Current chemotherapeutics pose many di sadvantages due to their lack of specificity and low therapeutic index. To overcome these challenges, research has focused its attention on the development of nano-based delivery systems that can penetrate the leaky vasculature of tumor endothelium, use site-directed ligands that can bind with high affinity and specific ity to tumor cells, physically entrap poorly soluble drugs, and deliver these cytotoxic agents directly to the tumor site. One approach to nanosystem drug delivery is with the use of peptide amphiphiles (PAs) that are conjugated with the Arginine-Glycine-Aspartic Acid (RGD) motif to actively target a αVβ3 integrin receptors on cancer cells or tumor endothelium. The current work is focused on mechanistic studies to evaluate the uptake of novel RGD amphiphi les with varying alkyl chain lengths (palmitic acid : Cl 6 and stearic acid: C 18) and hydrophilic linkers, 8-amino- 3,6-dioxaoctonoic acid (ADA) or glucose, as micellar delivery systems of hydrophobic anticancer agents. PAs were confirmed for their self-assembling properties and further evaluated for their RGD-mediated binding specificity to purified αVβ3 integrin through a competitive binding fluorescence polarization assay (with novel RGD micelles displacing an integrin-bound fluorescent RGD probe by as much as 63.03%). Ultimately, these nanocarriers were assessed for their ability to deliver phys ically entrapped fluorescein isoth iocyanate (FITC) to A2058 cells overexpressing αVβ3 integrin receptors. Results from confocal microscopy indicate that uptake of RGD micelles was driven by an energy-dependent mechanism, as statistically significant levels of FITC internalization was seen at 37°C versus 4°C (p-value<0.05 for all treatment groups); moreover, intracellular fluorescence was notably higher (as much as 4-fold) when delivered through novel RGD conjugates as opposed to its free form. Regardless of chain length and the number of hydrophilic linkers, all RGD PAs showed promising results as micellar carriers that can effectively deliver their payload to the target tumor site via receptor mediated endocytosis.

Cell migration performs a critical function in numerous physiological processes, including tissue homeostasis or wound healing after tissue injury, as well as pathological processes that include malignant progression of cancer. The efficiency of cell migration and invasion appears to be based on the mechano-phenotype of the cytoskeleton. The properties of the cytoskeleton depend on internal cytoskeletal and external environmental factors. A reason for this are connections between the cell and its local matrix microenvironment, which are established by cell-matrix adhesion receptors. Upon activation, focal adhesion proteins such as PINCH1 are recruited to sites where focal adhesions form. PINCH1 specifically couples through interactions with ILK, which binds to cell matrix receptors and the actomyosin cytoskeleton. However, the role of PINCH1 in cell mechanics regulating cellular motility in 3D collagen matrices is still unclear. PINCH1 is thought to facilitate 3D motility by regulating cellular mechanical properties, such as stiffness. In this study, PINCH1 wild-type and knock-out cells were examined for their ability to migrate in dense extracellular 3D matrices. Indeed, PINCH1 wild-type cells migrated more numerously and deeper in 3D matrices, compared to knock-out cells. Moreover, cellular deformability was determined, e.g., elastic modulus (stiffness). PINCH1 knockout cells are more deformable (compliable) than PINCH1 wild-type cells. Migration of both PINCH1−/− cells and PINCH1fl/fl cells was decreased by Latrunculin A inhibition of actin polymerization, suggesting that actin cytoskeletal differences are not responsible for the discrepancy in invasiveness of the two cell types. However, the mechanical phenotype of PINCH1−/− cells may be reflected by Latrunculin A treatment of PINCH1fl/fl cells, as they exhibit resembling deformability to untreated PINCH1−/− cells. Moreover, an apparent mismatch exists between the elongation of the long axis and the contraction of the short axis between PINCH1fl/fl cells and PINCH1−/− cells following Latrunculin A treatment. There is evidence of this indicating a shift in the proxy values for Poisson’s ratio in PINCH1−/− cells compared with PINCH1fl/fl cells. This is probably attributable to modifications in cytoskeletal architecture. The non-muscle myosin II inhibitor Blebbistatin also reduced the cell invasiveness in 3D extracellular matrices but instead caused a stiffening of the cells. Finally, PINCH1 is apparently essential for providing cellular mechanical stiffness through the actin cytoskeleton, which regulates 3D motility.

TGF-β isoforms are important signalling molecules in wound repair in the skin. Transforming growth factor β3 (TGF-β3) has been implicated in scarless healing. In both animal and human models the application of exogenous TGF-β3 causes a reduction in the inflammatory response and improves the architecture of the neodermis. Research into the influence of TGF-β on scarring has tended to focus on fibroblasts. However, keratinocytes play a major role in scarring both indirectly, as a result of their influence over the behaviour of fibroblasts and also by directly influencing wound contraction. Thus, experiments were carried out to investigate the influence of TGF-β3 on the behaviours of a keratinocyte cell line (HaCaT). Incubation with TGF-β3 increased cell spreading and appeared to reduce cell-surface contacts indicated by both SPR imaging and a detachment assay. TGF-β3 also caused a decreased cell alignment response to microcontact printed protein patterns, in part due to the deposition of laminin which is associated with the TGF-β induced cell migration. There is evidence that TGF-β isoforms differentially influence the outcome of wound healing. Similar to the results produce following addition of exogenous TGF-β3, the neutralisation of TGF-β1 and 2 has been shown to reduce scar formation in the adult wounds. During reepithelialisation keratinocytes experience a dynamic environment. Both extracellular matrix proteins and growth factors influence the progression of wound repair which includes both cell migration and proliferation. Few studies have examined collective cell behaviour in response to TGF-β isoforms and ECM coated substrates. Thus both wound closure and cell proliferation assays were conducted for different ECM proteins fibronectin, laminin and collagen type I and for TGF-β1, 2 and 3. Rates of wound closure were significantly reduced on laminin coated substrates while cell proliferation rates were increased. TGF-β2 and 3 induced significant increases in wound closure rates. This appeared to correspond with an increase in the number of cells independently migrating out from the wound margins. Only TGF-β3 caused a significant decrease in cell proliferation over a 4 day period. Laminin332 deposition is central to the reepithelialisation process and is known to be induced in response to TGF-β. Thus experiments were carried out to investigate HaCaT cell laminin332 deposition in response to TGF-β1, 2 and 3. Both an immunofluorescence staining technique and an ELISA based semi-quantification method was used. Following 4 day incubation all TGF-β isoforms significantly increased laminin332 deposition; however TGF-β2 and 3 caused the most significant increases. Integrin receptors enable cell-matrix interactions during wound repair. TGF-β is known to influence the expression of integrin subunits. Thus, experiments were carried out to compare the influence of each TGF-β isoform on the expression of subunits α3, α2, α5, β1 and β4. All TGF-β isoforms significantly increased all subunit expression. TGF-β3 caused the most significant increase in β4 and both TGF-β2 and 3 caused the most significant increase in α2. While there were differences in cell responses to each isoforms, TGF-β3 did not stand out from the other two isoforms. Interestingly, TGF-β2 shared more similarities with TGF-β3 than it did with TGF-β1, in its role in enhancing wound closure and LN332 deposition. These comparative studies have shown that differences exist in the way TGF-β isoforms influence HaCaT cell behaviour, namely migration, laminin deposition and integrin expression. / EPSRC and DTA grant

Integrins and discoidin domain receptors (DDRs) 1 and 2 promote cell adhesion and migration on both fibrillar and non fibrillar collagens. Collagen I contains DDR and integrin selective binding motifs; however, the relative contribution of these two receptors in regulating cell migration is unclear. DDR1 has five isoforms (DDR1a-e), with most cells expressing the DDR1a and DDR1b isoforms. We show that human embryonic kidney 293 cells expressing DDR1b migrate more than DDR1a expressing cells on DDR selective substrata as well as on collagen I in vitro. In addition, DDR1b expressing cells show increased lung colonization after tail vein injection in nude mice. DDR1a and DDR1b differ from each other by an extra 37 amino acids in the DDR1b cytoplasmic domain. Interestingly, these 37 amino acids contain an NPxY motif which is a central control module within the cytoplasmic domain of β integrins and acts by binding scaffold proteins, including talin. Using purified recombinant DDR1 cytoplasmic tail proteins, we show that DDR1b directly binds talin with higher affinity than DDR1a. In cells, DDR1b, but not DDR1a, colocalizes with talin and integrin β1 to focal adhesions and enhances integrin β1-mediated cell migration. Moreover, we show that DDR1b promotes cell migration by enhancing Rac1 activation. Mechanistically DDR1b interacts with the GTPase-activating protein (GAP) Breakpoint cluster region protein (BCR) thus reducing its GAP activity and enhancing Rac activation. Our study identifies DDR1b as a major driver of cell migration and talin and BCR as key players in the interplay between integrins and DDR1b in regulating cell migration.

Cancer presents as a highly heterogeneous disease with partly overlapping and partly distinct (epi)genetic characteristics. These characteristics determine inherent and acquired resistance, which need to be overcome for improving patient survival. In line with the global efforts in identifying druggable resistance factors, extensive preclinical research of the Cordes lab and others designated the cancer adhesome as a critical and general therapy resistance mechanism with multiple druggable cancer targets. In our study, we addressed pancancer cell adhesion mechanisms by connecting the preclinical datasets generated in the Cordes lab with publicly available transcriptomic and patient survival data. We identified similarly changed differentially expressed genes (scDEGs) in nine cancers and their corresponding cell models relative to normal tissues. Those scDEGs interconnected with 212 molecular targets from Cordes lab datasets generated during two decades of research on adhesome and radiobiology. Intriguingly, integrative analysis of adhesion associated scDEGs, TCGA patient survival and protein-protein network reconstruction revealed a set of overexpressed genes adversely affecting overall cancer patient survival and specifically the survival in radiotherapy-treated cohorts. This pancancer gene set includes key integrins (e.g. ITGA6, ITGB1, ITGB4) and their interconnectors (e.g. SPP1, TGFBI), affirming their critical role in the cancer adhesion resistome. In summary, this meta-analysis demonstrates the importance of the adhesome in general, and integrins together with their interconnectors in particular, as potentially conserved determinants and therapeutic targets in cancer.

Abstract While most of the in vitro cultures are carried out on bi-dimensional (2D) substrates, most of the in vivo extracellular matrices are threedimensional (3D). Consequently cells behave differently on 2D substrates as a way to self-adaptation to a non-physiological environment. This fact has encouraged the development of more relevant culture conditions seeking to provide more representative models for biomedicine (e.g. cancer, drug discovery and tissue engineering) and further insights into any dimension-dependent biological mechanism. Different 3D culture systems have been established though their variability and complexity hinder their standardisation in common cell culture procedures. So, this thesis deals with the dimensionality issue in cell/material interactions and introduces sandwich-like microenvironments as a versatile tool to study cell behaviour. Cells cultured within this system use both dorsal and ventral receptors to adhere and spread, undergoing important changes with respect to the 2D cultures and approaching to 3D conditions. Stimulation of dorsal receptors has been previously addressed by overlaying a protein gel on cells already attached on a 2D surface. Here we propose a sandwich-like system that consists of two 2D surfaces so that wider spectra of conditions can be investigated by changing the nature of the substrate (material, topography…) and the protein coatings of both ventral and dorsal sides. Since sandwich culture provides an altered cellular adhesion compared to the traditional 2D substrates by the excitation of the dorsal receptors, changes in the intracellular signalling are expected, which might alter important processes such as proliferation, morphology, migration and differentiation. Hence this thesis evaluates the effect of different sandwich culture parameters in cell behaviour. First, cell fate upon adhesion was evaluated in terms of morphology, proliferation and adhesion. Different conditions were studied such as materials with different properties or protein coatings (dorsal and ventral substrates), as well as the effect of sandwiching cells just after seeding or after been allowed to adhere to the ventral substrate. Interesting results were obtained such as the relationship between the ability of cells to reorganise the ECM with cell morphology, proliferation and adhesion, similarly as observed in 3D hydrogels (degradable vs nondegradable systems). Then, cell migration within sandwich culture was studied by live imaging of a wound healing assay. Results revealed the key effect of both ventral and dorsal substrates in determining the migration rate as well as the migration mode used by cells. Moreover cells within the sandwich culture migrating in the wound healing assay adopted an elongated cell morphology that resembled cells migrating in other 3D systems. Beyond differences in cell morphology and migration, dorsal stimulation promoted cell remodelling of the extra-cellular matrix (ECM) over simple ventral receptor activation in traditional 2D cultures. Finally the effect of sandwich culture on cell differentiation was evaluated. First we showed an increase in C2C12 myogenic differentiation when cultured within the sandwich system. This enhancement was shown to be dorsal stimulation dependent and related to an alteration of the signalling pathway and the growth factor release. To determine if sandwich culture leads only to myogenic differentiation or whether it allows differentiation to other lineages, 4 different human mesenchymal stem cells (hMSCs) lines were cultured under the same conditions. Results showed the same sandwich environment triggered different cell differentiation. This points out the importance of the microenvironment cell niche in vivo, which highly influence cell fate, and thus the need of mimicking it properly in vitro. Overall, sandwich-like microenvironments switch cell behaviour towards 3D-like patterns, demonstrating the importance of this versatile, simple and robust approach to mimic cell microenvironments in vivo. / Ballester Beltrán, J. (2014). Sandwich-like systems to engineer the cellular microenvironment [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48166

Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that link the extracellular matrix to the actin cytoskeleton. Uniquely, these adhesion receptors mediate inside-out signal transduction, whereby extracellular adhesion is activated from within the cell by talin, a large cytoskeletal protein that binds to the cytoplasmic tail of the β integrin subunit via its PTB-like F3 domain. Features of the interface between talin1 and small β3 fragments only have been described previously. Through NMR studies of full-length integrin β tails, we have found that β tails differ widely in their interactions with different talin isoforms. The muscle-specific β1D/talin2 complex exhibited particularly high affinity, leading to the X-ray crystal structure of the β1D tail/talin2 F2-F3 complex. Further NMR and biological experiments demonstrated that integrin activation is induced by a concerted series of interactions between the talin F3 domain and the β tail and between the talin F2 domain and the cell membrane. Additional studies revealed the structural determinants of tight talin2/β1D binding and the basis of more general differences between β1 and β3 talin binding. NMR studies were also performed on tyrosine-phosphorylated integrin tails binding to the PTB domains of talin1 and Dok1, an inhibitor of integrin activation; these revealed that phosphorylation can inhibit integrin activation by increasing the affinity of the β tail for talin competitors. Key residues governing this switch were identified, and proteins were engineered with reversed affinities, offering potentially useful biological tools. Taken together, these results reveal the remarkable complexity of structural features that enable talin and its competitors to mediate this important form of transmembrane signalling.

Made available in DSpace on 2015-03-04T18:50:57Z (GMT). No. of bitstreams: 1 Dissertacao_Monika.pdf: 24724342 bytes, checksum: a8d2da36e8294d281f856fd1f9f4fca1 (MD5) Previous issue date: 2008-02-28 / The production of mechanical force on plasma membrane is mediated by integrins, connecting ECM components and cell cytoskeleton. This allows cells to generate traction during migration and tension during ECM remodeling. Integrins are membrane-spaning adhesion receptors that mediate dynamic linkages between intracellular actin cytoskeleton and the extracelullar adhesive matrix, outside-in/inside-out signaling, migration and detachment. Several proteins with diferent functions have already been identified as integrin ligands, and some important candidates as disintegrin-like and cystein-rich domains present in the snake venon metalloproteinases and ADAM (A Disintegrin and Metaloprotease) become important as they interfere in cell signaling pathways mediated by these transmembrane receptors. Thus, the isolation, characterization and structure determination of disintegrin-like domains o_er valuable tools for the development of new therapeutic compounds for a wide range of diseases. These compounds may provide new treatments for diseases such as cancer and inflammation pathologies. However, the mechanisms of ADAM-Integrin interaction have not been well clarified, yet. In this perspective, this study aims to analyze the molecular structure of the alfa6beta1 integrin and the disintegrin-like domain of human ADAM2 and ADAM9. Computational biology methods such as homology modeling and molecular dynamics were used in order to study the dynamics of the interaction of these proteins. Using in silico experimentation, detailed models of human alfa6beta1 and human ADAM 2 and 9 were obtained. Based on these models, the molecular basis of alfa6beta1-ADAMdsld interactions was assessed, and the most important structural components in ligand recognition/discrimination were identified. Using the collected structural information, we designed different small peptide based inhibitors, based on the structure of the interaction loop of human ADAM 9 disintegrin-like domain. Here proposed A9a inhibitor was tested in vitro , showing satisfactory results in blocking cell adhesion on specific substrates by alfa6beta1- laminin affnity inhibition in nanomolar concentrations. Our results also show the effcacy of the constructed models, the power of computational biology tools in new drug-design technologies, and clearly suggest that here presented alfa6beta1 inhibitors are good candidates for further development of new therapeutic agents against inflammation pathologies. / A integração entre o citoesqueleto celular e a MEC mediada pelas integrinas gera a produção de força mecânica sobre a membrana plasmática. Isto permite às células gerar tração durante sua migração e tensão durante o remodelamento da MEC. Várias proteínas com diferentes funções já foram identificadas como ligantes das subunidades a e b das integrinas. O estudo de proteínas capazes de se ligar e interferir na sinalização via integrina, como as desintegrinas-like e cisteina-rich presentes nos venenos de serpente e proteínas conhecidas como ADAM (A Disintegrin And Metaloprotease), torna-se cada vez mais importante. Assim, o isolamento, a caracterização e a determinação da estrutura de várias desintegrinas oferecem valiosas ferramentas para o desenvolvimento de novos compostos terapêuticos para um vasto número de doenças, sendo excelentes candidatos-protótipo para o desenvolvimento de novos fármacos que interfiram nas funções celulares moduladas por proteínas de adesão. Entretanto, as formas como a integrina e a ADAM interagem ainda não foram bem esclarecidas. Neste contexto, este trabalho visa analisar em escalar molecular a estrutura da integrina alpha6beta1 e do domínio desintegrina-like das ADAMs 2 e 9 humanas, e a forma como estas proteínas interagem, aplicando metodologias de biologia computacional estrutural como modelagem e dinâmica molecular. Com o objetivo de estudar a interação destas proteínas, modelos estruturais foram construídos por homologia a partir das estruturas 3D de proteínas obtidas por cristalografia de raio-X, e realizaram-se simulações de dinâmica molecular com solvente explícito para as proteínas isoladas e em complexo. Através do estudo estrutural e funcional pelo método in silico da integrina alpha6beta1 e ADAMs 2 e 9 humanas, as análises dos resultados das simulações e da flutuação dos resíduos de contato entre as duas proteínas durante a dinâmica molecular, foram desenhados e caracterizados novos candidatos peptídicos para inibição da integrina alpha6beta1. Nas simulações da movimentação angular do domínio b A/Hybrid, visando a possível ativação da integrina alpha6beta1 através da interação com o domínio desintegrina-like de ADAM9 e ligantes peptídicos, obtivemos resultados positivos para os peptídeos A9b e A9d. Este estudo aponta para o desenvolvimento de inibidores protéicos viáveis da integrina alpha6beta1 com base nestas estruturas. Nossos resultados ainda comprovam pelas metodologias in silico a eficácia dos modelos construídos, conseguindo reproduzir o comportamento das proteínas em estudo.

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 .

O aparelho propulsor mandibular é utilizado na odontologia para modular o crescimento da cartilagem condilar, por meio de forças geradas pela alteração postural da musculatura. Neste estudo foi avaliado o papel de integrinas ligantes de fibronectina (FN) na transdução das forças mecânicas geradas pelo aparelho, em ratos. Por meio de imuno-histoquímica e PCR em tempo real verificou-se que, in vivo, o uso do aparelho modulou a expressão das subunidades <font face=\"symbol\">1, <font face=\"symbol\">5, e <font face=\"symbol\">v de integrinas, FN e PCNA, um marcador de proliferação celular. In vitro, forças distensivas cíclicas aplicadas sobre células da cartilagem condilar aumentaram a expressão de mRNA para FN, fatores de crescimento IGF-I e IGF-II e PCNA. A adição do peptídeo GRGDSP, que bloqueia a ligação de algumas integrinas à FN, inibiu todos os efeitos, exceto na expressão de IGF-II. Esses resultados sugerem que integrinas ligantes de FN desempenham papel importante na mecano-transdução neste sistema e contribuem para o entendimento das bases moleculares envolvidas na ortopedia funcional dos maxilares / The mandibular propulsor appliance is widely used in dentistry to modulate the growth of the condylar cartilage, through forces generated by postural changes in the orofacial musculature. The aim of this study was to evaluate the role of fibronectin (FN)-binding integrins in the transduction of mechanical forces generated by the appliance in rats. By immunohistochemistry and real time PCR it was observed that, in vivo, the appliance´s use modulated the expression of the integrin subunits <font face=\"symbol\">1, <font face=\"symbol>\"5, and <font face=\"symbol\">v, FN and PCNA, a cell proliferation marker. In vitro, the application of cyclic distension forces on condylar cartilage cells increased the expression of FN, IGF-I, IGF-II and PCNA mRNA. Addition of the peptide GRGDSP, which blocks the binding of some integrins to FN, inhibited all the effects except the increase in IGF-II mRNA. These results suggest that FN-binding integrins play an important role in mechanotransduction in this system, contributing to the understanding of the molecular basis involved in maxillary functional orthopedic therapy.