigation of the precocious puberty.

L’acide γ-aminobutyrique ou GABA est le principal neurotransmetteur inhibiteur présent dans le système nerveux central (CNS). Afin d’obtenir un nouveau dérivé cyclobutanique du GABA, le cis-3,4CB-GABA, sous forme énantiomériquement pure, deux stratégies de synthèses efficaces et reproductibles ont été mises au point. Ces deux voies de synthèse impliquent toutes les deux une étape-clé de photocycloaddition [2+2] qui permet de créer le cycle à 4 chaînons. La première consiste en une homologation de l’acide cis-2-aminocyclobutanique (cis-ACBC), et la deuxième est une synthèse multi-étape qui utilise le caprolactame comme composé de départ.D’autre part, grâce à une synthèse stéréosélective du (1R,2S)-cis-2,3CB-GABA, quelques oligomères C- et N-protégés – di, tri, et tétra-peptides – de cet aminoacide ont été préparés. Ceux-ci ont été caractérisés par les techniques de RMN 1D et 2D, IR, RX. Les analyses ont montré qu’il n’existe pas d’interactions non-covalentes (liaisons hydrogène) inter-résidu au sein de ces structures moléculaires. En revanche, la propriété de gélification de ces oligomères dans différents solvants organiques a été mise en évidence. Des solutions et des gels formés à partir de ces peptides ont été analysés par microscope électronique à balayage et des clichés ont été obtenus montrant une organisation du dipeptide et du tetrapeptide en fibrilles. Le tripeptide lui n’a présenté aucun assemblage intermoléculaire régulier. / The γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). In order to obtain new enantiomerically pure cyclobutanic derivative of GABA, the cis-3,4CB-GABA, two efficient synthetic strategies have been established. Both synthetic routes employed a photocycloaddition [2 +2] protocol, which provided the cyclobutanic ring. The first route involved the homolgation of the cis-2-aminocyclobutanecarboxylic acid (cis-ACBC), whereas the second route is a multi-step synthesis using caprolactam as starting material.On the other hand, the (1R,2S)-cis-GABA-2,3CB was synthetized, and a series of N- and C-protected oligomers of di, tri, and tetrapeptides of this amino acid were prepared. These oligomers were characterized by NMR (1D and 2D) techniques, IR, and X-ray. The analyses have shown that there are no non-covalent interactions (hydrogen bonds) between the residues of each oligomers. However, the gelation property of these oligomers in various organic solvents was demonstrated. Solutions and gels formed from these peptides were analyzed by scanning electron microscopy, and the obtained images showed a fibrous organization of the di- and tetrapeptide, while the tripeptide showed no regular intermolecular assembly.

Η παρούσα διατριβή ασχολήθηκε με τη μελέτη της έκφρασης των υπομονάδων των υποδοχέων του γλουταμινικού οξέος και του γ-αμινοβουτυρικού οξέος (GABA) στα βασικά γάγγλια και τον φλοιό των εγκεφαλικών ημισφαιρίων του μυός weaver. Παράλληλα, μελετήθηκε η έκφραση των νευροπεπτιδίων εγκεφαλίνης και δυνορφίνης στα βασικά γάγγλια του μυός weaver. O μυς weaver χαρακτηρίζεται από προοδευτική, γενετικά επαγόμενη εκφύλιση των ντοπαμινεργικών κυττάρων του μεσεγκεφάλου, κυρίως αυτών οι οποίοι καταλήγουν στο ραβδωτό σώμα. Για αυτόν τον λόγο, θεωρείται ένα καλό μοντέλο της νόσου Parkinson και η μελέτη των νευροχημικών μεταβολών που συμβαίνουν στον εγκέφαλο του παραπάνω μυός, αποτελεί πολύτιμο εργαλείο για τη διερεύνηση των παθογενετικών μηχανισμών της νόσου. Mε την τεχνική του υβριδισμού in situ, προσδιορίστηκαν τα επίπεδα mRNA των υπομονάδων z1, ε1 και ε2 του υποδοχέα NMDA, των υπομονάδων KA2 και GluR6 του υποδοχέα καϊνικού οξέος, των υπομονάδων α1, α2, α4, β2 και β3 του υποδοχέα GABAA, καθώς και των πρόδρομων πολυπεπτιδίων προ-προεγκεφαλίνη και προδυνορφίνη. Η μελέτη πραγματοποιήθηκε σε φυσιολογικούς μύες (+/+) και μύες weaver (wv/wv), στις ηλικίες των 26 ημερών, 3 μηνών και 6 μηνών μετά τη γέννηση. Όσον αφορά στους υποδοχείς του γλουταμινικού οξέος, τα αποτελέσματά μας υπέδειξαν αύξηση στην έκφραση των υπομονάδων z1, ε2, ΚΑ2 και GluR6 στο ραβδωτό σώμα των μυών weaver, σε σχέση με τους φυσιολογικούς. Η αύξηση στο mRNA των υπομονάδων z1, ε2 και GluR6 παρατηρήθηκε μόνο στην ηλικία των 6 μηνών, ενώ το mRNA της υπομονάδας KA2, παρουσίασε αύξηση και στις τρεις ηλικίες που μελετήθηκαν. Οι αυξήσεις της έκφρασης των υπομονάδων z1, ε2, ΚΑ2 και GluR6 συμφωνούν και πιθανόν εξηγούν τις αυξήσεις στα επίπεδα των θέσεων δέσμευσης για τους υποδοχείς NMDA και μη-NMDA, οι οποίες έχουν βρεθεί από παλαιότερες μελέτες του εργαστηρίου μας στο ραβδωτό σώμα των μυών weaver ηλικίας 6 μηνών. Με βάση βιβλιογραφικά δεδομένα, υποστηρίζουμε ότι η καθυστερημένη αύξηση στην έκφραση των υπομονάδων z1, ε2 και GluR6 κατά πάσα πιθανότητα συντελείται μέσω επαγωγής του μεταγραφικού παράγοντα ΔfosB, σε απόκριση προς τη μείωση της ντοπαμίνης. Στον σωματοαισθητικό φλοιό των μυών weaver ηλικίας 26 ημερών, παρατηρήθηκε αύξηση στην έκφραση των υπομονάδων z1, ε1, ε2 και KA2, η οποία θα μπορούσε να οφείλεται στη μειωμένη θαλαμοφλοιϊκή γλουταμινεργική είσοδο. Όσον αφορά στους υποδοχείς GABAA, παρατηρήθηκε αύξηση στα επίπεδα mRNA των υπομονάδων α4 και β3, στο ραβδωτό σώμα των μυών weaver ηλικίας 6 μηνών, η οποία συμφωνεί και μπορεί να εξηγήσει την αύξηση στα επίπεδα των θέσεων δέσμευσης για τους υποδοχείς GABAA, η οποία έχει βρεθεί σε προηγούμενη μελέτη του εργαστηρίου μας, στο ραβδωτό σώμα των μυών weaver ηλικίας 6 μηνών. Σκοπεύουμε να ελέγξουμε την πιθανότητα, η αύξηση της έκφρασης της υπομονάδας α4, να υποδεικνύει μία αύξηση του αριθμού των εξωσυναπτικών υποδοχέων GABAA στους νευρώνες προβολής του ραβδωτού σώματος. Στην ωχρά σφαίρα των μυών weaver ηλικίας 6 μηνών, παρατηρήθηκε μείωση των επιπέδων mRNA των υπομονάδων α1 και β2, υποδεικνύοντας μία μείωση του αριθμού των υποδοχέων GABAA, η οποία ήταν αναμενόμενη, λόγω της αυξημένης GABAεργικής εισόδου στην εν λόγω εγκεφαλική περιοχή του μυός weaver. Στον σωματοαισθητικό φλοιό, παρατηρήθηκε μείωση στην έκφραση των υπομονάδων α2 και β2 και ταυτόχρονα αύξηση στην έκφραση των υπομονάδων α4 και β3. Με βάση βιβλιογραφικά δεδομένα, προτείνουμε ότι οι μεταβολές αυτές μπορεί να αντανακλούν μείωση στον αριθμό των συναπτικών και αύξηση στον αριθμό των εξωσυναπτικών υποδοχέων GABAA, σε απόκριση προς τη μειωμένη GABAεργική είσοδο προς τους νευρώνες του σωματοαισθητικού φλοιού του μυός weaver. Όσον αφορά στην έκφραση των πολυπεπτιδίων, το mRNA της προ-προεγκεφαλίνης, παρουσίασε αύξηση στο ραβδωτό σώμα των μυών weaver, μόνο στην ηλικία των 6 μηνών, ενώ το mRNA της προδυνορφίνης, παρουσίασε μείωση στην παραπάνω περιοχή, στην ηλικία των 26 ημερών και αύξηση στις μεγαλύτερες ηλικίες. Σύμφωνα με τα βιβλιογραφικά δεδομένα υποστηρίζουμε ότι: α) η καθυστερημένη αύξηση της έκφρασης της προ-προεγκεφαλίνης στο ραβδωτό σώμα του μυός weaver, οφείλεται στη μείωση της τονικής ανασταλτικής ρυθμιστικής δράσης της ντοπαμίνης στην έκφραση του εν λόγω γονιδίου και πιθανώς συντελείται μέσω του μεταγραφικού παράγοντα ΔfosB, β) ο παραπάνω μεταγραφικός παράγοντας είναι κατά πάσα πιθανότητα υπεύθυνος και για την καθυστερημένη επαγωγή της έκφρασης της προδυνορφίνης στο ραβδωτό σώμα των μυών weaver και γ) η μείωση του παραπάνω mRNA στην ηλικία των 26 ημερών οφείλεται στη μείωση της τονικής διεγερτικής δράσης της ντοπαμίνης στην έκφραση του εν λόγω γονιδίου. Τέλος, το γεγονός ότι οι μεταβολές των mRNA των διαφόρων υπομονάδων και νευροπεπτιδίων δεν ήταν οι ίδιες στις διάφορες ηλικίες που μελετήθηκαν υποδεικνύει ότι κατά την πρόοδο της ντοπαμινεργικής εκφύλισης των ντοπαμινεργικών νευρώνων του μεσεγκεφάλου διαφορετικοί μηχανισμοί ευθύνονται για την πρόκληση των αλλαγών στην έκφραση των υπό μελέτη γονιδίων. / In the present study we investigated the expression of the subunits of glutamate and γ-aminobutyric acid (GABA) receptors in basal ganglia and cerebral cortex of the weaver mouse. We also studied the expression of striatal neuropeptides, which are important neuromodulators of the synaptic transmission in the basal ganglia circuitry. The weaver mouse is characterized by a progressive, genetically induced degeneration of the mesencephalic dopaminergic neurons, especially those that project to the striatum. For this reason, the weaver mouse is a useful model for clarifying the pathogenetic mechanisms that underly Parkinson’s disease. Using the in situ hybridization method, the mRNA levels of the ΝΜDA subunits z1, ε1 and ε2, the kainate subunits KΑ2 and GluR6, the GABAA subunits α1, α2, α4, β2 and β3, as well as the mRNA levels of the precursor polypeptides pre-proenkephalin and prodynorphin, were estimated. The study was performed using wild-type (+/+) and weaver mice (wv/wv) of the following ages: 26 days, 3 months and 6 months. Concerning the glutamate receptors, an increase in the mRNA levels of z1, ε2, KA2 and GluR6 subunits was indicated in the weaver striatum, compared to the wild type. The z1, ε2 and GluR6 mRNA increases were observed only at the age of 6 months, whereas the KA2 mRNA increase was observed at all three ages studied. The increases in z1, ε2, ΚΑ2 and GluR6 mRNA expression are in agreement and probably explain the increased levels of ΝΜDA- and non-NMDA-sensitive binding sites that we had previously found in the 6 months old weaver striatum. Based on bibliographic data, we suggest that the delayed increases in z1, ε2 and GluR6 mRNA levels, are probably mediated by the delayed induction of the ΔfosB transcription factor, in response to the reduction of striatal dopamine levels. In the somatosensory cortex of 26 day old weaver mice, an increase in the levels of z1, ε1, ε2 and ΚΑ2 mRNAs was observed. The above increases can be attributed to the decreased thalamocortical glutamatergic imput. Concerning the GABAA receptors, the observed increases of the α4 and β3 mRNA levels in the 6 months old weaver striatum are in agreement and probably explain the increased levels of GABAA binding sites that we had previously found in the 6 months old weaver striatum. We are going to test the hypothesis, that the α4 mRNA increase might indicate an increase in the number of extrasynaptic GABAA receptors in striatal projection neurons. In the 6 months old weaver globus pallidus, the observed decrease in α1 and β2 mRNA levels was expected, since the GABAergic transmission is increased in the above region of the weaver brain. In the weaver somatosensory cortex, a decrease in the α2 and β2 mRNA levels and an increase in the α4 and β3 mRNA levels were observed. Based on bibliographic data, we suggest that the above alterations probably indicate a differential regulation of the synaptic versus extrasynaptic cortical GABAA receptors, in response to the decreased GABAergic presynaptic input to the weaver cortical neurons. Concerning the expression of the striatal neuropeptides, the pre-proenkephalin mRNA was increased in the weaver striatum, only at the age of 6 months. In contrast, prodynorphin mRNA was decreased in the 26 day old weaver striatum, whereas it was increased in the 3 and 6 months old weaver striatum. Based on bibliographic data, we suggest that: a) the delayed increase in the expression of pre-proenkephalin could be caused by the reduction of the tonic dopaminergic inhibitory control on the expression of the above gene in the dopamine-depleted weaver striatum and is probably mediated by the ΔfosB transcription factor; b) the above transcription factor could be responsible for the delayed induction of the prodynorphin expression in the weaver striatum as well, and c) the decrease of prodynorphin mRNA in the 26 day old weaver striatum could be attributed to the reduction of the dopaminergic stimulatory control on the expression of the above gene. Finally, the different pattern of expression alterations among the three ages studied indicates that distinct mechanisms are responsible for the observed changes, during the progress of the dopaminergic degeneration of the weaver brain.

No description available.

La compréhension des enzymes et de leurs mécanismes catalytiques est d'une grande importance dans le développement de médicaments plus efficaces Pour mieux appréhender ces phénomènes, différentes approches théoriques comme les méthodes QM, MM-MD et QM/MM, peuvent être utilisées. L'objectif principal de cette thèse est d'obtenir une meilleure compréhension des mécanismes de réactivité et de la dynamique de l'enzyme GABA-AT (y-aminobutyric acid aminotransferase), un modèle d'enzyme dépendante au phosphate pyridoxal (PLP). Notre travail a consisté en 5 étapes vers une plus grande compréhension de GABA-AT. 1) la réaction et le mode d'attachement du substrat naturel GABA ont été étudié pour différents isomères à l'aide de systèmes modèles et de la DFT. 2) l'enzyme a été simulée par dynamique moléculaire classique dans les cas de l'apoenzyme, l'holoenzyme et l'holoenzyme inactivée. Nos résultats montrent que plusieurs résidus du site actif jouent un rôle important et que leur état de protonation ainsi que celui du PLP sont cruciaux dans l'activité de GABA-AT. 3) l'influence des résidus du site actif sur la réactivité a été étudiée par la modélisation quantique de clusters moléculaires. Le plus gros cluster comprenait 165 atomes entouré d'un solvant implicite. 4) de nouvelles routines de diagonalisation pour SEBOMD ont été incorporées dans la suite AMBER à travers l'utilisation des bibliothèques LAPACK et SCALAPACK. Ces nouvelles routines ont été testées et leur efficacité a été évaluée. 5) des énergies libres de réaction ont été évaluées par dynamiques SEBOMD sur des intermédiaires réactionnels GABA-PLP / Understanding enzymes and their catalytic mechanisms is very important in order to develop more effective drugs having little to no side effects. In order to decipher the catalytic behavior of enzymes, different approaches such as QM, MM-MD, and QM/MM can be used and their results can be correlated. The main aim of this thesis is to get a deeper understanding of the mechanistic insights of the reactivity and of the dynamics of the pyridoxal-5-phosphate (PLP) dependent enzyme y-aminobutyric acid aminotransferase (GABA-AT). Because GABA-AT resembles many other PLP-dependent enzymes, understanding it could be of importance for the broad community of biochemists and computational chemists who study such class of proteins. Our work has consisted of five stages to pursuit the comprehension of GABA-AT. First, the reaction and the preferred binding mode of the natural substrate GABA has been elucidated with different isomers by means of model systems with DFT. Second, the dynamics and the behavior of the enzyme has been studied with MM-MD through the use of apoenzyme, holoenzyme and holoenzyme with an inactivator. Third, the effect of the active site residues in the inactivation mechanism has been investigated with the modelling of clusters at the QM level involving key residues. Fourth, new diagonalization routines for the SEBOMD (SemiEmpirical Born-Oppenheimer Molecular Dynamics) approach implemented in the Amber suite of programs, have been incorporated using LAPACK and SCALAPACK libraries, tested and evaluated to optimize the diagonalization procedure of the Fock matrix. Fifth, reaction free energies of PLP containing systems have been investigated with SEBOMD simulations