Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / O câncer colorretal (CCR) é o terceiro tipo de câncer mais incidente no mundo para o sexo masculino, o segundo para o sexo feminino e a radioterapia é um dos tratamentos de primeira linha no combate a este tipo de câncer. Durante a progressão do CCR as células sofrem alterações morfogenéticas, sendo a desorganização do complexo juncional apical (CJA) um dos eventos iniciais desse processo. As junções oclusivas (JTs) são um dos principais componentes da CJA e desempenham papel importante no controle do fluxo paracelular, na determinação da polaridade celular e na transdução de sinais relacionados com a progressão tumoral. As claudinas são proteínas transmembrana, constituintes das JTs e cumprem um importante papel no controle desses eventos. Alterações na expressão das claudinas são observadas em tumores de diferentes órgãos e têm sido relacionadas com a progressão tumoral. No entanto os mecanismos que regulam essas alterações e sua consequência na progressão do CCR são poucos conhecidos. Desta forma, o presente estudo teve como objetivo avaliar a influência da superexpressão da claudina-3 na radiorresposta de células CCR. Nossos resultados mostraram que a superexpressão de claudina-3 minimiza alterações morfológicas causadas pela radiação, causa diminuição da resistência elétrica transepitelial e não tem efeito na permeabilidade a macromoléculas após a irradiação. Além disso, observamos que a superexpressão de claudina-3 aumenta o potencial proliferativo das células e que esta característica torna as células mais sensíveis a radiação. Porém quando avaliamos eventos celulares relacionados a progressão tumoral observamos que apesar da radiação diminuir a capacidade migratória das progênies, as células que superexpressam claudina-3 apresentam migração mais elevada. Além disso, verificamos que a superexpressão de claudina-3 diminui a invasão e a capacidade de formação de colônias frente ao tratamento com a radiação. Em seguida fomos avaliar o efeito da inibição das vias de proliferação (MEK/ERK) e sobrevivência (PI3K-Akt) na resposta das células que superexpressam claudina-3 frente a radiação. Observamos que a inibição de MEK é capaz de sensibilizar as células que superexpressam claudina-3 à radiação no ensaio de proliferação celular, no entanto a inibição de MEK e PI3K antes da exposição à radiação é capaz aumentar a migração e a capacidade de formação de colônias de células que superexpressam claudina-3 contribuindo para o aumento do potencial maligno. Em conjunto nossos resultados mostram que a superexpressão de claudina-3 contribui para um fenótipo mais maligno, no entanto frente ao tratamento com a radiação é capaz de sensibilizar as células. / Colorectal cancer (CRC) is the third more incident cancer for males, the second for females worldwide and radiotherapy is one of the first-line treatments to fighting this type of cancer. During the progression of CRC cells undergo morphogenetic alterations and the apical junctional complex disorganization (AJC) is one of the initial events of this process. The tight junctions (TJs) is a major component of AJC and play an important role in paracellular flux control, determination of cell polarity and in signal transduction related to tumor progression. Claudins are transmembrane proteins, members of TJs and play an important role on these events. Changes on claudins expression are found in tumors of different organs and have been associated with tumor progression. However the mechanisms that regulate these changes and their consequences in the CRC progression are not completely understood. Thus, this study aimed to evaluate the influence of claudin-3 overexpression on cellular response after radiation treatment of CRC cells. Our results show that claudin-3 overexpression minimizes morphological changes caused by the radiation, decrease transepithelial electrical resistance, and has no effect on macromolecules permeability after irradiation. Moreover, we observed that claudin-3 overexpression increases the proliferation rate of cells and that this feature makes the cells more sensitive to radiation. However, when evaluating the cellular events associated tumor progression we observed that despite decrease on migratory capacity caused by radiation, cells that overexpress claudin-3 have higher migration. In addition, we found that claudin-3 overexpression decreases the invasion and the capacity to form colonies after treatment with radiation. Then we evaluate the effect of inhibition of proliferation (MEK / ERK) and survival (PI3K-Akt) pathways in cells that overexpress claudin-3 in the response to radiation. We observed that inhibition of MEK could sensitize cells overexpressing claudin-3 radiation on cell proliferation assay, but the inhibition of MEK and PI3K before radiation exposure can increase the migration and colony forming ability cells overexpressing claudin-3 contributes to the increase of malignant potential. Altogether, our results show that claudin-3 overexpression contributes to a more malignant phenotype, however, claudin-3 overexpression is able to sensitize the cells to radiation.

Abstract Acute pancreatitis is a common disease, Finland being among the countries with the highest incidence. The majority of patients have a mild, self-limiting disease. However, 20% of these patients develop severe necrotizing pancreatitis with a mortality rate of 7 to 25%. The mechanisms for developing the severe disease are not known, it is not possible to accurately forecast the severity of the disease and there is no curative treatment yet. This study was aimed at analyzing the early phase of acute experimental porcine oedematous and necrotizing pancreatitis. In Study I, the pancreatic microcirculatory changes were measured and the expression of tight junction proteins (claudins-2, -3, -4, -5 and -7) and the rate of apoptosis in the pancreas were all measured. In Study II, bacterial translocation to the blood in the portal vein blood or to the mesenteric lymph nodes was analyzed and the intestinal expression of tight junction proteins (claudins-2, -3, -4, -5 and -7) and the intestinal apoptosis/ proliferation rates were measured. The basic histology of the jejunum and colon were analyzed. Study III analyzed which cytokines are released from the pancreas to the portal venous blood. In Study IV, the ultrastructure of the epithelium of the jejunum and colon was analyzed and the expression of adherens junction proteins, E-cadherin and β-catenin, were measured from both jejunum and colon. The first study (I) showed that membranous immunoreactivity of claudin-2 in acinar cells appeared in the pancreas during acute oedematous and necrotizing pancreatitis. The expressions of claudins -3, - 4, - 5 and 7 were unaffected. The second study (II) showed that bacterial translocation from the gut was not present at the beginning of acute porcine pancreatitis. The expressions of claudins-2 and -5 do not become altered; however, there might be some decrease in claudin-3 expression in the colon and decrease in the expression of claudins-4 and -7 in the jejunum in necrotizing pancreatitis. Performing the laparotomy itself caused increased apoptosis in the colon and the jejunum. In the third study (III), the initial inflammatory process was diverse in oedematous and necrotizing pancreatitis. Increased monocyte count in combination with elevated PDGF and IL-6 are characteristic of necrotizing pancreatitis in our model. The fourth study (IV) indicated that necrotizing pancreatitis caused damage to the epithelial and endothelial cells of the colon in the early stages of the disease. The expression of E-cadherin immunoreactivity showed a decreasing trend in the colon in both oedematous and necrotizing pancreatitis. The results of this study suggest that claudin-2 increases in acinar cells during acute porcine pancreatitis. Bacterial translocation is not present during the early phase of acute porcine pancreatitis. Increased monocyte count and elevated PDGF and IL-6 are characteristic of early phase necrotizing porcine pancreatitis and necrotizing porcine pancreatitis causes damage to the epithelial and endothelial cells of the colon. / Tiivistelmä Akuutti haimatulehdus on yleinen sairaus, jonka ilmaantuvuus Suomessa on verrattain suuri. Suurimmalla osalla potilaista tauti on lievä ja itsestään paraneva. Kuitenkin 20 %:lle potilaista kehittyy vaikea haimatulehdus, johon liittyy 7–25 %:n kuolleisuus. On epäselvää, miksi toisinaan kehittyy vaikea tautimuoto. Taudin vaikeusastetta ei voida etukäteen tarkasti ennustaa, eikä tautiin ole parantavaa hoitoa. Väitöskirjatyön tarkoituksena oli tutkia lievän ja vaikean haimatulehduksen varhaisvaihetta kokeellisessa sikamallissa. Työssä I mitattiin haiman mikroverenkierron muutoksia, tutkittiin tiivisliitosproteiinien klaudiini-2:n, -3:n, -4:n, -5:n ja -7:n ilmenemistä sekä apoptoosin määrää haimassa. Toisessa työssä tutkittiin mahdollista bakteeritranslokaatiota porttilaskimovereen ja vatsaontelon imusolmukkeisiin, mitattiin suoliston tiivis liitos-proteiinien klaudiinien-2, -3, -4, -5 ja -7 ilmenemistä ja suoliston apoptoosin ja soluproliferaation määrää. Mahdollisia muutoksia ohut- ja paksusuolen perushistologiassa analysoitiin. Kolmannessa työssä mitattiin sytokiinipitoisuuksia porttilaskimoverestä. Neljännessä työssä analysoitiin ohut- ja paksusuolen mikrorakennetta elektronimikroskopian avulla ja mitattiin vyöliitosproteiinien E-cadherin ja β-catenin määrää. I työssä todettiin klaudiini-2:n ilmaantuvan haiman asinaarisolujen solukalvoille lievässä ja vaikeassa kokeellisessa haimatulehduksessa. Klaudiinien 3,- 4,- 5 ja 7 esiintyminen haimassa ei muuttunut. II työssä todettiin, että bakteeritranslokaatiota ei tapahtunut seuranta-aikana. Suolistossa klaudiinien-2 ja -5 ilmenemisessä ei tapahtunut muutoksia. Klaudiini-3:n ilmenemisessä paksusuolessa ja klaudiinien -4 ja -7 ilmenemisessä ohutsuolessa saattaa tapahtua vähenemistä vaikeassa haimatulehduksessa. Tutkimustoimenpide itsessään aiheutti ohut- ja paksusuolen apoptoosin lisääntymistä. III työn mukaan tulehdusvaste oli erilainen akuutissa lievässä ja vaikeassa kokeellisessa haimatulehduksessa. Monosyyttimäärän sekä PDGF:n ja IL-6:n pitoisuuksien lisääntyminen, olivat tyypillisiä vaikealle haimatulehdukselle tässä mallissa. IV työssä todettiin, että vaikea haimatulehdus vaurioittaa paksusuolen epiteeli- ja endoteelisoluja. E-cadherin: n määrässä todettiin jonkin verran vähentymistä sekä lievässä että vaikeassa haimatulehduksessa. Näiden tulosten mukaan klaudiini-2 lisääntyy sian haiman asinaarisoluissa akuutissa haimatulehduksessa. Sialla ei tapahdu bakteerien translokaatiota haimatulehduksen varhaisvaiheessa. Sian vaikeaan haimatulehdukseen liittyy monosyyttien, PDGF:n ja IL-6:n lisääntyminen. Kokeellisessa vaikeassa haimatulehduksessa paksusuolen epiteeli- ja endoteelisolut vaurioituvat jo varhaisvaiheessa.

Polymorphic light eruption (PLE) is the most common photodermatosis, affecting ~17% of the population. PLE is a delayed-type hypersensitivity response to an antigen induced by solar ultra-violet radiation (UVR). Its effects vary between patients, but the main symptom is a non-scarring, red papular rash in areas exposed to UVR. An effective therapy is low dose ultra-violet B (NBUVB) phototherapy. It is thought that NBUVB phototherapy desensitises the skin to further UVR exposure, but the mechanism by which this happens is unknown. Current immune based studies have been unable to clarify a mechanism as to how PLE arises. However, research in other skin diseases, such as psoriasis and atopic dermatitis, has shown that the barrier function of the skin is compromised by these disorders. Furthermore, research in lesional PLE skin showed an increase in barrier permeability of the skin. Recent research has specifically linked claudin proteins of tight junctions to the barrier dysfunction. Therefore, this study used quantitative immunofluorescent staining to measure tight junction (TJ) proteins and other barrier proteins of interest. Barrier function was also measured by transepidermal water loss (TEWL); a tracer dye penetration assay was used to measure TJ barrier function specifically. All measurements were made in non-lesional PLE skin, as compared to skin from healthy human volunteers. In photoprotected PLE skin the TJ protein claudin-1 was significantly reduced compared to healthy skin. The use of a tracer dye highlighted there was a reduction in TJ barrier function in PLE skin compared to healthy individuals. PLE and healthy skin were then exposed to ultra-violet B (UVB) and 24h later TJ proteins and TJ barrier function were measured. There was no change to claudin-1 after UVB exposure in PLE skin, but claudin-7 was reduced and claudin-12 increased. In contrast, in UVB-irradiated skin in normal controls after UVB exposure claudin-7 and claudin-12 were both increased, whilst claudin-1 was reduced. In PLE patients there was no further change to TJ barrier function, however, in normal controls, skin TJ barrier function was reduced post UVB. Both in healthy and PLE skin TEWL was unchanged before and after UVB exposure. Lastly TJ proteins were investigated after NBUVB in PLE patients. There was a further reduction in claudin-1 in PLE patients as well as a reduction in the TJ protein occludin, however the stratum corneum was significantly thickened. It could be suggested that this is a compensatory measure for the reduction seen in TJ barrier proteins, however further studies are needed to understand this. These data show significant differences in the TJ skin barrier in patients with PLE as compared to healthy human volunteers before and after UVB exposure. Furthermore, in PLE skin there is a significant change to the epidermis after NBUVB phototherapy. These data demonstrate that TJ protein expression and function is altered in PLE skin and may contribute to aetiology of the disorder, however the role of TJ barrier in aetiology is yet to be firmly established.

The mammalian skin provides a necessary barrier between the organism and the environment, defending against loss of water and solutes, preventing the invasion of pathogens as well as protecting against chemical and physical assault. Claudin (Cldn)-based Tight Junctions (TJs) are the main functional part of the skin barrier. In particular, Cldn6 through its cytoplasmic tail has been shown to be important for barrier function. In other to further investigate the role of the Cldn6 tail in TJ-function, we developed Cldn6 mouse mutants carrying varying truncations of the Cldn6 tail. Both of these mice present with epidermal differentiation perturbations and delayed barrier function that is repaired later in life. These studies support the importance of the tail portion of the Cldn molecules in epidermal differentiation and barrier function. In addition, both of these mouse models are useful for the study of barrier function in preterm infants and in aging, with the hope of developing novel therapeutics for the alleviation of barrier dysfunction. Cdx is a family of homeodomain (HD) transcription factors (TFs) essential for many key developmental processes. In particular, Cdx2 is important for the establishment and maintenance of posterior identity in the developing endoderm. In spite of this, only a few Cdx targets in the developing endoderm have been discovered. In addition, the interplay between Cdx and its targets within the endoderm is poorly understood. In this study, we show that the forkhead box transcription factor, Foxa2 is a Cdx2 target. We also show that Foxa2 and Cdx2 physically and genetically interact to regulate a subset of genes that are implicated in endodermal development. These studies help to further our understanding of endoderm biology with the goal of developing new strategies to diagnose and treat diseases associated with defective endoderm development.

The immunoglobulin superfamily (IgSF) is a large protein superfamily of membrane and soluble proteins that influence recognition, binding, and adhesion. Among members of this family are cell adhesion molecules (CAMs), which form cell-cell contact points that play key roles in development, cell polarization, and cellular fate. Cadherins (CADs) are calcium-dependent proteins of the adherens junction (AJ), and polarize epithelium and endothelium. The tight junction (TJ) is a multiprotein junctional complex whose function is to control the permeability of the paracellular pathway. At the membrane level, TJs are composed of three types of proteins: claudins (CLDNs), occludin (OCLN) and junctional adhesion molecules (JAMs). JAMs are members of the IgSF while CLDN and OCLN are 4-α-helix membrane proteins. Although JAMs are part of the TJ and reside in the same ultrastructure, they are similar to CADs in their secondary, tertiary, and quaternary protein structure. Crystallographic studies of CADs in the presence of calcium yielded trans interactions that resulted in cell-cell contacts. In the absence of calcium, CADs form cis interactions that do not form cell-cell interactions. The crystal structure of JAM-A, has a quaternary organization of a cis dimer. In spite of the many similarities, a link between CADs and JAMs remains unclear. Beyond this point, the association between JAMs, CLDNs, and OCLN in the TJ is vaguely understood. The JAM family (JAM-A, -B, -C and 4) and their tissue-specific distribution indicate that they are key to understanding the TJ’s function and the interplay with the AJ. JAM-A has been used as a prototype for the other three members of the family, but based on current evidence we hypothesized that these proteins may display unique properties to support TJ’s function in a given tissue. Are JAMs affected by calcium just as CADs? Do CLDNs and OCLN make direct contact with JAMs? Do JAMs coordinate the interplay between TJ and AJ? We designed a strategy based on recombinant proteins and biophysical methods to answer these questions. First, we fused the extracellular domain of each JAM to maltose-binding protein (MBP). Our results indicate that JAM proteins have similar secondary structures, but unique tertiary structures. Surface Plasmon Resonance experiments showed that JAM proteins favored heterotypic compared to homotypic interactions. Second, we addressed the effects of cations (Ca2+, Mg2+, Cu2+, Fe2+, Fe3+, and Zn2+) on JAM-A. The exposure of JAM-A to the resulted in changes in its secondary, tertiary structure, and homotypic binding affinity. Finally, we addressed whether cations had an effect on the other TJ components and if there is an interplay with E-CAD. We determined that in the assembly of a simple TJ and AJ, JAM-A and E-CAD are calcium-dependent, while CLDN1 and OCLN are calcium independent. We conclude that TJ components such as CLDN1 and OCLN may work as anchors to maintain cell-cell interactions while JAM-A and E-CAD would be regulated by cations in order to accommodate other homeostatic functions.

The blood-brain barrier (BBB) maintains brain homeostasis by limiting entry of substances to the central nervous system through interaction of transmembrane and intracellular proteins that make up endothelial cell tight junctions (TJs). Recently it was shown that the BBB can be modulated by disease pathologies including inflammatory pain. This study examined the effects of chronic inflammatory pain on the functional and molecular integrity of the BBB. Inflammatory pain was induced by injection of complete Freund's adjuvant (CFA) into the right plantar hindpaw in female Sprague-Dawley rats under halothane anesthesia; control animals were injected with saline. Edema and hyperalgesia were assessed by plethysmography and infrared pawwithdrawal latency. At 72 h postinjection, significant edema formation and hyperalgesia were noted in the CFA-treated rats. Examination of permeability of the BBB by in situ perfusion of [14C]sucrose while rats were under pentobarbital anesthesia demonstrated that CFA treatment significantly increased brain sucrose uptake. Western blot analysis of BBB TJ proteins showed no change in expression of zonula occludens-1 (an accessory protein) or actin (a cytoskeletal protein) with CFA treatment. Expression of the transmembrane TJ proteins occludin and claudin-3 and -5 significantly changed with CFA treatment with a 60% decrease in occludin, a 450% increase in claudin-3, and a 615% increase in claudin-5 expression. This study demonstrates that during chronic inflammatory pain, alterations in BBB function are associated with changes in specific transmembrane TJ proteins.

No description available.

Die Tight Junction (TJ) reguliert den parazellulären Transport von Ionen, Wasser und Soluten an Epithelien und Endothelien und ist von entscheidender Bedeutung für die Aufrechterhaltung der Funktion von Organen und Geweben. Obwohl Claudin-3 zu den zuerst identifizierten und ubiquitär exprimierten Komponenten der TJ gehört, konnte seine spezifische Funktion bislang nicht geklärt werden. Für die funktionelle Charakterisierung des humanen Claudin-3-Proteins wurden stabile Überexpressionsklone der lecken Nierenepithel-Zelllinie MDCK II generiert. Die Überexpression von Claudin-3 führte zu einer deutlichen Änderung des TJ-Strangmusters sowie zu einer starken Zunahme des transepithelialen Widerstandes und einer verminderten Permeabilität für Ionen und Moleküle der Größe 332 Da und 4000 Da. Der parazelluläre Durchtritt von Wasser war unverändert. Claudin-3 konnte eindeutig als abdichtende Komponente der TJ identifiziert werden. Anhand des endothelialen Zellkulturmodells HUVEC wurden Expression und Regulation von Claudin-3 und anderen TJ-Proteinen unter dem Einfluss mechanischer Strömungsverhältnisse und des Sauerstoffpartialdrucks analysiert. Die Behandlung mit fehlender Wandschubspannung führte zur Hochregulation der abdichtenden TJ-Proteine Occludin, Claudin-3, Claudin-5 und Claudin-11, nicht aber Claudin-23. Die Regulation der einzelnen TJ-Komponenten wurde durch unterschiedliche Signalwege vermittelt, wobei der verstärkten Proteinexpression jeweils eine Hochregulation auf mRNA-Ebene zugrunde lag. Die kombinierte Behandlung mit fehlender Wandschubspannung und Hypoxie resultierte in einer sehr stark erhöhten Expression von Claudin-3. Durch die Hochregulation abdichtender TJ-Komponenten unter Bedingungen fehlender Wandschubspannung und Hypoxie, wie sie in verschiedenen physiologischen und pathologischen Situationen auftreten, könnte einem unerwünschten Durchtritt von Substanzen aus dem Blut in das umliegende Gewebe vorgebeugt werden. / The tight junction (TJ) regulates the paracellular transport of ions, water and solutes in epithelia and endothelia and is of particular importance for a correct function of organs and tissues. Although claudin-3 is one of the first identified and ubiquitously expressed TJ components, its specific function was unsolved as yet. For functional characterization, human claudin-3 was stably overexpressed in the leaky epithelial cell line MDCK II. Overexpression of claudin-3 led to a marked alteration of TJ meshwork pattern, a strong increase in transepithelial resistance and a decrease in permeability for ions and paracellular tracers (332 or 4000 Da). Paracellular water transport was not affected. It was proved that claudin-3 acts as a „tightening“ TJ component. The endothelial cell culture model HUVEC was used for analysis of expression and regulation of claudin-3 and several other TJ proteins under different conditions of wall shear stress and oxygen saturation. Treatment with lacking wall shear stress led to an upregulation of the “tightening” TJ proteins occludin, claudin-3, claudin-5, and claudin-11, but not claudin-23. Upregulation of all proteins was due to increased mRNA levels. Apparently, different signaling pathways were involved in regulation of particular TJ components. Combined treatment with lacking shear stress and hypoxia resulted in drastically increased claudin-3 expression. Upregulation of tightening TJ components under lacking shear stress and hypoxic conditions as occuring in different physiological or pathological situations would limit the passage of solutes from the blood into the surrounding tissue.

Les claudines sont des protéines des jonctions serrées qui déterminent la perméabilité ionique paracellulaire. La branche large ascendante corticale de l’anse de Henle (cBLAH) exprime plusieurs claudines (en particulier claudine-10b, -14, -16 et -19). Les mutations inactivatrices des claudines-16 et -19 sont responsables du syndrome FHHNC chez l’Homme, caractérisé par une perte de calcium et de magnésium par un défaut de réabsorption dans la BLAH. En l’absence de modèle in vitro adapté, les fonctions des claudines dans la cBLAH restent méconnues, ainsi que l’action des déterminants de la réabsorption du calcium et du magnésium qui y sont exprimés : récepteur de la PTH (PTH1R) et Casr. L’objectif de ce travail était de contribuer à combler ces lacunes. Nous avons montré que la claudine-16 était requise pour une perméabilité paracellulaire aux cations divalents normale dans la cBLAH murine, grâce à l’étude des souris claudine-16-/-. La claudine-16 n’affecte pas les autres perméabilités ioniques, ce qui est concordant avec le phénotype d’inactivation de la claudine-16 chez l’Homme. En condition basale, la claudine-14 est indétectable. Les flux calciques ne sont pas augmentés chez les souris claudine-14-/-. Nous avons décrit le premier cas de mutation inactivatrice de la claudine-10b chez l’Homme, à l’origine d’un syndrome de Bartter et de déficits de sécrétion salivaire et sudorale et conclu que la claudine-10 augmente la perméabilité paracellulaire au sodium. Nous avons également étudié le rôle de Casr et du récepteur de la PTH (PTH1R) dans le contrôle de la perméabilité ionique de la cBLAH murine. La stimulation de la réabsorption du calcium par l’inhibition du Casr ou l’activation du PTH1R ne nécessite pas la présence de la claudine-16. En revanche, la claudine-16 est nécessaire pour le contrôle de la réabsorption de magnésium par ces deux déterminants. Ces résultats sont en faveur d’un contrôle séparé des absorptions de calcium et magnésium, et remettent en cause le concept du rôle central de claudine-14. Notre projet nous a également permis de décrire un nouveau mécanisme d’atteinte des jonctions serrées, d’origine auto-immune, par anticorps anti-claudine-16 chez un patient. Si l’origine de cette atteinte est inconnue, ce modèle soulève de nouvelles questions sur les conséquences de l’inactivation de la claudine-16 dans la BLAH, le phénotype du patient étant différent du syndrome FHHNC (absence de néphrocalcinose en particulier). Ce travail nécessite d’être complété par une étude plus approfondie des mécanismes contrôlant la fonction des claudines en réponse au calcium et au magnésium extracellulaires et à la PTH. / Claudins are integral membrane proteins expressed at tight junctions, which determine paracellular permeability to ions. The thick ascending limb of the loop of Henle (TALH), expresses several claudins (including claudin-10b, -14, -16, -19). Inactivating mutation of claudin-16 and -19 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), a renal tubular disorder featuring a severe defect in paracellular divalent cation transport in the TALH. Due to the lack of appropriate study model, both the function of claudins and the role of potential regulatory factors, such as the PTH receptor PTH1R and the calcium receptor CaSR, are only partially known. The purpose of the study was to contribute to fill this gap. We studied several models of claudin inactivation (claudin-16-/- and claudin-14-/- mice, human inactivating mutation of claudin-10b). We demonstrated that claudin-16 was required for normal paracellular permeability to calcium and magnesium in the TALH, but did not affect the permeability to other ions; this was consistent with the known features of FHHNC syndrome. In basal conditions, claudin-14 is not detectable and calcium reabsorption is not increased in claudin-14-/- mice. We described the first inactivating mutation of claudin-10b in Humans, causing a Bartter syndrome and defects in sweat and saliva secretions; we concluded that claudin-10b increases paracellular permeability to sodium. We studied the role of Casr and PTH1R, as determinants of paracellular permeability in the mouse TALH. Casr inactivation or PTH1R activation could increase calcium absorption in the absence of Claudin-16. By contrast, both conditions did not change magnesium transport in the absence of Claudin-16. These results suggest that calcium and magnesium absorptions can be differentially controlled in the TALH; in addition, they challenge the hypothesis of a central role of Claudin-14. Finally, we described for the first time an autoimmune disease affecting tight junction in the TALH, due to anti-claudine-16 autoantibody. The phenotype of this patient differed from that of patients with FHHNC syndrome (no nephrocalcinosis) and raised new questions about the pathogenesis of claudin-16 inactivation. More data on the effect of extracellular calcium and magnesium, PTH1R and Casr on the function of tight junction in the TALH are warranted.

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 »