La sclérose tubéreuse de Bourneville (STB) est une maladie génétique et multi-systémique à transmission autosomique dominante due à des mutations d’un gène TSC1 ou TSC2 qui codent respectivement pour hamartine et tuberine ayant une action inhibitrice sur la voie de signalisation mTOR. L’épilepsie précoce et pharmacorésistante est la manifestation neurologique la plus fréquente et la plus délétère de la STB. Elle débute souvent dans la première année de vie par des spasmes infantiles qui évoluent avec l’âge et en absence de traitement vers des crises toniques ou tonico-cloniques. Bien que les crises soient supposées être générées dans des tubers corticaux, les mécanismes de l’épilepsie ne sont pas bien élucidés et le traitement reste souvent inefficace. Des études morphologiques ont montré une altération de l’expression ARNm des récepteurs au glutamate dans les cellules géantes et les neurones dysplasiques des tubers, mais leur implication fonctionnelle restait à montrer. Les différentes sous-unités NMDA ont une expression âge-dépendante et région-spécifique, les plus grands changements survenant au début de la vie quand l’épilepsie de la STB apparaît. Ce travail avait pour but d’étudier à l’aide de méthodes électrophysiologiques in vitro et in vivo l’expression fonctionnelle des sous-unités NMDA aberrantes et de déterminer leur rôle dans l’épileptogènese chez les souris hétérozygotes Tsc1+/- et sur le tissu humain STB post-opératoire. Nous avons pu démontrer que : i) Les souris hétérozygotes pour le gène Tsc1 sont spontanément épileptiques in vivo et in vitro dans une courte fenêtre dévelopmentale de P9 à P18. ii) Elles présentent une altération d’expression des récepteurs NMDA couche-spécifique et mTOR dépendante avec une surexpression des sous-unités GluN2C/D dans la couche 4 et 2/3 et GluN2B dans les couches 2/3. Cette expression anormale est prévenue par l’administration d’un inhibiteur de la voie mTOR, la rapamycine. iii) Les mêmes altérations d’expression des récepteurs NMDA, sont montrées sur les tissus post-opératoires, non seulement de tubers de STB mais aussi des dysplasies corticales focales (DCF), ces deux malformations ayant des similarités étiologiques et physiopathologiques. iv) La RT-PCR quantitative confirme une expression excessive de GluN2C dans le cortex de souris Tsc1+/- et sur le tissu humain des tubers et DCF. v) Les décharges épileptiques chez la souris Tsc1+/- sont générées dans la couche granulaire 4 du cortex avant de se propager vers les couches superficielles et les couches profondes, empruntant ainsi les microcircuits corticaux. vi) L’expression excessive de la sous-unité GluN2C dans le cortex contribue à l’hyperexcitabilité neuronale chez la souris Tsc1+/- et sur des tissus humains de tubers et de DCF puisque les crises et les décharges sont bloquées par les antagonistes sélectifs de GluN2C/D. vii) Les crises chez la souris Tsc1+/- suivent une séquence âge-dépendante évoluant du type «spasms-like» vers «tonic-clonic like», rappelant celle de l’épilepsie humaine, avec deux pics de haute incidence de crises à P13 et P16 correspondant chez l’homme respectivement l’âge des spasmes infantiles et celui des crises toniques. L’évolution avec l’âge du délai de propagation inter-hémisphérique pourrait contribuer à ce changement de types de crises. Ces résultats montrent donc pour la première fois qu’une happloinsuffisance pour le gène Tsc1 chez les souris Tsc1+/- sans tubers suffit à produire une altération de l’expression des récepteurs NMDA de manière mTOR dépendante et contribuer ainsi à l’épileptogènese dans la STB. La souris Tsc1+/- est le premier modèle génétique sans anomalies morphologiques présentant une épilepsie spontanée qui évolue des spasmes vers des crises toniques et tonico-cloniques. Néanmoins cette épilepsie diffère de l’épilepsie humaine de la STB par l’absence de crises focales et de pharmacorésistance, ce qui pourrait être expliqué par l’absence de tubers chez la souris Tsc1+/-. (...) / Tuberous sclerosis complex (TSC) is a genetic multisystemic disease with autosomal dominant transmission due to mutations in a gene TSC1 or TSC2 respectively which encode hamartin and tuberin proteins having an inhibitory action on the mTOR signaling pathway. Early refractory epilepsy is the most common and most deleterious neurological manifestation. The epilepsy often begins in the first year of life by infantile spasms that change in the lack of treatment to tonic or tonic-clonic seizures in age-dependent manner. Although seizures are thought to be generated in cortical tubers, epilepsy mechanisms are not well understood and treatment is often ineffective. Morphological studies showed the altered expression of glutamate receptor mRNA in the giant cells and dysplastic neurons of tubers, but their functional involvement remains unknown. The different NMDA subunits have an age-dependent and region-specific expression, the greatest changes occurring early in life when the TSC epilepsy appears. This work aimed to study the functional expression of aberrant NMDA subunits expression and their role in the epileptogenesis in heterozygous Tsc1+/- mice and post-surgical human tissue of TSC patients using in vitro and in vivo electrophysiological methods. The study reveal that: i) Heterozygous tuber-free Tsc1+/- mice show spontaneous epilepsy in vivo and in vitro in a short developmental window from P9 to P18. ii) These mice exhibit an altered NMDA receptor expression in mTOR dependent and layer-specific manner with GluN2C/D subunits overexpression in layers 4 and 2/3, and GluN2B ovexpression in layers 2/3. This abnormal NMDA receptors expression is prevented by the administration of an mTOR inhibitor, rapamycin. iii) The same alterations of NMDA receptors’ expression are shown in post-surgical tissues not only in tubers from TSC patients, but also in focal cortical dysplasia (FCD), these two malformations sharing etiological and pathophysiological similarities. iv) Quantitative RT-PCR confirms the excessive GluN2C subunit expression in Tsc1+/- mouse cortex and human tissue of tubers and DCF. v) Epileptic discharges in Tsc1+/- mice are generated in the granular layer 4 of the cortex before spreading to the superficial and then to deep layers, thus borrowing the cortical microcircuits. vi) Excessive expression of GluN2C subunit in the cortex contributes to neuronal hyperexcitability in Tsc1+/- mice, as well as in human tubers and DCF tissues, since epileptic discharges are blocked by selective GluN2C/D antagonists. vii) Seizures in Tsc1+/- mice follow the age-dependent sequence, evolving from "spasms-like" to "tonic-clonic like" thus reminding the human epilepsy, with two peaks of highest seizure incidence at P13 and P16 corresponding respectively to age of infantile spasms and of tonic seizures in human. The age-dependent evolution of interhemispheric propagation delay could contribute to this change in seizure type. These results show for the first time that TSC1 happloinsuffisancy in tuber-free Tsc1+/- mice is sufficient to produce an alteration in NMDA receptor expression in an mTOR dependent manner, and thus contributes to epileptogenesis in TSC. The Tsc1+/- mouse line is the first genetic model of TSC without morphological abnormalities presenting with early spontaneous seizures which evolves from “spasms-like” to “tonic-clonic like” seizures. However, the epilepsy in Tsc1+/- mice differs from human TSC epilepsy by the absence of focal seizures and of drug-resistance. Both could be explained by the lack of tubers in the Tsc1+/- mice. It remains to determine whether the expression of GluN2C subunit is also transitional in Tsc1+/- mice and whether other factors contribute to determine the age-dependent epilepsy. This study opens new therapeutic perspectives of TSC epilepsy targeting GluN2C subunit of NMDA receptors.

The thesis titled Educational Specifics of Children with Epilepsy aims to find out what are the specifics of education of children with epilepsy. The theoretical part of the thesis defines the characteristics of epilepsy, contains information about epilepsy and epileptic seizure, it deals with etiology of epilepsy, classification of epileptic and non epileptic seizures. It deals with the provocative factors of epileptic seizure and informs about the possibilities of treating this disease. The diploma thesis further outlines the current topic of today, and this is the education of children with epilepsy in the family and especially at school. In the thesis one can read about the issues of family education and healthy siblings of a child with disabilities, as well as some prejudices that an individual with epilepsy can encounter today. The work highlights the educational specifics of the education of children with epilepsy and does not forget to mention the legislative framework and the possibilities of education. It explains the support measures and the concept of inclusion. In the practical part, it contains the questions and the objectives that have been answered by qualitative research. Four case studies of epilepsy children aged between 5 and 16 years, a semi-structured interview with the family...

O autor aborda a conformidade das execuções fiscais de anuidades aos princípios do Devido Processo Legal e da Capacidade Contributiva, especialmente após o advento da Lei nº 12.514/2011, que alterou a definição do fato gerador da Contribuição devida aos Conselhos de Fiscalização Profissional. Valendo-se de pesquisa jurisprudencial sobre a forma como vêm sendo utilizados os meios para salvaguardar os créditos tributários, a presente dissertação questiona os parâmetros adotados para o deferimento de penhoras eletrônicas no âmbito destas execuções, considerando as peculiaridades da constituição do crédito tributário e da extração do título executivo pelas diversas projeções regionais dos Conselhos de Fiscalização. Com base no postulado da razoabilidade, apresenta critérios alternativos aptos a mitigar o caráter confiscatório da ordem de bloqueio e proteger o mínimo existencial dos executados, minimizando o risco de indisponibilidade de valores diretamente comprometidos com despesas de alimentação, saúde e moradia. / The author discusses the compliance of the procedure prior to tax foreclosure to the Due Process of Law and Contributory Capacity, especially after the advent of Law 12.514/2011, which changed the definition of the taxable event of the contribution due to the Supervisory Boards of Professional Activity. Drawing on jurisprudential research on how has been used the tools to safeguard the tax credits, the author also analyzes the parameters that have been used for the acceptance of electronic attachment under these executions, considering the peculiarities of the constitution of the tax credit and the extraction of the enforcement by the various projections of regional councils. Based on the postulate of reasonableness, the author presents alternative proposals able to mitigate the confiscatory nature of the freezing order and protect the vital minimum of the debtors, minimizing the risk of embrancing amounts directly committed to food expenses, health and housing.

Made available in DSpace on 2016-04-26T20:22:13Z (GMT). No. of bitstreams: 1 Leonardo Leal Peret Antunes.pdf: 1227750 bytes, checksum: f3b40ebf7bd780592f56d42067dc6467 (MD5) Previous issue date: 2013-10-28 / This study aims to analyze the institutes of search and seizure within the constitutional criminal procedure, understood as such once it is guided by the constitutional values that permeate the Federal Constitution of 1988. Even though they represent serious restrictions to the fundamental rights of the citizen who suffers a criminal prosecution, searches and the seizures have been trivialized nowadays. They are performed without any limit or criteria, leading to unjustified trespass and indelible losses. Considering that factual reality, we sought, from a methodological focus of empirical and applied nature, to approach some of the everyday situations involving searches and seizures. Furthermore, with views to the future, we pursued to establish their limits within the Democratic State of Law which is based on human dignity and seeks to further the fundamental rights of the citizens. After a thorough study of the constitutional criminal procedure, the institutes of search and seizure and an analysis of some practical situations related to the issue at stake, our findings showed that there is a need to reconcile the effectiveness of criminal prosecution with the fundamental rights, so as to set up the limits of the search and the seizure in the constitutional criminal procedure. In this case, it is now absolutely clear that it is possible, in the effectiveness of prosecution, to conciliate public with private interest, respecting the fundamental rights, in order to establish the ideal of a democratic and ethical criminal procedure, grounded on human dignity / O presente trabalho tem por fim analisar os institutos da busca e da apreensão no âmbito do processo penal constitucional, assim entendido uma vez pautado pelos valores constitucionais que permeiam a Constituição Federal de 1988. Embora representem sérias restrições aos direitos fundamentais do cidadão que sofre uma persecução penal, as buscas e as apreensões têm sido banalizadas nos dias de hoje, realizadas sem qualquer parâmetro ou limite, ensejando, com isso, devassas indevidas e prejuízos indeléveis. Considerando essa realidade fática, procuramos, a partir de um enfoque metodológico de natureza empírica e aplicada, abordar algumas situações cotidianas envolvendo buscas e apreensões e, sempre pensando no futuro, estabelecer seus limites no âmbito de um Estado Democrático de Direito, fundado na dignidade da pessoa humana e que busca efetivar os direitos fundamentais de seus cidadãos. Os resultados obtidos após o estudo do processo penal constitucional, dos institutos da busca e da apreensão e analisando algumas situações práticas atinentes à questão apontam para a necessidade de compatibilizar a efetividade da persecução penal com os direitos fundamentais, de forma a estabelecer os limites da busca e da apreensão no processo penal constitucional. Nesse sentido, ficou absolutamente claro ser possível harmonizar o interesse público, na efetividade da persecução penal, com o interesse privado, no respeito aos direitos fundamentais, para concretizar o ideal de um processo penal democrático e ético, pautado na dignidade da pessoa humana

As crises não epilépticas psicogênicas (CNEP) podem ser definidas como episódios de alteração de movimentos, sensações ou experiência similar à epilepsia causada por processo psicológico e sem associação com descarga elétrica cerebral anormal. Estima-se que o número de casos de pacientes com CNEP seja de 2 a 33 por 100.000 habitantes. O índice de CNEP corresponde ainda a aproximadamente 5% dos pacientes tratados como epilépticos. Os pacientes com CNEP são tratados como possuindo epilepsia refratária, chegando ao limite máximo do tratamento medicamentoso e sem a obtenção de resultados satisfatórios. Objetivos: relatar os efeitos do tratamento psicanalítico individual em pacientes com CNEP. Método: A casuística foi composta por 37 pacientes com diagnóstico prévio de CNEP realizado por meio da monitorização por vídeo-EEG. Foram realizadas sessões individuais de tratamento psicanalítico, com frequência semanal, com duração aproximada de 50 minutos e duração total de 48 sessões em 12 meses. Resultados: Este estudo constatou elevado índice de sucesso no tratamento dos pacientes com CNEP: 29,7% (n=11) de cessação/cura dos sintomas e 51,4% (n=19) redução das crises convulsivas. Foi constatada associação entre cessar ou reduzir as crises e sexo (p < 0,01), religião (p < 0,01) e término do tratamento (p < 0,01). Conclusão: Este estudo apontou a eficácia do tratamento psicanalítico individual realizado com pacientes com CNEP, podendo ser considerada uma forma de assistência essencial para que haja decréscimo ou cessação das crises. / Psychogenic non-epileptic seizures (PNES) can be defined as episodes of alteration of movement, feeling or a similar experience to epilepsy caused by a psychological process and with no association with abnormal electrical discharges in the brain. It is estimated that the number of cases of patients with CNEP is from 2 to 33 in 100.000 inhabitants. The number of patients with PNES reaches 5% of those treated as epileptics. Patients with PNES are treated as if they had intractable epilepsy, with unsatisfactory results even after medication treatment is used to its maximum limits. Objectives: to present the effects of individual psychoanalytical treatment in patients with PNES. Methods: The case base was composed of 37 patients with PNES. The diagnosis was reached with video-EEG monitoring. . Psychoanalytical treatment was carried out through 12 months of weekly sessions of around 50-minutes each, in a total of 48 individual sessions. Results: This study found a high success rate in the treatment of PNES patients. 29,7% (n=11) of patients saw cessation or cure of symptoms and 51,4% (n=19) saw a decrease in the number of episodes. There is an association between cessation or decrease in the number of episodes and sex (p < 0,01), religion (p < 0,01) and concluding treatment (p < 0,01). Conclusions: Individual psychoanalytical treatment applied to patients with PNES is considered effective and can be considered as an essential form of assistance for the reduction of cessation of episodes. Psychogenic non-epileptic seizures, Conversion disorder, Psychoanalysis, Treatment

Em tempos correntes, importantes processos de comunicação (escrita ou verbal) e armazenamento de informações aperfeiçoam-se por intermédio dos meios eletrônicos. À medida que o acesso à rede mundial de computadores (internet) se intensifica em progressões geométricas, multiplicam-se também os dados intercambiados por internautas e emergem técnicas cada vez mais avançadas de coleta e processamento de informações. Atividades rotineiras como a navegação e o envio de mensagens eletrônicas realizam-se apenas à custa de imenso trânsito de informações; como pegadas deixadas pelo caminho, os dados comutados nestas atuações permitem reconstituir os caminhos e atividades empreendidos na rede. A salvaguarda destas informações afigura-se imprescindível à vida privada e demanda rígida disciplina normativa. Não se trata apenas de impedir que dados de tráfego ou de conteúdo sejam empregados contrariamente ao Direito, mas de assegurar também que, em situações excepcionais descritas pelo legislador, sirvam para corroborar investigações criminais. Pretende-se neste trabalho um exame sistemático dos principais meios de busca da prova digital, com o escopo de delimitar o regime jurídico das intervenções nas comunicações eletrônicas e das medidas de apreensão de dados automatizados. Neste mister, inafastável analisar a disciplina constitucional do sigilo da comunicação de dados (CF, art. 5o, inciso XII) e investigar o fundamento legal das autorizações judiciais para a obtenção de informações eletrônicas. Sob perspectiva eminentemente interdisciplinar, cumpre discorrer sobre noções de Cibernética, telecomunicações, Informática, liberdade como autonomia recíproca de acesso à informação e comunicações eletrônicas, imprescindíveis à compreensão deste novo espaço do agir humano: o entorno digital. No campo da hermenêutica constitucional, necessário perquirir relevantes aspectos da vida privada e da proteção da intimidade antecedentes históricos, direito à privacy, hodierna projeção como autodeterminação informativa, teoria das três esferas e inviolabilidade das comunicações que permitirão opinar sobre a constitucionalidade das interceptações de dados em processos informacionais. Em sequência, devem ser conceituados os dados digitais e suas respectivas categorias, as técnicas de investigação penal e o resguardo de fontes de provas digitais. Sem deixar de contribuir para o aperfeiçoamento da normativa legal vigente, de rigor o exame de duas ordens distintas de incorporação dos dados ao processo: a intervenção no fluxo comunicativo destinada a captar dados e a apreensão física do dispositivo informático que alberga as informações. Como meios de busca de prova, esses procedimentos devem ser estudados de maneira individualizada, a partir de aspectos como conceito, regulação, natureza jurídica, finalidade, condicionantes legais (pressupostos, requisitos e limites), direito de defesa, juízo de proporcionalidade e controle. Por fim, tecidas as necessárias considerações sobre a conservação, eficácia probatória e valoração dos conteúdos automatizados, impõe-se indagar acerca dos efeitos decorrentes de operações ilícitas perpetradas sobre dados digitais. / In these days, important communication process (written or verbal) and information storage improve through electronic means. While the access to the computer worldwide web (internet) grows in geometrical progression, it also increases the number of webusers data and more and more advanced technics of gathering and processing information emerge. Routine activities such as sailing or sending electronic messages only happen due to the vast transit of information; like footprints left on the way, the data commutated in these actions allow to re-establish the ways and activities undertaken in the web. The security of these information figures indispensable to private life and demands a severe normative discipline. It is not only a matter of preventing that traffic or contents data may be used against the law. But also to assure that, in exceptional situation described by the legislator, it can be useful to confirm criminal investigation. This work intends a systematic examination of the main ways of searching digital evidence, with the purpose of delimitate the judiciary system of the intervention in electronic and apprehension extent of automation data. So, it must be analysed the constitutional discipline of communication data secrecy (CF, art. 5º, inciso XII) and even to investigate the legal foundation of judicial authorization to obtain electronic information. Under a strictly multidiscipline perspective, one must consider some notions of Cybernetics, telecommunication, Informatics, freedom as reciprocal autonomy access to electronic information and communication, which are essential to understand this new area of human act; the digital place. In the field of constitutional interpretation of law, it is necessary to scrutinize considerable aspects of private life and intimacy protection historical antecedents, privacy right, actual projection such as informative selfdetermination, three sphere theory and inviolability of communication so that they will permit to express an opinion about the constitutionality of interception data in informative proceedings. Sequentially, conceptualize the digital data and their respective categories, penal investigation technics and the protection of digital evidence sources. There may be a cooperation to a better improvement of the effective legal normative, an accurate examination of two different disposition of data incorporation to the process, the intervention in the communicative flow just to receive data and physical apprehension of the informatic device which contains information. As a quest in resources of proof, these proceedings must be examined in a individual way, starting with the aspects such as concept, regulation, juridical nature, finality, legal conditioning (presupposed, requisite and limits), right of defence, judgement of proportionality and control. At last, taken into everything about the conservation, evidential efficiency and the value of automatize contents still we have to enquire about the results of illicit operations perpetrated on digital data.

La lutte contre la délinquance de lucre et de trafic repose aujourd’hui en France sur une stratégie fondée principalement sur l’appréhension des avoirs illicites ainsi générés par cette activité. En pratique, la loi française a conféré aux magistrats, et sous certaines conditions aux enquêteurs, des prérogatives très offensives permettant de placer sous-main de justice des biens patrimoniaux très en amont de la procédure, au plus tôt, et ce indépendamment du droit de propriété et de la présomption d’innocence. Cependant, si la loi a grandement réformé le droit des saisies en créant les saisies pénales spéciales, elle n’a pas donné le cadre à un droit de l’exécution des confiscations. En effet, la confiscation reste le pivot de l’enquête pénale patrimoniale, même si les saisies pénales spéciales en sont aujourd’hui le moteur. Pourtant, l’absence d’une procédure post-sentence est de nature à ouvrir un nouvel espace d’opportunité pour permettre aux condamnés de dissiper une partie de leurs biens. C’est la raison pour laquelle le droit pénal patrimonial français devrait connaître une mise à jour pour conférer à la justice un cadre complet pour garantir que le crime ne paie pas / In France, the money-laundering and trafficking fight is based on a apprehension of the illicit assets strategy. French law give to magistrates, and under conditions to investigators, very offensive prerogatives to seize property assets very early in the proceedings and regardless of the property and the presumption of innocence rights. However, the law largely reformed law of seizures by creating special criminal seizures, it did not provide a framework for confiscation enforcement. Indeed, confiscation remains the pivot of criminal property investigation, although special criminal seizures are now the driving force. The absence of a post sentential procedure is likely to open a new space of opportunity for the convicteds to dissipate part of their property. This is the reason why French criminal law should be updated to give justice a complete framework to ensure that crime does not pay

Notre système juridique laisse apparaître un déséquilibre entre droits et devoirs qui place le créancier dans une situation inconfortable face à un débiteur de mauvaise foi. L’ampleur de la crise économique mondiale, qui persiste depuis près d’une décennie, a institué un climat d’incertitude. Le manquement à l’obligation d’exécution des contrats en est une des conséquences principales, notamment l’inexécution du débiteur envers son créancier. De ce fait, le règlement est devenu la préoccupation majeure. Certes, l’inexécution est condamnée par le législateur, dans le but de conserver un équilibre entre les parties au contrat. Toutefois, malgré les nombreuses et fructueuses évolutions, une observation plus minutieuse de la situation fait émerger une surprotection sous-jacente du débiteur qui s’est vu octroyer de nombreux privilèges. A contrario, la place du créancier présente des signes d’affaiblissement dans notre système juridique et fonctionnement économique. Cette situation est préoccupante, dans la mesure où elle induit un ralentissement de l’économie nationale, dû à une fuite avérée des investissements en dehors de notre territoire, au profit d’autres pays européens. Cet état de fait se présente comme un problème subjectif mais également objectif, et il est nécessaire d’étudier les divers obstacles qui se dressent devant le créancier lors de la procédure de saisie du logement familial du débiteur. Le terme « obstacles » renvoie aux contraintes que le créancier doit prendre en compte, en amont mais aussi tout au long de la procédure de saisie, qui l’empêchent de recouvrer sa créance et par conséquent de réinvestir. Il est donc essentiel de proposer des solutions afin de répondre à un besoin de rééquilibrage des forces en présence, dans un contexte économique dégradé, qui suppose une adaptation du cadre législatif. / Our legal system in France reveals a discrepancy between rights and obligations whereby the creditor is plunged into a much more critical situation as compared to the bad faith debtor. The magnitude of the economic crisis that has lasted for more than a decade has causes a climate of considerable uncertainty. The failure to comply with the obligation of execution of contracts proves to be one of the main consequences, particularly the violation of the debtor’s duty to the creditor. Therefore regulations, in which lawmakers condemn the failure to meet contracts in order to secure a balance between parties, have become the main issue. However, despite a drastic and positive evolution in our legal system it appears when following the situatio more carefully that the debtor was in fact granted with numerous privileges. In fact, in our current legal system and economic situation the creditor’s position shows signs of weakening. This is an issue of concern. Investments are thus moving abroad, benefiting other European member states and resulting in a dip in our own national economy. As this is an eestablished fact, it is necessary to consider the various main obstacles in the legal framework, which the creditor has to overcome. One example is the case of a debtor’s family property seizure. The term « obstacles »refers to the contraint satisfaction process, before and during the seizure procedure, preventing the creditor from recovering her/his outstanding debt. As a matter of fact, the lasting unfavorable economic situation supposes to consider solutions in order to better balance forces, including changes in the legal framework.

No description available.

Epilepsy is characterized by the hyperexcitability of individual neurons and hyper synchronization of groups of neurons (networks). The acquired changes that take place at molecular, cellular and network levels are important for the induction and maintenance of epileptic activity in the brain. Epileptic activity is known to alter the intrinsic properties and signaling of neurons. Understanding acquired changes that cause epilepsy may lead to innovative strategies to prevent or cure this neurological disorder. Advances in in vitro electrophysiological techniques together with experimental models of epilepsy are indispensible tools to understand molecular, cellular and network mechanisms that underlie epileptiform activity. The aim of the study was to investigate the epileptiform activity induced alterations in Ca2+ dynamics in apical dendrites of hippocampal subicular pyramidal neurons in slices and changes in network properties of cultured hippocampal neurons. We have also made attempts to develop an in vitro model of epilepsy using organotypic hippocampal slice cultures. In the first part of the present study, investigations on the basic properties of dendritic Ca2+ signaling in subicular pyramidal neurons during epileptiform activity are described. Subiculum, a part of the hippocampal formation is present, adjacent to the CA1 subfield. It acts as a transition zone between the hippocampus and entorhinal cortex. It receives inputs directly from the CA1 region, the entorhinal cortex, subcortical and other cortical areas. Several forms of evidences support the role of subiculum in temporal lobe epilepsy. Pronounced neuronal loss has been reported in various regions of the hippocampal formation (CA1 and CA3) leaving the subiculum generally intact in human epileptic tissue. It has been observed that epileptic activity is generated in subiculum in cases where the CA3 and CA1 regions are damaged or even absent. However, it is not clear how subicular neurons protect themselves from epileptic activity induced neuronal death. It is widely accepted that epileptiform activity induced neuronal damage is a result of an abnormally large influx of Ca2+ into neuronal compartments. In the present study, combined hippocampus / entorhinal cortical brain slices were exposed to zero Mg2+ + 4-amino pyridine artificial cerebrospinal fluid (ACSF) to generate spontaneous epileptiform discharges. Whole cell current-clamp recordings combined with Ca2+ imaging experiments (by incorporating Oregon green BAPTA-1 in the recording pipette) were performed on subicular pyramidal neurons to understand the changes in [Ca2+]i transients elicited in apical dendrites, in response to spontaneous epileptic discharges. To understand the changes occurring with respect to control, experiments were performed (in both control and in vitro epileptic conditions) where [Ca2+]i transients in dendrites were elicited by back propagating action potentials following somatic current injections. The results show clear distance-dependent changes in decay kinetics of [Ca2+]i transients (τdecay), without change in the amplitude of the [Ca2+]i transients, in distal parts (95–110 µm) compared to proximal segments (30–45 µm) of apical dendrites of subicular pyramidal neurons under in vitro epileptic condition, but not in control conditions. Pharmacological agents that block Ca2+ transporters viz. Na+/Ca2+ exchangers (Benzamil), plasma membrane Ca2+-ATPase pumps (Calmidazolium) and smooth endoplasmic reticulum Ca2+-ATPase pumps (Thapsigargin) were applied locally to the proximal and distal part of the apical dendrites in both experimental conditions to understand the molecular aspects of the Ca2+ extrusion mechanisms. The relative contribution of Na+/Ca2+ exchangers in Ca2+ extrusion was higher in the distal apical dendrite in in vitro epileptic condition. Using computer simulations with NEURON, biophysically realistic models were built to understand how faster decay of [Ca2+]i transients in the distal part of apical dendrite associated with [Ca2+]i extrusion mechanisms affect excitability of the neurons. With a linear increase in the density of Na+/Ca2+ exchangers along the apical dendrite, the decrease in τ decay values of [Ca2+]i transients in distal regions seen in experimental epileptic condition was reproduced in simulation. This linear increase in Na+/Ca2+ exchangers lowered the threshold for firing in response to consecutive synaptic inputs to the distal apical dendrite. Our results thus, show the existence of a novel neuroprotective mechanism in distal parts of the apical dendrite of subicular pyramidal neurons under in vitro epileptic condition with the Na+/Ca2+ exchangers being the major contributors to this mechanism. Although the enhanced contribution of Na+/Ca2+ exchangers helps the neuron in removing excess [Ca2+]i loads, it paradoxically makes the neuron hyperexcitable to synaptic inputs in the distal parts of the apical dendrites. Thus, the Na+/Ca2+ exchangers may actually protect subicular pyramidal neurons and at the same time contribute to the maintenance of epileptiform activity. In the second part of the study, neuronal network topologies and connectivity patterns were explored in control and glutamate injury induced epileptogenic hippocampal neuronal networks, cultured on planar multielectrode array (8×8) probes. Hyper synchronization of neuronal networks is the hallmark of epilepsy. To understand hyper synchronization and connectivity patterns of neuronal networks, electrical activity from multiple neurons were monitored simultaneously. The electrical activity recorded from a single electrode mainly consisted of randomly fired single spikes and bursts of spikes. Simultaneous measurement of electrical activity from all the 64 electrodes revealed network bursts. A network burst represents the period (lasting for 0.1–0.2 s) of synchronized activity in the network and, during this transient period, maximum numbers of neurons interact with each other. The network bursts were observed in both control and in vitro epileptic networks, but the frequency of network bursts was more in the latter, compared to former condition. Time stamps of individual spikes (from all 64 electrodes) during such time-aligned network burst were collected and stored in a matrix and used to construct the network topology. Connectivity maps were obtained by analyzing the spike trains using cross-covariance analysis and graph theory methods. Analysis of degree distribution, which is a measure of direct connections between electrodes in a neuronal network, showed exponential and Gaussian distributions in control and in vitro epileptic networks, respectively. Quantification of number of direct connections per electrode revealed that the in vitro epileptic networks showed much higher number of direct connections per electrode compared to control networks. Our results suggest that functional two-dimensional neuronal networks in vitro are not scale-free (not a power law degree distribution). After brief exposure to glutamate, normal hippocampal neuronal networks became hyperexcitable and fired a larger number of network bursts with altered network topology. Quantification of clustering coefficient and path length in these two types of networks revealed that the small-world network property was lost once the networks become epileptic and this was accompanied by a change from an exponential to a Gaussian network. In the last part of the study, we have explored if an excitotoxic glutamate injury (20 µM for 10 min) that produces spontaneous, recurrent, epileptiform discharges in cultured hippocampal neurons can induce epileptogenesis in hippocampal neurons of organotypic brain slice cultures. In vitro models of epilepsy are necessary to understand the mechanisms underlying seizures, the changes in brain structure and function that underlie epilepsy and are the best methods for developing new antiseizure and antiepileptogenic strategies. Glutamate receptor over-activation has been strongly associated with epileptogenesis. Recent studies have shown that brief exposure of dissociated hippocampal neurons in culture to glutamate (20 µM for 10 min) induces epileptogenesis in surviving neurons. Our aim was to extend the in vitro model of glutamate injury induced epilepsy to the slice preparations with intact brain circuits. Patch clamp technique in current-clamp mode was employed to monitor the expression of spontaneous epileptiform discharges from CA1 and CA3 neurons using several combinations of glutamate injury protocols. The results presented here represent preliminary efforts to standardize the glutamate injury protocol for inducing epileptogenesis in organotypic slice preparations. Our results indicate that glutamate injury protocols that induced epileptogenesis in dissociated hippocampal neurons in culture failed to turn CA1 and CA3 neurons of organotypic brain slice cultures epileptic. We also found that the CA1 and CA3 neurons of organotypic brain slice cultures are resilient to induction of epileptogenesis by glutamate injury protocols with 10 times higher concentrations of glutamate (200µM) than that used for neuronal cultures and long exposure periods (upto 30 min). These results clearly show that the factors involved in induction of epileptiform activity after glutamate injury in neuronal cultures and those involved in making the neurons in organotypic slices resilient to such insults are different, and understanding them could give vital clues about epileptogenesis and its control. The resilience of CA1 and CA3 neurons seen could be due to differences in homeostatic plasticity that operate in both these experimental systems. However, further studies are required to corroborate this hypothesis.