O objetivo desse estudo foi investigar a participação de integrina &alpha;1&beta;1 e microRNAs (miRs) no potencial osteogênico de superfícies de titânio (Ti) com nanotopografia. Discos de Ti previamente polidos foram tratados quimicamente com H2SO4/H2O2 para obtenção de nanotopografia, que foi observada por microscopia eletrônica de varredura. Para o estudo da participação da integrina &alpha;1&beta;1, células-tronco mesenquimais (CTMs) de ratos foram cultivadas em condições osteogênicas e não osteogênicas sobre superfícies de Ti com nanotopografia e sem tratamento químico (controle). O resultados mostraram que a nanotopografia de Ti aumentou a proliferação celular, a atividade de fosfatase alcalina (Alp) e regulou positivamente a expressão gênica de marcadores da diferenciação osteoblástica em CTMs cultivadas tanto em condições osteogênicas quanto em condições não osteogênicas. Além disso, uma maior expressão gênica para as integrinas &alpha;1 e &beta;1 foi observada em culturas crescidas sobre nanotopografia em condições não osteogênicas em relação ao Ti controle. O uso de obtustatina, um inibidor de integrina &alpha;1&beta;1, reduziu os efeitos da nanotopografia sobre os marcadores osteoblásticos, indicando a participação da via de sinalização dessa integrina nos efeitos da nanotopografia sobre CTMs. Para investigar a participação de miRs no efeito osseoindutor da nanotopografia de Ti, foram utilizadas CTMs humanas e células préosteoblásticas de camundongos da linhagem MC3T3-E1. A análise em larga escala da expressão de miRs revelou que 60 miRs foram regulados positivamente (no mínimo, 2x maior), enquanto 58 miRs foram regulados negativamente (no mínimo, 2x menor) em CTMs crescidas sobre a nanotopografia. Três desses miRs, miR-4448, -4708 e -4773, cuja expressão foi significativamente reduzida pela nanotopografia de Ti (no mínimo, 5x menor), afetaram a diferenciação osteoblástica de CTMs. Esses miRs atuam diretamente sobre SMAD1 e SMAD4, proteínas transdutoras da sinalização da proteína óssea morfogenética 2 (Bmp-2), conhecida por sua capacidade osseoindutora. Além disso, verificou-se que a sobreexpressão de miR-4448, -4708 e -4773 em células pré-osteoblásticas MC3T3-E1 inibiu a expressão gênica e proteica de SMAD1 e SMAD4 e, consequentemente, a expressão gênica de marcadores ósseos. Esses dados sugerem a influência do circuito miR-SMAD-Bmp-2 sobre o efeito osseoindutor da nanotopografia. Conjuntamente, os achados do presente estudo mostraram que o efeito da nanotopografia de Ti sobre a diferenciação osteoblástica resulta de um mecanismo regulatório complexo, do qual fazem parte as vias de sinalização da integrina &alpha;1&beta;1 e da Bmp-2, com a participação de miRs. Esses resultados podem representar um avanço para o desenvolvimento de novas modificações de superfície, com o objetivo de acelerar e/ou melhorar o processo de osseointegração. / The aim of this study was to investigate the role of the &alpha;1&beta;1 integrin and microRNAs (miRs) on the osteogenic potential of titanium (Ti) with nanotopography. Polished Ti discs were chemically treated with H2SO4/H2O2 to generate nanotopography, which was observed under scanning electron microscopy. For the study related to the &alpha;1&beta;1 integrin, rat mesenchymal stem cells (MSCs) were cultured under osteogenic and non-osteogenic conditions on Ti with nanotopography and non-treated Ti discs (control). Nanotopography increased cell proliferation and alkaline phosphatase (Alp) activity and upregulated the gene expression of bone markers in cells cultured under osteogenic and non-osteogenic conditions. Furthermore, the gene expression of &alpha;1 and &beta;1 integrins was higher in cells cultured on nanotopography under non-osteogenic conditions compared with control. Obtustatin, an inhibitor of &alpha;1&beta;1 integrin, reduced the higher gene expression of the bone markers induced by nanotopography. These results indicate that &alpha;1&beta;1 integrin signaling pathway determines the osteoinductive effect of nanotopography on MSCs. The role of miRs in the osteogenic potential of Ti with nanotopography was evaluated using human MSCs and MC3T3-E1 mouse pre-osteoblastic cells. The miR sequencing analysis revealed that 60 miRs were upregulated (> 2 fold), while 58 miRs were downregulated (< 2 fold) in MSCs grown on nanotopography. Three miRs, miR-4448, -4708 and -4773, which were significantly downregulated (< 5 fold) by nanotopography, affected the osteoblast differentiation of MSCs. These miRs directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (Bmp-2) osteogenic signal, which were upregulated by nanotopography. Overexpression of miR-4448 - 4708 and 4773 in MC3T3-E1 cells noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and by targeting them, these miRs repressed gene expression of key bone markers. These results suggest that a miR-SMAD-Bmp-2 circuit acts in the Ti nanotopography-mediated osteoblast differentiation. Taken together, our data showed that the osteoblast differentiation induced by Ti with nanotopography is governed by a complex regulatory network involving a crosstalk between &alpha;1&beta;1 integrin and Bmp-2 signaling pathways with participation of miRs.

Résumé : LRP-1 est un récepteur d’endocytose qui fut d’abord associé à des propriétés anti-tumorales via l’internalisation et le catabolisme de protéases matricielles. Cependant, malgré ses capacités à limiter le remodelage de la matrice extracellulaire, LRP-1 peut également coordonner la balance adhérence/dé-adhérence des cellules tumorales afin de favoriser l’invasion. LRP-1 fonctionne ainsi en régulant l’organisation du cytosquelette et le renouvellement des structures d’adhérence grâce à l’activation de la voie MEK/ERK et l’inhibition concomitante de la voie MKK7/JNK. Au cours de ce travail, nous avons cherché à déterminer comment LRP-1 peut réguler le protéome membranaire des cellules tumorales. Nos données révèlent que le taux d’intégrine β1 à la surface de carcinome thyroïdien FTC-133 est augmenté en présence de RAP, un antagoniste de LRP-1. Des immunoprécipitations et des analyses par imagerie confocale montrent que LRP-1 et l’intégrine β1 coexistent au sein des mêmes complexes biomoléculaires. Des tests d’endocytose démontrent que LRP-1 constitue un récepteur d’endocytose de l’intégrine β1 dans les FTC 133 car le nombre d’endosomes contenant l’intégrine β1 est diminué de 30% quand l’endocytose dépendante de LRP-1 est inhibée. Par ailleurs, nos données indiquent que LRP-1 est principalement impliqué dans le recyclage de l’intégrine β1 mais pas dans son ciblage au lysosome. Nous avons ainsi identifié une relation moléculaire privilégiée et originale entre LRP-1 et l’intégrine β1 dans le contexte tumoral. Ces travaux nous ont également incités à initier le développement d’un traceur bimodal original (fluorescence/Raman) permettant de suivre l’endocytose dépendante de LRP 1. / LRP-1 is a large multifunctional endocytic receptor first associated to anti-tumor properties by carrying the uptake and catabolism of extracellular matrix-associated proteinases. However, despite its ability to limit extracellular matrix remodeling, LRP-1 may also coordinate the adhesion/deadhesion balance in malignant cells to support invasion. LRP-1 acts so by regulating the cytoskeleton organization and adhesion structure turnover through the activation of MEK/ERK and concomitant inhibition of MKK7/JNK pathway.During this study, we investigated how LRP-1 is able to regulate the cell-surface proteome in malignant cells. Our data revealed that β1-integrin level is significantly increased at the cell surface of FTC-133 thyroid carcinoma upon treatment with RAP, used as LRP-1 antagonist. Immunoprecipitation experiments and confocal analysis highlight that LRP-1 and β1 integrin coexist at the same biomolecular complexes. Biochemical endocytosis assays demonstrate LRP-1 as a mediator of β1-integrin endocytosis in FTC-133 because the number of endosomes-containing β1-integrin decreases by 30% when LRP-1-mediated endocytosis is inhibited. Moreover, our data indicate that LRP-1 is mainly involved in β1-integrin recycling, but not in lysosome targeting.Overall, we identified an original molecular way between LRP-1 and β1-integrin in the tumor context. These works also allowed to initiate the development of an original bimodal molecular tracker (using both fluorescence and Raman) to study LRP-1-mediated endocytosis.

La cardiomyopathie dilatée (CMD) est caractérisée par une fraction d’éjection (FE) réduite, une fonction systolique altérée, une désorganisation de la matrice extracellulaire et des défauts métaboliques. Dans différents modèles de CMD, le niveau d’expression de la nicotinamide riboside kinase 2 (Nmrk2) impliquée dans la synthèse du NAD, un coenzyme majeur du métabolisme énergétique et une molécule de signalisation, est augmenté. NMRK2 est identique à « Muscle Integrin Binding Protein » se liant à l’hétérodimère intégrine β1/α7. Le rôle de Nmrk2 dans le cœur n’est pas connu. Les souris Nmrk2-KO jeunes développent une réponse hypertrophique concentrique normale en réponse à l’angiotensine II et à la constriction aortique. Les échocardiographies jusque 8 semaines post-TAC et au cours du vieillissement à l’état basal, montrent une diminution plus sévère de la FE et un développement de CMD. Les RT-QPCR montrent une augmentation du niveau d’expression de l’isoforme lente β de myosine. NMRK2 n’est pas requise pour maintenir le taux de NAD dans le cœur en réponse aux traitements pro-hypertrophiques et à un âge jeune. Par contre, au cours du vieillissement, les niveaux d’expression de Nmrk1 et Nampt sont diminués et à 24 mois, le NAD myocardique est réduit de 50% chez les souris Nmrk2-KO. A ce même âge, le complexe α7β1 est réduit. Les analyses histologiques montrent un défaut du dépôt de la laminine, la présence d’une fibrose et un élargissement de l’espace intercellulaire chez le mutant Nmrk2-KO. NMRK2 est requise pour préserver la fonction et la structure cardiaque et l’homéostasie du NAD à un âge avancé. Des composants moléculaires modulant sa voie pourraient être une option thérapeutique. / Dilated cardiomyopathy (DCM) is a severe heart disease characterized by reduced ejection fraction, altered systolic function, extracellular matrix disorganization and metabolic defects. In different mice models of DCM, the expression of the nicotinamide riboside kinase 2 (Nmrk2) implicated in the synthesis of NAD, a major coenzyme in energy metabolism and a signaling molecule, is increased. NMRK2 is similar to the muscle integrin binding protein (MIBP) that binds to the integrin α7β1 heterodimer. The role of Nmrk2 in the heart is unknown. Young Nmrk2-KO mice develop a normal cardiac hypertrophic response to angiotensin-II exposure and transverse aorta constriction (TAC) but follow-up echocardiography until 8 weeks post-TAC and during aging from 5 to 24 months revealed a more severe decrease in the EF and the development of a DCM phenotype. RT-qPCR analysis of cardiac mRNAs showed an increase in the slow, cardiac, β myosin heavy chain isoform starting at 12 months. NMRK2 was not essential to maintain myocardial NAD levels in response to pro-hypertrophic treatments and in young adults. However Nmrk1 and Nampt expression level declined strongly with aging and Nmrk2-KO mice displayed a 50% reduction in myocardial NAD levels at 24 months. The α7β1 integrin complex was repressed at this age. Immunofluorescent analyses and electron microscopy revealed a defect in laminin deposition and enlarged intercellular space in the Nmrk2-KO heart. The Nmrk2 gene is required to preserve cardiac function and structure during aging and becomes indispensable for maintaining NAD at late age. Molecular characterization of compounds modulating this pathway could give future therapeutic prospect.

Mild Traumatic Brain Injuries (mTBIs) are non-penetrating brain injuries that do not result in gross pathological lesions, yet they may cause a spectrum of cognitive and behavioral deficits. mTBI has been placed in the spotlight because of increased awareness of blast induced and sports-related concussions, but the underlying pathophysiological mechanisms are poorly understood. Several studies have implicated neuronal membrane poration and ion channel dysfunction as the primary mechanism of injury. We hypothesized that injury forces utilize mechanically-sensitive, transmembrane integrin proteins, which are coupled to the neuronal cytoskeleton (CSK) and distribute injury forces within the intracellular space, disrupting CSK organization and reducing intercellular neuronal functionality. To test this, magnetic beads were coated with adhesive protein, allowing them to bind to integrins in the neuronal membrane in vitro. To apply forces to the neurons via the bound beads, we built custom magnetic tweezers and demonstrated that focal adhesions (FACs) formed at the site of bead binding. We showed that the beads were coupled to the CSK via integrins by measuring the disparate adhesion of the soma and neurite to their underlying substrate. The soma also required more force to detach than neurites, correlating with the FAC density between each neuronal microcompartment and substrate. We then utilized the magnetic tweezers to test whether beads bound to integrins injured neurons more than beads that bound to neurons nonspecifically. Integrin-bound beads injured neurons more often and the injury was characterized by the formation of focal swellings along axons, reminiscent of Diffuse Axonal Injury. While integrin-bound beads initiated swellings throughout neurons, beads bound nonspecifically only caused local injury where beads were attached to neurons. To demonstrate the electrical dysfunction of integrin-mediated injury forces, we adapted Magnetic Twisting Cytometry to simultaneously apply injury forces to beads bound to multiple cells within neuronal networks in vitro. The formation of focal swellings resulted in reduced axonal electrical activity and decreased coordinated network activity. These data demonstrate that the mechanical insult associated with mTBI is propagated into neurons via integrins, initiating maladaptive CSK remodeling that is linked to impaired electrical function, providing novel insight into the underlying mechanisms of mTBI. / Engineering and Applied Sciences

Traumatic Brain Injury (TBI) is linked to a diverse range of diffuse pathological damage for which there is a severe lack of therapeutic options. A major limitation to drug development is the inability to identify causal mechanisms that link head trauma to the multitude of secondary injury cascades that underlie neuropathology. To elucidate these relationships, it is important to consider how physical forces are transmitted through the brain across multiple spatial scales ranging from the whole head to the sub-cellular level. In doing so, the mechanical behavior of the brain is typically characterized solely by its material properties and biological structure. Alternatively, forces transmitted through distinct cellular and extracellular structures have been shown to influence physiological processes in multiple cell types through the transduction of mechanical forces into cellular chemical responses. As an essential component of various biological processes, these mechanotransduction events are regulated by mechanical cues directed through extracellular matrix (ECM) and cell adhesion molecules (CAM) to mechanosensitive intra-cellular structures such as focal adhesions (FAs). Using a series of in vitro models, we have implicated FAs in the cellular mechanism of traumatic axonal injury by showing that forces directed through these structures potentiate injury levels and, moreover, that inhibition of FA-mediated signaling pathways may be neuroprotective. In addition, we show that localizing trauma forces through specific brain ECM results in differential injury rates, further implicating mechanosensitive cell-ECM linkages in the mechanism of TBI. Therefore, we show that FAs play a major role in axonal injury at low strain magnitudes indicating that cellular mechanotransduction may be an important mechanism underlying the initiation of cell and sub-cellular injuries ultimately responsible for the diffuse pathological damage and clinical symptoms observed in diffuse axonal injury. Furthermore, since these mechanisms may present the earliest events in the complex sequelae associated with TBI, they also represent potential therapeutic opportunities. / Engineering and Applied Sciences

Les Virus de l'Immunodéficience Humaine (VIH) sont le résultat de plusieurs transmissions inter-espèces de SIV (Virus de l'Immunodéficience Simienne) de Primates Non Humains (PNH) à l'Homme. Les SIV les plus proches du VIH-1 sont le SIVcpz et le SIVgor qui infectent naturellement les chimpanzés et les gorilles. Les SIVsmm retrouvés chez les mangabés enfumés d'Afrique de l'Ouest sont les plus proches du VIH-2. Actuellement, au moins 13 transmissions du singe à l'Homme ont été documentées, 4 à l'origine des 4 groupes du VIH-1 (groupe M, N, O et P) et 9 pour le 9 VIH-2 (A-I). La question du réservoir à l'origine du VIH-1 chez l'Homme est partiellement résolue. Les chimpanzés, Pan troglodytes troglodytes, du sud-est et centre sud du Cameroun sont respectivement les réservoirs du VIH-1 M pandémique chez l'Homme ainsi que du VIH-1 groupe N. En ce qui concerne les groupes O et P, il n'y a actuellement pas de réponse définitive. Les SIVgor sont bien les virus les plus proches phylogénétiquement des VIH-1 O et P. Cependant, de plus amples recherches sont nécessaires pour identifier les ancêtres directs des variants O et P. Ces recherches supplémentaires aideront aussi à élucider l'origine du SIVgor chez les gorilles, et à savoir si ce sont les gorilles qui ont transmis les virus O et/ou P à l'Homme, ou s'il existe toujours un réservoir des ancêtres O et P chez les chimpanzés. Des études supplémentaires sont aussi nécessaires afin de mieux comprendre les mécanismes d'adaptation à un nouvel hôte et l'impact des infections SIV chez les grands singes. Dans ce but, l'étude du récepteur accessoire pour le VIH, l'intégrine α4β7, pourrait aussi jouer un rôle pour l'infection du SIV/VIH. Cette intégrine facilite également la migration du virus vers l'intestin. Une étude récente a montré des substitutions d'acides aminés chez les Primates du Nouveau Monde (PNM) qui empêche l'adhérence du liant. Ainsi, les polymorphismes de cette intégrine et son rôle dans l'infection SIV chez les Primates de l'Ancien Monde (PdAM) sont encore inconnus. L'objectif majeur de cette thèse était de mieux documenter et mieux comprendre l'infection SIV chez les gorilles sauvages en Afrique Centrale. Sur plus de 6.000 échantillons testés, nous avons constaté que seuls les gorilles (Gorilla gorilla gorilla) du sud Cameroun sont infectés par le SIVgor. Parmi eux, nous avons identifié les ancêtres du VIH-1 P chez des populations du sud-ouest Cameroun. Nous avons aussi mis en évidence que les gorilles sont à l'origine du VIH-1 groupe O. Les analyses fonctionnelles du facteur de restriction APOBEC3G ont montré que celui-ci protège les gorilles des infections SIVcpz, expliquant en partie la faible prévalence de SIVgor. Nous avons évalué une nouvelle technologie sérologique, le Luminex®, en utilisant des antigènes spécifiques de la lignée SIVgor. Ces résultats ont été comparés avec ceux que nous pouvons obtenir avec l'INNO-LIATM qui est une technique de référence basée sur des réactions croisées entre anticorps SIV et antigènes VIH-1. Nous avons aussi évalué la faisabilité de la technologie de séquençage de deuxième génération Illumina® pour étudier les viromes de deux gorilles. Nous n'avons pas pu obtenir la séquence du SIVgor dans l'échantillon de l'individu infecté. Cependant, en comparant les résultats obtenus entre les deux gorilles étudiés, nous avons pu constater un probable déséquilibre de la réplication des virus entériques seulement pour le gorille infecté par le SIVgor. Enfin, nous avons décrit la diversité de la sous-unité α4 de l'intégrine α4β7 chez les PdAM. En conclusion, ces travaux de thèse ont apporté de nouvelles connaissances majeures sur l'infection SIV chez les gorilles et ont contribués à élucider l'origine des quatre groupes VIH-1. / Human Immunodeficiency Viruses (HIV) are the result of numerous interspecies transmissions of different SIV (Simian Immunodeficiency Virus) from Non-Human Primates (NHP) to humans. SIVcpz and SIVgor from chimpanzees and gorillas are most closely related to HIV-1, and SIVsmm from sooty mangabeys in West Africa to HIV-2. At least 13 cross-species transmissions from NHP to humans have been reported, 4 leading to the 4 HIV-1 group (M, N, O and P) and 9 for the 9 HIV-2 groups (A-I). Today the origin of HIV-1 group M and N is elucidated and their simian ancestors, have been identified in chimpanzee (Pan troglodytes troglodytes) populations in southeast and south-central Cameroon, respectively. HIV-1 group O and P are most closely related to SIVgor from gorillas but their direct ancestors have not been identified yet. More studies are thus needed to clarify the origin of HIV-1 group O and P in humans as well as on the origin of SIVgor in gorillas. These studies will also elucidate whether HIV-1 group O and P have been transmitted by chimpanzees or gorillas and whether simian ancestors of these HIV groups and the ancestor of SIVgor still circulates in today's chimpanzee populations. More studies are also needed to understand viral and host factors related adaptation in the new host and the impact of SIV infection in general in apes. As such, α4β7 integrin has been recently described as a new HIV-1 receptor that facilitates virus migration to the Gut-Associated Lymphoid Tissue (GALT). In a recent study, amino acid substitutions were observed in the α4 binding site in New World Primates (NWP), that can reduce the activity of this receptor. The impact of the genetic diversity of this integrin in Old World Primates (OWP) and its role in SIV infection is still unknown. Therefore, characterizing the polymorphisms profiles in OWP could bring new insights into progression of the pathogenic and non pathogenic SIV infections. The main objective of this thesis was to better characterize and understand SIV infection in wild gorillas in Central Africa. On more than 6,000 fecal samples from gorillas collected across Central Africa, we showed that only gorillas from southern Cameroon are infected with SIVgor and we identified the ancestors of HIV1 group P in gorilla populations from southwest Cameroon. We also provided evidence that gorillas are at the origin of HIV-1 group O in humans. Functional analysis of the restriction factor APOBEC3G showed that its protects gorillas from SIVcpz infections and can explain the low prevalence in gorillas. We evaluated a new antibody detection approach in faecal samples, based on Luminex® technology that use SIVgor specific antigens, comparing with the actual serological test INNO-LIATM HIV confirmatory assay, based on cross-reactive SIV antibodies with HIV antigens. We also evaluated the feasibility of virus sequencing in faecal samples with the Illumina® technology to study viromes of gorillas. We studied two samples, one of a SIVgor infected individual and one from an uninfected gorilla. Although the SIVgor sequence was not retrieved from the infected individual, we observed a tendency to enteric virus replication disorder in the infected animal that has not been seen in the uninfected one. Finally, we also documented here the genetic diversity of the α4 subunit from OWP. In this thesis we documented more in detail different aspects of SIV infection in gorillas and contributed to elucidate the origin of all HIV-1 groups.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第22671号 / 人博第957号 / 新制||人||227(附属図書館) / 2020||人博||957(吉田南総合図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 川本 卓男, 教授 宮下 英明, 教授 高田 穣 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

Melanoma remains an aggressive form of skin cancer with limited treatment options. Novel methods to treat primary tumors and prevent metastatic disease can lead to improved survival for those diagnosed with melanoma. Through this work, we have evaluated the antineoplastic effects of Rhodiola crenulata (R. crenulata) root extracts on B16-F10 melanoma both in vitro and in vivo. In this study, we observed that R. crenulata treatment resulted in an increased cell death as well as a reduced cell growth, proliferation and migration in vitro. Additionally, we observed that R. crenulata decreased the expression of integrin β1 and vimentin, and increased expression of E-cadherin upon in vitro treatment. Further, we observed in a topical R. crenulata based cream therapy, a more radial growth pattern of tumors as well as a reduced mitotic activity and increased tumor necrosis. Markedly, we observed that mice supplemented with R. crenulata orally in their drinking water also displayed reduced establishment of metastatic foci in a disseminated model of melanoma. Collectively, these findings reveal that R. crenulata exhibits striking anti-tumorigenic and anti-metastatic properties, and that this extract may increase survival and harbor potential novel adjuvant therapy for the treatment of melanoma.

Idiopathic pulmonary fibrosis (IPF) is one of the most aggressive and severe interstitial lung diseases (ILDs) for which there is no cure. IPF is characterized by an excessive accumulation of fibroblasts which secrete an abundance of extracellular proteins such as collagen. These processes lead to repetitive tissue scarring and fibrosis in the lung parenchyma. As a result, lungs become rigid limiting oxygen intake and gas exchange. Once diagnosed, IPF is fatal within 2-3 years. There is no known cause or proven treatment that significantly improves outcomes. Although the cause is unknown, the current model of IPF suggests that an overactive epithelial repair mechanism caused by genetic and epigenetic factors as well as environmental exposures is responsible for the chronic fibrosis and scarring characteristic of IPF. The transforming growth factor beta (TGF-B) signaling pathway has been implicated as a major contributor in activating this chronic fibrosis. An upstream activator of the TGF-B pathway, avB6, has been identified as a potential therapeutic target. My collaborators in Dr. David Baker's lab at the University of Washington have created a novel avB6 integrin inhibitor (BP2_disulf) whose efficacy in improving IPF outcomes has yet to be tested. In my study, I test the ability of BP2_disulf to combat IPF through the use of the standard IPF murine model and translatable end points like non-invasive uCT scans, pulmonary function tests, bronchoalveolar lavage fluid (BALF) profiles, and histology. With these methods, I demonstrate that intraperitoneal injection of BP2_disulf in bleomycin-injured mice has the ability to decrease rate of fibrotic progression and pulmonary function decline compared to mice treated with bleomycin alone. These results prove that BP2_disulf is a promising therapeutic not only for IPF but other ILDs as well. Further efficacy validation and investigation into an aerosolized delivery method will advance this drug to clinical trials and make it accessible to those in need.

L’étude de l’intégrine alphaE(CD103)beta7, exprimée à la surface des lymphocytes T cytotoxiques (CTL) et ayant pour ligand la E cadhérine, marqueur des cellules épithéliales, a permis de démontrer son rôle dans le recrutement des CTL dans la tumeur, la formation de la synapse immunologique et l'induction de la mort des cellules tumorales épithéliales par les CTL spécifiques. Dans ce contexte, nous avons étudié la signalisation de CD103, et ainsi démontré que la paxilline, une protéine des points d'adhésion focaux, interagit avec CD103. De plus, son inhibition diminue les capacités d'adhésion et d'étalement des CTL sur un tapis d'E cadhérine recombinante humaine. Nous avons ensuite identifié le domaine cytoplasmique de CD103 nécessaire à son activation. Une protéine de fusion CD103-GFP a été construite, ainsi que plusieurs mutants du domaine cytoplasmique de CD103. Nous avons identifié le motif ESIRKAQL, nécessaire à l'activation des lymphocytes T. La sérine en position 1163, présente dans ce motif, semble indispensable à la relocalisation de CD103, des lysosomes et de la paxilline, à la zone de contact avec la E-cadhérine. / The alphaE(CD103)beta7 integrin plays a crucial role in the formation of a functional immune synapse (IS) between T cells and epithelial target cells. Indeed, the interaction of CD103 on tumor-infiltrating T lymphocyte (TIL) with its ligand, the epithelial cell marker E cadherin on tumor cells, promotes tumor cell killing. We studied CD103 signaling, and we showed that paxillin, a protein found in focal adhesion, interacts with CD103, and its inhibition leads to a decrease in T cell adhesion and spreading of T cell upon activation with immobilized recombinant E-cadherin-Fc. Secondly, we investigated CD103 cytoplasmic domains involved in its activation. Therefore, we used wild-type (WT)-CD103-GFP or various mutant CD103-GFP fusion proteins. Our results showed that ESIRKAQL intracellular motif is necessary for T cell activation, and in particular the serine in position 1163 that is essential for CD103 clustering and paxillin recruitment.