Carcinona epidermóide é um prevalente tumor de cabeça e pescoço relacionado a altas taxas de mortalidade. Neste trabalho, verificamos se AG73 (RKRLQVQLSIRT, cadeia a1), peptídeo derivado da laminina-111, regula migração, invasão e secreção de protease em células de carcinoma epidermóide oral (OSCC). Cadeia a1 da laminina e MMP9 estão expressas neste tumor in vivo e in vitro. AG73 induziu aumento da taxa migratória de células OSCC em ensaios de ferida e migração, e também estimulou invasão em ensaio em câmaras bipartites com Matrigel. Células OSCC crescidas sobre AG73 exibiram aumento dose-dependente de MMP9, detectado por zimografia. Buscamos receptores de AG73 que regulariam atividade nesta linhagem. Células OSCC crescidas sobre AG73 exibiram colocalização de sindecana-1 e integrina b1, e silenciamento desses receptores com RNA de interferência promoveu diminuição de migração e invasão dependente de AG73 nestas células. Esses resultados sugerem que sindecana-1 e integrina b1, ativados por AG73, podem regular migração, invasão e secreção de MMPs em células OSCC. / Oral squamous cell carcinoma is a prevalent head and neck tumor, related to high mortality rates. Here we studied the role played by AG73 (RKRLQVQLSIRT, a1 chain) on migration, invasion and protease secretion of a cell line (OSCC) from human oral squamous cell carcinoma. Laminin a1 chain and MMP9 are expressed in oral squamous cell carcinoma cells in vivo and in vitro. AG73 increased migratory activity of OSCC cells, as shown by monolayer wound assays and migration assays. This peptide also stimulated cell invasion in chemotaxis chambers coated with Matrigel. OSCC cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, detected by zymography. We searched for AG73 receptors regulating activities in this cell line. OSCC cells grown on AG73 exhibited colocalization of syndecan-1 and b1 integrin, and siRNA knockdown of these receptors decreased AG73-dependent migration and invasion of OSCC cells. Our results suggest that syndecan-1 and b1 integrin signaling downstream of AG73 regulate migration, invasion and MMP secretion by OSCC cells

Estudamos indução da atividade de MMP pelo peptídeo da laminina a1 AG73 em linhagem celular (CAC2) de carcinoma adenóide cístico. CAC2 cultivadas em laminina-111 com AG73 geraram espaços pseudocísticos. Inibidor de MMP diminuiu tais espaços, sugerindo ação de MMPs. CAC2 crescidas sobre AG73 mostraram aumento dose-dependente de MMP9. RNAi para MMP9 diminuiu remodelação em cultura 3D. Buscamos receptores de AG73 ligados à atividade de MMP9. CAC2 crescidas sobre AG73 exibiram colocalização de sindecan-1 e integrina b1. RNAi para sindecan-1 ou para integrina b1 geraram, isolados, redução na adesão a AG73 e nas atividades de remodelação e de protease. Duplo RNAi estudou a cooperação entre os receptores e promoveu diminuição na adesão a AG73 e na atividade de MMP. Distinção de receptores foi feita por cromatografia de afinidade e espectrometria de massa, através de colunas de afinidade com AG73 acoplado, que resultou em possíveis receptores, como integrinas b1 e aV. Sugerimos que AG73 regula adesão e secreção de MMP em células CAC2 através de sindecan-1 e integrina b1. / We studied induction of MMP activity by b1-laminin peptide AG73 in adenoid cystic carcinoma cell line (CAC2). Cells grown inside AG73-enriched laminin-111 exhibited pseudocystics spaces. MMP inhibitor decreased those spaces, suggesting MMPs action. Cells grown on AG73 showed a dose-dependent increase of MMP9 secretion. MMP9 siRNAi decreased remodeling in 3D culture. We searched for AG73 receptors regulating MMP9 activity. CAC2 grown on AG73 exhibited colocalization of syndecan-1 and b1 integrin. Syndecan-1 siRNA or siRNA b1 integrin showed reduction in adhesion to AG73 and in remodeling and protease activities. Double-knockdown explored syndecan-1 and 1 integrin cooperation and showed decrease in adhesion to AG73 and in MMP activity. Receptors characterization was made by affinity chromatography followed by mass spectrometry through AG73-affinity columns and showed putative receptors, like b1 and aV integrins. We suggest that AG73 peptide regulates adhesion and MMP secretion in CAC2 cells through syndecan-1 and b1 integrin.

L’objectif de cette thèse est de développer l’imagerie de transfert de saturation des agents de contraste lipoCEST pour la détection de l’angiogenèse dans un modèle souris de tumeur cérébrale U87. Un lipoCEST offrant un seuil de sensibilité in vitro de 100 pM est optimisé afin de répondre aux contraintes de l’imagerie CEST in vivo. Grâce à la mise en place d’un dispositif expérimental dédié à l’imagerie CEST, nous évaluons les performances des lipoCEST pour détecter de façon spécifique l’angiogenèse tumorale. Nous montrons pour la première fois qu’il est possible de détecter un lipoCEST in vivo dans un cerveau de souris suite à une injection intraveineuse. De plus, l’utilisation d’un lipoCEST fonctionnalisé avec un peptide RGD permet de cibler spécifiquement l’intégrine ανβ3 surexprimée lors de l’angiogenèse tumorale. L’association spécifique du RGD-lipoCEST est confirmée grâce à des données d’immunohistochimie et de microscopie de fluorescence. Enfin, dans le but de tendre vers un protocole d’imagerie moléculaire par IRM-CEST, nous mettons en place un outil de quantification des lipoCEST. Cet outil repose sur la modélisation des processus d’échange de protons in vivo. Grâce à la prise en compte des inhomogénéités de champs B0 et B1 qui peuvent se révélées être délétères pour le contraste CEST, nous démontrons que la précision de notre outil de quantification est de 300 pM in vitro. La quantification des données CEST acquises chez la souris U87 permet d’estimer à 1,8 nM la concentration maximale en RGD-lipoCEST liés à leur cible moléculaire. / The study aimed at developing saturation transfer imaging of lipoCEST contrast agents for the detection of angiogenesis in a U87 mouse brain tumor model. A lipoCEST with a sensitivity threshold of 100 pM in vitro was optimized in order to make it compatible with CEST imaging in vivo. Thanks to the development of an experimental setup dedicated to CEST imaging, we evaluated lipoCEST to detect specifically tumor angiogenesis. We demonstrated for the first time that lipoCEST visualization was feasible in vivo in a mouse brain after intravenous injection. Moreover, the integrin ανβ3 overexpressed during tumor angiogenesis can be specifically targeted using a functionalized lipoCEST with RGD peptide. The specific association between the RGD-lipoCEST and its target ανβ3 was confirmed by immunohistochemical data and fluorescence microscopy. Finally, in order to tend to a molecular imaging protocol by CEST-MRI, we developed a quantification tool of lipoCEST contrast agents. This tool is based on modeling of proton exchange processes in vivo. By taking into account both B0 and B1 fields inhomogeneities which can dramatically alter CEST contrast, we showed that the accuracy of our quantification tool was 300 pM in vitro. The tool was applied on in vivo data acquired on the U87 mouse model and the maximum concentration of RGD-lipoCEST linked to their molecular targets was evaluated to 1.8 nM.

L’élucidation des mécanismes permettant l’optimisation de la réponse immunitaire antitumorale correspond à un enjeu majeur pour le développement de stratégies d’immunothérapie efficace. En effet, les réponses immunitaires antitumorales se traduisent rarement par l’éradication de la tumeur. Dans ce contexte, les travaux antérieurs de mon équipe ont démontré que l’interaction de l’intégrine αE(CD103)β7, souvent exprimée par les lymphocytes infiltrant la tumeur (TIL), avec son ligand E-cadhérine, à la surface des cellules tumorales épithéliales, joue un rôle majeur dans la potentialisation de l’activité lytique des cellules T en induisant la polarisation et l’exocytose des granules cytotoxiques. Nos résultats ont indiqué aussi que le TGF-β1, souvent abondant dans les tumeurs, joue un rôle déterminant dans cette induction suite à l’engagement du récepteur des cellules T. Dans ce contexte, nous avons cherché à comprendre les mécanismes de régulation du gène ITGAE qui codent la sous-unité alphaE de l’intégrine CD103. Nos résultats ont montré que les facteurs transcriptionnels Smad2, Smad3 et NFAT-1 sont impliqués dans la régulation de l’expression de la sous-unité αE(CD103). En effet, une costimulation avec du TGF-β1 recombinant et un anticorps anti-CD3 d’un clone T CD103- induit l’expression de cette intégrine qui est accompagnée d’une translocation dans le noyau de Smad2, Smad3 et NFAT-1 qui sont cytoplasmiques à l’état basal. L’inhibition spécifique de ces facteurs transcriptionnels inhibe l’expression de CD103 et abroge le potentiel lytique du clone T vis à vis de sa cible tumorale autologue. De plus, nous avons identifié deux séquences régulatrices du gène ITGAE humain, un promoteur proximal et un enhancer. Par ailleurs, mon équipe a récemment montré que l’interaction de CD103 à la surface des TIL avec une molécule E-cadhérine recombinante est suffisante pour induire la polarisation des granules cytolytiques par un mécanisme dépendant de la PLC-g1 et ERK et que cette intégrine possède non seulement une fonction d’adhérence, mais aussi une fonction de costimulation du signal TCR des TIL antitumoraux. Nous avons cherché à mieux comprendre la signalisation de l’intégrine CD103, en identifiant les domaines intracytoplasmiques de la sous-unité αE impliqués dans son activation. Nous avons ainsi construit une protéine de fusion CD103-GFP et plusieurs mutants du domaine intracytoplasmique de la sous-unité αE qui ont été ensuite transfectés dans la lignée Jurkat Tag CD103-/beta7+. Nos résultats ont montré que le domaine intracytoplasmique de la chaîne alphaE n’est pas nécessaire à la reconnaissance du ligand, la E-cadhérine. Par contre, nous avons montré que ce domaine est impliqué dans le phénomène de clustering de l’intégrine et dans sa polarisation à la zone de contact avec des billes couvertes avec la E-cadhérine-Fc. Nous avons identifié un domaine de 8 acides aminés (ESIRKAQL), contenant une sérine en position 1163 potentiellement phosphorylable, et qui est indispensable pour la signalisation de l’intégrine. De plus, nos travaux ont montré que ce domaine ESIRKAQL, est nécessaire pour la phosphorylation de la ERK1/2 et PLC-g1. Ainsi, une meilleure compréhension des mécanismes moléculaires régulant les fonctions de CD103 pourrait contribuer au développement et à l’amélioration de la réponse antitumorale exercée par les CTL. / The elucidation of mechanisms for optimizing the antitumor immune response is a major challenge for the development of strategies for effective immunotherapy. Indeed, the anti-tumor immune responses rarely result in the eradication of the tumor. In this context, the previous work of my team have shown that the interaction of integrin αE(CD103)β7, often expressed by tumor infiltrating lymphocytes (TIL) with its ligand E-cadherin at the cell surface tumor epithelial cells, plays a major role in the potentiation of the lytic activity of T cells by inducing polarization and exocytosis of cytotoxic granules. Our results also indicated that TGF-β1, often abundant in tumors, plays a key role in the induction due to the commitment of the T cell receptor. In this context, we sought to understand the mechanisms regulating ITGAE gene encoding the subunit αE of integrin. Our results showed that the transcription factors Smad2, Smad3 and NFAT-1 are involved in regulating the expression of subunit αE(CD103)β7. Indeed, costimulation with recombinant TGF-β1 and anti-CD3 antibody induces on T cell clone CD103- the expression of this integrin ant the translocation into the nucleus of Smad2, Smad3 and NFAT-1 that are cytoplasmic at baseline. Specific inhibition of these transcription factors inhibits the expression of CD103 and repeals the lytic potential of cloned T with respect to the autologous tumor target. In addition, we identified two regulatory sequences of human ITGAE gene, proximal promoter and enhancer. In addition, my team has recently shown that the interaction of CD103 on the surface of TIL with a recombinant molecule E-cadherin is sufficient to induce the polarization of cytolytic granules by ERK and PLC-γ1 pathway thus this integrin has not only a function of adherence, but also a function of costimulatory signal TCR of TIL. We sought to better understand the signaling of integrin CD103, by identifying the cytoplasmic domains of the subunit αE involved in its activation. We have constructed a fusion protein CD103-GFP and several mutants of intracytoplasmic domain of the subunit αE which were then transfected into the Jurkat Tag cell line CD103-/ β7+. Our results showed that the intracytoplasmic domain of CD103 is not necessary for ligand recognition, E-cadherin. By cons, we have shown that this area is involved in the phenomenon of clustering of integrin and its polarization to the contact area with balls covered with E-cadherin-Fc. We have identified a range of 8 amino acids (ESIRKAQL) containing a potentially phosphorylatable serine in position 1163, which is essential for integrin signaling. In addition, our work has shown that this area ESIRKAQL is necessary for the phosphorylation of ERK1/2 and PLC-g1. Thus, a better understanding of the molecular mechanisms that regulate the functions of CD103 may contribute to the development and improvement of the antitumor response exerted by CTL .

Avaliamos a capacidade de indução de migração, invasão e secreção de protease pelo peptídeo derivado da laminina, C16 (KAFDITYVRLKF, cadeia g1) em linhagem celular (CAC2) de carcinoma adenóide cístico humano. Laminina g1 foi imunolocalizada no carcinoma adenóide in vivo e in vitro. Ensaio de ferida, em câmara bipartite e em vídeo microscopia (time-lapse) mostraram que C16 estimula migração em células CAC2. C16 também estimulou invasão em ensaio com câmaras bipartites cobertas com Matrigel. Invasão depende de atividade de protease. Zimografia mostrou que C16 aumentou secreção de MMPs 2 e 9. Diferentes vias de sinalização podem estar relacionadas com os efeitos de C16. Immunoblot revelou que C16 aumentou a fosforilação de AKT e ERK. Para o estudo de possíveis receptores do peptídeo, preparações de membrana foram passadas em colunas de afinidade com C16 acoplado. Banda de 40kDa foi eluída e analisada por espectrometria de massa (LC-MS/MS) que identificou a cadeia a1 do colágeno. O fragmento de colágeno eluído poderia ser parte de um complexo protéico envolvendo C16. Integrinas são receptores de colágeno e candidatas a fazerem parte desse complexo. Células CAC2 expressaram as integrinas av, a5, b3 and b1. Silenciamento dessas integrinas promoveu redução da migração e secreção de protease induzidas por C16. Sugerimos que C16 estimularia migração, invasão e secreção de protease em células de carcinoma adenóide cístico através de integrinas a5b1 e avb3. O sinal gerado por C16 seria transduzido pelas vias AKT e ERK1/2. / We studied induction of migration, invasion and protease activity by laminin-derived peptide C16 (KAFDITYVRLKF, g1 chain) in a cell line (CAC2) from adenoid cystic carcinoma. Laminin g1 was immunolocalized in adenoid cystic carcinoma in vivo and in vitro. C16 increased migratory activity of CAC2 cells, as shown by monolayer wound assay, Transwell migration assay and time-lapse video microscopy. This peptide also stimulated cell invasion in Transwell chambers coated with Matrigel. Invasion depends on protease activity. Zymograms showed that C16 increased secretion of MMPs 2 and 9. Different signaling pathways could be related to C16 regulation in CAC2 cells. Immunoblot showed that C16 increased phosphorylation of both AKT and ERK compared to controls. To study putative receptors of this peptide we used affinity chromatography. Membrane preparations were run through C16-affinity columns. A 40kDa band was eluted and analyzed by mass spectrometry (LC-MS/MS) identifying a collagen a1 chain. The collagen fragment eluted could be part of a protein complex involving C16. This protein complex may include integrins, which are collagen receptors. CAC2 cells exhibited av, a5, b3 and b1 integrins. siRNA knockdown of these integrins inhibited both C16-induced migration and protease activity. We propose that C16 increases migration, invasion and protease activity of a human salivary gland adenoid cystic carcinoma cell line through a5b1 and avb3 integrins. The signal generated by C16 is transduced by AKT and ERK1/2 signaling pathways.

Crk associated substrate (Cas) is an adaptor protein that becomes phosphorylated upon integrin signaling and influences regulation of cell processes such as migration, proliferation and survival. It consists of multiple domains and regions that can interact with several signaling proteins involved in different signaling pathways. Cas was first discovered as a highly phosphorylated protein in v-Src and v-Crk transformed cells, showing involvement of this protein in cell transformation High level of Breast cancer antiestrogen resistance protein (BCAR-1), a homologue to Cas has shown to correlate with rapid reoccurrence of breast cancer and also create resistance towards Tamoxifen, the widely used medicine for receptor positive breast cancer patients. We have defined boundaries of two regions of Cas termed serine rich region (SRR) and Src binding domain (SBD) respectively and have isolated these segments for biochemical and structural studies. The structure of the serine rich part of Cas has been determined by NMR spectroscopy and reveals a four-helix bundle with unusually long loops. The 14-3-3 protein binds to Cas in a phospho-serine dependent manner and our study suggests that the binding site is located between two helices. The SH2-SH3 domain of a Src family kinase, Lck has also been crystallized in complex with a nine residue long peptide corresponding to the region in Cas that binds to SH2 domains. The structure of this complex has been solved at 2.7Å and shows that Cas binds Src family kinases (SFK) with high affinity suggesting a specific interaction between these two molecules. The biochemical studies on the specific binding site of these molecules show that SFK can bind to any of the phosphorylated tyrosines on the SH2 binding domain of Cas and only one phospho-tyrosine is enough to establish the binding. This binding assay does also indicate that SH3 binding domain of Cas is not essential for SFK binding.

Certain hydrogel forming de novo proteins that utilize different crosslinking methods are studied experimentally on a rheometer. The stress reaxation modulus of CRC, a telechelic, triblock protein, is shown to be that of a stretched exponential function with a value of β ≅ 0.5. The insertion of an integrin binding domain and changes in pH within the range 6.5–8.5 are shown not to significantly affect the resulting rheological behavior. A selective chemical crosslinker is used on CRC hydrogel systems and is shown to change the rheological behavior of the system to that of a combination of a chemically and physically crosslinked system. Chemically crosslinked hydrogels composed of W6, a wheat gluten-based protein, demonstrate a storage modulus weakly dependent on the angular frequency that is much greater than the loss modulus, with a modulus concentration dependence of c^9/4.

Certain hydrogel forming de novo proteins that utilize different crosslinking methods are studied experimentally on a rheometer. The stress reaxation modulus of CRC, a telechelic, triblock protein, is shown to be that of a stretched exponential function with a value of β ≅ 0.5. The insertion of an integrin binding domain and changes in pH within the range 6.5–8.5 are shown not to significantly affect the resulting rheological behavior. A selective chemical crosslinker is used on CRC hydrogel systems and is shown to change the rheological behavior of the system to that of a combination of a chemically and physically crosslinked system. Chemically crosslinked hydrogels composed of W6, a wheat gluten-based protein, demonstrate a storage modulus weakly dependent on the angular frequency that is much greater than the loss modulus, with a modulus concentration dependence of c^9/4.

Cell adhesion plays an important role in inflammation and immunological responses. Adhesion molecules (e.g., selectins and integrins) are key modulators in mediating these cellular responses, such as leukocyte trafficking under shear stress. In this thesis, we use a bio-membrane force probe (BFP) to study force regulation on kinetics and conformations of selectin and LFA-1 integrin. A new BFP was built up, and a new assay, using thermal fluctuation of the BFP, was developed and used to monitoring selectins and their ligands association and dissociations. The new BFP was also used to investigate the force and force history dependence of selectin-ligand interactions. We found tri-phasic transition of force-dependent off-rates and force-history dependence of selectin/ligaind interactions. The BFP was also used to characterize force-dependent lifetimes of the LFA-1-ICAM-1 interaction. We found that LFA-1/ICAM-1 bonds behaved as catch bond and that LFA-1-ICAM-1's catch bonds were abolished blocking the downward movement of αA domain α7 helix. Finally, the BFP was applied to dynamically probe the global conformational changes of LFA-1 and to characterize force-regulated transitions among different conformational states on a living cell. We observed dynamic transitions of LFA-1 between extended and bent conformations on living cells. The observed average distance change of LFA-1's extensions was about 18nm, while that of the bending was only about 14nm. We also found that forces could facilitate extension but they slow down the bending of LFA-1. The observed transition time of extension was less than 0.1s, while that of contraction was longer than 0.2s. Our observations here are the first in-situ evidence to demonstrate how integrins dynamically transit different conformations and how force regulates these transitions.

No description available.