Ever since the discovery of DNA, there has been many pathologies identified effecting mankind. With the development in technology, there are many methods to alleviate these pathologies. One such is gene therapy or gene delivery. It is a process of introducing some foreign material into the body to correct the effected cells. In principle, it is a modern method to cure cells or a method to transfer nucleic acid into a cell to treat specific cells in the body. The process of delivering a genetic material is carried out using vectors, namely, viral vectors and non-viral vectors. In viral vectors, viruses are modified to make it efficient for delivery into the host cells. This method has high transduction rate as compared to non-viral method. Non-viral methods include chemical and physical transfection methods, which are used to deliver the gene of interest into the host cell unlike viral methods. In this study, a physical method using high voltage is used to deliver a genetic material into cells. High voltages are used to permeabilize the cell to allow the foreign material into it and to express it in the host cell. This process is termed as Electroporation. In specific, in this research, studying a process of charging a region that mimics skin and trying to localize the presence of electric fields on the surface where the strongest uptake of genetic material is found. In other words, region where the gene expression is strongest at a specific region if performed on skin is studied by localizing electric fields on the surfaces. My work is to characterize and develop where this effect takes place on the surface based on both positive and negative electric fields. A physical method is useful as it is a non-toxic way to get a DNA/protein into someone’s body without making them sick, unless if not using a virus to deliver. This is all done using high voltages up to 8kV and the electric fields produced due to high voltages are localized, visualized and characterized with both positive and negative polarities of voltages. In this study, experiments with high voltages are performed and the spread of charges at specific regions are collected using a needle. This needle goes into corona, which may be called as a secondary corona. It might be called a secondary corona because the flat conductor is being charged by a metal finger but not directly by the power supply. Here, the conductor is charged by a metal finger of high input voltage, which ionizes the air molecules above the flat conductor to form a conductive region. As the input voltage is increased further, electrons escape from the needle to air or from molecules to needle forming negative or positive ions respectively. The outputs at needle were measured on the oscilloscope. In this study, repeated sets of experiments are carried out to collect consistent and reliable data. Visualizing/characterizing these fields are important as maximum delivery takes place at high voltage regions, with a condition that permeability of the cells should be known for proper transfection to occur, otherwise cells would die due to high voltages or no transfection takes place due to poor permeability of cell membrane.

In the last decade, a tremendous gain in knowledge concerning the molecular events of apoptosis signaling and execution has been achieved. The aim of this thesis was to clarify the role of lysosomal membrane permeabilization and lysosomal proteases, cathepsins, in signaling for apoptosis. We identified cathepsin D as an important factor in staurosporine-induced human fibroblast cell death. After release to the cytosol, cathepsin D promoted mitochondrial release of cytochrome c by proteolytic activation of Bid. Cathepsin D-mediated cleavage of Bid generated two fragments with the apparent molecular mass of 15 and 19 kDa. By sequence analysis, three cathepsin D-specific cleavage sites, Phe24, Trp48, and Phe183, were identified. Moreover, we investigated the mechanism by which cathepsins escape the lysosomal compartment, and found that Bax is translocated from the cytosol to lysosomes upon staurosporine treatment. In agreement with these data, recombinant Bax triggered release of cathepsins from isolated rat liver lysosomes. Conceivably, the Bcl-2 family of proteins may govern release of pro-apoptotic factors from both lysosomes and mitochondria. The importance of lysosomal cathepsins in apoptosis signaling was studied also in oral squamous cell carcinoma cells following exposure to the redox-cycling drug naphthazarin or agonistic anti-Fas antibodies. In this experimental system, cathepsins were released to the cytosol, however, inhibition of neither cathepsin D, nor cysteine cathepsin activity suppressed cell death. Interestingly, cysteine cathepsins still appeared to be involved in activation of the caspase cascade. Cathepsins are often overexpressed and secreted by cancer cells, and it has been reported that extracellular cathepsins promote tumor growth and metastasis. Here, we propose that cathepsin B secreted from cancer cells may suppress cancer cell death by shedding of the Fas death receptor. Defects in the regulation of apoptosis contribute to a wide variety of diseases, such as cancer, neurodegeneration and autoimmunity. Increased knowledge of the molecular details of apoptosis could lead to novel, more effective, treatments for these illnesses. This thesis emphasizes the importance of the lysosomal death pathway, which is a promising target for future therapeutic intervention. / In the last decade, a tremendous gain in knowledge concerning the molecular events of apoptosis signaling and execution has been achieved. The aim of this thesis was to clarify the role of lysosomal membrane permeabilization and lysosomal proteases, cathepsins, in signaling for apoptosis. We identified cathepsin D as an important factor in staurosporine-induced human fibroblast cell death. After release to the cytosol, cathepsin D promoted mitochondrial release of cytochrome c by proteolytic activation of Bid. Cathepsin D-mediated cleavage of Bid generated two fragments with the apparent molecular mass of 15 and 19 kDa. By sequence analysis, three cathepsin D-specific cleavage sites, Phe24, Trp48, and Phe183, were identified. Moreover, we investigated the mechanism by which cathepsins escape the lysosomal compartment, and found that Bax is translocated from the cytosol to lysosomes upon staurosporine treatment. In agreement with these data, recombinant Bax triggered release of cathepsins from isolated rat liver lysosomes. Conceivably, the Bcl-2 family of proteins may govern release of pro-apoptotic factors from both lysosomes and mitochondria. The importance of lysosomal cathepsins in apoptosis signaling was studied also in oral squamous cell carcinoma cells following exposure to the redox-cycling drug naphthazarin or agonistic anti-Fas antibodies. In this experimental system, cathepsins were released to the cytosol, however, inhibition of neither cathepsin D, nor cysteine cathepsin activity suppressed cell death. Interestingly, cysteine cathepsins still appeared to be involved in activation of the caspase cascade. Cathepsins are often overexpressed and secreted by cancer cells, and it has been reported that extracellular cathepsins promote tumor growth and metastasis. Here, we propose that cathepsin B secreted from cancer cells may suppress cancer cell death by shedding of the Fas death receptor. Defects in the regulation of apoptosis contribute to a wide variety of diseases, such as cancer, neurodegeneration and autoimmunity. Increased knowledge of the molecular details of apoptosis could lead to novel, more effective, treatments for these illnesses. This thesis emphasizes the importance of the lysosomal death pathway, which is a promising target for future therapeutic intervention.

Electric field mediated gene delivery modalities have preferable safety profiles with the ability to rapidly transfect cells in vitro and in vivo with high efficiency. However, the current state of the art has relied on trial and error studies that target the average cell within a population present in treated tissue to derive electric pulse parameters. This results in fixed gene electrotransfer (GET) parameters that are not universally optimum. Slow progress towards the validation of a mechanism that explains this phenomena has also hindered its advancement in the clinic. To date, GET methods utilizing feedback control as a means to optimize doses of electric field stimulation have not been investigated. However, with modern electric components the electric characteristics of tissue exposed to electric pulses can be measured in very short time scales allowing for a near instantaneous assessment of the effect these pulses have on cells and tissue. This information is ideal for use in optimizing GET parameters to ensure the conditions necessary for gene delivery can be created regardless of anisotropic tissue architecture and electrode geometry. Bioimpedance theory draws parallels between cell structures and circuit components in an attempt to use circuit theory to describe changes occurring at a cellular and tissue level. In short, a reduction in tissue impedance indicates a reduction to the opposition of current flow in a volume conductor indicating new pathways for current. It has been purported these new pathways exist in the cell membrane and indicate a degree of membrane permeability/destabilization that either indicates or facilitates the uptake of exogenous molecules, such as nucleic acids or plasmid DNA. This study evaluated the use of relative impedance changes from 10 Hz – 10 kHz that occur in tissue before and after GET to indicate relative increase in tissue and membrane permeability. An optimum reduction in impedance was then identified as an indicator of the degree of membrane permeability required to significantly enhance exogenous DNA uptake into cells. This study showed the use of impedance-based feedback control to optimize GET pulse number in real time to target 80% or 95% reduction in tissue impedance resulted in an 12 and 14 fold increase in transgene expression over controls and a 6 and 7 fold increase in transgene expression over fixed pulse open loop protocols.

This thesis describes the use of antibodies and immunofluorescence for subcellular localization of proteins. The key objective is the creation of an open-source atlas with information on the subcellular location of every human protein. Knowledge of the spatial distribution and the precise location of a protein within a cell is important for its functional characterization, and describing the human proteome in terms of compartment proteomes is important to decipher cellular organization and function.   Immunofluorescence and confocal microscopy of cultured cells were used for high-resolution detection of proteins on a high-throughput scale. Critical to immunofluorescence results are sample preparation and specific antibodies. Antibody staining of cells requires fixation and permeabilization, both of which can result in loss or redistribution of proteins and masking of epitopes. A high-throughput approach demands a standardized protocol suitable for the majority of proteins across cellular compartments. Paper I presents an evaluation of sample preparation techniques from which such a single fixation and permeabilization protocol was optimized. Paper II describes the results from applying this protocol to 4000 human proteins in three cell lines of different origin.   Paper III presents a strategy for application-specific antibody validation. Antibodies are the key reagents in immunofluorescence, but all antibodies have potential for off-target binding and should be validated thoroughly. Antibody performance varies across sample types and applications due to the competition present and the effect of the sample preparation on antigen accessibility. In this paper application-specific validation for immunofluorescence was conducted using colocalization with fluorescently tagged protein in transgenic cell lines. / <p>QC 20160509</p>

Les ondes térahertz s’étendent de 0.1 à 10x1012 Hz, à la frontière entre les domaines de l’optique et des radiofréquences. Cette position intermédiaire originale en a longtemps rendu l’accès difficile : les technologies térahertz n’ont pris leur essor qu’au cours des années 90. Le domaine n’a pas encore atteint la maturité des domaines des microondes ou de l’infrarouge qui le jouxtent. Cependant, les motivations exploratoires sont fortes, de par la sensibilité spectroscopique du térahertz aux états moléculaires (rotationnels, vibrationnels..) et aux liaisons faibles établies dans et entre les molécules. Dans le cas des objets biologiques, le térahertz est particulièrement sensible à l’eau : sa quantité, son état physico-chimique et ses solutés.Nous avons mis en œuvre un montage d’imagerie en réflexion interne totale atténuée (ATR) pour pouvoir distinguer des cellules vivantes de leur milieu physiologique. Au cours de ce travail, le montage d’imagerie ATR a été caractérisé théoriquement, puis expérimentalement. La première démonstration de l’origine du contraste sur ces images térahertz a été réalisée. Il provient du contenu intracellulaire, plus spécifiquement des protéines et peptides dissouts dans le cytoplasme.Une analyse fine des mécanismes sous-jacents à la nature protéique du contraste térahertz a également été développée. Elle donne accès à des informations spectroscopiques inédites sur l’eau, les protéines dissoutes et la couche de solvatation les entourant.Mettant à profit cette compréhension de notre montage térahertz, nous l’avons proposé comme outil non invasif de suivi quantitatif de la perméabilisation de cellules en conditions physiologiques. Lors de la perméabilisation, augmentation des transferts moléculaires à travers la membrane, notre outil permet de quantifier le passage des peptides et protéines. La perméabilisation de cellules vivantes a une gamme d’application vaste, de l’entrée de fluorochromes pour l’imagerie ou de médicaments à la thérapie génique. Afin d’assurer ces passages à travers la membrane des cellules, il est nécessaire d’altérer ses propriétés, sans pour autant compromettre la viabilité cellulaire. L’étude de deux types de perméabilisation avec notre outil térahertz est proposée : la perméabilisation chimique et l’électroporation. Dans les deux cas, des mécanismes d’effet dose ont été caractérisés quantitativement. Notre outil térahertz a démontré de grands avantages devant les méthodes actuellement utilisées pour quantifier ces dynamiques de perméabilisation et en caractériser la réversibilité. / Lying between 0.1 to 10x1012 Hz, the terahertz radiation occupies a middle ground between microwaves and infrared light waves, sometimes named “the terahertz gap” for technologies relevant to generation and detection have only risen at the beginning of the 90’s and aren’t fully developed yet. Nevertheless, there are strong exploratory incentives because of terahertz spectroscopic sensitivity to molecular states (rotational, vibrational…) and weak bounds in and between molecules. In the case of biological object, terahertz waves are especially sensitive to water: its quantity, physico-chemical state and solutes. We implemented an Attenuated Total internal Reflection (ATR) imaging setup in order to distinguish live cells from their physiological bathing medium. Throughout this work, we characterized both experimentally and experimentally the ATR setup. The first demonstration of the contrast origin in the terahertz images obtained was done. It arises from the intracellular content, more specifically the proteins and peptides dissolved in the cytoplasm.A precise analysis of the underlying mechanism of this proteinaceous terahertz contrast has also been developed. It gives access to original spectroscopic information about water, dissolved proteins and the hydration shell around them.Taking advantage of our whole setup comprehension, we proposed it as a non-invasive tool for quantitative live-cell permeabilization assessment in physiological conditions. During permeabilization, aka increased molecular transfers through the cell membrane, our tool allows to quantify the transfer of peptides and proteins. Live-cell permeabilization has a large application range, from fluorochrome entry in imaging, to drugs or gene therapy. In order to ensure molecules crossing the cell membrane, it’s necessary to alter its properties without compromising cell viability.A study of two permeabilization methods is proposed: chemical permeabilization and electroporation. In both cases dose effect mechanisms were quantitatively characterized. Our terahertz tool demonstrated great advantages over classical permeabilization quantification methods and permeabilization reversibility assessment methods.

Utilizada amplamente como agente terapêutico no tratamento da leucemia linfoblástica aguda (LLA), a L-Asparaginase II (ASNase) é uma enzima que atua diminuindo a concentração de asparagina livre no plasma. Dessa forma, impede o fornecimento de asparagina para a proliferação de células malignas, as quais ao contrário das células saudáveis, não conseguem sintetizar a asparagina. A ASNase utilizada atualmente no Brasil é importada, o que gera problemas com custo e abastecimento. Sendo assim, é notavelmente atrativa a procura por sistemas que apresentem níveis elevados de expressão de asparaginase e o encontro de formas de produzir tal enzima para um fácil acesso e, se possível, com menor potencial alérgico. Isso nos incentiva a estudar a produção biotecnológica de ASNase produzida em Escherichia coli BL21 (DE3) recombinante que super expressa esta enzima. O objetivo deste trabalho foi estabelecer, em agitador orbital e sistema descontínuo, os parâmetros do cultivo e indução da bactéria Escherichia coli BL21 visando à produção de ASNase, os quais serão úteis para futuros estudos em sistema descontínuo-alimentado. Nosso trabalho avaliou fatores que influenciam a fase de crescimento e/ou a fase de indução da E. coli BL21 (DE3): meio de cultivo baseado na composição elementar, controle do pH, uso de glicose ou glicerol como fonte de carbono, formação de acetato, tempo inicial e final da indução, permeabilização celular para secreção da ASNase, concentração de indutor, temperatura de pós-indução. Nós apresentamos uma estratégia para produção extracelular de ASNase em E. coli BL21 (DE3) pelo crescimento em meio Luria Bertani (LB) modificado para permeabilização celular. A produtividade volumétrica de ASNase extracelular foi 484 IU L h-1 em agitador orbital, correspondendo a 89 % de secreção após 24h de pós-indução com IPTG a 37 ºC. Isto representou rendimento 50 % maior para a ASNase total e 15,5 vezes mais secreção de ASNase em relação ao uso do meio LB modificado. Entretanto no cultivo em biorreator de 3 L nas mesmas condições (exceto a forma de aeração: 500 rpm de agitação e 1 vvm de vazão de ar, kLa = 88 h-1) operado em regime descontínuo foram obtidos resultados semelhantes aos cultivos em agitador orbital, sendo a produtividade volumétrica da ASNase extracelular igual a 525 IU L h-1 após 20 h de pós-indução. A biomassa obtida para agitador orbital e biorreator foi 3,26 e 2,63 g L-1, respetivamente. Por esse motivo, esses resultados foram considerados promissores para aumentar a produtividade nos futuros ensaios em biorreator operado em regime descontinuo-alimentado. / Widely used as a therapeutic agent in the treatment of acute lymphoblastic leukemia (ALL), L-Asparaginase II (ASNase) is an enzyme that works by reducing the concentration of free asparagine in plasma. Thus, it prevents the delivery of asparagine to the proliferation of malignant cells, which unlike healthy cell, cannot synthesize asparagine. ASNase currently used in Brazil is imported, which causes problems with cost and supply. Thus, the search for systems with high levels of asparaginase expression and the finding of ways to produce this enzyme for easy access and, if possible, with a lower allergic potential, are strikingly attractive. This encourages us to study the biotechnological production of ASNase in recombinant Escherichia coli BL21 (DE3) which super expresses this enzyme. The objective of this work was to establish, in shaker and batch bioreactor system, growth and induction parameters of the Escherichia coli BL21 aiming the production of ASNase, which will be useful for future studies in a fed-batch system. Our work evaluated factors that influenced the growth and induction phase of E. coli BL21 (DE3): culture medium based on elemental composition, pH control, use of glucose or glycerol as carbon source, formation of acetate, initial and final induction time, cellular permeabilization for ASNase secretion, inducer concentration, post-induction temperature. We performed a strategy for extracellular production of ASNase in E. coli BL21 (DE3) by growing in modified Luria Bertani (LB) medium for cell permeabilization. The volumetric productivity of extracellular ASNase was 484 IU L h-1 on shaker, which reached 89% secretion at 24 h of post-induction with IPTG at 37°C. This represented an increase yield of 50 % regarding to the total ASNase formed and 15.5 times the ASNase secretion as compared to that attained with LB modified. While in batch 2L-bioreactor cultivation under the same conditions (except for the aeration employed: 500 rpm of stirring and 1 vvm of air flow, kLa = 88 h-1) it was obtained similar results in relation to shaker cultures. The volumetric productivity of extracellular ASNase was 525 IU L h-1 at 20 h of post-induction. The biomass obtained for shaker and bioreactor were 3.26 and 2.63 g L-1, respectively. For this reason, we consider these promising results to increase productivity in future studies in bioreactor operated as fed-batch regimen.

Utilizada amplamente como agente terapêutico no tratamento da leucemia linfoblástica aguda (LLA), a L-Asparaginase II (ASNase) é uma enzima que atua diminuindo a concentração de asparagina livre no plasma. Dessa forma, impede o fornecimento de asparagina para a proliferação de células malignas, as quais ao contrário das células saudáveis, não conseguem sintetizar a asparagina. A ASNase utilizada atualmente no Brasil é importada, o que gera problemas com custo e abastecimento. Sendo assim, é notavelmente atrativa a procura por sistemas que apresentem níveis elevados de expressão de asparaginase e o encontro de formas de produzir tal enzima para um fácil acesso e, se possível, com menor potencial alérgico. Isso nos incentiva a estudar a produção biotecnológica de ASNase produzida em Escherichia coli BL21 (DE3) recombinante que super expressa esta enzima. O objetivo deste trabalho foi estabelecer, em agitador orbital e sistema descontínuo, os parâmetros do cultivo e indução da bactéria Escherichia coli BL21 visando à produção de ASNase, os quais serão úteis para futuros estudos em sistema descontínuo-alimentado. Nosso trabalho avaliou fatores que influenciam a fase de crescimento e/ou a fase de indução da E. coli BL21 (DE3): meio de cultivo baseado na composição elementar, controle do pH, uso de glicose ou glicerol como fonte de carbono, formação de acetato, tempo inicial e final da indução, permeabilização celular para secreção da ASNase, concentração de indutor, temperatura de pós-indução. Nós apresentamos uma estratégia para produção extracelular de ASNase em E. coli BL21 (DE3) pelo crescimento em meio Luria Bertani (LB) modificado para permeabilização celular. A produtividade volumétrica de ASNase extracelular foi 484 IU L h-1 em agitador orbital, correspondendo a 89 % de secreção após 24h de pós-indução com IPTG a 37 ºC. Isto representou rendimento 50 % maior para a ASNase total e 15,5 vezes mais secreção de ASNase em relação ao uso do meio LB modificado. Entretanto no cultivo em biorreator de 3 L nas mesmas condições (exceto a forma de aeração: 500 rpm de agitação e 1 vvm de vazão de ar, kLa = 88 h-1) operado em regime descontínuo foram obtidos resultados semelhantes aos cultivos em agitador orbital, sendo a produtividade volumétrica da ASNase extracelular igual a 525 IU L h-1 após 20 h de pós-indução. A biomassa obtida para agitador orbital e biorreator foi 3,26 e 2,63 g L-1, respetivamente. Por esse motivo, esses resultados foram considerados promissores para aumentar a produtividade nos futuros ensaios em biorreator operado em regime descontinuo-alimentado. / Widely used as a therapeutic agent in the treatment of acute lymphoblastic leukemia (ALL), L-Asparaginase II (ASNase) is an enzyme that works by reducing the concentration of free asparagine in plasma. Thus, it prevents the delivery of asparagine to the proliferation of malignant cells, which unlike healthy cell, cannot synthesize asparagine. ASNase currently used in Brazil is imported, which causes problems with cost and supply. Thus, the search for systems with high levels of asparaginase expression and the finding of ways to produce this enzyme for easy access and, if possible, with a lower allergic potential, are strikingly attractive. This encourages us to study the biotechnological production of ASNase in recombinant Escherichia coli BL21 (DE3) which super expresses this enzyme. The objective of this work was to establish, in shaker and batch bioreactor system, growth and induction parameters of the Escherichia coli BL21 aiming the production of ASNase, which will be useful for future studies in a fed-batch system. Our work evaluated factors that influenced the growth and induction phase of E. coli BL21 (DE3): culture medium based on elemental composition, pH control, use of glucose or glycerol as carbon source, formation of acetate, initial and final induction time, cellular permeabilization for ASNase secretion, inducer concentration, post-induction temperature. We performed a strategy for extracellular production of ASNase in E. coli BL21 (DE3) by growing in modified Luria Bertani (LB) medium for cell permeabilization. The volumetric productivity of extracellular ASNase was 484 IU L h-1 on shaker, which reached 89% secretion at 24 h of post-induction with IPTG at 37°C. This represented an increase yield of 50 % regarding to the total ASNase formed and 15.5 times the ASNase secretion as compared to that attained with LB modified. While in batch 2L-bioreactor cultivation under the same conditions (except for the aeration employed: 500 rpm of stirring and 1 vvm of air flow, kLa = 88 h-1) it was obtained similar results in relation to shaker cultures. The volumetric productivity of extracellular ASNase was 525 IU L h-1 at 20 h of post-induction. The biomass obtained for shaker and bioreactor were 3.26 and 2.63 g L-1, respectively. For this reason, we consider these promising results to increase productivity in future studies in bioreactor operated as fed-batch regimen.

Los biotensioactivos son compuestos producidos por microorganismos que poseen actividad superficial, baja toxicidad, carácter biodegradable y efectividad a temperaturas y valores de pH extremos. Los ramnolípidos, son un tipo de biotensioactivos con interesantes actividades biológicas, que parecen estar relacionadas con su interacción con las membranas. En esta tesis doctoral se estudia la interacción de la fracción dirramnolipídica purificada de cultivos de Pseudomonas aeruginosa con membranas modelo y membranas biológicas, la interacción con albúmina de suero bovino, utilizada como proteína modelo, y por último, se evalúa el papel de este biotensioactivo en la estabilización de liposomas sensibles a pH y su aplicación en la introducción de compuestos en células animales. Para el desarrollo de este trabajo de investigación se han utilizado técnicas como calorimetría, FT-IR, difracción de rayos X, dispersión de luz dinámica y microscopía electrónica. / Rhamnolipids are bacterial biosurfactants produced by Pseudomonas spp. These compounds have been shown to present several interesting biological activities. It has been suggested that the interaction with the membrane could be the ultimate responsible for these actions. This thesis presents a detailed molecular level study of the dirhamnolipid interactions with artificial phospholipids membranes and biological membranas, and the dirhamnolipid interactions with bovine serum albumin, used as a general globular protein model. Furthemore, this work includes the characterization of phosphatidylethanolamine/diRL pH-sensitive liposomes described as efficient systems for cytoplasmic delivery of foreign compounds into living cells. It has been employed a number of physical techniques such as calorimetry, FTIR, small angle X-ray (SAX) diffraction and dynamic light scattering.

Depuis plusieurs années, de nouvelles méthodologies basées sur l’utilisation du champ électrique pour agir ou caractériser les cellules ou les tissus cellulaires génèrent de nombreuses avancées et apportent des nouvelles promesses dans les laboratoires de recherche et dans l'industrie : diagnostic de cancer, ElectroChimioThérapie (insertion d’un médicament en perméabilisant les membranes des cellules), thérapie génique (insertion d’un gène thérapeutique), immunothérapie (vaccins anti-tumoraux obtenus par électrofusion de cellules dendritiques et cellules cancéreuses pour réactiver le système immunitaire).L’application d’ impulsions électriques à des cellules ou dans des tissus cellulaires induit un changement sur leurs propriétés, en particulier sur leurs membranes qui deviennent transitoirement perméables, laissant temporairement le passage aux ions et macro-molécules. Les phénomènes induits lors d’une perméabilisation par application de champ électrique ont été partiellement caractérisés en microscopie epi-fluorescence. Pour effectuer un suivi en temps réel de la dynamique du processus de l’électroperméabilisation, une voie prometteuse consiste à caractériser électriquement l’échantillon. Dans cet objectif, mon travail de thèse consiste à mettre en oeuvre le suivi en temps réel de l’évolution des caractéristiques électriques sur une large bande de fréquences d’un tissu cellulaire ou d’une cellule isolée, avant, pendant et après la sollicitation par un champ électrique pulsé.Dans le cadre de ma thèse un modèle du système biologique et de son environnement a été élaboré, afin de mieux décrire des phénomènes observés expérimentalement: effet des sollicitations électriques sur la viabilité cellulaire, sur la perméabilité de la membrane externe, effets induits sur les composés intracellulaires, dynamique de fusion membranaire. Le degré de perméabilisation de l’objet biologique (cellule ou tissu) dépend de manière fortement non-linéaire de nombreux paramètres, ce qui rend complexe l’élaboration de ce modèle et son interprétation. La détection de ce niveau de perméabilisation est effectuée en temps réel (mesure du niveau de perméabilisation avant, pendant et après l’application de l’impulsion électrique). In fine cette approche devrait permettre d’optimiser le taux de perméabilisation cellulaire en fonction de l’application considérée. Ce système de contrôle individuel du niveau de perméabilisation cellulaire pourrait à terme être parallélisé massivement sur une puce dédiée à l’électroporation d’un grand nombre de cellules. Afin d’avoir une vision multi-échelle des effets, l’étude a été menée sur plusieurs modèles expérimentaux: qui vont du tissu (échelle millimétrique) à la cellule unique, en passant par les échelles intermédiaires (caractérisation de spéroides cellulaires).Dans ces deux derniers cas (sphéroide, cellule unique) l’objet biologique est isolé dans une biopuce microfluidique équipée d’électrodes de mesure et d’application du champ (échelle micrométrique).Les micro-dispositifs que j’ai réalisé pour caractériser en temps réel la perméabilisation de cellules, intègrent une géométrie spécifique d’électrodes, ainsi que d'un réseau de canaux microfluidiques pour contrôler le débit de cellules Le degré de miniaturisation de ces puces permet de travailler au niveau de la cellule unique, et appliquer des champs électriques de forte amplitude, de forte fréquence, localisés spatialement. / The increasing interest for new methodologies based on the use of the electric field to characterize the cells or tissue cells and generate brought promising development in research laboratories and industry: cancer diagnosis, electrochemotherapy (insertion of a drug after cell membranes permeabilization), gene therapy (insertion of a therapeutic gene), immunotherapy (anti-tumor vaccines obtained by electrofusion of dendritic cells and cancer cells to activate the immune system).The application of electrical pulses to cells or cell tissues induces a change in their properties, in particular on their membranes which become transiently permeable, and temporarily allow the passage of ions and macromolecules. Effect linked to the permeabilization phenomenon have been partially characterized by epi-fluorescence microscopy. Nevertheless, in order to perform the real-time monitoring of the electroporation process and know its dynamics, the electrical sample characterization is employed. Thus the aim of this work is to implement a real-time monitoring of dielectrical characteristics changes, on a wide frequency range, of a cellular tissue or a single cell, before, during and after the pulsed electric field application.As part of my thesis a model of the biological system has been developed to better describe the phenomena observed experimentally: effect of electrical stress on cell viability, on the permeability of the outer membrane, induced effects on the intracellular compounds, dynamics of membrane fusion.The degree of permeabilization of the biological sample (cells or tissues) is non linearly dependent of several parameters, which makes complicated the development of the model and its interpretation.The detection of a specific level of permeabilization is done in real time (measure of the level of permeabilization before, during and after the electric pulses application). This cell permeabilization level control could eventually be parallelized on a chip dedicated to the electroporation of a large number of cells. The latter can be used to optimize the electric pulses parameters in order to reach the desired permeabilization level. In order to have a multi-scale overview of the phenomenon, the study was performed on different size-level: from the tissue level (millimeter scale) to the single cell model through the intermediate scales (cell spéroides characterization).In the latter two cases (spheroid, single cell) the biological sample is isolated in a microfluidic biochip where the electric field solicitation are applied (micrometer scale).The microdevice designed and fabricated during this work, allows the real time characterization of the cell permeabilization. Furthermore the miniaturization of the system is crucial to work at the level of the single cell, and make possible the application of electrical fields of high amplitude, high frequency and spatially localized.

La résistance aux antibiotiques est responsable de nombreuses maladies et décès depuis plusieurs décennies. L'augmentation de la résistance bactérienne a donc encouragé les chercheurs à développer de nouveaux antibiotiques, et de nouvelles stratégies pour contrer les différents mécanismes de résistance. L'un des mécanismes de résistance les plus notables est la formation de biofilms. Par conséquent, notre groupe de recherche s'est concentré sur différents types de mécanismes d'action des antibiotiques, plus particulièrement sur la perméabilisation de la membrane cellulaire. Ceci est réalisé par la formation de pores, d'agrégats, de canaux ou de micelles à travers celle-ci. En réponse à cela, nous avons synthétisé des composés antibactériens possédant deux cations benzimidazolium, deux chaînes apolaires hydrophobes et un échafaudage phényl- ou pyridyl-phényléthynylène, ayant la capacité de former des agrégats supramoléculaires via des interactions π-π et des liaisons hydrogène à travers la bicouche lipidique. Ces composés perturbateurs de la membrane agissent par un mécanisme rapide et efficace et ont montré de bons résultats contre les souches MRSA (Methicilin Resistant Staphylococcus Aureus), ce qui en fait des candidats prometteurs pour combattre les infections bactériennes et la formation des biofilms. / Antibiotic resistance has been responsible for multiple diseases and deaths for several decades. The rise of bacterial resistance has therefore encouraged researchers to develop new antibiotics, and new strategies to counter their various resistance mechanisms. One of the more notable resistance mechanics is the formation of biofilms. Consequently, our research group focused on the different types of antibiotics mechanisms of action, more particularly on the permeabilization of the cell membrane. This is achieved through the formation of pores, aggregate, channels, or micelles through it. In response to this, we have thus synthesized antibacterial compounds with a benzimidazolium cation, a hydrophobic apolar chain and a phenyl- or pyridyl-phenylethynylene scaffold with the capacity to form supramolecular aggregates via π-π interaction and hydrogen bonding through the lipid bilayer. These membrane-disrupting compounds act via a rapid and effective mechanism and have shown good results against strains of MRSA, thus making promising candidates to combat bacterial infections and biofilms formation.