Melanoma é uma neoplasia maligna com comportamento agressivo considerado o câncer mais comum da cavidade bucal de cães. Ele pode ser classificado quanto a morfologia celular em epitelióide, fusiforme e misto ou quanto ao fenótipo: melânico ou amelânico. Estudos têm sugerido que os melanomas amelânicos são mais agressivos. O objetivo deste estudo foi avaliar o comportamento dos melanomas da cavidade bucal dos cães, quantificando a expressão das Cx26 e 43, caderina-E, MMPs 2 e 9 e proliferação celular. Para tanto foram coletados 25 melanomas provenientes de cães atendidos no HOVET FMVZ-USP (16 melânicos e 9 amelânicos). Após a cirurgia os animais foram acompanhados até a morte, sendo que 5 animais foram eutanasiados (2 antes da cirurgia e 3 após o procedimento cirúrgico, devido ao sofrimento físico). Os tumores eram diagnosticados através do histopatológico e classificados de acordo com OMS (1998). Para confirmação dos melanomas amelânicos foi utilizado à técnica de imuno-histoquímica com o perfil de anticorpos pré-estabelecidos. A proliferação celular foi quantificada através do uso de PCNA, índice apoptótico e caspase-3. O comportamento tumoral foi avaliado através da técnica de imuno-histoquímica para caderina-E e MMPs 2 e 9. E a expressão das Cxs foram avaliadas através da imunofluorescência e western blot. Cães com melanma amelânico apresentaram menor sobrevida, com aumento do número de metástase, fraca marcação para caderina-E e alta intensidade para as MMPs 2 e 9, aumento de células positivas para PCNA e índice mitótico, e diminuição da caspase-3, não havendo diferença significante quanto aos tipos histotógicos. Houve diminuição de expressão das Cxs entre os amelânicos, no entanto, aumento da síntese do gene de CX 26 entre o mesmo grupo, o que se verificou através do PCR em Tempo Real. Nossos achados sugerem que os melanomas da cavidade bucal de cães apresentam um comportamento mais agressivo / Melanoma is a malignant neoplasm with aggressive behavior considered the most common cancer of the buccal cavity in dogs. It can be classified as cell morphology in epithelioid, spindle and mixed or on the phenotype, in melanotic or amelanotic. Studies have suggested that amelanotic melanomas are more aggressive. The aim of this study was to evaluate the behavior of melanoma of the buccal cavity of dogs, quantifying the expression of Connexins 26 and 43, E-cadherin, MMPs 2 and 9 and cell proliferation. For both melanomas were collected from 25 dogs attended at HOVET FMVZ (USP) (16 melanotic and 9 amelanotic melanomas). After the surgery, the animals were followed until death, so 5 animals were euthanized (2 before surgery and 3 after surgery, due to physical suffering). The tumors were diagnosed by histopathology and classified according to WHO (1998). For confirmation of amelanotic melanomas it was used the technique of immune-histochemistry with the antibody profile pre-established. Cell proliferation was quantified by using PCNA, apoptotic index and caspase-3. The tumor behavior was evaluated using the technique of immune-histochemistry for E-cadherin and MMPs 2 and 9. And the expression of Cxs was evaluated by immune-fluorescence and western blot. Dogs with amelanotic melanoma had lower survival with increasing number of metastatic, weak immunoreactivity for E-cadherin and high intensity for MMP 2 and 9, an increase of cells positive for PCNA and mitotic index, and decreased caspase-3, no significant difference in the morphologic subtypes. There was a decrease of expression of Cxs between amelanotic, however, increased synthesis of CX 26 gene among the same group, which was verified by real time PCR. Our findings suggest that melanomas of the buccal cavity of dogs exhibited more aggressive behavior.

A eletroporação é um fenômeno que promove a formação de poros na membrana citoplasmática quando esta é submetida à exposição de um campo elétrico específico. Dentre as principais aplicações da eletroporação podemos citar a eletroquimioterapia, uma nova terapia contra o câncer, e a terapia gênica, ainda em desenvolvimento. As conexinas são unidades formadoras das junções gap, canais transmembrana responsáveis pela comunicação intercelular e que participam de uma serie de eventos fisiológicos como o crescimento e diferenciação celular. A importância desta família de genes na oncologia está cada vez mais clara dadas às evidencias de seu comportamento de supressor de tumor ou como facilitador da disseminação e metástases de acordo com a fase de em que o câncer se apresenta. A eletroporação como um fenômeno que promove perturbação em membrana poderia interferir de alguma forma na expressão das conexinas. O objetivo deste trabalho foi avaliar os possíveis efeitos de interferência na expressão gênica das células promovidos pela eletroporação em especial na família de genes das conexinas. Para tanto as células foram expostas a um campo semelhante a o utilizado para eletroquimioterapia e avaliadas em diferentes tempos após a exposição. Três linhagens, duas neoplásicas (melanoma B16/BL6 e E9) e uma linhagem não neoplásica (E10) foram utilizadas como modelo experimental para que pudesse ser feita uma analise comparativa e a avaliação dos possíveis diferentes comportamentos das mesmas frente à eletroporação. O estudo da expressão gênica foi realizado pelas técnicas de imunofluorescência, western blot e PCR em tempo real. A Cx 26 foi avaliada na linhagem B16/BL6 e a Cx 43 nas linhagens E9 e E10. Os resultados da imunofluorescência apontaram uma marcação no citoplasma em B16/BL6, predominantemente em núcleo para a linhagem E9 e em núcleo e citoplasma em E10. Somente a linhagem E9 apresentou diferentes marcações entre os grupos, com uma marcação em citoplasma no grupo t1/2 (30 minutos apos a eletroporacao) O western blot mostrou diminuição transiente para as Cxs apenas nas linhagens neoplásicas nos grupos t1/2 e t1 (1 hora apos a eletroporacao). A linhagem E10 apresentou aumento de expressão nos mesmos grupos. O PCR em tempo real apresentou diferenças significativas nos tempos t1/2 e t1 para as linhagens B16/BL6 e E10. A linhagem B16/BL6 teve diminuição de RNAm enquanto a linhagem E10 apresentou aumento de transcrição de RNAm. A linhagem E9 apresentou padrão de expressão bastante heterogêneo. Frente aos resultados apresentados podemos concluir que a eletroporação interferiu de maneira transiente na expressão de conexinas. / Electroporation is a phenomenon that promotes the formation of pores in the cytoplasmic membrane when it is exposed to a specific electric field. The main applications of electroporation include the electrochemotherapy, a new cancer therapy, and gene therapy, still in development. Connexins are forming units of gap junctions, transmembrane channels responsible for intercellular communication and participating in a series of physiological events such as cell growth and differentiation. The importance of this gene family in oncology is increasing. The evidence of its behavior as a tumor suppressor or facilitator of dissemination and metastases according to the stage where the cancer presents itself. Electroporation as a phenomenon that promotes membrane disruption could interfere somehow in the expression of connexins. The aim of this study was to evaluate the possible effects of interference in gene expression. of cells promoted by electroporation in particular gene family of connexins. For this purpose cells were exposed to a electric field similar to that used for Electrochemotherapy and evaluated at different times after exposure. Three cell lines, two neoplastic (melanoma B16/BL6 and E9) and one line non-neoplastic (E10) were used as experimental models that could provide a comparative analysis between the lines and an assessment of the possible different behaviors due to electroporation. Its expression was assessed by immunofluorescence, western blot and real-time PCR. Cx 26 was evaluated in the melanoma cell line B16/BL6 and Cx 43 in the cell lines E9 and E10. The results showed a cytoplasm immunofluorescence staining pattern in the in B16/BL6, predominantly in the nucleus in line E9 and in the nucleus and cytoplasm for cell line E10. Only the cell line E9 had different immunoflurescence stainings between the groups, with a positivity in the cytoplasm in the group t1/2. The western blot showed transient decrease of Cxs only in cell lines neoplastic in the times t1/2 and t1. The lung cell line E10 showed increased expression in the same time points. The real-time PCR showed significant differences in t1/2 and t1 for the cell lines B16/BL6 and E10. The melanoma cell line B16/BL6 had mRNA decreased while the cell line E10 showed increased transcription of mRNA. The cell line E9 showed a very heterogeneous expression pattern. According to our results, we can conclude that electroporation interfered transiently in the expression of connexins.

Gap junctions are a class of membrane proteins that facilitate cell-to-cell communication by forming channels that directly couple the cytoplasm of neighboring cells. The channels are composed of monomers called connexins. Humans express 21 connexin isoforms in a cell-type specific fashion, and each isoform has distinct mechanisms of permeation and regulation. Co-assembly of multiple isoforms into a single intercellular channel can change channel properties, such as conductance and selectivity to substrates (e.g., ions, metabolites and signaling molecules). However, the mechanistic basis for this functional diversity has remained poorly understood. This lack of mechanistic insight has been due in large part to the lack of high-resolution (atomic-level) structural knowledge on this class of proteins. Prior to this work, the only high-resolution information available on gap junction structure came from a single connexin isoform, connexin-26 (cx26). CryoEM has recently transformed from a low-resolution technique into one capable of rivaling the atomic-level resolutions achieved by x-ray crystallography -- but without the necessity for crystal formation, which has hindered progress towards understanding many classes of proteins (ie, membrane proteins, intrinsically disordered cell signaling complexes and other structurally dynamic systems). For my thesis research, I applied novel methods in single particle electron cryo-microscopy (CryoEM) to study a class of membrane proteins called gap junctions isolated from native lens tissue, as well as two signaling complexes not amenable to other structural techniques. I determined the structure of the lens gap junction, which contains connexin-46 (cx46) and connexin-50 (cx50), to a resolution of 3.4 Å and generated atomic models for both connexin isoforms. Structural analysis paired with molecular dynamics gave insight into energetic features of these channels that determine their isoform-specific conductance and selectivity to electrically charged ions. The cx46/50 gating domain was found to be stabilized by hydrophobic anchors, and also seems to adopt a more stable open state than found in cx26. Genetic mutations associated with congenital cataract formation were found to map to hot-spots of conserved structural and functional importance, rationalizing their disease-causing effects. As part of collaborative efforts, I used methods in single particle EM to characterize two separate signaling complexes that had proven difficult to study with x-ray crystallography and NMR spectroscopy. One system, Ca2+/Calmodulin Kinase II (CaMKII), is a signaling complex in the brain involved in memory formation. Characterization of the CaMKII complex by single particle EM revealed an extended state, which was also shown to be prevalent in cells -- giving more depth to our understanding of how this signaling molecule functions. The second collaboration characterized the multimeric binding sites of the hub protein LC8, which interacts with the disordered region of a transcription factor (ASCIZ). This provided support for a novel model of transcription regulation, wherein LC8 fine-tunes its own transcription levels through multi-valent binding to the disordered region of its own regulatory transcription factor.

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

Plyšinių jungčių kanalai yra sudaryti iš koneksinų baltymų, kurie yra randami įvairiuose audiniuose ir dėka jų ląstelės gali keistis metabolitais bei elektriniais signalais. Plyšinių jungčių kanalai pasižymi dideliu jautrumu viduląsteliniam pH. Šiame darbe tyrėme įvairių inhibitorių poveikį homotipinių plyšinių jungčių, sudarytų iš Cx26, Cx30.2, Cx36, Cx40, Cx43, Cx45, Cx46, Cx47 ir Cx50 laidumui bei šio poveikio priklausomybę nuo viduląstelinio pH.. Plyšinių jungčių blokatoriais buvo pasirinktos kelias skirtingas farmakologines grupes atstovaujančios medžiagos: ilgagrandžiai alkoholiai (heksanolis, oktanolis, nonanolis), izofluranas, meflokvinas, flufenaminė rūgštis, karbenoksolonas ir arachidono rūgštis. Pademonstravome, kad pHi pakėlimas panaudojant NH4Cl iki ~8 didino plyšinių jungčių laidumą tarp ląstelių, turinčių Cx30.2 ar Cx45, tačiau neturėjo jokios įtakos ląstelėms su Cx26, Cx40, Cx46, Cx47 ir Cx50 ir mažino laidumą tarp ląstelių su Cx36 ar Cx43. pHi padidinimas žymiai susilpnino ilgagrandžių alkoholių, izoflurano ir meflokvino poveikį Cx45 plyšinių jungčių laidumui ir neturėjo įtakos flufenaminės rūgšties, karbenoksolono ir arachidono rūgšties poveikiui. Taip pat parodėme, kad nuo pHi priklauso oktanolio poveikio Cx45 plyšinių jungčių laidumui galingumas, t.y., pusinė blokavimo koncentracija IC50 buvo 0.1, 0.25 ir 2.68 mM atitinkamai prie pHi 6.9, 7.2 ir 8.1. Manoma, kad pH poveikis plyšinių jungčiu laidumui pasireiškia protonams jungiantis su koneksino baltymo... [toliau žr. visą tekstą] / Gap junction channels formed from connexin proteins provide a direct pathway for electrical and metabolic cell-cell communication exhibiting high sensitivity to intracellular pH. We examined pHi-dependent modulation of junctional conductance of GJs formed of Cx26, Cx30.2, Cx36, Cx40, Cx43, Cx45, Cx46, Cx47 and Cx50 by reagents representing several distinct groups of uncouplers, such as long carbon chain alkanols, arachidonic acid, carbenoxolone, isoflurane, flufenamic acid and mefloquine. We demonstrate that alkalization by NH4Cl to pH≈8 increased coupling in cells expressing Cx30.2 and Cx45, yet did not affect coupling of Cx26, Cx40, Cx46, Cx47 and Cx50 and decreased it in Cx43 and Cx36 gap junctions. Unexpectedly, cells expressing Cx45, but not other connexins, exhibited full coupling recovery after alkalization with NH4Cl under the continuous presence of alkanols, isoflurane and mefloquine. There was no coupling recovery by alkalization in the presence of arachidonic acid, carbenoxolone and flufenamic acid. In cells expressing Cx45, IC50 for octanol was 0.1, 0.25 and 2.68 mM at pHis of 6.9, 7.2 and 8.1, respectively. Histidine modification of Cx45 protein by N-bromosuccinimide proportionally reduced the coupling-promoting effect of NH4Cl as well as uncoupling effect of octanol. This suggests that long chain alkanols and some other uncouplers may act through the formation of hydrogen bonds with the as-of-yet unidentified histidine/s of the Cx45 gap junction channel protein... [to full text]

Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 120-134).

Orientador: Edi Lucia Sartorato / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T04:28:58Z (GMT). No. of bitstreams: 1 Silva-Costa_SueliMatildeda_M.pdf: 4568147 bytes, checksum: d8aadad3b82357deab2dec0ee176e6ed (MD5) Previous issue date: 2009 / Resumo: Mutações no gene GJB2 (Cx26) são as causas mais comuns de perda auditiva não-sindrômica autossômica recessiva. Contudo, 10 a 40% dos casos com mutações no gene da Cx26 são detectadas em apenas um dos alelos, causando um problema no diagnóstico molecular. Estes achados podem ser atribuídos à existência de mutações em regiões não codificantes do gene ou mutações em outros genes cujos produtos protéicos estão envolvidos em interações com a Cx26. O principal objetivo deste trabalho foi esclarecer a causa genética da perda auditiva de 45 pacientes com surdez sensorioneural não-sindrômica monoalélicos para mutações patogênicas na região codificante do gene GJB2. Mutações incluindo deleções e duplicações nos genes GJB2, GJB3 e GJB6 foram investigadas nesses pacientes. A mutação IVS1+ lG>A no sítio de splice na região não codificante do gene GJB2, que pode contribuir para o fenótipo de surdez, foi também investigada em todos os 45 pacientes. A fim de avaliar a freqüência da mutação IVS1+1G>A em pacientes brasileiros foram analisados 142 pacientes com perda auditiva sensorioneural não-sindrômica que não apresentavam nenhuma mutação patogência na região codificante do gene GJB2. Além disso, o método de MLP A (Multiplex Ligationdependent Probe Amplification) foi utilizado neste estudo para avaliar sua eficiência em detectar deleções e duplicações em genes envolvidos na perda auditiva. Analisando todos os pacientes, dez diferentes mutações no gene GJB2 e uma deleção no gene GJB6 foram encontradas.. Esses achados explicaram a perda auditiva de aproximadamente 27% dos pacientes monoalélicos para mutações no gene GJB2. A mutação IVSl+IG>A foi encontrada em dois pacientes monoalélicos. Essa alteração não foi encontrada nos pacientes sem mutações na região codificante do gene GJB2. Essa mutação parece ser rara entre pacientes surdos brasileiros. Na região promotora do gene, em um paciente, uma deleção foi encontrada e no outro uma duplicação foi observada por meio do método de MLP A. Essas possíveis alterações na região não codificante do gene ainda não foram descritas. Outras análises moleculares e estudos funcionais são necessários para confirmar uma possível associação desses achados com a perda auditiva. A técnica de MLP A se mostrou eficiente para detectar deleções e duplicações no genoma. Portanto, demonstramos no presente estudo que essa técnica pode contribuir para explicar a surdez em pacientes monoalélicos. / Abstract: Mutations in the GJB2 gene (Cx26) are the most common cause of autosomal recessive nonsyndromic hearing loss. However, in 10 to 40% of these cases, mutations in Cx26 gene are detected in on1y one allele which causes a problem in molecular diagnostico These findings could be attributed to the existence of mutations in non-coding regions of the gene or mutations in other genes of which protein products are involved in interaction with Cx26. The aim of this study was to clarifying the genetic cause of the hearing loss of 45 patients with non-syndromic sensorioneural deafness, monoallelics for pathogenic mutations in the coding region of the GJB2 gene. Mutations including deletions and duplications in the GJB2, GJB3 and GJB6 genes were investigated in these patients. 'The IVSl+ 1G>A splice site mutation in the non-coding region of the GJB2 gene which can contribute to the deafuess phenotype was also investigated in all 45 patients. In order to evaluate the frequency of the IVSl+ 1G>A mutation were tested 142 Brazilian patients with non-syndromic sensorineural hearing loss without any pathogenic mutation ín the GJB2 coding region. Furthermore, the MLP A method (Multiplex Ligation-dependent Probe Amplification) was used to evaluate its usefulness in detecting deletions/duplications in genes involved in hearing loss. Ten different mutations in the GJB2 gene and one deletion in the GJB6 gene were found. These findings eXplained the hearing loss about 27% ofGJB2 monoallelic patients. The IVSl+ 1G>A mutation was found in two monoallelics patients. This alteration was not found in patients without mutations in the GJB2 coding region. This mutation seems to be rare in deaf Brazilian patients. One patient a deletion was found in the promoter region of GJB2 gene and in another patients a duplication was observed by the MLP A method. These possible alterations were not described in noncoding region of GJB2 gene yet. Additional molecular and functional studies are needed to assess a possible association of these findings with hearing loss. MLP A proved to be a highly accurate method to detected deletions and duplications in the genome. Therefore, we have shown in the present study that this technique can explain the deafness in monoallelics patients. / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Introduction Cutaneous scars represent a common surgical complication, yet no effective drug therapy for scar treatment currently exists despite huge patient and physician demand. A connexin 43 (Cx43) carboxyl terminus (CT) mimetic peptide, alpha Connexin Carboxy-Terminus 1 (αCT1), has demonstrated efficacy in improving long-term scar appearance in pre-clinical and clinical trials. However, current understanding of the mechanism-of-action by which αCT1 improves long-term scar appearance with early intervention treatment is not well understood. Methods In vivo: Scar biopsies from 1) human, 2) Sprague-Dawley rat, and 3) IAF Hairless guinea pig trials of αCT1 were examined for collagen matrix structure at 4 weeks (all models), and 2 and 6 weeks (rat and guinea pig models only). Collagen matrix variables examined included local disorganization of the fibers, a variable that is higher in unwounded skin compared to scar tissue, and density of the fibers, which is higher in scar tissue but can also be used as an early temporal marker of the rate of healing. In vitro: Primary murine dermal fibroblasts were isolated from the whole dermis of 3-4 week old transgenic mice expressing collagen 1(α2) GFP-tpz. Cells were sorted for expression via FACS and plated on prealigned collagen substrate for 7 days under conditions favorable to generating extracellular matrix. Results: All in vivo scar biopsies demonstrated some level of altered collagen matrix structure with αCT1 treatment. Treated scars had higher local disorganization of the collagen fibers within the wound, and an increase in collagen matrix density compared to control at certain earlier timepoints that tended to decrease or disappear at later timepoints. The IAF Hairless guinea pig, a novel splinted wound healing model presented herein, was found to closely replicate the human dermal collagen profile and changes in collagen profile spurred by αCT1, significantly outperforming the traditional rat model. Primary dermal murine fibroblasts treated in vitro with αCT1 significantly increased synthesis of procollagen 1, the precursor of collagen 1 necessary for constructing the extracellular matrix, suggesting that at least part of the reason for higher collagen density at early in vivo timepoints is due to increased collagen synthesis by fibroblasts. Conclusion: αCT1 treatment in the early stages of wound healing prompts individual fibroblasts to increase their output of collagen and create a more disorganized early collagen matrix. These early changes potentially spur the long-term scar appearance improvements seen in clinical trials, and provide a basis for future work to discover the cellular pathways to alter in order to improve wound healing and cutaneous scarring outcomes. / Doctor of Philosophy / Skin wounds frequently result in scars that can range from barely visible to enormous eyesores. Almost everyone will experience at least one skin wound in their lifetime leading to a scar that they wish were less visible, feeding the multi-billion dollar market for anti-scarring agents. However, many of the products on store shelves that claim to reduce scar appearance have not proven those claims. Most of the therapies that do have some degree of scientific evidence to support their claims are difficult to use properly, such as silicone sheeting, and often result in only minor improvements to scar appearance. Alpha Connexin Carboxy-Terminus 1 (αCT1), marketed in clinical trials as Granexin® gel, is a protein-based therapy that works on the cellular level to fundamentally alter the skin's initial reaction to wounding and improving long-term scar appearance. This dissertation explores the link between cellular processes altered by αCT1 and long-term clinical improvements in scar appearance by studying both the extracellular matrix present in the scar in human and animal models and the creation of that extracellular matrix by dermal fibroblasts. In both human and animal models, topical application of αCT1 had no effect on skin surface appearance at early timepoints of 2-6 weeks, correlating with previous research that found scar appearance only improved at 3+ months post-injury. However, deep within the newly constructed tissue of the scar, these studies show the collagen organizational structure of αCT1-treated scars is more similar to unwounded skin and slightly more dense at early timepoints, suggesting αCT1 marginally improved the speed of healing. These findings in humans and animals were also verified in part in cell culture experiments that found dermal fibroblasts increased collagen output in response to αCT1 treatment. A novel wound healing model in the hairless guinea pig, superior at replicating human skin than established models like the rat, is also presented and shown to have effects strongly similar to the human with αCT1 treatment. These results provide a fundamental insight into the mode-of-action by which αCT1 may improve long term scar appearance and identifies early collagen structure as a target for future therapeutics to modify, as well as a new animal model in which to test them.

La Neuromyélite Optique (NMO) est une maladie auto-immune démyélinisante, rare et grave, du système nerveux central (SNC). Elle est caractérisée par une démyélinisation et une perte axonale ciblant principalement le nerf optique et la moelle épinière. La découverte d'un auto-anticorps (IgG-NMO) dirigé contre l'aquaporine-4 (AQP4), un canal hydrique exprimé par l'astrocyte, a été une étape clé dans la compréhension de la physiopathologie de la NMO, actuellement définie comme une astrocytopathie. La pathogénicité de l'IgG-NMO a été démontrée : il induit une internalisation d'AQP4 et des transporteurs au glutamate, provoquant une altération de la fonction astrocytaire. Cependant les mécanismes permettant de lier la dysfonction astrocytaire aux altérations caractéristiques de la NMO, notamment la démyélinisation, restent méconnus. Les astrocytes sont des cellules gliales essentielles à l'établissement et au maintien de l'homéostasie du SNC. Ils permettent la régulation des flux hydriques et ioniques, le contrôle extracellulaire des neuromédiateurs ainsi que l'apport de métabolites énergétiques aux neurones et aux oligodendrocytes. Ils sont aussi caractérisés par une très forte expression de connexines (Cx), des molécules transmembranaires s'assemblant sous une forme hexamérique : le connexon. Les connexines forment soit des hémicanaux, permettant l'échange de petites molécules entre les milieux intra- et extra-cellulaires, soit des jonctions communicantes par la juxtaposition de connexons appartenant à deux cellules, assurant le couplage intercellulaire avec le passage de petites molécules et d'ions (ATP, glutamate, lactate, calcium). Les fonctions hemicanal et jonction communicante sont fortement régulées en condition physiologique et altérées en condition pathologique, notamment en contexte neuroinflammatoire. Nous émettons l'hypothèse que les IgG-NMO altèrent l'expression et la fonction des connexines, et conduisent ainsi à la production d'un environnement toxique pour les oligodendrocytes et la myéline, et délétère pour le fonctionnement neuronal. Mon projet de thèse avait trois objectifs : i) la caractérisation du phénotype astrocytaire induit par les IgGNMO ; ii) l'identification d'altérations des connexines et leur implication dans la pathologie ; iii) la mise en évidence d'altérations de la transmission synaptique induites par les IgG-NMO et l'implication de connexines dans cet effet. Des modèles de cultures primaires gliales traitées par des IgG-NMO issue d'une cohorte de patients m'ont permis de caractériser le phénotype acquis par les astrocytes, et de proposer le concept d'un astrocyte réactif spécifique de pathologie. Les astrocytes réactifs spécifiques de la NMO induisent un milieu inflammatoire spécifique et toxique, provoquant une démyélinisation. Grâce au développement d'une coculture gliale et neuronale produisant des neurones myélinisés, et à l'utilisation de peptides inhibiteurs des Cx, j'ai pu montrer que les NMO-IgG ont un effet démyélinisant et que celui-ci implique les Cx. La démyélinisation est en effet associée à des modifications structurales et fonctionnelles des Cx astrocytaires, observées à la fois in vitro et dans notre modèle in vivo, le rat-NMO. Enfin, la mise en place d'une étude électrophysiologique en potentiel de champs local sur des tranches d'hippocampe de rats m'a permis d'étudier l'effet des IgG-NMO sur la transmission glutamatergique basale. J'ai pu mettre en évidence un effet dépresseur des IgG-NMO, partiellement bloqué par un inhibiteur de connexines, la carbenoxolone. Comme il a déjà été démontré par des études cliniques dans des pathologies neurodégénératives, l'utilisation de modulateurs de Cx semble être une voie thérapeutique prometteuse afin de prévenir la démyélinisation et les altérations du fonctionnement neuronal de la NMO / Neuromyelitis Optica (NMO) is a rare and severe auto-immune demyelinating disease of the central nervous system (CNS). It is characterized by demyelination and axonal loss targeted to the optic nerve dans the spinal cord. The identification of a specific autoantibody (NMO-IgG) directed against the astrocytic protein AQP4 was a key step in the understanding of NMO physiopathology: it is now considered as an astrocytopathy. NMO-IgG is also a biomarker of NMO, and its pathogenicity has been demonstrated. NMO-IgG induce an internalization of AQP4 together with other membrane proteins such à glutamate transport GLT1. This could alter astrocyte functions but the mechanisms connecting astrocytopathy and demyelination remain unclear. Astrocytes are abundant glial cells crucial for the establishment and the maintenance of CNS homeostasis. They regulate water flux and ion homeostasis and control extracellular volume and neurotransmitter concentrations. They also provide neurons and oligodendrocytes with energy substrates. Astrocytes are characterized by a high expression of connexins (Cx), transmembrane proteins assembling in hexameric form, called connexon. Cx form either hemichannels, unopposed connexon at the membrane, allowing the exchange of small molecules (<1,2kDa e.g. glutamate, ATP) and ions (Ca2+, K+) between extra- and intra-cellular compartments. Cx also form gap junctions, formed by the juxtaposition of two connexons at the membrane of two different cells, and allow the quick cell to cell exchange of small molecules, metabolites and ions (e.g. glucose, lactate, Ca2+). Hemichannel and gap junction functions are tightly regulated under physiological conditions and can be altered in pathological condition for example during neuroinflammation. We proposed that NMO-IgG by altering connexins expression and/or function could lead to the production of a toxic environment for oligodendrocytes and myelin but also for neuronal functioning. This feature of astrocyte dysfunction could participate to NMO alterations. My thesis project had three main goals: i) the characterisation of astrocyte phenotype induced buy NMO-IgG, ii) the identification of connexins alterations and their implication NMO physiopathology, iii) the highlight of synaptic alterations induced by NMO-IgG and the involvement of connexins in this effect. Primary glial cell cultures treated with NMO-IgG from a cohort of NMO patients, were used to characterize astrocyte phenotype and we proposed the concept of a specific reactive dysfunctioning astrocyte induced by NMO-IgG. Those astrocytes, called “NMO-astrocytes” are responsible for the production of a proinflammatory toxic microenvironment for oligodendrocytes and leading to demyelination. With the development of a myelinated culture model, composed of glial cells and neurons with myelinated axons, together with the use of specific inhibitors of Cx functions, we showed that NMO-IgG induced demyelination involved connexin dysfunction. In fact, demyelination was associated with structural and functional alterations of astrocytic connexins observed both in vitro and in vivo in the NMO-rat model. Electrophysiological recording of basal glutamatergic synaptic activity in the rat hippocampus showed a strong depression of synaptic responses induced by NMO-IgG. Connexins could be implicated in this alteration since blocking all connexins with carbenoxolone blocked NMO-IgG effect