Il est possible de perméabiliser la membrane plasmique des cellules par des agents chimiques (tels que les polyéthylènes glycols ou le diméthylsulfoxyde) ou par des agents physiques (tels que les ultrasons ou les impulsions électriques). Cette perméabilisation peut être réversible ou non, ce qui signifie qu’après la perméabilisation, la membrane retrouve son intégrité et ses propriétés d’hémi-perméabilité ou pas. Ces techniques peuvent être utilisées pour faire rentrer des médicaments ou des acides nucléiques dans les cellules ou pour générer des fusions cellulaires. Une approche récente, la dynamique moléculaire, utilise des simulations numériques pour prédire les effets des agents perméabilisants sur les membranes à l’échelle moléculaire, et permet d’apporter de nouvelles données pour comprendre les mécanismes moléculaires, encore peu connus à ce jour.Les impulsions dites « classiques » en électroperméabilisation, de l’ordre de la dizaine de millisecondes à la centaine de microsecondes et d’amplitude de champ de l’ordre de 100 kV/m, perméabilisent la membrane plasmique uniquement. Cependant, récemment, des impulsions plus courtes, dites impulsions nanoseconde (quelques nanosecondes) et de plus grande amplitude de champ (de l’ordre de 10 MV/m) ont été utilisées et permettent d’affecter également les membranes des organites cellulaires. Les travaux de cette thèse portent dans un premier temps sur les effets perméabilisants d’un agent chimique (le diméthylsulfoxyde, DMSO) en comparant les modèles prédictifs de la dynamique moléculaire avec des expériences in vitro sur des cellules. Le modèle numérique prédit trois régimes d’action en fonction de la concentration du DMSO. Utilisé à faible concentration, il y a déformation de la membrane plasmique. L’utilisation d’une concentration intermédiaire entraîne la formation de pores membranaires et les fortes concentrations de DMSO ont pour conséquence la destruction de la membrane. Les expériences in vitro faites sur des cellules ont confirmé ces résultats en suivant l’entrée de marqueurs de perméabilisation. Cette étude a été comparée avec la perméabilisation par un agent physique (les impulsions électriques). Dans un deuxième temps, ces travaux traitent du développement et de l’utilisation d’un nouveau dispositif d’exposition des cellules aux impulsions nanoseconde qui permet d’appliquer des champs électriques très élevés et d’observer par microscopie leurs au niveau cellulaire. Pour finir, ce dispositif a été utilisé avec des impulsions nanoseconde pour générer des pics calciques dans de cellules souches mésenchymateuses qui présentent des oscillations calciques spontanées liées à leur état de différenciation. Ces pics induits sont dus à la libération de calcium stocké dans les organites et/ou à la perméabilisation de la membrane plasmique permettant l’établissement d’un flux de calcium intramembranaire. Il est aussi possible d’utiliser des impulsions microseconde pour générer des pics calciques dans ces cellules. Dans ce cas, les pics calciques ne sont dus qu’à la perméabilisation de la membrane plasmique. En jouant sur l’amplitude des champs électriques appliqués et sur la présence ou l’absence de calcium externe, il est possible de manipuler les concentrations calciques cytosoliques en mobilisant le calcium interne ou externe. Une des particularités de ces nouveaux outils est de pouvoir être déclenchés et arrêtés instantanément, sans réminiscence, contrairement aux molécules chimiques permettant de produire des pics calciques. Ces outils pourraient donc permettre de mieux comprendre l’implication du calcium dans des mécanismes comme la différenciation, la migration ou la fécondation. / It is possible to permeabilize the cellular plasma membrane by using chemical agents (as polyethylen glycols or diméthylsulfoxyde) or physical agents (as ulstrasounds or electric pulses). This permeabilization can be reversible or not, meaning that after the permeabilization, the membrane recovers its integrity and its hemi-permeable properties. These techniques can be used for the uptake of medicines or nucleic acids or to generate cellular fusions. A recent approach, the molecular dynamics, uses numerical simulations to predict the effects of permeabilizing agents at the molecular scale, allowed generating of new data to understand the molecular mechanisms that are not completely known yet.The pulses so called “classical” in electropermeabilization, from the range of the ten of milliseconds to the hundred of microseconds and with a field amplitude in the range of 100 kV/m, can only permeabilize the plasma membrane. However, more recently, shorter pulses, so called nanopulses (few nanosecondes) and with an higher field amplitude (in the range of 10 MV/m) have been used and allow to affect also cellular organelles membranes.This thesis is, in a first time, about the permeabilizing effects of a chemical gent (the diméthylsulfoxyde, DMSO) by comparing predictive models from molecular dynamics with experiments in vitro on cells. The numerical model predicts three regimes of action depending on the DMSO concentration. Used at low concentration, there is a plasma membrane deformation. The use of an intermediate concentration lead to membrane pores formation and higher DMSO concentrations resulted in membrane destruction. The experiments done in vitro on cells confirmed these results using the following of permeabilization markers. This study has been compared to permeabilization due to a physical agent (electric pulses).Secondly, it is about the development and the use of a new cell exposure device for nanopulses that permit to apply very high electric fields and to observe induced cellular effects simultaneously by microscopy.To finish, this device has been used with nanopulses to generate calcium peaks in mesenchymal stem cells that are presenting spontaneous calcium oscillations in correlation to their differentiation state.. These induced peaks are due to the release of the calcium stored in organelles and/or to plasma membrane permeabilization leading to a intramembrane calcium flux establishment. It is also possible to use microsecond pulses to generate calcium peaks in these cells. In this case, the calcium peaks are due to the plasma membrane permeabilization . By changing the amplitude of the applied electric fields and the presence or the absence of external calcium, it is possible to manipulate cytosolic calcium concentrations by mobilizing internal or external calcium. One feature of these new tools is to be triggered and stopped instantly without reminiscence, unlike chemical molecules permitting the production of calcium peaks. These tools could therefore lead to a better understanding of the involvement of calcium in mechanisms such as differentiation, migration or fertilization.

Made available in DSpace on 2016-12-12T20:27:39Z (GMT). No. of bitstreams: 1 Heric Denis Farias.pdf: 2196819 bytes, checksum: ebe776ba26727b6ab81d9c4b1fb49e5d (MD5) Previous issue date: 2016-09-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The application of high electric fields on biological cells causes the formationof pores in the cell membrane, thereby causing an increase in their permeability. This phenomenon, called electropermeabilization have attracted increasing attention due to its wide application in biotechnology. Even being known for decades, the pore opening process in biological cell membranes is still not fully understood, nor was it even properly modeled. In this paper, two types of modeling are presented, which allow characterizing the electropermeabilization in biological cell suspensions. One of the methods is based on the analysis of the electrical impedance spectra of suspensions using genetic algorithm to determine parameters of a generic model dielectric dispersion. The other method uses instantaneous voltage and current values applied to a cell suspension during the electroporation experiment to determine the variation of the medium conductivity and thus, through the analytical model proposed by Ramos and colleagues (RAMOS et al., 2012), determine the cell conductivities. By modeling the impedance spectrum, it was observed the change in the dielectric dispersion of the sample due to the electropermeabilization process, in addition to obtaining the electrical conductivity and permittivity of the involved media. Using the electroporated cell model proposed by Ramos and colleagues (RAMOS et al., 2012), it was possible to determine the change of membrane conductance during the electropermeabilization. The validity of this model is assessed using finite element simulations, which showed good agreement with the analytical model used. Genetic algorithms are used in obtaining the parameters of the various models presented, showing great robustness in obtaining parameters based on the git between experimental and theoreticalcurves. / A aplicação de campos elétricos intensos em células biológicas provoca a formação de poros na membrana celular, causando assim o aumento de sua permeabilidade. Este fenômeno, denominado de eletropermeabiliza ção têm atraído cada vez mais atenção devido a sua ampla aplicação em biotecnologia. Mesmo sendo conhecido há várias décadas, o processo de abertura de poros em membranas de células biológicas ainda não é plenamente entendido e nem foi ainda corretamente modelado. Neste trabalho, apresenta-se dois tipos de modelagem que permitem a caracterização da eletropermeabilização em suspensões de células biológicas. Um dos métodos baseia-se na análise do espectro de impedância elétrica de suspensões com o uso de algoritmo genético para determinar parâmetros de um modelo genérico de dispersão dielétrica. O outro método utiliza valores instantâneos de tensão e corrente aplicados em uma suspensão de células durante um experimento de eletropermeabiliza ção para determinar a variação da condutividade do meio e com isso, através do modelo analítico proposto por Ramos e colaboradores (RAMOS et al., 2012), determinar a condutividade das células. Através da modelagem do espectro de impedância, foi possível verificar a alteração da dispersão dielétrica da amostra devido ao processo de eletropermeabiliza ção, além da obtenção das condutividades e permissividades elétricas dos meios envolvidos. Utilizando o modelo de célula eletropermeabilizada proposto por Ramos e colegas (RAMOS et al., 2012), foi possível obter a variação da condutância de membrana durante a eletropermeabilização. A validade deste modelo é avaliada utilizando simulações em elementos infinitos, as quais apresentaram grande concordância com o modelo analítico utilizado. Algoritmos genéticos são utilizados na obtenção dos parâmetros dos diversos modelos apresentados, mostrando grande robustez na obtenção de parâmetros baseada no ajuste entre curvas experimentais e teóricas.

Made available in DSpace on 2016-12-12T17:38:36Z (GMT). No. of bitstreams: 1 Carlos Toshiyuki Matsumi.pdf: 2530177 bytes, checksum: d45251b727305a9062002cf939cb57eb (MD5) Previous issue date: 2009-07-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Electropermeabilization is the process of transient increase in the permeability of biological membranes of cells subjected to intense electric fields. This technique is currently still in development and has important clinical and technological applications such as electrochemotherapy and gene transfer. Electroporator is the equipment used in the generation and application of such intense fields. This dissertation presents the design, construction and testing of an electroporator for use in biomedical research. The proposed equipment presents versatility and performance appropriate to allow for different types of experiments with biological tissues or cell suspensions. It consists of a voltage generator with programmable wave form, a high voltage amplifier with high output current capability and a system for transduction of voltage and current in the sample. Both the generation of signal as the measured values in the load are monitored by a program built in LabVIEW® environment that triggers a data acquisition card with 16 bits of resolution. The performance of the electronic system developed fully meets the requirements of project. The amplifier can deliver up to 500V and 5A to the load during a time interval enough for the testing of electropermeabilization. The bandwidth, slightly higher than 100kHz and the step response time of the order of 1μs are suitable for performing experiments with different waveforms and different sizes of cells. An important application of the equipment built is demonstrated with an experiment of electropermeabilization in suspension of red cells of rats. This result demonstrated the occurrence of increased conductivity of the sample during stimulation with high-intensity electric field. / Eletropermeabilização é o processo de aumento transitório da permeabilidade das membranas de células biológicas submetidas a campos elétricos intensos. Esta é uma técnica atualmente ainda em desenvolvimento e que possui importantes aplicações clínicas e tecnológicas tais como a eletro quimioterapia e a transferência genética. Os eletropermeabilizadores são equipamentos usados na geração e aplicação desses campos intensos. Esta dissertação apresenta o projeto, construção e teste de um eletropermeabilizador para uso em pesquisa biomédica. O equipamento proposto apresenta versatilidade e desempenho adequados para permitir a realização de diferentes tipos de ensaios com tecidos biológicos ou suspensões de células. É constituído de um gerador de tensão com forma de onda programável, um amplificador de alta tensão e alta corrente de saída e um sistema de transdução de tensão e corrente na amostra analisada. Tanto a geração de sinal quanto os valores medidos na carga são monitorados por um programa construído em ambiente LabVIEW® que aciona uma placa de aquisição de dados com 16 bits de resolução. O desempenho do sistema eletrônico desenvolvido atende completamente os requisitos de projeto. O amplificador pode fornecer até 500V de amplitude de tensão com 5A de corrente de carga durante intervalos de tempo suficientes para os ensaios de eletropermeabilização. A banda passante pouco maior que 100KHz e os tempos de resposta ao degrau da ordem de 1μs são adequados para a realização de experimentos com diferentes formas de onda e diferentes tamanhos de células. Uma importante aplicação do equipamento construído é exemplificada com um experimento de eletropermeabilização em suspensão de hemácias de rato, sendo demonstrada a ocorrência de aumento da condutividade da amostra durante a estimulação com campo elétrico de alta intensidade.