L'endothelium vasculaire constitue la principale barrière entre le sang et les tissus régulant le passage de macromolécules et de cellules circulantes. Longtemps considéré comme une monocouche passive, l'endothélium joue d'importants rôles dans la régulation de la pression sanguine, de l'hémostase, des réponses immunitaires et inflammatoires. L'adhérence cellule/cellule est initiée dans l'endothélium vasculaire par des interactions homophiliques entre molécules de VE-cadhérine (= jonctions adhérentes). La dynamique de la jonction et du cytosquelette est importante pour le remodelage des jonctions intercellulaires qui a lieu au cours l'angiogenèse, de la vasculogenèse et lors de la réparation de l'endothélium. C'est pourquoi la détermination des mécanismes moléculaires sous-jacents est indispensable à la comprehension de phénomènes physiopathologiques (angiogenèse et progression tumorales, inflammation...). D'après la littérature, la protéine EPLIN intervient dans la formation du complexe E-cadhérine/alpha-caténine/EPLIN et stabilise l'actine corticale. Actuellement décrite comme spécifique des modèles épithéliaux, EPLIN peut-elle intervenir dans la liaison du complexe à base de VE-cadhérine au cytosquelette d'actine? De plus, il paraît essentiel de comprendre le rôle de cette protéine dans les cellules car son expression est fortement diminuée dans la plupart des cancers alors qu'à l'inverse sa surexpression bloque la prolifération cellulaire. / The endothelium forms the main barrier regulating the passage of macromolecules and circulating cells between the blood and tissue. Historically viewed as a passive vascular lining, vascular endothelium plays important roles in the regulation of vascular pressure, hemostasis, immune and inflammatory responses. In vascular endothelium, cell/cell adhesion is mediated by homophilic interactions of VE-cadherin molecules (= adherens junctions). Cytoskeleton and junction dynamics are important for intercellular junctions remodelling that occurs during angiogenesis, vasculogenesis and endothelium repair. So, determining the underlying molecular mechanisms is essential for the comprehension of pathologic phenomena such as angiogenesis, tumor progression or inflammation. We learn from the literature that EPLIN is involved in E-cadherin/α-catenin/EPLIN complex formation and cortical actin stabilization. Usually described as a protein specific of epithelial models, we wondered if EPLIN is able to link VE-cadherin complex to actin cytoskeleton. Furthermore, it seems essential to understand its cellular role since it is downregulated in many cancers while in contrast its overexpression blocks cell proliferation.

O carcinoma epidermóide da mucosa oral (CEMO) é uma neoplasia maligna comum; no Brasil, são estimados, para 2016, 15.490 novos casos. A invasão óssea ocorre em casos avançados.; esta é classificada em erosiva e infiltrativa. Aparentemente, o processo de transição epitélio-mesenquimal, com o envolvimento da E-caderina, é implicado. Foi investigada a expressão de E-caderina, por meio da imunoistoquímica em 15 casos avançados de CEMO e avaliada sua correlação com as características clínicas e histológicas da invasão óssea. A imunoexpressão da E-caderina foi estudada nos 15 casos de CEMO com evidência histológica de invasão óssea. A maioria dos pacientes eram homens (10 pacientes) e apresentavam invasão em mandíbula (9 casos). A expressão de E-caderina foi negativa em CEMOs com invasão erosiva e positiva nos casos que apresentavam infiltração óssea. A expressão de E-caderina na invasão óssea sugere que a participação do fenômeno de transição epitélio-mesenquimal é um fator diretamente envolvido com o tipo de invasão óssea. / Oral squamous cell carcinoma (OSCC) is a common malignancy; in Brazil it is estimated, in 2016,15.490 new cases. Bone invasion occurs in advanced cases; it is classified in erosive and infiltrative patterns. Apparently, the epithelial-mesenchymal phenomenon, with important participation of E-cadherin is implicated. We investigated the expression of E-cadherin in advanced OSSC and correlated its expression with the clinical characteristics and histologic patterns of bone invasion. Immunoexpression of E-cadherin was studied in 15 cases of OSCC with histological evidence of bone invasion. Most patients were men (10 patients) and presented mandible invasion (9 cases). The expression of E-cadherin was negative in OSCC in erosive bone invasion and positive in the infiltrative bone invasion. E-cadherin expression in bone invasion suggests that participation of epithelial-mesenchymal phenomenon is dependent on the patterns of tumour bone invasion.

Os mastocitomas são formações cutâneas neoplásicas que mais acometem os cães, por isso inúmeras pesquisas estão sendo direcionadas no descobrimento de novas opções de tratamento, diagnóstico e prognóstico desta doença. O objetivo deste trabalho foi avaliar a expressão de um conjunto de proteínas que estão interligadas ou interligam vias de sinalização, na tentativa de identificar proteínas que se apresentem diferencialmente expressas nos mastocitomas caninos de diferentes graus. Realizamos um estudo da expressão deste conjunto de proteínas em 18 tumores oriundos dos arquivos do Serviço de patologia animal do Departamento de Patologia da FMVZ-USP. Realizamos coleta de material fresco de outras 18 amostras de mastocitomas cutâneos caninos, as quais foram submetidas ao cultivo celular, e então foram estabelecidas 10 linhagens de mastocitomas cutâneos caninos a fim de se avaliar este mesmo grupo de marcadores moleculares in vitro. As amostras do arquivo foram submetidas à imunomarcação das seguintes proteínas: PTEN, c-kit, E-caderina, β-catenina, α-catenina, p120-catenina, MMP-7. Nas linhagens tumorais estabelecidas analisamos o ciclo celular, ploidia de DNA, proliferação celular pelo CFSE, análise ultraestrutural pela microscopia eletrônica de transmissão, análise mutacional do gene c-Kit, e análise por imunocitoquímica e imunofluorescência dos seguintes marcadores moleculares: PTEN, c-kit, E-caderina, β-catenina, α-catenina, p120-catenina, MMP-7, CX32, CX43, vimentina, triptase. Os resultados demonstram a alteração da expressão das proteínas do complexo E-caderina/catenina, do c-Kit, da proteína PTEN e MMP-7 de acordo com o grau do mastocitoma canino. Observamos além da redução de expressão uma localização subcelular de todas estas proteínas nos tumores mais agressivos como nos mastocitomas de grau 3. O mesmo foi observado para as proteínas Cx 43 e 32. Realizamos levantamento do histórico clínico dos 18 casos de mastocitoma caninos oriundos do arquivo, e os parâmetros clínicos avaliados foram: idade, raça, gênero, localização, tempo de evolução, alteração linfonodo, metástases, tempo sobrevida, intervalo recidiva, óbito. Foram associados a um pior prognóstico os pacientes que apresentaram os seguintes parâmetros: animais idosos, presença de metástase, localização no tórax e graduação tumoral. Nas linhagens tumorais estabelecidas, a análise da ploidia revelou que todas as linhagens de mastocitomas são diploides e o CFSE mostrou que a proliferação máxima ocorre dentro de 24hs de cultivo. A análise ultraestrutural comprova que as células das linhagens são mastócitos tumorais. A análise pela imunocitoquimica dos marcadores em estudo revelaram padrões similares aos encontrados na imunoistoquimica. Pela expressão da vimentina e da triptase confirmamos mais uma vez se tratar de linhagens de mastócitos em cultivo. Na análise mutacional do gene c-kit encontramos mutações no éxon 8 e 11, mas não no éxon 17. Nossos resultados revelam a ocorrência simultânea de inúmeras alterações moleculares nos mastocitoma caninos. As proteínas avaliadas têm funções e vias distintas, mas, que se interligam podendo regular ou serem reguladas, dependendo do momento em que se encontra a célula. A desestabilização do complexo E-caderina-cateninas parece ser o programa efetor na progressão dos mastocitomas caninos. A finalidade maior de se realizar estudos morfológicos, funcionais e moleculares das neoplasias é contribuir, mais cedo ou mais tarde, para o controle destas doenças. Esperamos, com este trabalho, ter fornecido informações importantes que favorecerão a busca por melhores tratamentos dos mastocitomas caninos. / Mast cell tumors are malignant skin formations that most affect dogs, so many research projects are being directed at the discovery of new treatment options, diagnosis and prognosis of this disease. The objective of this study was to evaluate the expression of a set of proteins that are interlinked or interconnected signaling pathways, in an attempt to identify proteins that show differentially expressed in canine mast cell tumors of different grades. We performed a study of the expression of this set of 18 proteins in tumors originating from the files of the Service of Animal Pathology, Department of Pathology of the FMVZ - USP. We collected other 18 new samples of canine cutaneous mast cell tumors , which were subjected to cell culture , and 10 strains of canine cutaneous mast cell tumors were established in order to evaluate in vitro this same group of molecular markers. The samples were subjected to immunostainings the following proteins: PTEN, c-kit, E-cadherin, β-catenin, α-catenin, p120-catenin, MMP-7. In established tumor cell lines we analyzed the cell cycle, DNA ploidy, cell proliferation by CFSE, ultrastructural analysis by transmission electron microscopy , mutational analysis of c-kit gene, and analysis by immunocytochemistry and immunofluorescence of the following molecular markers: PTEN, c-kit, E-cadherin, β-catenin, α-catenin, p120-catenin, MMP-7, CX32, Cx43, vimentin, tryptase. The results demonstrate the altered expression of the proteins, c- Kit, MM7 and PTEN proteins according to the level of the canine mastocytoma E-caderina/catenina complex. It has been observed a reduced expression as well as alterations in subcellular localization of all these proteins in more aggressive tumors as in grade 3 mast cell tumors. The same was observed for Cx 43 and 32 proteins. It has been performed a survey of the medical records of 18 cases of canine mast cell tumors retrieved from the archives, and clinical parameters evaluated were age, race, gender, location, time of evolution, change lymph node metastasis, survival time, recurrence interval, death. Older animals, metastasis, and tumor location in the chest, and mast cell tumor grade: patients who had the following parameters were associated with a worse prognosis. In the established tumor cell lines, ploidy analysis revealed that all lines are diploid mastocytoma and CFSE proliferation showed that the maximum occurs within 24 hours of cultivation. The ultrastructural analysis showed that the tumor cells are mast cell lineages. Analysis by immunocytochemistry markers studied showed similar patterns to those found in immunohistochemistry. By expression of vimentin and tryptase confirmed once again the case of mast cell lines in culture. In mutational analysis of the c kit, mutations were found in exon 8 and 11, but not in exon 17. Our results show the simultaneous occurrence of numerous molecular alterations in canine mast cell tumors. Proteins have different functions and evaluated pathways, but that interlock may regulate or be regulated, depending on the moment of the cell. The destabilization of the complex E-cadherin-catenins seems to be the effector program in the progression of canine mast cell tumors. The main purpose of performing morphological, functional and molecular studies of tumors is to contribute, sooner or later, to the control of these diseases. Hopefully, with this work, we have provided important information which will facilitate the search for better treatment of canine mast cell tumors

Os mastocitomas são formações cutâneas neoplásicas que mais acometem os cães, por isso inúmeras pesquisas estão sendo direcionadas no descobrimento de novas opções de tratamento, diagnóstico e prognóstico desta doença. O objetivo deste trabalho foi avaliar a expressão de um conjunto de proteínas que estão interligadas ou interligam vias de sinalização, na tentativa de identificar proteínas que se apresentem diferencialmente expressas nos mastocitomas caninos de diferentes graus. Realizamos um estudo da expressão deste conjunto de proteínas em 18 tumores oriundos dos arquivos do Serviço de patologia animal do Departamento de Patologia da FMVZ-USP. Realizamos coleta de material fresco de outras 18 amostras de mastocitomas cutâneos caninos, as quais foram submetidas ao cultivo celular, e então foram estabelecidas 10 linhagens de mastocitomas cutâneos caninos a fim de se avaliar este mesmo grupo de marcadores moleculares in vitro. As amostras do arquivo foram submetidas à imunomarcação das seguintes proteínas: PTEN, c-kit, E-caderina, β-catenina, α-catenina, p120-catenina, MMP-7. Nas linhagens tumorais estabelecidas analisamos o ciclo celular, ploidia de DNA, proliferação celular pelo CFSE, análise ultraestrutural pela microscopia eletrônica de transmissão, análise mutacional do gene c-Kit, e análise por imunocitoquímica e imunofluorescência dos seguintes marcadores moleculares: PTEN, c-kit, E-caderina, β-catenina, α-catenina, p120-catenina, MMP-7, CX32, CX43, vimentina, triptase. Os resultados demonstram a alteração da expressão das proteínas do complexo E-caderina/catenina, do c-Kit, da proteína PTEN e MMP-7 de acordo com o grau do mastocitoma canino. Observamos além da redução de expressão uma localização subcelular de todas estas proteínas nos tumores mais agressivos como nos mastocitomas de grau 3. O mesmo foi observado para as proteínas Cx 43 e 32. Realizamos levantamento do histórico clínico dos 18 casos de mastocitoma caninos oriundos do arquivo, e os parâmetros clínicos avaliados foram: idade, raça, gênero, localização, tempo de evolução, alteração linfonodo, metástases, tempo sobrevida, intervalo recidiva, óbito. Foram associados a um pior prognóstico os pacientes que apresentaram os seguintes parâmetros: animais idosos, presença de metástase, localização no tórax e graduação tumoral. Nas linhagens tumorais estabelecidas, a análise da ploidia revelou que todas as linhagens de mastocitomas são diploides e o CFSE mostrou que a proliferação máxima ocorre dentro de 24hs de cultivo. A análise ultraestrutural comprova que as células das linhagens são mastócitos tumorais. A análise pela imunocitoquimica dos marcadores em estudo revelaram padrões similares aos encontrados na imunoistoquimica. Pela expressão da vimentina e da triptase confirmamos mais uma vez se tratar de linhagens de mastócitos em cultivo. Na análise mutacional do gene c-kit encontramos mutações no éxon 8 e 11, mas não no éxon 17. Nossos resultados revelam a ocorrência simultânea de inúmeras alterações moleculares nos mastocitoma caninos. As proteínas avaliadas têm funções e vias distintas, mas, que se interligam podendo regular ou serem reguladas, dependendo do momento em que se encontra a célula. A desestabilização do complexo E-caderina-cateninas parece ser o programa efetor na progressão dos mastocitomas caninos. A finalidade maior de se realizar estudos morfológicos, funcionais e moleculares das neoplasias é contribuir, mais cedo ou mais tarde, para o controle destas doenças. Esperamos, com este trabalho, ter fornecido informações importantes que favorecerão a busca por melhores tratamentos dos mastocitomas caninos. / Mast cell tumors are malignant skin formations that most affect dogs, so many research projects are being directed at the discovery of new treatment options, diagnosis and prognosis of this disease. The objective of this study was to evaluate the expression of a set of proteins that are interlinked or interconnected signaling pathways, in an attempt to identify proteins that show differentially expressed in canine mast cell tumors of different grades. We performed a study of the expression of this set of 18 proteins in tumors originating from the files of the Service of Animal Pathology, Department of Pathology of the FMVZ - USP. We collected other 18 new samples of canine cutaneous mast cell tumors , which were subjected to cell culture , and 10 strains of canine cutaneous mast cell tumors were established in order to evaluate in vitro this same group of molecular markers. The samples were subjected to immunostainings the following proteins: PTEN, c-kit, E-cadherin, β-catenin, α-catenin, p120-catenin, MMP-7. In established tumor cell lines we analyzed the cell cycle, DNA ploidy, cell proliferation by CFSE, ultrastructural analysis by transmission electron microscopy , mutational analysis of c-kit gene, and analysis by immunocytochemistry and immunofluorescence of the following molecular markers: PTEN, c-kit, E-cadherin, β-catenin, α-catenin, p120-catenin, MMP-7, CX32, Cx43, vimentin, tryptase. The results demonstrate the altered expression of the proteins, c- Kit, MM7 and PTEN proteins according to the level of the canine mastocytoma E-caderina/catenina complex. It has been observed a reduced expression as well as alterations in subcellular localization of all these proteins in more aggressive tumors as in grade 3 mast cell tumors. The same was observed for Cx 43 and 32 proteins. It has been performed a survey of the medical records of 18 cases of canine mast cell tumors retrieved from the archives, and clinical parameters evaluated were age, race, gender, location, time of evolution, change lymph node metastasis, survival time, recurrence interval, death. Older animals, metastasis, and tumor location in the chest, and mast cell tumor grade: patients who had the following parameters were associated with a worse prognosis. In the established tumor cell lines, ploidy analysis revealed that all lines are diploid mastocytoma and CFSE proliferation showed that the maximum occurs within 24 hours of cultivation. The ultrastructural analysis showed that the tumor cells are mast cell lineages. Analysis by immunocytochemistry markers studied showed similar patterns to those found in immunohistochemistry. By expression of vimentin and tryptase confirmed once again the case of mast cell lines in culture. In mutational analysis of the c kit, mutations were found in exon 8 and 11, but not in exon 17. Our results show the simultaneous occurrence of numerous molecular alterations in canine mast cell tumors. Proteins have different functions and evaluated pathways, but that interlock may regulate or be regulated, depending on the moment of the cell. The destabilization of the complex E-cadherin-catenins seems to be the effector program in the progression of canine mast cell tumors. The main purpose of performing morphological, functional and molecular studies of tumors is to contribute, sooner or later, to the control of these diseases. Hopefully, with this work, we have provided important information which will facilitate the search for better treatment of canine mast cell tumors

Aiming to identify immune molecules with a novel function in cancer pathogenesis, we found the cluster of differentiation 177 (CD177), a known neutrophil antigen, expression to be positively correlated with relapse-free (RFS), metastasis-free (MFS) or overall survival (OS) in several solid cancers including those from breast, prostate, cervix, and lung. To study the role of CD177 in breast cancer, we generated a total body Cd177 knockout mouse. These mice had no profound phenotype at 3 - months of age or younger. The only phenotype found at this age was reduced peripheral neutrophil counts, but no difference in their ability to clear infections. Upon further analysis these mice developed an age dependent hyperproliferative mammary gland phenotype at 10 - months of age that was lost in mice 15 - months and older. Focusing on breast cancer, we found that CD177 is expressed in normal breast epithelial cells and is significantly reduced in invasive cancer. We found that CD177 suppresses breast cancer pathogenesis. To understand the mechanism behind CD177 mediated suppression of breast cancer, we performed mass spectrometry on the purified CD177 complex. Mass spectrometry and co-immunoprecipitation results revealed CD177 interacts with β-Catenin and glycolytic enzymes PFK, aldolase A, GAPDH and enolase-ɑ. Further studies revealed that mechanistically CD177 forms a complex with ECadherin and β-Catenin at adherens junctions. This physical interaction between CD177, E-Cadherin and β-Catenin prevents β-Catenin activation via the canonical WNT. We also found CD177 suppressed WNT/β-Catenin signaling independent of E-Cadherin with an unknown protein. Thus, we identified a novel protein complex involving CD177 and proteins from adherens junctions that can suppress cancer formation via inhibiting the WNT/β-Catenin signaling pathway, a key cellular biological process relevant to the oncogenesis of multiple cancer types and tissue development. The lack of WNT/β- Catenin signaling control explains how mice without CD177 develop hyperproliferation of mammary epithelium in the mouse mammary gland. Interestingly, this phenotype is lost with age, possibly due to a decrease in WNT/β-Catenin signaling resulting from a decrease in progesterone and estrogen. In addition to CD177’s role in the regulation of WNT/β-Catenin signaling we also identified that CD177 plays a role in cancer cell metabolism. Since metabolism plays a significant role in cancer and CD177 interacts with glycolytic enzymes, we sought to determine if CD177 plays a role in metabolism. CD177 appears to interact with glycolytic enzymes, PFK, aldolase A, GAPDH, and ɑ-enolase and ultimately suppresses their mRNA expression. Furthermore, we found novel localization of CD177 at the mitochondrion, thus providing a potential explanation as to how an extracellular membrane bound protein such as CD177 interacts with glycolytic enzymes. Metabolic analysis of CD177 expression on cancer cells revealed that CD177 leads to a decrease in glucose uptake and a slight decrease in basal glycolysis, but an increase in lactate concentration. Further metabolic profiling also revealed that CD177 expression results in a significant decrease in glycolytic capacity (ECAR). Expression of CD177 also resulted in a significant decrease in basal respiration, ATP production, maximal respiration, and spare capacity (OCR) as well as an increase in reactive oxygen species. These data reveal that CD177 plays a novel role in cancer cell metabolism.

L’angiogenèse, indispensable à la mise en place d’un réseau vasculaire fonctionnel, est au cœur des stratégies thérapeutiques des pathologies ischémiques. L’hypoxie, caractérisant ces tissus ischémiques, est un stimulus majeur de l’angiogenèse, en induisant l’expression de facteurs de croissance tels que le VEGF et de protéines de la matrice extracellulaire endothéliale. Nous avons identifié la protéine ANGPTL4, comme une cible majeure de l’hypoxie et ayant des effets opposés au VEGF sur la perméabilité vasculaire. Le but de cette thèse a consisté en l’analyse du rôle d’ANGPTL4 sur la formation de capillaire et l’organisation des jonctions adhérentes dans un contexte dépendant du VEGF. J’ai démontré que le VEGF stimule la formation d’un dense réseau capillaire 3D alors qu’ANGPTL4 induit la formation de capillaires étroits et peu ramifiés. ANGPTL4 réduit la taille du réseau de capillaire induit par le VEGF en limitant le nombre de bourgeons, de branchements et la largeur des capillaires. ANGPTL4 renforce l’intégrité des capillaires formés en présence de VEGF en préservant des jonctions adhérentes stables. J’ai démontré qu’ANGPTL4 limite les processus de migration 3D et de prolifération induits par le VEGF. L’analyse de la voie de signalisation VEGF/ANGPTL4 a montré une potentialisation par ANGPTL4 de la phosphorylation Y1175 du VEGFR2, impliqué dans l’internalisation de VEGFR2. En conclusion, ce modèle révèle un effet d’ANGPTL4 dépendant du contexte 3D, qui stimule les processus d’angiogenèse en absence de VEGF et qui contrecarre la morphogenèse induite par le VEGF en renforçant l’intégrité des jonctions adhérentes et en régulant la signalisation en aval du VEGFR2. / Angiogenesis, by promoting new functional capillaries, is a main target of therapeutic strategies of ischemic pathologies. Ischemic tissues are characterized by hypoxic environment, which stimulates angiogenesis by inducing expression and secretion of growth factors such as VEGF and by remodeling endothelial extracellular matrix. Our team identified ANGPTL4 as a hypoxia-induced target and characterized its counteracting effect on VEGF-induced vascular permeability. This PhD study therefore aimed to decipher the role of ANGPTL4 on angiogenesis, capillary architecture and adherens junction (VE-cadherin) organization in a VEGF-dependent context. I demonstrated that VEGF induced formation of branched capillaries forming a dense 3D network while ANGPTL4 enhanced the formation of unbranched and tight capillaries. Remarkably, ANGPTL4 reduces VEGF-induced angiogenesis, by limiting branching and widening of the capillaries. Furthermore, ANGPTL4 regulates the local VE-cadherin patterning during the sprouting process by maintaining lateral linear structures and limiting the VEGF-induced formations involved in the migratory capacities. I demonstrated that ANGPTL4 limited VEGF-induced 3D endothelial cell migration and proliferation. Analysis of VEGF/ANGPTL4 signaling pathway pointed out that ANGPTL4 enhanced phosphorylation of Y1175 VEGFR2, known to enhance internalization of VEGFR2. In conclusion, this study modeled the 3D context-dependent effect of ANGPTL4 that stimulates angiogenesis in absence of VEGF whereas it counteracts VEGF-induced endothelial morphogenesis by regulating VEGFR2 trafficking and strengthening adherens junctions.

MUCDHL est une cadhérine atypique encore peu étudiée. Les données obtenues à ce jour suggèrent que ce gène joue un rôle suppresseur de tumeurs dans l’intestin, notamment par son interaction et son effet inhibiteur sur la β-caténine, et que son expression est fréquemment diminuée dans les cancers colorectaux (CCR). Parallèlement à cette fonction anti-oncogénique, d’autres travaux ont suggéré que MUCDHL est impliquée dans la structuration de la bordure en brosse (BB) intestinale, en contribuant à la formation d’un complexe d’interaction inter-microvillositaire. Notre objectif était de déterminer la fonction et le mode d’action de MUCDHL dans le système digestif. Par la caractérisation détaillée de l’interaction avec la β-caténine, nous avons montré que le mode d’action anti-oncogénique de MUCDHL est plus complexe qu’une simple séquestration membranaire de la β-caténine. De plus, nous avons confirmé le rôle suppresseur de tumeurs de MUCDHL sur une cohorte importante de CCR humains et montré pour la première fois que sa perte amplifie la tumorigenèse intestinale dans un model murin. Par ailleurs, l’étude phénotypique des souris Mucdhl-/- a démontré son importance dans l’homéostasie du système digestif. En effet, l’absence de MUCDHL cause des altérations morphologiques de la BB intestinale, mais également de nombreuses perturbations métaboliques. Ces travaux apportent donc des informations inédites sur la fonction et le mode d’action de MUCDHL dans le système digestif. / MUCDHL is an atypical cadherin that has been poorly studied. The data obtained so far suggest that this gene has tumor suppressive activity in the intestine, namely by its interaction and inhibitory effect on β-catenin, and that its expression is frequently decreased in colorectal cancers (CCR). In parallel to this anti-oncogenic function, other studies have suggested that MUCDHL is involved in the assembly of the intestinal brush border (BB), by contributing to the formation of an inter-microvilli interaction complex. Our objective was to determine the function and mode of action of MUCDHL in the digestive system, Through a detailed characterization of the interaction with β-catenin, we showed that the anti-oncogenic mode of action of MUCDHL is more complex than a simple membrane sequestration of β-catenin. In addition, we confirmed the tumor suppressive function of MUCDHL on a very large cohort of human CCR and showed for the first time that its loss amplifies intestinal tumorigenesis in a murine model. Moreover, the study of the phenotype of Mucdhl-/- mice allowed us to demonstrate the importance of MUCDHL in the homeostasis of the digestive system. Indeed, the absence of MUCDHL causes morphological alterations of the intestinal BB, but also numerous metabolic disturbances. Thus, this work provides new information on the function and mode of action of MUCDHL in the digestive system.

Par sa fréquence et sa gravité, le cancer colorectal (CCR) demeure un problème de santé publique. Notre objectif global est de mieux comprendre les mécanismes impliqués dans l'homéostasie intestinale au travers de Mucdhl, une cadhérine atypique méconnue mais qui semble jouer un rôle très particulier dans l'épithélium intestinal et être impliquée dans les CCR. De manière intéressante, son expression semble fréquemment perdue dans les CCR, tandis que son maintien dans les cellules cancéreuses coliques diminue leur potentiel tumoral.Pour mieux appréhender le mode d'action de Mucdhl, une caractérisation fonctionnelle de son interaction avec β-caténine oncogénique a été réalisée et de nouveaux partenaires ont été identifiés dans les cellules intestinales. Afin de comprendre le rôle de Mucdhl dans la physiologie intestinale, encore inconnu à ce jour, un modèle murin déficient pour Mucdhl a été étudié. L'analyse des conséquences de la perte d'expression de Mucdhl indique qu'il est impliqué dans la structure et le fonctionnement de l'intestin chez la souris, mais également au niveau de processus métaboliques. De plus, cette perte d'expression de Mucdhl augmente la sensibilité des souris au développement de certaines tumeurs intestinales. Ces travaux ont donc permis de générer des informations inédites sur les fonctions physiopathologiques de Mucdhl, une cadhérine atypique encore mal connue, mais potentiellement impliquée dans les CCR. / Because of its frequency and severity, Colorectal Cancer (CRC) remains an important public health issue. Our objective is to understand mechanisms contributing to intestinal homeostasis through Mucdhl, a poorly characterized atypical cadherin that may play a unusual role in the intestinal , epithelium and be implicated in CRC. lnterestingly, its expression seems to be frequently reduced in CRC, while its retention in colon cancer cells decreases their tumorigenic potential.To better apprehend the mode of action of Mucdhl, a functional characterization of its interaction with oncogen,iç β-catenin was performed and new partners have been identified in intestinal cells.To understand the role of Mucdhl in intestinal physiology, mice genetically-invalidated at the Mucdhl locus were studied. Analysis of the consequences of Mucdhl loss of expression indicates that it is involved in the morphology and function of the mouse intestine, but also in metabolic processes. Moreover, Mucdhl loss of expression increases the sensibility of mice to the development of certain intestinal tumors. Thus, we generated new information on the physiopathological functions of Mucdhl, an intriguing atypical cadherin potentially involved in CRC.

Spatial and temporal tracking of passively diffusing functionalized colloids continues to be an improving and auspicious approach to measuring weak specific and non-specific biomolecular interactions. Evidence of this is given by the recent increase in published studies involving the development and implementation of these methods. The primary aim of the work presented in this dissertation was to modify and optimize video microscopy (VM) and total internal reflection microscopy (TIRM) methods to permit the collection of equilibrium binding and sampling data from interaction of surface-immobilized biomolecules. Supported lipid bilayers were utilized as model systems for functionalizing colloid and wall surfaces. Preliminary results measuring calcium-specific protein-protein interactions between surface immobilized cadherin fragments demonstrate the potential utility of this experimental system and these methods. Additionally, quantum dot-modified colloids were synthesized and evanescent wave-excited luminescence from these particles was used to construct potential energy profiles. Results from this work demonstrate that colloids can be used as ultra-sensitive probes of equilibrium interactions between biomolecules, and specialized probes, such as those modified with quantum dots, could be used in a spectral multiplexing mode to simultaneously monitor multiple interactions.

Le cancer du col utérin est précédé par des lésions prénéoplasiques. Celles-ci sont associées dans plus de 95% des cas à une infection par un papillomavirus (HPV). Un phénomène fréquent durant la cancérogenèse cervicale est l'intégration du génome dun HPV oncogène dans lADN cellulaire. Celle-ci entraîne une expression sélective de gènes codant pour des oncoprotéines virales (appelées E6 et E7) capables d'inactiver les produits de certains gènes suppresseurs de tumeurs (p53, p21, pRb) ou dinteragir avec dautres protéines cellulaires impliquées dans le contrôle du cycle cellulaire. Des travaux antérieurs du laboratoire daccueil suggèrent que le développement du cancer du col utérin est associé à une faible capacité de présentation dantigènes au système immunitaire, comme le démontre la rareté et le déficit fonctionnel des cellules de Langerhans (LC, cellules dendritiques ayant une fonction professionnelle de présentation antigénique au niveau de la peau et des muqueuses) dans les lésions (pré)cancéreuses cervicales. Ces altérations pourraient empêcher une réponse immunitaire efficace et faciliter la persistance du virus ainsi que la progression tumorale. Il est actuellement bien admis que les kératinocytes (cellules cibles de linfection par HPV) sont susceptibles dinfluencer les réactions immunitaires au niveau de la peau et des muqueuses épidermoïdes par lintermédiaire de facteurs solubles, les chémokines (CCL20, contrôlant linfiltration des LC immatures au sein de lépithélium) ou de contacts membranaires (E-cadhérine). Les kératinocytes infectés par HPV pourraient se différencier des cellules normales pour la production de ces facteurs, ce qui pourrait contribuer aux altérations des cellules de Langerhans/cellules dendritiques (LC/DC) observées dans les lésions (pré)cancéreuses cervicales. Le fait que la molécule dadhésion E-cadhérine intervienne dans lattachement des LC aux kératinocytes suggère limportance de cette molécule dadhésion dans la rétention des CL au sein de lépithélium cervical. Les objectifs de ce travail ont été détudier linfluence des oncogènes viraux sur lexpression de facteurs immunitaires et dexaminer les conséquences de linhibition de E6 et de E7 sur lexpression de la E-cadhérine et de CCL20, qui jouent un rôle important dans limmunosurveillance au niveau des épithélia via leur action sur les cellules de Langerhans. En accord avec notre hypothèse, nous avons montré une diminution de lexpression de la E-cadhérine dans les lésions (pré)néoplasiques du col par rapport à lépithélium exocervical normal (Hubert et coll. 2005). Par des expériences dARN interférence (siRNA), nous avons également démontré limplication de loncoprotéine virale E7 dans linhibition de lexpression de la E-cadhérine membranaire (Caberg et coll. 2008) et limplication des oncoprotéines virales E6 et E7 dans la diminution de la sécrétion de la chémokine CCL20 dans des kératinocytes transformés par HPV16 (Caberg et coll. 2008).