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L'utilisation de champs électriques pulsés (PEF) dans les secteurs de la médecine et de la biotechnologie est devenue de plus en plus courante au cours des dernières décennies. La recherche a montré qu'en ajustant la durée du PEF, nous pouvons prédire quels effets seront observés. Alors que les PEF dans la gamme micro - milliseconde ont été utilisés pour perméabiliser la membrane cellulaire et améliorer l'absorption de médicament ou de protéine, le PEF nanoseconde (nsPEF) a démontré des effets uniques sur les organites intracellulaires. Les deux PEF et nsPEF ont démontré un potentiel thérapeutique pour une variété de pathologies humaines, y compris le traitement du cancer. Utilisant l'imagerie des cellules vivantes, cette thèse a étudié in vitro les effets de champs pulsés d'une durée de 10 ns à 10 ms sur des lignées cancéreuses (U87 glioblastome multiforme) et non cancéreuses (neurones hippocampes de souris (HT22) et cellules ovariennes du hamster chinois (CHO)). Des résultats publiés antérieurement ont démontré que les cellules cancéreuses sont plus sensibles aux champs électriques que les cellules saines. Nos résultats sont en accord avec ces résultats, dans la mesure où les cellules U87 ont subi une dépolarisation significativement plus importante de leur potentiel transmembranaire après une seule impulsion électrique à toutes les durées. Dans un ensemble d'expériences parallèles, malgré des seuils de champ électrique similaires pour la perméabilisation membranaire, les cellules U87 ont démontré une absorption significativement améliorée de YO-PRO par rapport aux autres lignées cellulaires. Bien que les cellules U87 aient subi le plus grand changement dans la dépolarisation membranaire et la perméabilisation membranaire, elles ont également montré la constante de rescellement de la membrane la plus rapide, qui était environ 30 secondes plus rapide que les autres lignées cellulaires. Pour élucider certains des mécanismes sous-jacents par lesquels les cellules U87 répondent aux champs électriques, une série d'expériences a examiné le rôle des canaux ioniques transmembranaires. Plusieurs études récentes ont rapporté que les PEF peuvent agir directement sur les canaux ioniques voltage-dépendants. En utilisant divers modulateurs de canaux ioniques pharmacologiques spécifiques et à action large, nous avons démontré que nous pouvions presque entièrement inhiber la dépolarisation membranaire induite par le champ électrique dans les cellules U87 en bloquant certains canaux cationiques. Ces résultats étaient assez spécifiques, tels que le canal de potassium de grande conductance (BK), les canaux calciques de type L et T, et le canal cationique non spécifique, TRPM8, étaient capables d'inhiber la dépolarisation tandis que le blocage d'autres canaux ioniques ne produisait aucun changement significatif. . Les travaux de cette thèse ont montré que la lignée cellulaire maligne U87 présentait une plus grande sensibilité aux champs électriques allant de 10 ns à 10 ms par rapport aux lignées cellulaires non cancéreuses étudiées. Des améliorations potentielles aux protocoles de traitement actuels ont été proposées sur la base des résultats présentés ici. / The use of pulsed electric fields (PEF) in medical and biotechnology sectors has become increasingly prevalent over the last few decades. Research has shown that by adjusting the duration of the PEF we can predict what effects will be observed. Whereas PEF in the micro-to-millisecond range have been used to permeabilize the cell membrane and enhance drug or protein uptake, nanosecond PEF (nsPEF) have demonstrated unique effects on intracellular organelles. Both PEF and nsPEF have demonstrated therapeutic potential for a variety of human pathologies, including the treatment of cancer. Using live-cell imaging, this thesis investigated, in vitro, the effects of pulsed fields ranging in duration from 10 ns to 10 ms on cancerous (U87 glioblastoma multiforme) and non-cancerous cell lines (mouse hippocampal neurons (HT22) and Chinese hamster ovary (CHO) cells). Previously published results have demonstrated that cancerous cells have a greater sensitivity to applied electric fields than healthy cells do. Our results are in agreement with these findings, insofar as the U87 cells underwent a significantly greater depolarization of their transmembrane potential following a single electric pulse at all durations. In a parallel set of experiments, despite having similar electric field thresholds for membrane permeabilization, the U87 cells demonstrated significantly enhanced YO-PRO uptake compared to the other cells lines. Although U87 cells underwent the greatest change in both membrane depolarization and membrane permeabilization, they also showed the fastest membrane resealing constant, which was approximately 30 seconds faster than other cell lines. To elucidate some of the underlying mechanisms by which U87 cells respond to electric fields, a series of experiments looked at the role of transmembrane ion channels. Several recent studies have reported that PEFs can act directly on voltage-gated ion channels. Using a variety of specific and broad acting pharmacological ion channel modulators, we demonstrated that we could almost entirely inhibit the electric field-induced membrane depolarization in U87 cells by blocking certain cationic channels. These results were quite specific, such that the big conductance potassium (BK) channel, L- and T-type calcium channels, and the non-specific cationic channel, TRPM8, were able to inhibit depolarization while blocking other ion channels produced no significant change. The work in this thesis showed that the malignant U87 cell line showed a greater sensitivity to electric fields from ranging from 10 ns – 10 ms when compared to the non-cancerous cell lines that were investigated. Potential improvements to current treatment protocols have been proposed based on the findings presented herein.

Made available in DSpace on 2016-04-04T18:21:44Z (GMT). No. of bitstreams: 1 Ana Carolina Davila Bitencourt-1.pdf: 6061377 bytes, checksum: 300ceb06e9e5a02bb5d81cccc0c24165 (MD5) Previous issue date: 2008-02-25 / This study researches the urban dynamic and the expansion of the city through the residential lot-like condominium form in the city of Valinhos, Campinas metropolitan area, state of S?o Paulo. It is analyzed how this new areas have provided a new culture in living, regarding the mentioned metropolitan area. In this sense, this study aims to show a new urban dynamic and analyses the emergence of this new way of living and its impact in the urban structures. Understanding this changes that occurred in the city, since the 70's of the last century, give a base to discuss the new urban spaces, which recreate the forms and uses of a traditional city, but in a different and excluding way. The contemporary city analyzed in its fragments, shows the peculiarity of urban growth and the sociability interaction in the space. / Neste trabalho investiga-se a din?mica urbana e a expans?o da cidade contempor?nea atrav?s dos condom?nios horizontais fechados no munic?pio de Valinhos - SP, discutindo como esses novos espa?os t?m propiciado uma nova cultura na forma de morar na escala metropolitana. Nesse sentido, esta disserta??o tem como objetivo apresentar uma nova din?mica urbana de produ??o da cidade e refletir sobre a emerg?ncia dessa nova forma de moradia e seus reflexos nas estruturas urbanas da cidade. Compreender as mudan?as que ocorreram na cidade, desde a d?cada de 1970, embasa as an?lises sobre os novos espa?os urbanos, que recriam as formas e os usos da cidade tradicional de forma excludente. A urbaniza??o contempor?nea analisada em seus fragmentos, evidencia as peculiaridades do espraiamento urbano e as formas de sociabilidade no espa?o.

Receptores purinérgicos e canais de cálcio voltagem-dependentes estão envolvidos em diversos processos biológicos como na gastrulação, durante o desenvolvimento embrionário, e na diferenciação neural. Quando ativados, canais de cálcio voltagem-dependentes e receptores purinérgicos do tipo P2, ativados por nucleotídeos, desencadeiam transientes de cálcio intracelulares controlando diversos processos biológicos. Neste trabalho, nós estudamos a participação de canais de cálcio voltagem-dependentes e receptores do tipo P2 na geração de transientes de cálcio espontâneos e sua regulação na expressão de fatores de transcrição relacionados com a neurogênese utilizando como modelo células tronco (CTE) induzidas à diferenciação em células tronco neurais (NSC) com ácido retinóico. Descrevemos que CTE indiferenciadas podem ter a proliferação acelerada pela ativação de receptores P2X7, enquanto que a expressão e a atividade desse receptor precisam ser inibidas para o progresso da diferenciação em neuroblasto. Além disso, ao longo da diferenciação neural, por análise em tempo real dos níveis de cálcio intracelular livre identificamos 3 padrões de oscilações espontâneas de cálcio (onda, pico e unique), e mostramos que ondas e picos tiveram a frequência e amplitude aumentadas conforme o andamento da diferenciação. Células tratadas com o inibidor do receptor de inositol 1,4,5-trifosfato (IP3R), Xestospongin C, apresentaram picos mas não ondas, indicando que ondas dependem exclusivamente de cálcio oriundo do retículo endoplasmático pela ativação de IP3R. NSC de telencéfalo de embrião de camundongos transgênicos ou pré-diferenciadas de CTE tratadas com Bz-ATP, o agonista do receptor P2X7, e com 2SUTP, agonista de P2Y2 e P2Y4, aumentaram a frequência e a amplitude das oscilações espontâneas de cálcio do tipo pico. Dados, obtidos por microscopia de luminescência, da expressão em tempo real de gene repórter luciferase fusionado à Mash1 e Ngn2 revelou que a ativação dos receptores P2Y2/P2Y4 aumentou a expressão estável de Mash1 enquanto que ativação do receptor P2X7 levou ao aumento de Ngn2. Além disso, células na presença do quelante de cálcio extracelular (EGTA) ou do depletor dos estoques intracelulares de cálcio do retículo endoplasmático (thapsigargin) apresentaram redução na expressão de Mash1 e Ngn2, indicando que ambos são regulados pela sinalização de cálcio. A investigação dos canais de cálcio voltagem-dependentes demonstrou que o influxo de cálcio gerado por despolarização da membrana de NSC diferenciadas de CTE é decorrente da ativação de canais de cálcio voltagem-dependentes do tipo L. Além disso, esse influxo pode controlar o destino celular por estabilizar expressão de Mash1 e induzir a diferenciação neuronal por fosforilação e translocação do fator de transcrição CREB. Esses dados sugerem que os receptores P2X7, P2Y2, P2Y4 e canais de cálcio voltagem-dependentes do tipo L podem modular as oscilações espontâneas de cálcio durante a diferenciação neural e consequentemente alteram o padrão de expressão de Mash1 e Ngn2 favorecendo a decisão do destino celular neuronal. / Purinergic receptors and voltage gated Ca2+ channels have been attributed with developmental functions including gastrulation and neural differentiation. Upon activation, nucleotide-activated P2 purinergic receptor and voltage-gated Ca2+ channel subtypes trigger intracellular calcium transients controlling cellular processes. Here, we studied the participation of voltage-gated calcium channels and P2 receptor activity in spontaneous calcium transients and consequent regulation expression of transcription factors related to retinoic acid-induced neurogenesis of mouse neural stem and embryonic stem cells (ESC). In embryonic pluripotent stem cells, proliferation is accelerated by P2X7 receptor activation, while receptor expression / activity needs to be down-regulated for the progress of neuroblast differentiation. Moreover, along neural differentiation time lapse imaging with means of a cytosolic calcium-sensitive fluorescent probe provided different patterns of spontaneous calcium transients (waves and spikes) showing that both, frequency and amplitude increased along differentiation. Cells treated with the inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor Xestospongin C showed spikes but not waves, indicating that waves exclusively depended on calcium release from endoplasmic reticulum by IP3R activation. Cells treated with the P2X7 receptor subtype agonist Bz-ATP and the P2Y2 and P2Y4 receptor 2-S-UTP increased frequency and amplitudes of calcium transients, mainly spikes, in embryonic telencephalon neural stem cells (NSC) and NSC pre-differentiated from ESC. Data obtained by luminescence time lapse imaging of stable transfected cells with Mash1 or Ngn2 promoter-protein fusion to luciferase reporter construct revealed increased Mash1 expression due to activation of P2Y2/P2Y4 receptor subtypes, while increased expression of Ngn2 was observed following P2X7 receptor activation. In addition, cells imaged in presence of the extracellular calcium chelator EGTA or following endoplasmic reticulum calcium store depletion by thapsigargin showed a decrease in Mash1 and Ngn2 expression, indicating that both are regulated by calcium signaling. Investigation of the roles of voltage gated Ca2+ channels in neural differentiation showed that Ca2+ influx in NSC pre-differentiated from ESC is due to membrane depolarization and L-type voltage gated Ca2+ channel activation, thereby controlling cell fate decision, by stabilizing the expression of MASH1 and inducing differentiation, by phosphorylation of the transcription factor CREB. Altogether these data suggest that P2X7, P2Y2, P2Y4 receptors and L-type voltage gated Ca2+ channels can modulate spontaneous calcium oscillations during neural differentiation and consequently change the Mash1 and Ngn2 expression patterns, thus favoring the cell fate decision to the neuronal phenotype.

Les politiques urbaines de « reconstruction » post-apartheid de ces dernières décennies n’ont pas permis de réduire les injustices spatiales du Gauteng et de Johannesburg en particulier. Sous l’impulsion des acteurs privés, les résidences fermées d’entrée de gamme, symbole de l’ascension sociale des classes moyennes, diffusent de nouvelles formes de relégation et de fragmentation qui questionnent fortement le lien social, l’étalement et la mobilité croissante. Le peu d’investigations menées sur cette ville « moyenne », qui se veut synonyme de progrès et de modernité, offre l’opportunité de mobiliser une réflexion nouvelle sur les interrelations qui existent entre production de la ville, pratiques et territoires. Loin d’être monolithique, le logement d’entrée de gamme s’est développé à travers maints arrangements institutionnels particuliers et géographiquement situés. Le rôle des acteurs privés impliqués dans la gouvernance urbaine, souvent méconnu et rarement étudié, est devenu la clé de voûte des transformations contemporaines de la ville. L’originalité de ce travail a été de révéler les principes de constitution de savoirs spécialisés et spatialisés, qui éclairent le processus de codification des pratiques et donc la naissance de l’urbanisme sécuritaire institutionnalisé au sein de la métropole.Le détour comparatif avec Delhi a été l’occasion de valider que ces résultats avaient une portée générale cumulable, tout en délocalisant le regard. / The post-apartheid urban policies of the last decades aiming at “rebuilding” the nation, have not led to reducing spatial injustice in Gauteng, and Johannesburg in particular. Spurred on by private actors, bottom-of-the-range closed residences, which symbolise the upward social mobility of the middle class, spread new forms of relegation and fragmentation, thereby challenging social links, urban sprawl and growing mobility. The little research carried out on this “average” city, which is meant to be synonymous with progress and modernity, is an opportunity to develop new thoughts on existing interrelations between urban production, practices and territories.Far from being monolithic, bottom-of-the-range housing is being developed through many specific and geographically located institutional arrangements. The role played by private actors involved in urban governance, which is often largely unknown and rarely being studied, has become the keystone of the city’s contemporary transformations. The novelty behind this research work is that it reveals the principles of what constitutes specialised and spatialized expertise, thereby shedding light on the codification process of practices and, as such, the birth of institutionalised security town planning within the metropolis.Comparing Johannesburg with Delhi was an opportunity to validate the fact that these results could be significantly drawn concurrently, while studying two different sites

在這篇研究朵莉絲．萊辛＜暴力之子＞系列第五冊＜四門之城＞裡空間和心靈互動關係的論文中，筆者所要探討的主題有二，第一點旨在分析作者如何透過空間意象呈現角色的內在心理狀態；第二點所要探討的是主角在城市空間和建築空間中的漫遊如何象徵其對心靈統合的追尋。以空間和心靈間的互動關係為主軸，筆者認為空間意象乃是內在心靈活動和外在環境刺激的語言化結晶，而空間意象所呈現的將不只是外在的物理表象更包含了小說人物的內在心理實態。於是本論文試圖以空間意象出發，探討如何以此些意象和容格的個體化理論說明主角的心靈旅行。 第一章，我將分析都市空間的主要兩個意象，疆界(boundary)與層疊漫渙(palimpsest)，以及主角瑪莎與心理原型面具(the archetype persona)的遭逢。在此我將探討主角作為一個城市的觀察者和批判者如何察覺那些可見或不可見分隔都市空間的疆界。同時也將說明主角在面對面具原型時如何解決內在退縮和成長的衝突。在下兩章，我將討論兩個建築空間和主角內在心靈的關係。首先，在傑克的房子裡，主角探索記憶和身體的連結並初探被深深壓抑的自我仇恨。其後，在科利奇的豪宅，主角則把自我狀態從防衛性的貝殼意象轉化為開放空間裡的流動中心點。同時，主角亦透過潛入瘋狂和內在暗影(the archetype shadow)的經驗，習得如何面對暗影與探掘其心靈的潛意識。 / In this study of Doris Lessing’s fifth and final volume of her Children of Violence series, The Four-Gated City, I would like to explore how the spatial imagery illustrates the inner landscape of the protagonist and how the protagonist’s wandering journey in the urban and architectural space symbolizes her inner quest for psychic integration. With an emphasis on the interchange between space and psyche, the spatial imagery is read as linguistic crystallization of psychic activities and environmental stimuli, which mirrors not only the physical appearance of the surroundings but also the psychological reality of the characters. As a result, with this spatial imagery as point of departure, I will also employ Jung’s individuation theory to account for the protagonist’s psychic journey. In the first chapter, I examine the two major urban spatial images, the boundary and palimpsest, and Martha’s encounter with the archetype persona. In this part, I would like to present the protagonist, Martha Quest, as an astute city observer and critic, who perceives the visible and invisible boundaries demarcating the urban space. I will also explicate how Martha is brought to confront the archetype persona and resolves the tension between regressive impulse and inner urge for growth. In the following two chapters, I will probe into how two architectures symbolize Martha’s intrapsychic space. In Jack’s house, Martha explores the nexus of body and memory and intimates the repressed self-hater. In Coldridge’s grand mansion she transforms her self-image from a defensive “shell” into a circulating center in a fluid open space. Then, in a symbolic descent to the madness and the inner shadow, Martha learns to confront the inner shadow and explore this unconscious aspect of her psyche.

Advances in digital signal processing (DSP) and computing technology have resulted in the emergence of increasingly adaptive radar systems. It is clear that the Electronic Attack (EA), or jamming, of such radar systems is expected to become a more difficult task. The reason for this research was to address the issue of jamming adaptive radar systems. This required consideration of adaptive jamming systems and the development of a methodology for outlining the features of such a system is proposed as the key contribution of this thesis. For the first time, game-based optimization methods have been applied to a maritime counter-surveillance/counter-targeting scenario involving conventional, as well as so-called ‘smart’ noise jamming.Conventional noise jamming methods feature prominently in the origins of radar electronic warfare, and are still widely implemented. They have been well studied, and are important for comparisons with coherent jamming techniques.Moreover, noise jamming is more readily applied with limited information support and is therefore germane to the problem of jamming adaptive radars; during theearly stages when the jammer tries to learn about the radar’s parameters and its own optimal actions.A radar and a jammer were considered as informed opponents ‘playing’ in a non-cooperative two-player, zero-sum game. The effects of jamming on the target detection performance of a radar using Constant False Alarm Rate (CFAR)processing were analyzed using a game theoretic approach for three cases: (1) Ungated Range Noise (URN), (2) Range-Gated Noise (RGN) and (3) False-Target (FT) jamming.Assuming a Swerling type II target in the presence of Rayleigh-distributed clutter, utility functions were described for Cell-Averaging (CA) and Order Statistic (OS) CFAR processors and the three cases of jamming. The analyses included optimizations of these utility functions, subject to certain constraints, with respectto control variables (strategies) in the jammer, such as jammer power and spatial extent of jamming, and control variables in the radar, such as threshold parameter and reference window size. The utility functions were evaluated over the players’ strategy sets and the resulting matrix-form games were solved for the optimal or ‘best response’ strategies of both the jammer and the radar.

Ion channels are fundamental molecules in the nervous system that catalyze the flux of ions across the cell membrane. There are mounting evidences suggesting that the kinetic properties of ion channels undergo activity-dependent changes in various pathophysiological conditions. Here such activity-dependent changes were studied in case of two different ion channels; the rat brain derived voltage-gated Na+ channel, rNav1.2 and the human background leak K+ channel, hTREK1 using the single channel patch-clamp technique. Our results on the voltage-gated Na+ channel (Chapter III) illustrated that sustained membrane depolarization, as seen in pathophysiological conditions like epilepsy, induced a defined non-linear variation in the unitary conductance, activation, inactivation and recovery kinetic properties of the channel. Signal processing tools attributed a pseudo-oscillatory nature to the non-linearity observed in the channel properties. Prolonged membrane depolarization also induced a “molecular memory” phenomenon, characterized by clustering of dwell time events and strong autocorrelation in the dwell time series. The persistence of such molecular memory was found to be dependent on the duration of depolarization. Similar plastic changes were observed in case of the hTREK1 channel in presence of saturating concentrations of agonist, trichloroethanol (TCE) (Chapter IV). TREK1 channel behaves similar to single enzyme molecules with a single binding site for the substrate K+ ion whereas TCE acts as an allosteric activator of the channel. We observed that with increasing concentration of TCE (10 M to 10 mM) the catalytic turnover rate exhibited progressive departure from monoexponential to multi-exponential distribution suggesting the presence of ‘dynamic disorder’ analogous to single enzyme molecules. In addition, we observed the induction of strong correlation in successive waiting times and flux intensities, exemplified by distinct mode switching between high and low flux activity, which implied the induction of memory in single ion channel. Our observation of such molecular memory in two different ion channels in different experimental conditions highlights the importance and generality of the phenomenon which is normally hidden under the ensemble behaviour of ion channels. In the final part of the work (chapter V) we observed strong negative cooperativity and half-of-sites saturation kinetics in the interaction of local anesthetic, lidocaine with hTREK1 channel. We also mapped the specific anesthetic binding site in the c-terminal domain of the channel. Further, single channel analysis and the heterodimer studies enabled us to propose a model for this interaction and provide a plausible paradigm for the inhibitory action of lidocaine on hTREK1.

Over the last few decades, computer simulation techniques have been established as an essential tool for understanding biochemical processes. This thesis deals mainly with the application of free energy calculations to ribosomal complexes and a cardiac ion channel. The linear interaction energy (LIE) method is used to explore the energetic properties of the essential process of codon–anticodon recognition on the ribosome. The calculations show the structural and energetic consequences and effects of first, second, and third position mismatches in the ribosomal decoding center. Recognition of stop codons by ribosomal termination complexes is fundamentally different from sense codon recognition. Free energy perturbation simulations are used to study the detailed energetics of stop codon recognition by the bacterial ribosomal release factors RF1 and RF2. The calculations explain the vastly different responses to third codon position A to G substitutions by RF1 and RF2. Also, previously unknown highly specific water interactions are identified. The GGQ loop of ribosomal RFs is essential for its hydrolytic activity and contains a universally methylated glutamine residue. The structural effect of this methylation is investigated. The results strongly suggest that the methylation has no effect on the intrinsic conformation of the GGQ loop, and, thus, that its sole purpose is to enhance interactions in the ribosomal termination complex. A first microscopic, atomic level, analysis of blocker binding to the pharmaceutically interesting potassium ion channel Kv1.5 is presented. A previously unknown uniform binding mode is identified, and experimental binding data is accurately reproduced. Furthermore, problems associated with pharmacophore models based on minimized gas phase ligand conformations are highlighted. Generalized Born and Poisson–Boltzmann continuum models are incorporated into the LIE method to enable implicit treatment of solvent, in an effort to improve speed and convergence. The methods are evaluated and validated using a set of plasmepsin II inhibitors.

Les ataxies épisodiques (EA) d’origine génétique sont un groupe de maladies possédant un phénotype et génotype hétérogènes, mais ont en commun la caractéristique d’un dysfonctionnement cérébelleux intermittent. Les EA de type 1 et 2 sont les plus largement reconnues des ataxies épisodiques autosomiques dominantes et sont causées par un dysfonctionnement des canaux ioniques voltage-dépendants dans les neurones. La présente étude se concentrera sur les mutations causant l'EA-1, retrouvées dans le senseur de voltage (VSD) de Kv1.1, un canal très proche de la famille des canaux Shaker. Nous avons caractérisé les propriétés électrophysiologiques de six mutations différentes à la position F244 et partiellement celles des mutations T284 A/M, R297 K/Q/A/H, I320T, L375F, L399I et S412 C/I dans la séquence du Shaker grâce à la technique du ‘’cut open voltage clamp’’ (COVC). Les mutations de la position F244 situées sur le S1 du canal Shaker sont caractérisées par un décalement des courbes QV et GV vers des potentiels dépolarisants et modifient le couplage fonctionnel entre le domaine VSD et le pore. Un courant de fuite est observé durant la phase d'activation des courants transitoires et peut être éliminé par l'application du 4-AP (4-aminopyridine) ou la réinsertion de l'inactivation de type N mais pas par le TEA (tétraéthylamonium). Dans le but de mieux comprendre les mécanismes moléculaires responsables de la stabilisation d’un état intermédiaire, nous avons étudié séparément la neutralisation des trois premières charges positives du S4 (R1Q, R2Q et R3Q). Il en est ressorti l’existence d’une interaction entre R2 et F244. Une seconde interface entre S1 et le pore proche de la surface extracellulaire agissant comme un second point d'ancrage et responsable des courants de fuite a été mis en lumière. Les résultats suggèrent une anomalie du fonctionnement du VSD empêchant la repolarisation normale de la membrane des cellules nerveuses affectées à la suite d'un potentiel d'action. / The genetic episodic ataxias form a group of disorders with heterogeneous phenotype and genotype, but share the common feature of intermittent cerebellar dysfunction. Episodic ataxia (EA) types 1 and 2 are most widely recognised amongst the autosomal dominant episodic ataxias and are caused by dysfunction of neuronal voltage-gated ion channels. The present study focuses on mutations causing EA-1 located in the voltage sensor domains (VSDs) of Kv1.1. A member of the Shaker channel family. Here, we have characterised the electrophysiological properties of six different mutations at the position of F244 and we also reported the partiality effects of these following mutations T284A/M, R297K/Q/A/H, I320T, L375F, L399I S412C/I on Shaker sequence using the cut open voltage clamp technique (COVC). We have shown that mutations of F244 in the S1 of the Shaker Kv channel positively shift the voltage dependence of the VSD movement and alter functional coupling between VSD and pore domain. The mutations causing immobilization of the VSD movement during activation and deactivation and responsible for creating a leak current during activation, are removed by the application of 4-AP (4-aminopyridine) or by reinsertion of N-type inactivation but not by TEA (tetraethylamonium). Insights into the molecular mechanisms responsible for the stabilization of the intermediate state have been investigated by separately neutralizing the first three charges (R1Q, R2Q and R3Q) in the S4 segment. The result suggests an interaction between R2 and F244 mutants. It was established that a second co-evolved interface exists between S1 and the pore helix near the extracellular surface and it acts as a second anchor point. It is also responsible for generation of leak currents. The results suggest a dysfunction of the VSD in which the affected nerve cells cannot efficiently repolarize following an action potential because of altered delayed rectifier function