No description available.

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.

No description available.

Inspired by the roles of hair cells in nature, this study aims to develop and characterize two new sets of novel flow sensors. One set of sensors developed and studied in this work are flow sensors fabricated using carbon nanomaterials. These sensors are made by embedding carbon nanotubes (CNT) and carbon nanohorns (CNH) into a polymeric substrate and then tested by flowing a conductive aqueous solution over the surface of the exposed CNT and CNH. In response, a flow-dependent voltage is generated. The surface coverage and the electrical relationship between the sensor and water is investigated and the voltage measurements of sensors with different levels of resistance were tested in varying fluid velocities. In response to these fluid velocities, the least resistive sensor showed small, but detectable changes in voltages, while higher resistance sensors showed less response. In addition, plasma treatment of the carbon nanomaterial/PDMS films were conducted in order to render the PDMS on the surface hydrophilic and in turn to pull more fluid towards the carbon material. This showed to improve the sensitivity of the flow sensors. This work also builds on previous research by investigating the flow dependent electrical response of a "skin"-encapsulated artificial hair cell in an aqueous flow. An artificial cell membrane is housed in a flexible polyurethane substrate and serves as the transduction element for the artificial hair cell. Flow experiments are conducted by placing the bio-inspired sensor in a flow chamber and subjecting it to pulse-like flows. This study demonstrates that the encapsulated artificial hair cell flow sensor is capable of sensing changes in flow through a mechanoelectrical response and that its sensing capabilities may be altered by varying its surface morphology. Furthermore, the sensor's response and dynamics as a function of its surface morphology and structural properties are investigated through synchronized motion tracking of the hair with a laser vibrometer and current measurements across the artificial cell membrane. / Master of Science

The many advances of nano technology extensively revolutionize mechanics. A tremendous need is growing to further bridge the gap between the classical mechanics and the nano scale for many applications at different engineering fields. For instance, the themes of interdisciplinary and multidisciplinary topics are getting more and more attention especially when the coherency is needed in diagnosing and treating terminal diseases or overcoming environmental threats. The fact that how mechanical, biomedical and electrical engineering can contribute to diagnosing and treating a tumor per se is both interesting and unveiling the necessity of further investments in these fields. This dissertation presents three different investigations in the area of nano mechanics and nano materials spanning from computational bioengineering to making mechanically more versatile composites. The first part of this dissertation presents a numerical approach to study the effects of the carbon nano tubes (CNTs) on the human body in general and their absorbability into the lipid cell membranes in particular. Single wall carbon nano tubes (SWCNTs) are the elaborate examples of nano materials that departed from mere mechanical applications to the biomedical applications such as drug delivery vehicles. Recently, experimental biology provided detailed insights of the SWCNTs interaction with live organs. However, due to the instrumental and technical limitations, there are still numerous concerns yet to be addressed. In such situation, utilizing numerical simulation is a viable alternative to the experimental practices. From this perspective, this dissertation reports a molecular dynamics (MD) study to provide better insights on the effect of the carbon nano tubes chiralities and aspect ratios on their interaction with a lipid bilayer membrane as well as their reciprocal effects with surface functionalizing. Single walled carbon nano tubes can be utilized to diffuse selectively on the targeted cell via surface functionalizing. Many experimental attempts have smeared polyethylene glycol (PEG) as a biocompatible surfactant to carbon nano tubes. The simulation results indicated that SWCNTs have different time-evolving mechanisms to internalize within the lipid membrane. These mechanisms comprise both penetration and endocytosis. Also, this study revealed effects of length and chirality and surface functionalizing on the penetrability of different nano tubes. The second part of the dissertation introduces a novel in situ method for qualitative and quantitative measurements of the negative stiffness of a single crystal utilizing nano mechanical characterization; nano indentation. The concept of negative stiffness was first introduced by metastable structures and later by materials with negative stiffness when embedded in a stiffer (positive stiffness) matrix. However, this is the first time a direct quantitative method is developed to measure the exact value of the negative stiffness for triglycine sulfate (TGS) crystals. With the advancements in the precise measuring devices and sensors, instrumented nano indentation became a reliable tool for measuring submicron properties of variety of materials ranging from single phase humongous materials to nano composites with heterogeneous microstructures. The developed approach in this chapter of the dissertation outlines how some modifications of the standard nano indentation tests can be utilized to measure the negative stiffness of a ferroelectric material at its Curie temperature. Finally, the last two chapters outline the possible improvements in the mechanical properties of conventional carbon fiber composites by introducing 1D nano fillers to them. Particularly, their viscoelastic and viscoplastic behavior are studied extensively and different modeling techniques are utilized. Conventional structural materials are being replaced with the fiber-reinforced plastics (FRPs) in many different applications such as civil structures or aerospace and car industries. This is mainly due to their high strength to weight ratio and relatively easy fabrication methods. However, these composites did not reach their full potential due to durability limitations. The majorities of these limitations stem from the polymeric matrix or the interface between the matrix and fibers where poor adhesion fails to carry the desired mechanical loadings. Among such failures are the time-induced deformations or delayed failures that can cause fatal disasters if not taken care of properly. Many methodologies are offered so far to improve the FRPs' resistance to this category of time-induced deformations and delayed failures. Several researchers tried to modify the chemical formulation of polymers coming up with stiffer and less viscous matrices. Others tried to modify the adhesion of the fibers to the matrix by adding different chemically functional groups onto the fibers' surface. A third approach tried to modify the fiber to matrix adhesion and at the same time improve the viscous properties of the matrix itself. This can be achieved by growing 1D nano fillers on the fibers so that one side is bonded to the fiber and the other side embedded in the matrix enhancing the matrix with less viscous deformability. It is shown that resistance to creep deformation and stress relaxation of laminated composites improved considerably in the presence of the nano fillers such as multiwall carbon nano tubes (MWCNTs) and zinc oxide nano wires (ZnO- NWs). The constitutive behaviors of these hybrid composites were investigated further through the use of the time temperatures superposition (TTS) principle for the linear viscoelastic behavior and utilizing phenomenological models for the viscoplastic behavior. / Ph. D.

No / We report on the development and on the first use of the widefield surface plasmon (WSPR) microscope in the examination of the cell surface interface at submicron lateral resolutions. The microscope is Kohler illuminated and uses either a 1.45 numerical aperture (NA) oil immersion lens, or a 1.65 NA oil immersion lens to excite surface plasmons at the interface between a thin gold layer and a glass or sapphire cover slip. Like all surface plasmon microscope systems the WSPR has been proven in previous studies to also be capable of nanometric z-scale resolutions. In this study we used the system to image the interface between HaCaT cells and the gold layer. Imaging was performed in air using fixed samples and the 1.45 NA objective based system and also using live cells in culture media using the 1.65 NA based system. Imaging in air enabled the visualisation of high resolution and high-contrast submicron features identified by vinculin immunostaining as component of focal contacts and focal adhesions. In comparison, imaging in fluid enabled cell surface interfacial interactions to be tracked by time-lapse video WSPR microscopy. Our results indicate that the cell surface interface and thus cell signalling mechanisms may be readily interrogated in live cells without the use of labelling techniques.

O desenvolvimento da nanociência e nanotecnologia promoveu uma nova fronteira no estudo da matéria, permitindo que materiais já conhecidos tivessem suas propriedades redescobertas ao serem manipulados em nível molecular. Vários materiais vêm apresentando relevância na nanociência e nanotecnologia, como os nanotubos de carbono (CNTs), nanopartículas (NPs) e óxido de grafeno, uma vez que os CNTs e óxido de grafeno são dotados de propriedades mecânicas, térmicas e elétricas que os tornam apropriados para o desenvolvimento e a aplicação em dispositivos, especialmente na área biotecnológica e de sensores. Diversas áreas se beneficiam com o uso da tecnologia em nanopartículas (NPs), por exemplo: alimentícia, médica, agronegócio, cosmética, etc. Uma possível perspectiva na utilização desses nanomateriais em sistemas biológicos torna muito interessante investigar como tais materiais interagem em nível molecular com modelos de membranas celulares e com células. Esta tese tem como objetivos: i) investigar detalhadamente a interação entre nanopartículas (Fe3O4/Dextran; Fe3O4/PDAC; PDAC; Dextran) e nanotubos de carbono com modelos de membranas celulares; e ii) desenvolver nanofibras poliméricas pela técnica de electrospinning para ser utilizada com óxido de grafeno como modelos mimetizados (scaffolds) para a diferenciação de células-tronco neurais. Os filmes ultrafinos foram fabricados utilizando as técnicas de Langmuir e Langmuir-Blodgett. Esses nanomateriais foram avaliados através da técnica de Espectroscopia vibracional por Geração de Soma de Frequências. A espectroscopia SFG é sensível a interfaces. Nanofibras de Poli(ε-Caprolactone) foram fabricadas pela técnica de electrospinning. Scaffolds com óxido de grafeno/Nanofibras de Poli(ε-Caprolactone) foram desenvolvidos como suportes sólidos para a diferenciação de células-tronco neurais de rato. Óxido de grafeno em diferentes concentrações foi incorporado nas nanofibras poliméricas. Os modelos deste sistema foram investigados por imagens de Microscopia Eletrônica de Varredura. Os resultados mostraram que a carga eletrostática de cada fosfolipídio utilizado pode influenciar nas interações com os nanomateriais (nanopartículas ou nanotubos de carbono), podendo resultar em uma desestruturação no modelo de membrana celular. Scaffolds contendo nanofibras de Poli(ε-Caprolactone) com óxido de grafeno representaram um eficiente modelo mimetizado para a interação/diferenciação de células-tronco neurais de rato conforme revelado por imagens de Microscopia Eletrônica de Varredura. Estas imagens mostraram que o sistema de nanofibras de Poli(ε-Caprolactone) com 1,0 mg/mL de óxido de grafeno foram ideais para a diferenciação de oligodendrócitos em células-tronco neurais de rato. / The development of nanoscience and nanotechnology promoted a new frontier on the study of matter, allowing conventional materials to exhibit novel or improved properties. Several materials show relevance in nanoscience and nanotechnology, such as carbon nanotubes (CNTs), nanoparticles (NPs) and graphene oxide. CNTs and graphene oxide, for example, exhibit unique mechanical, thermal and electrical properties, which make them appropriate to the development and application in devices, especially in biotechnology and sensors areas. Many areas are benefited from the use of nanoparticles (NPs), such as food, medical, agrobusiness, cosmetic etc. The perspective regarding the use of nanomaterials in biological systems requires the understanding on how these materials interact at the molecular level with cell membrane models and with cells. The objectives of this thesis are: i) to investigate the interaction between nanoparticles (Fe3O4/Dextran; Fe3O4/PDAC; PDAC; Dextran) and carbon nanotubes with cell membrane models; and ii) to develop polymeric nanofibers via electrospinning technique, to be used with graphene oxide as mimic models (scaffolds) in the differentiation of neural stem cells. The cell membrane models were manufactured using Langmuir and Langmuir-Blodgett techniques. These nanomaterials were evaluated through Sum Frequency Vibrational Spectrosocopy (SFG). Poly(ε-Caprolactone) nanofibers were manufactured by electrospinning technique. Scaffolds with graphene oxide/Poly(ε-Caprolactone) were developed as solid supports for differentiation of rats neural stem cells. This biosystem was investigated via Scanning Electron Microscopy and biochemical essays. The results showed that the charge of each phospholipid influenced the interactions with the nanomaterials (nanoparticles or carbon nanotubes), in some cases, resulting in a disruption of the cell membrane model. Scaffolds with Poly(ε-Caprolactone) nanofibers obtained via electrospinning with graphene oxide represented an efficient mimic model for interaction/differentiation of neural stem cells as shown via Scanning Electron Microscopy. The images revealed that the PCL nanofibers system with 1.0 mg/mL of graphene oxide were ideal to the differentiation of oligodendrocytes in neural stem cells.

Em pacientes portadores de insuficiência coronariana com prognóstico desfavorável pela presença de disfunção ventricular significativa, a pesquisa de viabilidade miocárdica traz contribuições ao predizer a possibilidade de recuperação contrátil após revascularização. Os segmentos miocárdicos com disfunção contrátil por hipoperfusão podem melhorar o desempenho contrátil restabelecido o aporte sanguíneo local, indicando que a revascularização miocárdica, quando bem indicada, é capaz de melhorar a sobrevida deste grupo de pacientes. Realizou-se pesquisa de tese de doutoramento do Departamento de Radiologia da Universidade de São Paulo na unidade de medicina nuclear da Nuclear Medcenter/Hospital SOCOR em Belo Horizonte, Minas Gerais. O objetivo do estudo foi verificar se a cintilografia de perfusão miocárdica com duplo-isótopo (99mTcsestamibi/ cloreto de tálio-201) como método radioisotópico para identificação do músculo viável, tem sua especificidade aumentada com a inclusão de informações sobre reserva contrátil miocárdica obtidas simultaneamente através do gatedSPECT (imagens tomográficas do coração sincronizadas ao eletrocardiograma) na vigência de baixas doses de dobutamina de forma semelhante ao ecocardiograma. Estudou-se 54 pacientes com infarto do miocárdio prévio, encaminhados ao serviço de medicina nuclear para realização de pesquisa de viabilidade miocárdica. Foram excluídos do estudo os pacientes que no seguimento não foram revascularizados ou que não realizaram controle cintilográfico pós-cirúrgico, uma vez que considerouse como critério de viabilidade a melhora da contratilidade miocárdica após a revascularização. Avaliou-se os parâmetros de viabilidade (integridade celular e reserva contrátil) e o desempenho contrátil pós-cirúrgico de 260 segmentos miocárdicos de treze pacientes revascularizados. Os pacientes estudados foram submetidos a cintilografia de perfusão miocárdica duoisotópica em protocolo de dois dias com imagens tomográficas do coração obtidas em gamma-camera de duas cabeças, modelo Varicam (Elscint) e processadas em estação de trabalho eNTEGRA(GE). As imagens de estresse foram adquiridas em sincronia com o ECG (gated SPECT) em condições basais e na vigência de baixa dose de dobutamina (10 a 15g/Kg/min) 45 minutos após a administração endovenosa do 99mTcsestamibi no pico do exercício isotônico (esforço) ou da ação de agentes farmacológicos (estresse farmacológico) e nas etapas de repouso e redistribuição do cloreto de tálio-201, 20 minutos e quatro a seis horas após a administração endovenosa do radioisótopo em condições basais. Os pacientes operados foram submetidos a um segundo estudo cintilográfico de perfusão miocárdica com gated SPECT, no período mínimo de três meses após o procedimento, para avaliação da performance contrátil pós-cirúrgica. Para análise dos achados cintilográficos, dividiu-se o coração em 20 segmentos que receberam diferentes escores, permitindo a quantificação da perfusão e função miocárdica pelo Cedars Sinai Quantitative Pefusion SPECT QPS/QGS(GE),. Analisou-se o padrão perfusional nas etapas de estresse, repouso e redistribuição e de função (análise do espessamento sistólico, motilidade parietal, valores de fração de ejeção e volumes cardíacos do ventrículo esquerdo) em condições basais e sob estímulo inotrópico. No tratamento estatístico a análise do espessamento sistólico foi o parâmetro considerado significativo para avaliação da reserva contrátil miocárdica pelo método. Houve incremento na especificidade da pesquisa de viabilidade miocárdica pelo método radioisotópico realizado, demonstrando valores de especificidade superiores aos encontrados na literatura. As contribuições do método se mostraram efetivas / In patients with coronariopathy in the setting of ventricular dysfunction having an unpromising prognostic, the myocardial viability must be assessed thus, bringing contribution as it can predict the myocardial dysfunction recovery after revascularization. The myocardial segments with contractile dysfunction as a consequence of hypoperfusion can improve wall motion after perfusion recovery, demonstrating that myocardial revascularization, whenever suggested, can improve survival to this group of patients. This research is a PHD thesis from Radiology Department of São Paulo University and was performed at a nuclear medicine unit - Nuclear Medcenter/SOCOR hospital - in Belo Horizonte, Minas Gerais. The aim of the study was to check if dual isotope perfusion myocardial gated SPECT (99mTc-sestamibi/thallium-201) as a nuclear medicine procedure to the identification of viable myocardium, can improve the method specificity with addition of contractile reserve information obtained simultaneously by gated SPECT with low dose of dobutamine, similar to the echocardiogram. 54 patients with myocardial stroke, referred to the nuclear medicine unit to seek myocardial viability have been studied. Patients that do not have been submitted to revascularization or that did not undergo the post surgery control were excluded, as the parameter considered for viability was the wall motion recovery after revascularization. 260 myocardial segments in 13 patients had their viability parameters (cellular integrity and contractile reserve) as the contractile performance after surgery evaluated. The images were acquired by a Varicam (Elscint) double head gamma camera and processed by eNTEGRA (GE) workstation. The gated SPECT stress images were performed in baseline conditions and with low-dose dobutamine (10 a 15g/Kg/min) 45 minutes after intravenous injection of 99mTc-sestamibi.on the peak of isotonic exercise or pharmacologic stress. The rest and redistribution images were acquired , 20 minutes and 4 or 6 hours after intravenous injection of thallium-201 at rest. The revascularizated patients were also submitted to a second gated SPECT study at least 3 months after surgery for evaluation of the contractile performance. In order to analyze the scintigraphic findings, the heart was divided into 20 segments that received different scores for quantification of myocardial perfusion and function by Cedars Sinai Quantitative Perfusion SPECT QPS/QGS(GE),. The perfusion pattern of stress, rest and redistribution and the parameters of function (wall thickening and motion, ejection fraction and cardiac volumes analysis) at baseline conditions and by inotropic effect. By the statistics analysis wall thickening was considered significant to evaluate the myocardial contractile reserve by this method. There was improvement in the specificity of the radioisotopic research showing specificity values larger than those found in literature. These method contributions were effective

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis, is one of the leading causes of death due to a single infectious agent and results in more than 1 million human deaths every year. M.tb infection of the host initiates a local inflammatory response, resulting in the migration of a number of host plasma protein and non-protein factors to the site of infection. In addition, some of these factors are also produced locally at the site of infection. It is envisaged that these host factors are likely to come in direct contact with M.tb and immune cells and may modulate the outcome of the infection. In this study, a number of host factors including transferrin, lactoferrin, fibrinogen, C-reactive protein, alpha-2-macroglobulin (α2M), vitronectin, plasminogen, low-density lipoprotein (LDL), high-density lipoprotein (HDL), serotonin, L-alpha dipalmitoyl phosphatidylcholine (DPPC) and platelet activating factor C-16 (PAF C-16) were screened in vitro for their direct effect on the growth of mycobacteria using M.smegmatis as a model. As a result of this screening, PAF C-16, a phospholipid compound was identified that directly inhibited the growth of M.smegmatis and M.bovis BCG in a dose and time-dependent manner. Use of a range of PAF C-16 structural analogues, including Lyso-PAF, PAF C-18, Hexanolamino PAF, 2-O-methyl PAF & Pyrrolidino PAF, revealed that small modifications in structure did not alter the direct growth inhibition property of PAF C-16 and similar levels of M.smegmatis and M.bovis BCG growth inhibition were observed as compared to PAF C-16. Structural dissection of PAF C-16 suggested that the attachment of carbon tail to the glycerol backbone via ether bond at sn-1 position was important for its direct growth inhibition activity against mycobacteria. Microscopy and flow cytometry with PAF C-16 treated M.smegmatis and M.bovis BCG showed damage to the bacterial cell membrane. The addition of membrane-stabilizing agents, α-tocopherol, tween-80 and tween-20, partially mitigated the growth inhibitory effect of PAF C-16. These results suggested that the growth inhibition activity of PAF C-16 against mycobacteria is most likely due to its detergent-like effect, resulting in damage to the bacterial cell membrane. PAF C-16 and its structural analogues were also investigated for their effect on the growth of intracellular M.smegmatis inside THP1 cells. In vitro, PAF C-16, PAF C-18 and Hexanolamino PAF inhibited the growth of intracellular M.smegmatis, whereas, analogues such as Lyso-PAF and 2-O-methyl PAF failed to show any growth inhibitory effect, suggesting that the presence of acetyl group at sn-2 position was important for growth inhibition of intracellular M.smegmatis. Use of PAF receptor antagonists partially mitigated the inhibitory effect of PAF C-16 on the growth of intracellular M.smegmatis, suggesting this inhibition was through receptor-mediated signalling pathways. Blocking of PAF C-16 signalling pathway components such as phospholipase C and phospholipase A2, resulted in the increased survival of intracellular M.smegmatis. Arachidonic acid, a product of PAF C-16 signalling pathway directly inhibited the growth of M.smegmatis. Furthermore, inhibition of iNOS enzyme and antibody-mediated neutralization of TNF-α partially mitigated the inhibitory effect of PAF C-16 on intracellular M.smegmatis growth, suggesting that the production of NO and TNF-α were also involved in PAF C-16 induced intracellular growth inhibition. Overall, this study has identified PAF C-16, its structural analogues such as Lyso-PAF, PAF C-18, Hexanolamino PAF and other compounds including 1-O-hexadecyl-sn-glycerol, miltefosine and hexadecyl lactate with novel anti-mycobacterial activity. Further investigations are needed to demonstrate their effectiveness against M.tb both in vitro and in animal models to assess their therapeutic potential as anti-TB drugs.

La membrane plasmique est un assemblage supramoléculaire qui délimite la cellule. C’est une structure fine, complexe et dynamique assurant des fonctions multiples et vitales pour la cellule. Sa rupture est un évènement physiologique pour les cellules soumises à des stress mécaniques fréquents et/ou importants, comme les cellules épithéliales, les cellules endothéliales ou les cellules musculaires. Dans des conditions physiopathologiques, la membrane plasmique peut également être endommagée par l’insertion de toxines bactériennes formant des pores (PFTs, pour « pore forming toxins »). Le processus de réparation membranaire et la machinerie protéique associée sont encore mal connus. Connaître les partenaires protéiques et comprendre les mécanismes mis en jeu durant le processus de réparation de la membrane plasmique sont deux enjeux fondamentaux majeurs. En effet, il a été établi qu’une défaillance du processus de réparation membranaire pour les fibres musculaires est la cause principale de certaines dystrophies musculaires. La machinerie protéique de réparation comprend des protéines comme la dysferline, la cavéoline-3 et certaines Annexines (Anx). Les Anx appartiennent à une superfamille de protéines répandue chez la plupart des eucaryotes, qui ont la propriété commune de se lier aux membranes biologiques en présence de calcium (Ca2+). Certaines Anx, comme l’AnxA5, une fois liées aux membranes biologiques s’auto-assemblent spontanément en réseau-2D. Lors de ce travail de thèse, nous avons étudié le rôle de l’AnxA5 dans la réparation membranaire des trophoblastes placentaires et des cellules du muscle squelettique humain. Pour les deux types cellulaires, nous avons montré que l’AnxA5 est un acteur indispensable du processus de réparation membranaire dans le cas de ruptures mécaniques. En associant des approches de microscopie de fluorescence et de microscopie électronique à transmission (MET), nous avons mis en évidence que dans ces cellules, le mécanisme de réparation est principalement basé sur la formation d’un « patch » lipidique. Dans les cellules musculaires, les expériences de MET ont mis en évidence qu’un pool d’AnxA5 endogène se lie aux bords du site de rupture quelques secondes après la lésion du sarcolemme. Ceci suggère qu’après rupture de la membrane plasmique, l’augmentation locale de la concentration calcique intracellulaire provoque la liaison de l’AnxA5 spécifiquement aux bords de la région membranaire lésée où elle forme un réseau-2D. Le réseau-2D stabiliserait localement la membrane et préviendrait sa déchirure, induite par les forces de tensions exercées par le cytosquelette cortical. Nous avons également montré que l’AnxA5 ne semble pas impliquée dans la réparation de la membrane plasmique après insertion de PFTs. Ceci suggère que différents mécanismes de réparation existent et que leur mise en place dépend probablement du type ou de l’importance des dommages. Finalement nous avons étendu notre étude à des lignées cellulaires établies à partir de patients diagnostiqués comme souffrant de dystrophies des ceintures de type 2B (déficience en dysferline) et 1C (déficience en cavéoline-3), respectivement. Nous avons montré, pour ces lignées, que la déficience en dysferline ou cavéoline-3 provoque un défaut de réparation dans le cas des ruptures mécaniques de la membrane plasmique. Dans ces cellules musculaires pathologiques intactes ou endommagées, l’AnxA5 a le même comportement, ce qui suggère que l’action de l’AnxA5 est indépendante de ces protéines. A la différence des cellules déficientes en dysferline, nous avons observé que les cellules déficientes en cavéoline-3 sont capables de réparer efficacement des lésions créées par l’insertion de PFTs dans le sarcolemme. Ce résultat supporte l’hypothèse de l’existence de plusieurs mécanismes de réparation. En conclusion, ce travail montre que l’AnxA5 est un composant clé de la machinerie de réparation dans le cas des ruptures mécaniques. / Plasma membrane is the supramolecular assembly that delimits the cell. It is a thin, dynamic and complex structure, ensuring multiple and vital cell functions. Its disruption is a physiological event occurring in cells submitted to frequent mechanical stresses, such as endothelial cells, epithelial cells and muscle cells. It is also a physiological event for cells exposed to pore forming bacterial toxins (PFTs). Membrane repair mechanisms and associated protein machinery are still poorly understood. This knowledge is, however, essential for obvious physiopathological issues. Indeed, a defect of membrane repair in muscle cells leads to some muscular dystrophies. Membrane repair machinery includes proteins such as dysferlin, MG-53, caveolin-3 and some Annexins (Anx). Anx belong to a superfamily of proteins widely spread in most of eukaryotes, which share the property of binding to biological membranes in the presence of calcium (Ca2+). Here, we investigated the role of AnxA5 in cell membrane repair of human trophoblastic and skeletal muscle cells. We showed that AnxA5 is required for membrane repair of mechanical damages in the two cell types. By combining fluorescence and transmission electron microscopy approaches, we evidenced that membrane repair mechanism in these cells is based on the formation of a lipid “patch”. In human muscle cells, TEM experiments revealed that a pool of endogenous AnxA5 binds to the edges of the torn sarcolemma as soon as a few seconds after membrane disruption. Our results suggest the following mechanism: triggered by the local increase in Ca2+ concentration, AnxA5 molecules bind to PS exposed at the edges of the torn membrane, where they self-assemble into 2D arrays. The formation of 2D arrays strengthens the damaged sarcolemma, counteracts the tensions exerted by the cortical cytoskeleton and thus prevents the expansion of the tear. We showed also that a pool of endogenous AnxA5 binds to intracellular vesicles that obstruct the wounding site. It is likely these vesicles, once associated one to each other, ensure membrane resealing. Our results suggest that sarcolemma repair of damages caused by PFTs is independent of AnxA5. Therefore, different membrane repair mechanisms may exist, their occurrence probably depending on the type and/or the size of damages. Finally, we performed studies on muscle cells established from patients diagnosed with limb girdle muscular dystrophies type 2B (dysferlin-deficient) and 1C (caveolin-3-deficient), respectively. We found that dysferlin or caveolin-3 deficiency leads to a defect of membrane repair, in the case of mechanical damages. AnxA5 behaved similarly in these damaged cells and wild-type cells, suggesting that its function is independent of dysferlin or caveolin-3. Unlike dysferlin-deficient cells, damages created by PFTs are efficiently repaired in caveolin- 3-deficient cells. This result supports the hypothesis that different mechanisms occur in muscle cells, depending on the type of damage. In conclusion, this work indicates that AnxA5 is a key component of the membrane repair machinery, in the case of mechanical disruptions. Our results enable to propose a detailed mode of action for AnxA5.