Blast neurotrauma represents a significant mode of traumatic injury to the brain. The incidence of blast neurotrauma is particularly high amongst military combat personnel and can be debilitating and endure clinically for years after injury is sustained. Mechanically, blast represents a unique and complex loading paradigm associated with compressive shock waves that propagate out from an explosive event and interact with the head and other organs through high-rate loading. When subjected to such insult, brain cells undergo characteristic injury responses which include neuroinflammation, oxidative stress, edema and persistent glial activation. These features of the injury have emerged as important mediators of the chronic brain damage that results from blast. Astrocytes have emerged as a potential therapeutic target because of their ubiquitous roles in brain homeostasis, tissue integrity and cognitive function. This glial subtype has a characteristic reactive response to mechanical trauma of various modes. In this work, custom in vitro injury devices were used to characterize functional models of astrocyte reactivity to high-rate insult to study mechano-stimulation mechanisms associated with the reactive phenotype. The working hypothesis was that high-rate overpressure exposure would cause metabolic aberrations, cell junction changes, and adhesion signal transduction activation, all of which would contribute to astrocyte response and reactivity. Astrocyte cultures were exposed to a 20 psi high-rate overpressure scheme using an underwater explosion-driven device. Astrocytes experienced dynamic energetic fluctuations in response to overpressure which were followed by the assumption of a classically defined reactive phenotype. Results indicated specific roles for cationic transduction, cell junction dynamics (gap junction and anchoring junctions) and downstream signal transduction mechanisms associated with adhesion alterations in onset of the astrocyte reactive phenotype. Investigation into adhesion signaling regulation by focal adhesion kinase in 2D and 3D cultures was also explored to better understand cellular reactivity as a function of extracellular environment. Additionally, another underwater in vitro device was built to study combination effects from overpressure and fluid shear associated with insult. Overall, the combined studies offer multiple mechanisms by which to explore molecular targets for harnessing astrocytes' potential for repair after traumatic injury to the brain. / PHD / Blast neurotrauma represents a significant mode of traumatic injury to the brain. The incidence of blast neurotrauma is particularly high amongst military combat personnel in which close to 80% of the injuries sustained in combat are attributed to explosive mechanisms. This injury, like other traumatic brain injuries, can be debilitating and result in altered quality of life for years after injury is sustained. There is a critical need to understand how brain cells are injured by and respond to blast loading in order to develop effective therapeutic strategies. The following work approaches this problem through the use of cellular models of blast-type insult. Custom injury devices were used to develop models of brain cell reactive response to high-rate insult based on experimental simulations of blast neurotrauma. In particular, a sub-type of brain cells called astrocytes were studied. Astrocytes have emerged as a potential therapeutic target because of their ubiquitous roles in brain homeostasis, tissue integrity and cognitive function. The working hypothesis was that high-rate overpressure exposure would cause metabolic aberrations, cell junction changes, and adhesion signal transduction activation, all of which would contribute to astrocyte response and reactivity. Astrocytes experienced dynamic energetic fluctuations in response to overpressure which were followed by the assumption of a classically defined reactive phenotype. Results indicated specific roles for cationic transduction, cell anchorage and downstream signaling mechanisms associated with adhesion alterations in onset of the astrocyte reactive phenotype. Investigation into adhesion signaling regulation by focal adhesion kinase in 2D and 3D cultures was also explored to better understand cellular reactivity as a function of extracellular environment. Additionally, another underwater cell injury device was built to study combination effects from overpressure and fluid shear associated with insult. Overall, the combined studies offer multiple mechanisms by which to explore molecular targets for harnessing astrocytes’ potential for repair after traumatic injury to the brain.

Claudin-5 is a transmembrane cell junction protein that is a component of tight junctions in endothelial cell layers. We have previously shown that claudin-5 also localizes to lateral membranes of murine cardiomyocytes at their junction with the extracellular matrix. Claudin-5 levels are specifically reduced in myocytes from a mouse model of muscular dystrophy with cardiomyopathy. To establish whether claudin-5 is similarly specifically reduced in human cardiomyopathy, we compared the levels of claudin-5 with other cell junction proteins in 62 cardiomyopathic end-stage explant samples. We show that claudin-5 levels are reduced in at least 60% of patient samples compared with non-failing controls. Importantly, claudin-5 reductions can be independent of connexin-43, a gap junction protein previously reported to be reduced in failing heart samples. Other cell junction proteins including α-catenin, β-catenin, γ-catenin, desmoplakin, and N-cadherin are reduced in only a small number of failing samples and only in combination with reduced claudin-5 or connexin-43 levels. We also show that reduced claudin-5 levels can be present independently from dystrophin alterations, which are known to be capable of causing and resulting from cardiomyopathy. These data are the first to show alterations of a tight junction protein in human cardiomyopathy samples and suggest that claudin-5 may participate in novel mechanisms in the pathway to end-stage heart failure.

Soluble form of Junctional adhesion molecule C (sJAM-C) has been identified to cause angiogenesis as well as chemotaxis in endothelial cells. However, the role of sJAM-C in the context of cancer has not been elucidated. Our atomic force microscopy (AFM) stiffness measurements of normal mammary epithelial cells (MCF 10A) have shown a two-fold decrease in cell's stiffness in response to sJAM-C. Changes in cell stiffness are indicative of modulations in a cell's mechanical properties. Our results indicated that sJAM-C increased the MCF 10A cell migration about two-fold and also promoted a three-fold increase in chemotaxis. Additionally, sJAM-C treatment resulted in considerable filamentous-actin loss and peripheral actin ring breakage. We also found activation of Rho signaling pathway to be the main mechanism behind sJAM-C mediated alterations in MCF 10A cell cytoskeleton and motility. Our data present for the first time that sJAM-C is a pro metastatic mediator for normal mammary epithelial cells. / by Anila Qureshi. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Infecções por Plasmodium spp. podem acarretar em complicações pulmonares (1 a 40% dos casos), que podem resultar no desenvolvimento da síndrome do desconforto respiratório agudo (SDRA). Esta síndrome é caracterizada por inflamação aguda, lesão do endotélio alveolar e do parênquima pulmonar, disfunção e aumento da permeabilidade da barreira alvéolo-capilar pulmonar e, consequente, formação de efusão pleural. Neste sentido, os mecanismos de regulação da permeabilidade das células endoteliais e as junções interendoteliais têm papel crítico na manutenção do endotélio pulmonar. O objetivo do estudo foi determinar precocemente o desenvolvimento da SDRA associada à malária por tomografia computadorizada por emissão de fóton único (SPECT/CT), além de identificar alterações nas junções interendoteliais das células endoteliais pulmonares primárias de camundongos DBA/2 (CEPP-DBA/2), após contato com os eritrócitos parasitados de Plasmodium berghei ANKA (EP-PbA). Os nossos resultados demonstraram que é possível identificar alterações na aeração pulmonar no 5° e 7° dia após a infecção e, consequentemente, diferenciar os animais que desenvolveriam SDRA daqueles que evoluiriam para hiperparasitemia (HP). Além disso, observamos em CEPP-DBA/2 que o contato direto com EP-PbA aumenta da abertura das junções interendoteliais e da permeabilidade vascular. Assim, avaliamos a diminuição da expressão das proteínas das junções interendoteliais que contribuem para o aumento da permeabilidade vascular, por imunofluorescência e Western Blot. Apesar da SDRA ter sido identificada há mais de 50 anos, ainda não se conhece formas de diagnóstico precoce e os mecanimos efetivos de desenvolvimento desta enfermidade, que permitam um tratamento efetivo e que evite a morte do paciente. Portanto, sugere-se que a técnica de SPECT/CT seja uma importante ferramenta de diagnóstico para identificação precoce de SDRA associado a malária. / Infections by Plasmodium spp. can lead to pulmonary complications (1 to 40% of the cases), that can result in the development of acute respiratory distress syndrome (ARDS). This syndrome is characterized by the acute inflammation, injury of the alveolar endothelium and pulmonary parenchyma, dysfunction and increased permeability of the pulmonary alveolar-capillary barrier and, consequently, formation of pleural effusion. In this aspect, mechanisms of regulation of endothelial cell permeability and interendothelial junctions play a critical role in the maintenance of the pulmonary endothelium. The present study aimed to determine the early development of single-photon emission computed tomography (SPECT/CT) associated malaria ARDS in addition to identifying changes in the interendothelial junctions of the primary pulmonary endothelial cells of DBA/2 mice (CEPP-DBA/2) after contact with erythrocytes infected with Plasmodium berghei ANKA (EP-PbA). Our results demonstrated that is possible to identify changes in lung aeration on the 5th and 7th day after infection and, consequently, differentiate the animals that should develop ARDS from those that would evolve to hyperparasitemia (HP). In addition, we observed in CEPP-DBA/2 that direct contact with EP-PbA increases the opening of the interendothelial junctions and vascular permeability. Thus, we evaluated that decrease the expression of interendothelial junction proteins contribute to the increase of vascular permeability, by immunofluorescence and Western Blot. Although ARDS was identified more than 50 years ago, it is not yet known what forms of early diagnosis, and the effective mechanisms of development of this disease, that allow an effective treatment and that prevent the death of the patient. Therefore, it is suggested that the SPECT/CT technique is a valuable tool to promote the early identification of ARDS associated with malaria.

Introduction: Cardiac cell-cell junctions play important roles in maintaining cardiac integrity linking single cardiomyocytes into a single functioning syncytium. There are three main types of cell junctions in the heart: gap junctions (GJ), desmosomes (D) and adherens junctions (AJ). Mutations in the proteins which make-up these junctions are known to cause arrhythmogenic right ventricular cardiomyopathy (ARVC). Pathological features include progressive replacement of right ventricular cardiac muscle with fibrofatty tissue. This can lead to heart failure and life threatening arrhythmias. During normal development of the mammalian heart, protein components of AJ and D gradually fuse to form composite junctions at the intercalated discs, also called areae compositae (singular, area composita, AC). In contrast, the adult heart of lower vertebrates, including the zebrafish, may have few or no AC type junctions. The detailed structure of cardiomyocyte cell-cell junctions in the adult zebrafish heart remain poorly defined and their role in normal development, growth and response to injury have yet to be studied. This thesis will examine the hypothesis that localisation and distribution of myocardial cell-cell junction proteins are crucial in normal myocardial development and in endogenous cardiac regeneration and repair following injury. This will be achieved by understanding the normal development of cell-cell junction proteins in zebrafish from embryonic to adulthood. These findings will then be analysed in comparison to cell-cell junction proteins localisation and distribution in early and late mammalian (mouse and human) myocardium. Once a normal pattern of cell-cell junction proteins will be established, the localisation of cell-cell junction proteins in plakologbin mutant zebrafish model for cardiomyopathy will be studied to understand the distribution and localisation of these proteins in disease manifestation. This model will then be used to test if localisation of cell-cell junction proteins plays an important in cardiac repair following injury by using embryonic laser injury model, this will be further tested by drug intervention study to investigate underlying pathways such as Wnt signalling pathway. Methods: Myocardial cell-cell junctions were assessed using immunohistochemistry in embryonic, juvenile and adult zebrafish hearts and in foetal and adult human hearts. The Plakoglobin mutant zebrafish line (UAS:Gal-4:Plakoglobin Naxos; named as PGNx) was characterised using various functional and morphological assessments including histology, echocardiography and MRI scanning. Similar studies were undertaken in PGNx mutants at different developmental stages. A pharmacological intervention study, using a GSK-3 inhibitor, was carried out in PGNx mutants followed by cardiac structural and functional assessments. Laser-induced cardiac trauma was used to assess the response to injury and repair in normal and PGNx embryos following treatment with the GSK3 inhibitor drug. Results: Cell-cell junction patterning in the embryonic, juvenile and adult zebrafish heart shows a characteristic pearl string appearance of desmoplakin and β-catenin labelled distinct disc shaped AJ. Human foetal heart showed small distinct D and AJ, while the adult human heart had features consistent with AC type junctions. PGNx fish showed reduced ventricle ejection fraction, dilatation of the atrium, reduced amplitude of wall motion and ventricle relaxation velocity compared to age-matched controls. Echocardiography and MRI imaging confirmed severe atrial dilatation and restrictive ventricle physiology in adult fish. The cell-cell junction proteins were over-expressed in the zebrafish PG mutant (PGNx) hearts compared to age-matched controls. Drug studies using a GSK-3β inhibitor showed complete recovery of cardiac function and partial recovery of heart structure. Cardiac injury studies, using laser, showed failure of repair in PGNx embryos compared to age-matched controls. The GSK3 inhibitor failed to improve the functional response following heart laser injury. Conclusions: Cell-cell junctions are distributed abundantly around cardiomyocytes in the zebrafish heart during early development and into adulthood. In contrast to previous studies in adult mammalian heart, there was no evidence of AC type junctions in adult zebrafish cardiomyocytes. The mutant zebrafish line showed restrictive cardiac physiology and abnormal cardiac structure confirming the key role played by plakoglobin in the normal heart development. This is further supported by evidence showing failure of repair in PGNx mutant embryos after injury. Drug treatment with a GSK-3 inhibitor highlights a potentially novel therapeutic pathway for treatment of ARVC involving Wnt signalling.

Lower respiratory diseases are currently the third leading cause of death worldwide. For many end-stage patients with these diseases, there is no cure and a shortage of donor organs available for transplant. A promising solution is to design regenerative scaffolds or complete bioengineered lungs, using decellularized lung tissues as a template for regeneration. Recent advances in the field have made significant strides towards developing a transplantable lung. However, the current technology has not produced a functional lung for in vivo transplant due to immature gas exchange barriers. The mechanisms driving alveolar barrier maturation and role that extracellular matrix (ECM) plays within the strengthening of each type of junction are not fully understood. This research has characterized and tailored a decellularized ECM (dECM) coating for the in vitro study of dECM component depletion and potential effects on cell barrier function, attachment, and survival. Adjustments to dECM digestion duration drastically changed the resulting structural and biochemical properties for each cellular microenvironment. Shorter digestion time resulted in a dense branching of the ECM architecture and biomimetic mechanical properties needed for epithelial culture. Also, through systematic supplementation of essential basement membrane (BM) proteins to dECM, we have found that supplementation with laminin enhanced barrier strength by ZO-1 junction stabilization. This indicates that dECM can promote barrier formation but may have lost vital proteins that need to be replenished. Laminin-mediated barrier function was determined to be caused by the upregulation of the Epac/Rap1 pathway. This pathway has previously been implicated in lung endothelial barriers but not alveolar epithelial junction strengthening. Finally, to establish the translatability of these findings to whole lung recellularization, the dECM coating was used to pre-treat the airways of decellularized lungs for recellularization. Culture of MLE12 mouse epithelial cells into dECM-coated lungs increased cell survival and distribution. In combination with dECM coatings, rotational cell seeding improved cell dispersal and viability. Altogether, these techniques, devised to promote healthy alveolar barriers, are vital to enhancing current lung recellularization strategies and the treatment of many edema-associated pulmonary diseases.

The Sertoli cell ectoplasmic specialization is a specialized domain of the calcium-dependent Sertoli-spermatid adherens junction. Structurally abnormal or absent Sertoli ectoplasmic specializations are associated with spermatid sloughing and subsequent oligospermia in conditions associated with reduced fertility potential, although the junctional strength between these cells is not known. Adjudin is a potential male contraceptive agent thought to interrupt testicular binding dynamics of adherens junctions, resulting in controlled spermatid sloughing.It was hypothesized that the mechanism of action of Adjudin, pertinent to its putative contraceptive effect, is the disruption of the Sertoli cell-spermatid junction. This was tested in vitro using primary isolates of germ cells and both primary and immortal Sertoli cells.This dissertation presents the examination of Sertoli-germ cell interactions in three parts, which address the overall aims of this dissertation project: (1) measurement of the junctional strength between Sertoli cells and spermatids in vitro, (2) determination of the efficacy of sk Sertoli cell lines in Sertoli-germ cell binding studies in vitro, and (3) assessment of Adjudin as a potential male contraceptive, by measuring the junctional binding strength between Sertoli cells and spermatids exposed to this chemical in vitro.For the first time, the strength of the Sertoli-spermatid junction has been measured, using a micropipette pressure transducing system (MPTS). Results reported in this dissertation demonstrate that the junctional strength between Sertoli cells and germ cells can be measured in vitro, support long held speculations regarding Sertoli-spermatid junctional interactions, and provide a technology to test proposed mechanisms of junctional binding dynamics between cells of the seminiferous epithelium (Chapter 2). Although the sk cell lines initially expressed mRNA for the FSH receptor, coculture results determined that these cell lines have limited value for investigating Sertoli-germ cell binding dynamics in vitro (Chapter 3). By utilizing the MPTS and primary cell isolates, Adjudin was determined to reduce the junctional strength between Sertoli cells and step-8 spermatids. In conclusion, results support the use of Adjudin as a potential reversible male contraceptive agent by a mechanism which alters the adhesion properties between the step-8 spermatid and the Sertoli cell (Chapter 4).

Les mutations MKS1 sont impliquées dans un groupe de ciliopathies récessives létales, telles que le syndrome de Meckel-Gruber (MKS) et le syndrome de Joubert (JBT), caractérisées par une dysplasie rénale kystique, des anomalies du système nerveux central (encéphalocèle occipitale), une polydactylie, une dysgénésie biliaire et une fibrose hépatique. MKS1 a été localisée dans la zone de transition du cil dans de nombreux types cellulaires où elle joue un rôle essentiel pour la structure et la fonction des cils, en particulier la régulation de plusieurs voies de signalisation telles que Wnt et Shh.Dans le présent travail, nous avons identifié la fonction pré-ciliaire de MKS1 dans des cellules épithéliales. Nous avons montré que la localisation subcellulaire de MKS1 varie au cours de la maturation de l’épithelium, passant du cytosol où MKS1 co-localise avec le réseau de kératine, aux jonctions cellulaires, où elle co-colocalise avec les caténines. De plus, la translocation de MKS1 des jonctions au cytosol s'est avérée être mécano-sensible, suggérant que MKS1 participe à l'homéostasie épithéliale en stabilisant les jonctions cellulaires, via la transduction des signaux mécaniques liés à la compaction de l’épithelium.L’analyse fonctionnelle a démontré que le « knockdown » de MKS1 désorganise le réseau de kératine, et déstabilise les jonctions adhérentes des cellules épithéliales en culture, avec une diminution de la β-caténine jonctionnelle et une libération de l’E-cadhérine, l’α-caténine et la vinculine dans le cytosol. De plus, la déplétion de MKS1 entraîne une diminution notable du réseau apical d’actine, ainsi que la désorganisation de la structure épithéliale et une transition partielle vers un état mésenchymateux. Ces résultats illustrent une fonction indépendante du cil de MKS1 dans l’homéostasie épithéliale, et apporte de nouvelles hypothèses quant à son rôle et celui des filaments intermédiaires dans les processus d’organogenèse des épitheliums, en particulier la tubulogenèse, qui repose à la fois sur l’équilibre de la transition épithelium/mesenchyme et la mécanotransduction des sollicitations mécaniques durant l’embryogenèseDans le but de caractériser les partenaires de MKS1, des expériences de Co-IP et d’analyses protéomiques ont permis d’identifier l’epiplakine comme un partenaire possible de MKS1. L'Epiplakine est un cytolinker capable de lier la kératine à la membrane et à l'actine ; l’interaction de MKS1 avec l’epiplakine pourrait ainsi rendre compte de la stabilisation à la fois du réseau de kératine et des jonctions cellulaires. Des analyses complémentaires de protéomique et des études fonctionnelles devront compléter ces résultats préliminaires.Finalement, ces travaux ont également permis de révéler le rôle de MKS1 dans la stabilisation des jonctions communicantes ; la déplétion de MKS1 conduisant à une diminution de la CX43 jonctionnelle et à une altération de la fonction de communication intercellulaire dans les cellules épithéliales en culture. Ces travaux, qui constituent la première mention d’une altération possible des jonctions communicantes dans ce type de maladies, devront être approfondis pour caractériser leur impact dans les processus de tubulogenèse.En conclusion, ce travail qui a permis de révéler un rôle pré-ciliaire de MKS1 dans l'homéostasie épithéliale, apporte de nouvelles hypothèses pour l’étiologie de ces maladies, jusqu’alors considérées comme essentiellement consécutives à des défauts de transduction de la signalisation ciliaire. Il propose également de nouveaux mécanismes pour rendre compte des anomalies du développement hépatique, telles que la dysgénésie des voies biliaires, et plus largement des processus de tubulogenèse impliqués dans le développement de nombreux organes. / MKS1 mutations are involved in a group of lethal recessive ciliopathies, such as Meckel-Gruber syndrome (MKS) and Joubert's syndrome (JBT), characterized by cystic renal dysplasia, central nervous system abnormalities (occipital encephalocele) , polydactyly, biliary dysgenesis and hepatic fibrosis. MKS1 has been located in the transition zone of the cilia in many cell types where it plays an essential role in the cilia structure and function, in particular in the regulation of signaling pathways such as Wnt and Shh.In the present work, we have identified the preciliary function of MKS1 in epithelial cells. We have shown that the subcellular localization of MKS1 varies during the maturation of the epithelium, from the cytosol where MKS1 co-localizes with the keratin network, to the cell junctions, where it co-localizes with the catenins. In addition, the MKS1 translocation to cytosol junctions proved to be mechano-sensitive, suggesting that MKS1 participates in epithelial homeostasis by stabilizing cell junctions, via the transduction of mechanical signals related to epithelial compaction.Functional analysis has shown that the knockdown of MKS1 disrupts the keratin network, and destabilizes the adherent junctions of epithelial cells in culture, with a decrease in the junctional β-catenin and a release of E-cadherin, the α-catenin and vinculin in the cytosol. In addition, the depletion of MKS1 results in a significant decrease in the apical actin network, as well as disorganization of the epithelial structure and a partial transition to a mesenchymal state. These results illustrate a ciliary-independent function of MKS1 in epithelial homeostasis, and provides new hypotheses regarding its role and that of intermediate filaments in epithelial organogenesis processes, in particular tubulogenesis, which is based both on the equilibrium of the epithelium / mesenchyme transition and the mechanotransduction of mechanical stresses during embryogenesisIn order to characterize MKS1 partners, Co-IP experiments and proteomic analyzes have identified epiplakin as a possible MKS1 partner. Epiplakin is a cytolinker capable of binding keratin to membrane and actin; the interaction of MKS1 with epiplakin could thus account for the stabilization of both the keratin network and cell junctions. Additional proteomic analyzes and functional studies will complement these preliminary results.Finally, this work has also revealed the role of MKS1 in the stabilization of gap junctions; the depletion of MKS1 leading to a decrease in the junctional CX43 and an alteration of the intercellular communication function in the epithelial cells in culture. This work, which constitutes the first mention of a possible alteration of gap junctions in this type of disease, will have to be further developed to characterize their impact on tubulogenesis processes.In conclusion, this work which revealed a pre-ciliary role of MKS1 in epithelial homeostasis, provides new hypotheses for the etiology of ciliopathies, previously considered as essentially consecutive to signal transduction defects. It also proposes new mechanisms to account for abnormalities of hepatic development, such as bile ducts dysgenesis, and more broadly tubulogenesis processes involved in the development of many organs.

Un défi majeur en cancérologie est le traitement des tumeurs mammaires dites triple-négatives (ER-, PR-, HER2-). Elles sont le plus souvent résistantes aux traitements conventionnels et présentent un haut risque de récidive. De plus, l’absence de cibles thérapeutiques ne permet pas le développement de thérapie spécifique. 78% de ces tumeurs expriment faiblement la claudine 1 et sont de très mauvais pronostic. Cette protéine est impliquée dans l'adhérence des cellules entre elles et pourrait jouer un rôle suppresseur de tumeur dans les cancers mammaires. Dans ce contexte, nous étudions si sa réexpression pourrait être une piste de traitement. Au laboratoire, nous avons développé des dérivés de la famille des thiazolidinediones (TZD) qui stimulent l’expression de la claudine 1 et induisent l’apoptose des cellules cancéreuses mammaires. Les objectifs de ma thèse ont consisté 1) à déterminer l’implication de la claudine 1 dans l’effet pro-apoptotique de ces composés 2) à l’étude de leurs mécanismes d’action 3) évaluer si l’expression de la claudine 1 pourrait sensibiliser les cellules cancéreuses triple-négatives aux agents de chimiothérapie. Au cours de cette thèse, nous avons montré que la surexpression de la claudine 1 et le composé Δ2-TGZ induisent l’apoptose des cellules triple-négatives « claudin 1-low » MDA-MB-231 et Hs578T. De plus, la claudine 1 est impliquée dans l’effet pro-apoptotique de la Δ2-TGZ dans les cellules MDA-MB-231. Par ailleurs, nous avons démontré que les dérivés TGZ, la Δ2-TGZ et l’AB186, agissent de manière précoce en modifiant la morphologie des cellules suivie d’une réexpression de la claudine 1 membranaire et d’une inhibition de la migration cellulaire avant même d’induire la mort cellulaire par apoptose. De plus, la surexpression de la claudine 1 inhibe la migration cellulaire associée à la perte des fibres de stress et la formation des jonctions intercellulaires. Nous avons également montré que la réexpression de la claudine 1 sensibilise les cellules MDA-MB-231 à l’agent de chimiothérapie, le 5-FU. L’ensemble des résultats de thèse a permis de mieux comprendre le mécanisme d’action de la Δ2-TGZ et de l’AB186 sur les cellules cancéreuses mammaires mais aussi d’identifier la claudine 1 comme cible potentielle prometteuse dans les cellules triple-négatives « claudin 1-low » / A major challenge in oncology is the treatment of triple-negative breast cancer (ER-, PR-, HER2-) as no targeted therapy are available. These tumors present often a chemotherapy resistance and a higher relapse incidence. 78% of them do not express claudin 1 and display a poor prognosis. Claudin 1 is involved in cell-cell adhesion and may be a tumor suppressor gene in breast cancer. In this context, we study if claudin 1 re-expression could be a possible approach. In the laboratory, we developed derivatives thaziolidinediones (TZD) compounds, which increase claudin 1 expression and lead to apoptosis of breast cancer cells. The goals of my thesis is 1) to characterize the involvement of claudin 1 in their pro-apoptotic effect 2) to study their mechanism of action 3) to determine if claudin 1 could sensitize the TNBC cells to the chemotherapy agents. During my thesis, we showed that claudin 1 overexpression and the compound Δ2-TGZ induce apoptosis of TNBC « claudin 1-low » MDA-MB-231 and Hs578T cells. Claudin 1 is involved in the pro-apoptotic effect of Δ2-TGZ in MDA-MB-231 cells. Then, we demonstrated that Δ2-TGZ and AB186 lead to early action through a modification of cell morphology followed an expression of claudin 1 at the membrane and an inhibition of cell migration before the apoptosis process. In addition, claudin 1 overexpression decreases the cell migration through the loss of stress fibers and the formation of cell junctions. We showed that claudin 1 overexpression potentialize the pro-apoptotic effect of Δ2-TGZ in MDA-MB-231 cells. Finally, we observed that claudin 1 sensitize the MDA-MB-231 cells to 5-FU. In fine, our data allowed a better understanding of Δ2-TGZ and AB186 mechanism of action and identification of claudin 1 as a promising target in TNBC « claudin 1-low »

Mechanical tension is an important regulator of cell proliferation, differentiation, migration and cell death. It is involved in the control of tissue architecture and wound repair and its improper sensing can contribute to cancer. The Hippo tumor suppressor pathway was recently shown to be involved in regulating cell proliferation in response to mechanical tension. The core of the pathway consists of the kinases MST1/2 and LATS1/2, which regulate the target of the pathway, the transcription co-activator YAP/ TAZ (hereafter referred to as YAP). When the Hippo pathway is inactive, YAP remains in the nucleus and promotes cell proliferation and stem cell maintenance. When the Hippo signaling pathway is turned on, MST1/2 phosphorylate and activates LATS1/2. LATS1/2 phosphorylates and inactivates YAP in the cytoplasm which is sequestered and degraded, stopping cell proliferation and promoting differentiation of stem cells. Mechanical forces are transmitted across cells and tissues through the cell-cell junctions and the actin cytoskeleton. However, the factors that connect cell-cell junctions to the Hippo signaling pathway were not clearly known. We identified a LIM domain protein called TRIP6 that functions at the adherens junctions to regulate the Hippo signaling pathway in a tension-dependent manner. TRIP6 responds to mechanical tension at adherens junctions and regulates LATS1/2 activity. Under high mechanical tension, TRIP6 sequesters and inhibits LATS1/2 at adherens junctions to promote YAP activity. Conditions that reduce tension at adherens junctions by inhibition of actin stress fibers or disruption of cell-cell junctions reduce TRIP6-LATS1/2 binding, which activates LATS1/2 to inhibit YAP. Vinculin has been shown to act as part of a mechanosensory complex at adherens junctions. We show that vinculin promotes TRIP6 inhibition of LATS1/2 in response to mechanical tension. Furthermore, we show that TRIP6 competitively inhibits MOB1 (a known LATS1/2 activator) from binding and activating LATS1/2. Together these findings reveal TRIP6 responds to mechanical signals at adherens junctions to regulate the Hippo signaling pathway in mammalian cells.