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Morphological development during crystallization of thin films of poly(ethylene oxide) (PEO) / poly(methyl methacrylate) (PMMA) blends has been reported. Studies have focused on the effects of the blend composition, PMMA molecular weight, film thickness, and crystallization temperature on the observed crystal morphology. As the blend composition was varied from 90 to 30 wt% PEO, the crystal morphology varied from spherulites to needles and dendrites. Variation of the crystallization temperature and PMMA molecular weight resulted in similar changes in morphology. A morphological map demonstrating the roles of the experimental controls on the observed crystal morphology has been developed. This map was used as a tool for more detailed studies of the observed morphologies and morphological transitions. The dendritic region of the map (~ 30 = 40 wt% PEO) was studied in detail. Changes in the diffusion length were achieved through variation of the PMMA molecular weight, and were shown to influence the secondary sidebranch spacing. Sidebranch spacing measurements revealed that coarsening of the dendritic microstructure occurred well after the competition between diffusion fields of neighboring dendrite arms vanished, indicating the existence of another coarsening mechanism. These studies of dendritic sidebranching indicate that polymer dendrites develop by mechanisms similar to those in small molecules and metals. A number of in-situ observations of morphological transitions have also been reported, including a dense-branched morphology (DBM)/dendrite transition, a DBM/stacked-needle/needle transition, and a transition from dendrites with 90o sidebranching to dendrites with 45o branching or a dense-branched morphology, both of which grow at 45o to the original dendrite trunk. The DBM/dendrite transition occurred over a range of crystallization temperatures, indicating that the transition is not sharp. Crystal growth rate measurements verified this result. The DBM/stacked-needle/needle transitions demonstrated distinct jumps in the crystal growth rate, indicating a change in the growth mechanism or direction. For the transition involving a change in the growth direction, the effective level of noise (fluctuation) was found to be important in morphological selection. The results of this work have helped to define new directions for the study of crystal morphologies, especially in the areas of spherulite formation and dendritic growth. / Ph. D.

La compréhension du comportement des matériaux en présence d'impuretés, durant le processus de solidification, nécessite le développement de méthodologies appropriées pour une analyse et un contrôle efficace des changements topologiques des microstructures (par exemple, la formation des dendrites) au cours des différentes phases de transformation. L'objectif de cette thèse est de construire un modèle pertinent de solidification d'alliages binaires sous l'action de champs magnétiques, d'analyser les systèmes issus du modèle mathématique ainsi développé, d'un point de vue théorique et numérique, et enfin de développer une méthode de contrôle optimal afin de contrôler la dynamique du front de solidification par l'action du champs magnétiques. Dans un premier temps, nous avons décrit la physique du problème et les lois fondamentales nécessaires à la modélisation, puis nous avons construit un nouveau modèle de champ de phase, qui tient compte de l'influence de l'action du champ magnétique sur le mouvement du front de solidification. Le modèle ainsi développé est caractérisé par le couplage de trois systèmes : un de type magnétohydrodynamique, un second de type Warren-Boettingger avec convection (représentant l'évolution du front de solidification et la concentration du mélange binaire) et un troisième représentant l'évolution de la température. Les équations du système complet décrivant le modèle, dans un domaine Ω inclus dans R^{n}, n ≤ 3, sont évolutives, non linéaires, couplées et anisotropes. Dans une seconde partie, nous avons effectué l'analyse théorique du modèle développé dans le cas isotherme et isotrope en dimension deux. Nous avons obtenu des résultats d'existence, de régularité, de stabilité et d'unicité d'une solution, sous certaines conditions sur des opérateurs non linéaires du système. Enfin, nous avons développé une méthode de contrôle optimal non linéaire : le champ magnétique (qui intervient sous forme multiplicative) joue le rôle de contrôle, et l'observation est l'état désiré de la dynamique du front. Nous avons démontré l'existence d'une solution optimale et obtenu la sensibilité de l'opérateur solution et les conditions d'optimalité en introduisant un problème adjoint. Cette partie théorique de la thèse est complétée par un important travail numérique. L'analyse et les simulations numériques ont été menées sur le problème complet bi-dimensionnel non linéaire (isotrope et anisotrope). Nous avons utilisé pour la discrétisation la méthode des lignes qui consiste à considérer séparément la discrétisation temporelle et spatiale. La discrétisation spatiale est effectuée par un schéma d'éléments finis mixtes et le système différentiel algébrique obtenu est résolu par l'utilisation du solveur DASSL. La discrétisation du domaine est effectuée par des mailles triangulaires non structurées. Dans le cas réaliste, elles correspondent à un maillage non uniforme et trés fin dans la zone de la dendrite et au niveau de l'interface. Nous avons obtenu des estimations d'erreur pour les différentes variables d'état du modèle et analysé la robustesse et la stabilité des schémas d'approximation. Ce code numérique a été validé sur différents exemples, et donne d'excellents résultats. Ensuite, nous avons exploité le code pour traiter un problème réaliste, à savoir la solidification dendritique d'un alliage binaire Nickel-Cuivre, et analyser l'influence de champs magnétiques sur l'évolution des dendrites. Les résultats obtenus montrent l'efficacité de l'approche à reproduire les observations expérimentales.

There have been a number of computational modelling studies that aim to replicate the cerebellar Purkinje cell, though these typically use the morphology of rodent cells. While many species, including rodents, display intricate dendritic branching, it is not a universal feature among Purkinje cells. This study uses morphological reconstructions of 24 Purkinje cells from seven species to explore the changes that occur to the cell through evolution and examine whether this has an effect on the processing capacity of the cell. This is achieved by combining several modes of study in order to gain a comprehensive overview of the variations between the cells in both morphology and behaviour. Passive and active computational models of the cells were created, using the same electrophysiological parameters and ion channels for all models, to characterise the voltage attenuation and electrophysiological behaviour of the cells. These results and several measures of branching and size were then used to look for clusters in the data set using machine learning techniques. They were also used to visualise the differences within each species group. Information theory methods were also employed to compare the estimated information transfer from input to output across each cell. Along with a literature review into what is known about Purkinje cells and the cerebellum across the phylogenetic tree, these results show that while there are some obvious differences in morphology, the variation within species groups in electrophysiological behaviour is often as high as between them. This suggests that morphological changes may occur in order to conserve behaviour in the face of other changes to the cerebellum.

Nous avons étudié in situ et en temps réel la solidification dirigée d'échantillons minces d'alliages binaires et d'un quasicristal en combinant radiographie et topographie X. Sur Al-3,5%pdsNi non-affiné, nous avons étudié la formation de l'état initial et la morphologie du front de solidification (croissance cellulaire puis dendritique). Les effets des contraintes mécaniques ont été mis en évidence. La transition colonnaire-équiaxe a été étudiée sur Al-3,5%pdsNi affiné. Nous avons décrit le blocage du front colonnaire et le régime de croissance équiaxe ultérieure. L'efficacité des affinants tend vers une limite. Une analyse de la morphologie des microstructures a été effectuée. L'étude du quasicristal i-AlPdMn montre la croissance de grains dodécaédriques facettés. Nous avons mis en évidence les effets de la convection thermosolutale. La visualisation des contraintes montre une forte déformation des grains. L'apparition de porosités a été observée au cours de la fusion des grains.

Les études sur les souris transgéniques SOD1 utilisées comme modèle de sclérose latérale amyotrophique (SLA) montrent que les signes cliniques apparaissent quand la majorité des motoneurones (MNs) est déjà déconnectée de la périphérie et la force contractile de certains muscles diminuée de 80%. L'identification des mécanismes pathologiques précoces est une étape importante pour améliorer notre compréhension de la maladie et expliquer l'atteinte préférentielle des MNs observée pendant la SLA. Notre objectif était de rechercher la présence d'anomalies précoces dès la naissance dans un modèle souris de SLA. L'analyse comportementale suggère que dès la période postnatale, la mutation SOD1-G85R perturbe la maturation du système sensori-moteur des souris transgéniques et ralentit l'expression de réflexes moteurs comme le montrent les retards observés lors des tests de placement et d'agrippement des membres postérieurs et lors du test de retournement. Ces retards pourraient principalement résulter d'un dysfonctionnement des réseaux lombaires de la moelle épinière pendant la 1ère semaine postnatale. Pendant cette période, sur les préparations in vitro de moelle épinière / tronc cérébral, les réseaux lombaires SOD1 sont difficilement activables lors d'applications pharmacologiques. Les enregistrements électrophysiologiques réalisés pendant cette période appuient principalement la possibilité que la composante glutamatergique possède un rôle clé dans cette difficulté. Nos données suggèrent la possibilité d'un déséquilibre entre les récepteurs AMPA et NMDA dans les MNs SOD1 avec un défaut lors de l'activation des récepteurs NMDA. En 2ème semaine postnatale, les MNs lombaires SOD1 sont hypoexcitables et présentent une résistance d'entrée ainsi qu'un gain plus faibles. Nous montrons que l'augmentation anormale de la surface dendritique de ces MNs suffit à expliquer cette diminution de la résistance d'entrée alors que la baisse du gain indique plutôt des modifications de conductances ioniques qui restent à caractériser. Enfin l'analyse morphologique montre que les dendrites des motoneurones SOD1 sont anormalement développées et complexes. Il est important de souligner dans le cadre de la maladie que les segments de la moelle sacrée et les interneurones lombaires qui sont épargnés à l'âge adulte ne sont pas concernés par ces différences. Cela renforce l'idée que les anomalies que nous décrivons sont spécifiques de la maladie et que dès le plus jeune âge les MNs sont la cible privilégiée de la SLA. Dans ce travail, nous montrons des dysfonctionnements des MNs bien avant la dénervation périphérique et la dégénérescence des axones moteurs. La modification de l'excitabilité des motoneurones pendant la période de maturation des unités motrices pourrait fragiliser les jonctions neuromusculaires qui sont une des cibles principales de la SLA.

The fundamental attribute of neurons is their cellular electrical excitability, which is based on the expression of a plethora of ligand- and voltage-gated membrane channels that give rise to prominent membrane currents and membrane potential variations that represent the biophysical substrate underlying the transfer and integration of information at the cellular level. Dendrites have both an electrical and a biochemical character, which are closely linked. In contrast, glial cells are non-electrically excitable but nevertheless display a form of excitability that is based on variations of the Ca2+ concentration in the cytosol rather than electrical changes in the membrane. Cytoplasmic Ca2+ serves as an intracellular signal that is responsible for controlling a multitude of cellular processes. The key to this pleiotropic role is the complex spatiotemporal organization of the [Ca2+]i rise evoked by extracellular agonists, which allows selected effectors to be recruited and specific actions to be initiated. Ca2+ handling in the cell is maintained by operation of multiple mechanisms of Ca2+ influx, internal release, diffusion, buffering and extrusion. Ca2+ tends to be a rather parochial operator with a small radius of action from its point of entry at the cytoplasm resulting in the concept of microdomains. Dendritic Ca2+ signaling have been shown to be highly compartmentalized and astrocytic processes have been reported to be constituted by hundreds of microdomains that represent the elementary units of the astrocyte Ca2+ signal, from where it can eventually propagate to other regions of the cell. The astrocyte Ca2+ elevation may thus act as intra and intercellular signal that can propagate within and between astrocytes, signaling to different regions of the cell and to different cells. The spatio-temporal features of neuron-to-astrocyte communication, results from diverse neurotransmitters and signaling pathways that converge and cooperate to shape the Ca2+ signal in astrocytes. Alterations in Ca2+ homeostasis have been shown to be associated with major pathological conditions of the brain such as epilepsy, ischemia and neurodegenerative diseases. Although there are evidences of Ca2+ rise in hippocampal neurons in in vitro models of epilepsy (Pal et al., 1999; Limbrick et al., 2001), there is no information on the Ca2+ regulatory mechanisms operating in discrete compartments of the epileptic neuron following Ca2+ influx through voltage gated calcium channels (VGCCs). In the first part of the work, the spatial and temporal profiles of depolarization induced changes in the intracellular Ca2+ concentration in the dendrites of cultured autaptic hippocampal pyramidal neurons rendered epileptic experimentally have been addressed. Our in vitro epilepsy model consisted of hippocampal neurons in autaptic culture that were grown in the presence of kynurenate and high Mg2+, and subsequently washing the preparation free of the blockers. To understand the differences in Ca2+ handling mechanisms in different compartments of a control neuron and the kynurenate treated neuron, a combination of whole-cell patch-clamp recording and fast Ca2+ imaging methods using the Ca2+ indicator Oregon Green 488 BAPTA-1 was applied. All our analysis was focused on localized regions in the dendrite that showed pronounced Ca2+ transients upon activation of high voltage activated (HVA) Ca2+ channels. The spatial extent of Ca2+ signals suggested the presence of distinct dendritic compartments that respond to the depolarizing stimulus. Further, the local Ca2+ transients were observed even in the presence of NMDA and AMPA receptor antagonists, suggesting that the opening of VGCCs primarily triggered the local Ca2+ changes. The prominent changes in intracellular Ca2+ observed in these dendritic regions appear to be sites where Ca2+ evoked dendritic exocytosis (CEDE) takes place. Since cellular Ca2+ buffers determine the amplitude and diffusional spread of neuronal Ca2+ signals, quantitative estimates of the time-dependent spread of intracellular Ca2+ in the dendritic compartments in the control and treated neurons were done using image processing techniques. Physiological changes in Ca2+ channel functioning were also induced by kynurenate treatment and one such noticeable difference was the observation of Ca2+ dependent inactivation in the treated neurons. We provide evidences of localized Ca2+ changes in the dendrites of hippocampal neurons that are rendered epileptic by kynurenate treatment, suggesting that these sites are more vulnerable (Padmashri et al., 2006). This might contribute to the epileptiform activity by local changes in cellular and membrane properties in complex ways that remains to be clearly understood. Status Epilepticus (SE), stroke and traumatic brain injury are all associated with large increases in extracellular glutamate concentrations. The concentration of glutamate in the extracellular fluid is around 3-4 µM and astrocytes are primarily responsible for the uptake of glutamate at the synapses. The extracellular levels of glutamate has been shown to increase dramatically (16 fold) in human SE suggesting an important role of glutamate in the mechanism of seizure activity and seizure related brain damage (Carlson et al., 1992). Several other studies have also shown a persistent increase in extracellular glutamate concentration to potentially neurotoxic concentrations in the epileptogenic hippocampus (During and Spencer, 1993; Sherwin, 1999; Cavus et al., 2005). We addressed the problem related to the effects of prolonged glutamate pretreatment on Ca2+ signaling in an individual astrocyte and its adjoining astrocyte (astrocyte pair), rather than on a syncytium of astrocytes in culture. Individual astrocytes may have functional domains that respond to an agonist through distinct receptor signaling systems. These are difficult to observe in studies that are done on glial syncytium because of spatial limits of image capture. This was examined with simultaneous somatic patch-pipette recording of a single astrocyte to evoke voltage-gated calcium currents, and Ca2+ imaging using the Ca2+ indicator Oregon Green 488 BAPTA-1 to identify the Ca2+ microdomains. Transient Ca2+ changes locked to the depolarization were observed in certain compartments in the astrocyte processes of the depolarized astrocyte and the responses were more pronounced in the adjoining astrocyte of the astrocyte pair. The Ca2+ transient amplitudes were enhanced on pretreatment of cells with glutamate (500 µM for 20 minutes). Estimation of local Ca2+ diffusion coefficients in the astrocytic processes indicated higher values in the adjoining astrocyte of the glutamate pretreated group. In order to understand the underlying mechanisms, we performed the experiments in the presence of different blockers for the metabotropic glutamate receptor, inositol 1,4,5 triphosphate (IP3) receptors and gap junctions. Ca2+ transients recorded on pretreatment of cells with glutamate showed attenuated responses in the presence of the metabotropic glutamate receptor (mGluR) antagonist α-Methyl(4-Carboxy-Phenyl) Glycine (MCPG). Intracellular heparin (an antagonist of IP3 receptor) introduced in the depolarized astrocyte did not affect the Ca2+ transients in the heparin loaded astrocyte, but attenuated the [Ca2+]i responses in the adjoining astrocyte suggesting that IP3 may be the transfer signal. The uncoupling agent 1-Octanol attenuated the [Ca2+]i responses in the adjoining cell of the astrocyte pair in both the control and glutamate pretreated astrocytes indicating the role of gap junctional communication. The findings of [Ca2+]i responses within discrete regions of astrocytic processes suggest that astrocytes may be comprised of microdomains whose properties are altered by glutamate pretreatment. The data also indicates that glutamate induced alterations in Ca2+ signaling in the astrocyte pair may be mediated through phospholipase C (PLC), IP3, internal Ca2+ stores, VGCCs and gap junction channels (Padmashri and Sikdar, 2006). Neuronal (EAAC-1) and glial (GLT-1 and GLAST) glutamate transporters facilitate glutamate reuptake after synaptic release. Transgenic mice with GLT-1 knockout display spontaneous epileptic activity (Tanaka et al., 1997) and loss of glial glutamate transporters using chronic antisense nucleotide administration was reported to result in elevated extracellular glutamate levels and neurodegeneration characteristic of excitotoxity (Rothstein et al., 1996). Dysfunction of glutamate transporters and the resulting increase of glutamate have been speculated to play an important role in infantile epilepsies (Demarque et al., 2004). We examined the effects of pretreatment with glutamate in the presence of the glutamate transport inhibitor threo-β-hydroxy-aspartate (TBHA) and in Na+-free extracellular medium to understand whether this resulted in any alteration in the astrocytic intracellular Ca2+ dynamics following activation of voltage gated calcium channels. The Ca2+ responses were found to be attenuated in both the cases indicating that the elevated levels of extracellular glutamate due to blockade of glutamate transporters may influence the responses mediated by the astrocytic glutamate receptors. Our studies indicate that the heightened extracellular glutamate concentration is not gliotoxic in our experimental system, although it may have a profound effect on altering the activity of surrounding neurons which was not addressed in the present work. Several studies have indicated that neurons control the level of gap junction mediated communication between astrocytes (Giaume and McCarthy, 1996; Rouach et al, 2000). All our earlier studies were done on process bearing astrocytes that were co-cultured with neurons. We have addressed the question as to whether the spatio-temporal changes in [Ca2+]i in astrocyte pairs differ if the astrocytes are cultured in the absence of neurons. The results indicate that there is indeed a significant reduction in the responses that are evoked in response to the depolarization pulse in the adjoining cell of the astrocyte pair. These experiments demonstrate that neurons in the cocultures may selectively enhance the Ca2+ responses possibly by increasing the coupling between the two cells.

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.

Orientador: Juno Gallego / Banca: Ruís Camargo Tokimatsu / Banca: Itamar Ferreira / Resumo: No Brasil, a maior parte da energia elétrica disponível é gerada por usinas hidrelétricas as quais contam com grande número de turbinas hidráulicas. Durante sua operação estas turbinas sofrem sérios danos provenientes tanto de natureza mecânica como também hidráulica. Fenômenos de perda de massa e também trincas e rachaduras são alguns dos principais problemas que ocorrem devido a um fenômeno chamado de erosão cavitacional, que comumente é recuperado por soldagem. Sendo assim, grandes níveis de resistência à cavitação vêm sendo obtidos com a deposição de aços inoxidáveis com a presença de cobalto pelo processo de soldagem a arco com proteção gasosa. Neste trabalho, seis amostras feitas de aço-carbono ASTM A36, as quais foram extraídas de retalhos de turbinas, foram usadas como metal de base para as amostras. Usando um processo a arco elétrico com proteção gasosa (GMAW) em posição plana, duas camadas de aço AWS E70-S6 (1,2 mm de diâmetro) foi depositada em todas as amostras, e, somente em três das amostras foram depositadas duas camadas de amanteigamento com o arame de aço inoxidável AWS E309-T1 (1,6 mm de diâmetro) usando o mesmo processo, onde foi utilizado como proteção gasosa uma mistura de 75% de dióxido de carbono e 25% de argônio. A energia de soldagem nominal nestes casos foi de 0,5 kJ/mm. Por final, mais duas camadas de aço inoxidável com cobalto, liga resistente à cavitação foram depositadas por arco pulsado, com energia de soldagem nominal de 0,5 a 0,8kJ/mm, com uma mistura de gases de 98% de argônio e 2% de oxigênio como proteção. Secções transversais das amostras foram preparadas, devidamente lixadas e depois polidas com alumina 1,0μm, seguido de ataque químico moderado com Villela para observação de microestruturas. Foi feito o estudo de microdureza Vickers com carga padrão de 0,4 N e espaçamento regular (0,4 mm) ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Nowadays most of the power supply used in Brazil is provided for a large number of hydraulic turbines. During its operation in hydroelectric power plants, these turbines usually have been damaged either mechanical or hydraulic reasons. So, catastrophic cracking and loss-of-mass due to cavitation erosion are main problems which are commonly repaired by welding. Higher levels of resistance to cavitation erosion have been attained with surface deposition of a cobalt-alloyed stainless steel coating by gas-protected arc welding techniques. In the present work six plates, which were machined from blades of hydraulic turbines made with ASTM A36 carbon steel grade, were used as base metal. Using manual gas-metal arc welding (GMAW) in flat weld position two layers of AWS E70-S6 carbon steel (1.2 mm diameter) were deposited on all samples. Only 3 samples two buttering layers were deposited with AWS E309-T1 flux-cored wire (1.6 mm diameter) using same processing, where were applied a 75% carbon dioxide - 25% argon mixture as protection gas. The nominal heat input used in all layers was approximately 0.5 kJ/mm. At the last welding deposition another two cobalt-alloyed, cavitation resistant, stainless steel cladding layers were deposited under pulsed arc with a nominal heat input of 0.5 to 0.8kJ/mm, being used for them a protective gas mixture of 98% argon - 2% oxygen. Transverse sections of weld deposit were prepared according standard grinding method (up to 1200-grit SiC paper) and final mechanical polishing using 1.0μm alumina, followed by moderate etching in Villela reagent for microstructural observation. Vickers microhardness measurements were carried out at standard load (0.4 N) and regular spacings (0,4mm) from surface to base metal. Light microscopy (LM) was used to determine average size of inclusions, which was measured from digitalized images using a freeware image analysis ...(Complete abstract, click electronic access below) / Mestre

Systematic investigations through several experimental techniques have revealed that hippocampal pyramidal neurons express voltage gated ion channels (VGICs) with well-defined gradients along their dendritic arbor. These actively maintained gradients in various dendritic VGICs effectuate several stereotypic, topographically continuous functional gradients along the topograph of the dendritic arbor, and have been referred to as intraneuronal functional maps. The prime goal of my thesis was to understand the emergence and homeostasis of the several coexistent functional maps that express within hippocampal pyramidal neurons. In the first part of the thesis, we focus only on spatial interactions between ion channels and analyzed the role of such interactions in the emergence of functional maps. We developed a generalized quantitative framework, the influence field, to analyze the extent of influence of a spatially localized VGIC cluster. Employing this framework, we showed that a localized VGIC cluster could have spatially widespread influence, and was heavily reliant on the specific physiological property and background conductances. Using the influence field model, we reconstructed functional gradients from VGIC conductance gradients, and demonstrated that the cumulative contribution of VGIC conductances in adjacent compartments plays a critical role in determining physiological properties at a given location. These results suggested that spatial interactions among spatially segregated VGIC clusters are necessary for the emergence of the functional maps. In the second part of the thesis, we assessed the specific roles of only kinetic interactions between ion channels in determining physiological properties by employing a single-compartmental model. In doing this, we analyzed the roles of interactions among several VGICs in regulating intrinsic response dynamics. Using global sensitivity analysis, we showed that functionally similar models could be achieved even when underlying parameters displayed tremendous variability and exhibited weak pair-wise correlations. These results suggested that that response homeostasis could be achieved through several non-unique channel combinations, as an emergent consequence of kinetic interactions among these channel conductances. In the final part of the thesis, we analyzed the combined impact of both spatial and kinetic interactions among ion channel conductances on the emergence and homeostasis of functional maps in a neuronal model endowed with extensive dendritic arborization. To do this, we performed global sensitivity analysis on morphologically realistic conductance-based models of hippocampal pyramidal neurons that coexpressed six functional maps. We found topographically continuous functional maps to emerge from disparate model parameters with weak pair-wise correlations between parameters. These results implied that individual channel properties need not be set at constant values in achieving overall homeostasis of several coexistent functional maps. We suggest collective channelostasis, where several channels regulate their properties and expression profiles in an uncorrelated manner, as an alternative for accomplishing functional map homeostasis. Finally, we developed a methodology to assess the contribution of individual channel conductances to the various functional measurements employing virtual knockout simulations. We found that the deletion of individual channels resulted in variable, measurement-and location-specific impacts across the model population.