A leptospirose é uma zoonose de distribuição mundial causada por espiroquetas patogênicas do gênero Leptospira. Resultados experimentais demonstraram que a patogênese pode estar relacionada com a capacidade destas bactérias em aderir a proteínas da matriz extracelular, escapar da resposta imune do hospedeiro e de produzir toxinas. Este trabalho teve como objetivo identificar proteínas secretadas por Leptospira interrogans sorovar Pomona estirpe Fromm kennewicki (patogênica) e Leptospira biflexa sorovar Patoc estirpe Patoc I (saprófita), através de análise proteômica. As leptospiras foram cultivadas em meio EMJH suplementado com soro de coelho ou albumina bovina. Os sobrenadantes foram filtrados, dialisados e liofilizados para aplicação das tecnologias de análise proteômica utilizando gel bidimensional e análise em solução. A análise dos peptídeos obtidos, nos dois procedimentos, foi realizada utilizando-se LC/MS/MS. Foi possível a identificação de 159 proteínas diferentes nas amostras de L.interrogans, entre as quais 64 foram positivas em pelo menos uma das ferramentas usadas para a predição. Em L. biflexa, 104 proteínas diferentes foram identificadas, entre elas 43 proteínas foram positivas pela análise in silico. Entre as proteínas identificadas, estão aquelas que possuem peptídeo sinal sec ou tat dependentes. Em outras, a predição da localização celular é desconhecida ou podem ter múltiplos sítios de localização, e ainda, proteínas que não possuem peptídeo sinal e que podem ser secretadas por mecanismos não convencionais. Muitos destas são proteínas hipotéticas sem domínios conservados detectados. No que diz respeito à atividade proteolítica, foi identificada a presença de metaloproteases no secretoma de L.interrogans. Não houve detecção da presença significativa de proteases bacterianas em amostras de L. biflexa. A identificação e a caracterização funcional de proteínas secretadas poderão contribuir para a elucidação dos mecanismos patogênicos e no desenvolvimento de novas estratégias para o tratamento e prevenção de leptospirose. / Leptospirosis is a zoonosis of worldwide distribution caused by pathogenic spirochetes of the genus Leptospira. The mechanisms by which leptospires invade the host and cause the disease are not yet fully understood. Experimental results have shown that the pathogenesis may be related to the ability of these bacteria to bind to extracellular matrix proteins, to escape hosts immune responses and to produce toxins. This work aimed to identify secreted proteins by Leptospira interrogans serovar Pomona strain Fromm kennewicki (pathogenic) and Leptospira biflexa serovar strain Patoc Patoc I (saprophyte) through proteomic analysis. The leptospires were grown in EMJH supplemented with rabbit serum or BSA. Supernatants were filtered, dialyzed and lyophilized to proteomic technology, two-dimensional gel and non-gel. The analysis of the obtained peptides in two procedures was performed using LC/MS/LC. It was possible to identify 159 different proteins in the samples of L.interrogans; among them, 64 were positive proteins in at least one of the tools used for prediction. In L. biflexa, 104 different proteins were identified; among them, 43 positive proteins were positive by in silico analysis. Among the identified proteins are those that possess sec or tat dependent signal peptide. In others, the prediction of the cellular location is unknown or may have multiple sites of localization, and even proteins which have no signal peptide can be secreted by unconventional mechanisms. Many of these are hypothetical proteins with no detected putative conserved domains. The presence of metalloproteases has been identified in the L.interrogans´ secretome, using proteolytic assay. There was no significant detection of the presence of bacterial proteases in samples of L. biflexa. The identification and functional characterization of secreted proteins may contribute to the elucidation of pathogenic mechanisms and in the developing of new strategies for the treatment and prevention of leptospirosis.

The extracellular matrix (ECM) is secreted by the host tissue and is an important key for mechanisms of cell responses. The main properties of the ECM materials include biodegradability, biocompatibility, and nanostructured in a 3D fibre network. In addition, ECM is composed of important molecules like growth factors, glycosaminoglycans (GAGs), collagens, fibronectin, and lamin, while final composition depends on the native tissue. We have selected for this study ECMs from cortical bone (B-ECM) and pericardium (P-ECM) tissue. These ECMs were digested by collagenase, pepsin and trypsin. Each of these digested ECMs was used to produce PLLA-ECM based electrospun scaffolds by two different methodologies (1) non-crosslinked (NCLK) hybrid electrospun scaffolds composed of PLLA and digested ECMs and (2) PLLA-collagen electrospun scaffolds crosslinked with digested ECMs (CLK scaffolds). This research proposes the characterization of the digestion promoted by collagenase, pepsin and trypsin on the ECMs, followed by the evaluation of the potential of the digested ECMs and of the PLLA-ECM scaffolds for bone regeneration. The proteinaceous mixture, produced from the ECM digestion, had compositions, which were dependent on the type of ECM, and on the enzymatic treatment, as shown by protein quantification, GAGs quantification, TGA, SDS-page and TPEF-SHG. All the results point to an extensive digestion caused by collagenase and pepsin and a milder digestion caused by trypsin. The digested ECMs were incorporated into nanofibrous scaffolds, and the products were characterized by SEM, TGA, DSC and TPEF-SHG. The porous nanofibrous mesh from non-crosslinked scaffolds exhibited fibres without beads and a uniform diameter. However, the crosslinked scaffolds presented non-organized agglomerates around the fibres making a less porous surface. TGA and DSC suggest the incorporation of the ECMs on the scaffolds. However, the distribution of the protein on the polymer was mostly dependent on the incorporation method, as showed by TPEF-SHG. To access the biomaterial ability for bone regeneration, bone marrow mesenchymal stem cells (BMMSCs) were cultured on the scaffolds over 21 days. Osteogenic markers such as ALP activity, mineral nodule formation by ARS staining, col1a2 immunostaining, and gene expression were analysed to access how the materials could induce BMMSCs osteodifferentiation. Comparing NCLK to CLK scaffolds the key factor for osteogenesis is the release of soluble factors, showing NCLK scaffolds with a higher ability to induce mineralization than CLK scaffolds. However, when comparing the effect of the enzymatic digestion on the mineralization of the scaffolds over the days, it is possible to establish that the effect of the enzymatic treatment is also related to the type of ECM. Despite all those differences, some PLLA-ECM scaffolds exhibited potential to induce earlier mineralization, observed by the analysis of bglap, runX2, Osx, sparc and col1a2 genes as osteogenic markers. / A matriz extracelular (ECM) é secretada pela células no tecido nativo e reúne propriedades chave para respostas celulares. Entre suas principais propriedades destacam-se: biodegradabilidade, biocompatibilidade e nanoestruturada tridimensionalmente. Além disso, é rica em sinalizadores celulares tais como: fatores de crescimento, glicosaminaglicanas (GAGs), colágeno, fibronectina e laminina, no entanto sua composição depende do tecido na qual se encontra. Para este estudo, foram selecionadas ECMs provenientes de osso cortical e de pericárdio. Estas ECMs foram digeridas por colagenase, pepsina e tripsina. Cada um dos produtos de digestão foi utlizado para a produção de suportes eletrofiados de PLLA-ECM, utilizando-se dois diferentes métodos de incorporação, (1) Suportes eletrofiados híbridos de PLLA-ECM obtidos a partir da eletrofiação da co-solução em 1,1,1,3,3,3-hexafluor-2-propanol, e (2) imobilização das ECM digeridas sobre suportes eletrofiados de PLLA-colágeno. O presente trabalho propõe-se a caracterizar as ECMs digeridas e a avaliar o potencial dos suportes eletrofiados de PLLA-ECM para a regeneração óssea. A mistura proteinácea obtida a partir da digestão das ECMs, mostrou que a sua composição é dependentes do tipo de ECM e da digestão enzimática, resultado este confirmado através da quantificação de proteínas, quantificação de glicosaminoglicanas, TGA, SDS-page e TPEF-SHG. A partir destes, foi observada que a colagenase é a enzima que promove a maior degradação das ECMs, enquanto que a tripsina promove uma degradação em menor escala. As matrizes digeridas foram incorporadas no material nanoestruturado, estes foram caraterizados por SEM, TGA, DSC e TPEF-SHG. Observou-se que a malha eletrofiada a partir da co-solução de PLLA-ECM exibiu a formação de fibras de diâmetro uniforme, enquanto que os suportes imobilizados apresentaram a formação de aglomerados sólidos ao redor das fibras, originando uma malha menos porosa. As análises de TGA e DSC confirmaram a incoporação das ECMs nas malhas eletrofiadas, e através da técnica de TPEF-SHG observou-se a distribuição das proteinas no polímero. O potencial dos materiais para a regeneração óssea foi avaliado através da cultura de células tronco mesenquimais de medula óssea sobre os suportes eletrofiados durante 21 dias, e em seguida, medidas de ALP, quantificação de coloração com vermelho de alizarina, imunofluorescência com anticorpo col1a2, e expressão de gênica foram analisadas para a avaliação de como os materiais eletrofiados de PLLA-ECM induzem a osteodiferenciação. Comparando-se materiais produzidos por co-solução e os materiais imobilizados foi possível observar que a resposta osteogênica é maior nos materiais híbridos devido a liberação de fatores solúveis dos suportes eletrofiados. No entanto, comparando-se o efeito da digestão enzimática na capacidade de mineralização dos suportes , é possível observar que o efeito da digestão enzimática é dependente do tipo de ECM. Em geral, foi possível observar que os suportes eletrofiados de PLLA-ECM exibem potencial para uso em engenharia de tecidos, em específico, regeneração óssea, uma vez que apresentaram-se regulados o conjunto de genes bglap, RunX2, Osx, sparc e col1a2.

Cells respond to their mechanical environment by changing shape and size, migrating, or even differentiating to a more specialized cell type. A better understanding of the response of cells to surrounding cues will allow for more targeted and effected designs for biomedical applications, such as disease treatment or cellular therapy. The spreading behavior of both human mesenchymal stem cells (hMSCs) and 3T3 fibroblasts is a function of substrate stiffness, and can be quantified to describe the most visible response to how a cell senses stiffness. The stiffness of the substrate material can be modulated by altering the substrate thickness, and this has been done with the commonly-used linearly elastic gel, polyacrylamide (PA). Though easy to produce and tune, PA gel does not exhibit strain-stiffening behavior, and thus is not as representative of biological tissue as fibrin or collagen gel. Fibroblasts on soft fibrin gel show spreading similar to much stiffer linear gels, indicating a difference in cell stiffness sensing on these two materials. We hypothesize cells can sense further into fibrin and collagen gels than linear materials due to the strain-stiffening material property. The goal of this work is to compare the material response of linear (PA) and strain-stiffening (fibrin, collagen gel) substrates through modulation of effective stiffness of the materials. The two-step approach is to first develop a finite element model to numerically simulate a cell contracting on substrates of different thicknesses, and then to validate the numerical model by quantifying fibroblast spreading on sloped fibrin and collagen gels. The finite element model shows that the effective stiffness of both linear and nonlinear materials sharply increases once the thickness is reduced below 10µm. Due to the strain-stiffening behavior, the nonlinear material experiences a more drastic increase in effective stiffness at these low thicknesses. Experimentally, the gradual response of cell area of HLF and 3T3 fibroblasts on fibrin and collagen gels is significantly different (p<0.05) from these cell types on PA gel. This gradual increase in area as substrate thickness decreases was not predicted by the finite element model. Therefore, cell spreading response to stiffness is not explained by just the nonlinearity of the material.

O presente estudo teve como objetivo caracterizar a expressão da família SIBLING (Small Integrin-Binding Ligand, N-linked Glycoproteins) após tratamento regenerativo de furca com enxerto de tecido reparativo de alvéolos dentários. Para isto, os 2os e 3os pré-molares superiores foram extraídos em quatro cães s.r.d. Cinco dias após as extrações, defeitos padronizados de furca classe II foram criados nos 2os, 3os e 4os pré-molares inferiores, bilateralmente. Estes defeitos foram tratados imediatamente com raspagem, alisamento e polimento corono-radicular (RAPCR) e retalho deslocado coronariamente (RDC) (Grupo Controle) ou com RAPCR + RDC + enxerto de tecido reparativo de alvéolos dentários (Grupo Teste) em um experimento de boca-dividida. Após um período de 6 semanas de reparação, os animais foram sacrificados e foi realizada análise imuno-histoquímica para avaliar a localização dos membros da família de proteínas SIBLING, composta pelas seguintes proteínas nãocolágenas da matriz extracelular: osteopontina (OPN), sialoproteína óssea (BSP), proteína da matriz dentinária 1 (DMP1), sialofosfoproteína da dentina (DSPP) e fosfoglicoproteína da matriz extracelular (MEPE). Não foram encontradas diferenças na expressão da família SIBLING entre os grupos teste e controle. Todas as proteínas foram expressas no novo osso, novo cemento e novo ligamento periodontal, em ambos os grupos. Os osteoclastos demonstraram imunolocalização intracelular intensa somente para a OPN. Cementócitos e o novo ligamento periodontal demonstraram, particularmente, marcação intensa para a MEPE. Houve uma diferença evidente entre o padrão de marcação entre o lado tratado (vestibular) e o não-tratado (lingual) de todos os espécimes, com presença de maior marcação do lado vestibular, para todos os anticorpos testados. Podemos concluir que não houve diferenças no padrão de expressão da família SIBLING após o uso do enxerto de tecido reparativo de alvéolos dentários. A família de proteínas SIBLING é expressa durante o processo de reparação de defeitos de furca, indicando possíveis papéis e funções para as proteínas OPN, BSP, DMP1, DSP e MEPE como moléculas alvo em terapias de regeneração periodontal. / The present study aimed in characterizing the expression of the SIBLING (Small Integrin- Binding Ligand, N-linked Glycoproteins) family in a regenerative treatment of furcation defects with a reparative tissue graft obtained from extraction sockets. The second and third upper premolars were extracted in four mixed breed dogs. Five days later, standardized class II furcation defects were created in the second, third and fourth mandibular premolars, bilaterally. The defects were immediately treated with either debridement and root planning (DRP) combined with a coronally positioned flap (CPF) (Control Group), or with DRP+CPF + a reparative tissue graft derived from the second and third premolar extraction sockets (Experimental Group) in a split-mouth design. After 6 weeks period of healing, the animals were sacrificed and immunohistochemistry was carried out to assess the localization of members of the SIBLING family of noncollagenous extracellular matrix proteins, namely osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE). No differences in the SIBLING family of proteins expression were noted between the control and experimental group. All proteins were expressed in new bone, new cementum and new periodontal ligament in both groups. Osteoclasts exhibited intense intracellular localization only for OPN. Cementocytes and the newly formed periodontal ligament demonstrated particularly intense staining for MEPE. There was an evident difference between the staining pattern between the treated (buccal side) and non-treated (lingual) side of the specimens, with a more intense staining pattern in the buccal side, for all the tested antibodies. In conclusion, there were no differences in the pattern of SIBLING expression following the use of a reparative tissue graft obtained from extraction sockets. The SIBLING family of proteins is expressed during the healing process of furcation defects indicating possible roles and functions of OPN, BSP, DMP1, DSPP and MEPE as target molecules in periodontal regeneration therapies.

Meckel-Gruber syndrome (MKS) is a universally lethal heritable human disease characterised by CNS malformations, cystic kidney, polydactyly, and liver fibrosis. MKS is classed as one of the ciliopathies due to its association with dysfunctional primary cilia, signalling organelles found on most cells in the human body. Some of the symptoms of MKS can be explained as a consequence of disrupted developmental signalling through the primary cilium, other defects are harder to explain, and evidence now exists for non-ciliary influences on ciliopathies. The nature of these influences, and the implications they may have for our understanding of ciliary function and the aetiology of MKS, remain unclear. In this thesis, defects in cell-extracellular matrix (ECM) interaction in MKS are investigated to determine whether MKS proteins have a role in this process, and if so, whether this role may be involved in MKS pathology. A combination of transcriptomic, proteomic, and cell imaging approaches are used to demonstrate that MKS patient cells produce a defective extracellular matrix, and that the MKS protein TMEM67 is present at the cell surface at sites of cell-ECM interaction. It is shown that the full-length TMEM67 protein is required for correct ECM morphology, and it is further shown that the abnormal extracellular matrix morphology in MKS cells underlies other defects, including failure to build cilia and alterations to the actin cytoskeleton. This represents the first set of causal relationships identified between the cellular defects in this complex disease. It is further shown that treatment with developmental signalling pathway antagonists can rescue these defects, potentially revealing a new avenue of therapeutic intervention for MKS. Finally, possible upstream defects are investigated that might underlie the ECM defect, including alterations to cell spreading behaviour and cell deformation resistance.

Physical factors in the environment of a cell affect its function and behavior in a variety of ways. There is increasing evidence that, among these factors, the geometric arrangement of receptor ligands plays an important role in setting the conditions for critical cellular processes. The goal of this thesis is to develop new techniques for probing the role of extracellular ligand geometry, with a focus on T cell activation. In this work, top-down molecular-scale nanofabrication and bottom-up selective self-assembly were combined in order to present functional nanomaterials (primarily biomolecules) on a surface with precise spatial control and single-molecule resolution. Such biomolecule nanoarrays are becoming an increasingly important tool in surface-based in vitro assays for biosensing, molecular and cellular studies. The nanoarrays consist of metallic nanodots patterned on glass coverslips using electron beam and nanoimprint lithography, combined with self-aligned pattern transfer. The nanodots were then used as anchors for the immobilization of biological ligands, and backfilled with a protein-repellent passivation layer of polyethylene glycol. The passivation efficiency was improved to minimize nonspecific adsorption. In order to ensure true single-molecule control, we developed an on-chip protocol to measure the molecular occupancy of nanodot arrays based on fluorescence photobleaching, while accounting for quenching effects by plasmonic absorption. We found that the molecular occupancy can be interpreted as a packing problem, with the solution depending on the nanodot size and the concentration of self-assembly reagents, where the latter can be easily adjusted to control the molecular occupancy according to the dot size. The optimized nanoarrays were used as biomimetic architectures for the study of T cell activation with single-molecule control. T cell activation involves an elaborate arrangement of signaling, adhesion, and costimulatory molecules organized into a stereotypic geometric structure, known as the immunological synapse, between T cell and antigen-presenting cell. Novel bifunctionalization schemes were developed to better mimic the antigen-presenting surfaces. Nanoarrays were functionalized by single molecules of UCHT1 Fab', and served as individual T cell receptor binding sites. The adhesion molecule ICAM-1 was bound to either static PEG background, or a mobile supported lipid bilayer. The minimum geometric requirements (receptor clustering, spacing and stoichiometry) for T cell activation was probed by systematic variation of the nanoarray spacing and cluster size. Out-of-plane spatial control of the two key molecules by way of nanopillar arrays was used to adjust the membrane bending and steric effects, which were essential for the investigation of molecular segregation in T cell activation. The results provide insights into the complicated T cell activation mechanism, with translational implications toward adoptive immunotherapies for cancer and other diseases. This single-molecule platform serves as a novel and powerful tool for molecular and cellular biology, e.g., receptor-mediated signaling/adhesion, especially when multiple ligands or membrane deformation are involved.

A Engenharia de Tecidos tem como objetivo desenvolver alternativas para o tratamento de doenças degenerativas e regeneração de tecidos lesionados. O princípio básico da Engenharia de Tecidos é promover o crescimento de células sobre um substrato. Esse substrato deve reproduzir a matriz extracelular, influenciando a diferenciação e as funções celulares. Para isso, neste trabalho, nanopartículas poliméricas de poli(3-hidroxibutirato-co- 3-hidroxivalerato) (PHBHV), com diferentes diâmetros hidrodinâmicos, foram sintetizadas pelo método de emulsão-evaporação do solvente. A concentração do surfactante, a velocidade de agitação durante a emulsificação e o tempo de agitação foram variados a fim de se analisar a influência desses parâmetros no diâmetro hidrodinâmico, índice de polidispersidade e estabilidade coloidal das emulsões. Os parâmetros que mais influenciaram no diâmetro hidrodinâmico e no índice de dispersidade foram a concentração de surfactante e a amplitude de sonicação da emulsão, respectivamente. Após a obtenção das dispersões com estabilidade coloidal, realizou-se a modificação química da superfície dessas partículas utilizando os aminosilanos, 3-aminopropiltrimetoxisilano (APTMS) e 3- aminopropiltrietoxisilano (APTES). Essa modificação química na superfície das nanopartículas teve como objetivo a mimetização da matriz extracelular, permitindo a adesão e proliferação das células. As partículas modificadas foram caracterizadas por espalhamento de luz dinâmico (DLS), microscopia de força atômica (AFM), calorimetria exploratória diferencial (DSC), Espectroscopia de Infravermelho por Transformada de Fourier (FTIR) e Ressonância Magnética Nuclear de Hidrogênio (RMN 1H). Os resultados de FTIR mostraram o aparecimento de pico referente ao grupo amino, que foi indicativo da modificação química da superfície das nanopartículas. Os resultados de RMN 1H mostraram o sinal dos grupos CH3 do silano APTMS no espectro das nanopartículas modificadas por APTMS, e nas partículas modificadas por APTES, foi possível identificar o sinal referente aos grupos CH2 do APTES. A partir desses resultados comprovou-se a modificação química da superfície das nanopartículas pelos aminosilanos. / Tissue engineering aims to develop alternatives to treat damaged tissues by promoting tissue regeneration. The basic principle of Tissue Engineering is to promote the growth of cells on a substrate. This substrate must reproduce the extracellular matrix, influencing particular cell functions and differentiation fate. For this purpose, at the present work, polymeric nanoparticles of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) with different hydrodynamic diameters were synthesized by emulsion-solvent evaporation technique. The concentration of the surfactant, stirring speed during emulsification and stirring time were varied in order to analyze the influence of these parameters on hydrodynamic diameter, polydispersity and colloidal stability. The parameters that most influenced the hydrodynamic diameter and polydispersity index were the variation in the surfactant concentration and the variation of emulsion sonication amplitude, respectively. After that, the nanoparticles had their surface modified by 3-aminopropyltrimethoxysilane (APTMS) and 3- aminopropyltriethoxysilane (APTES). The aim of this chemical modification was to mimic the extracellular matrix, allowing the adhesion and proliferation of cells. The modified particles were characterized by dynamic light scattering (DLS), atomic force microscopy (AFM), differential scanning calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR). The FTIR results showed a peak of the amino group, which was an indicative of the nanoparticles surface chemical modification. 1H NMR results showed the signal of the CH3 groups of the APTMS silane in the spectrum of the APTMS-modified nanoparticles and in the APTES-modified particles it was possible to identify signal relating to the CH2 groups of the APTES. From these results, it was verified the nanoparticles surface chemical modification by the aminosilanes.

A conversão da proteína príon celular (PrPc) em sua isoforma anormal PrPsc está associada a uma série de doenças neurodegenerativas, genericamente designadas por doenças priônicas. Embora a literatura tenha enfatizado o estudo do PrPsc e o mecanismo de propagação das doenças de príon, pouco tem sido feito para o entendimento do papel fisiológico do PrPc. Em 1997 nosso grupo descreveu um receptor/ligante para o PrPc utilizando o princípio da hidropaticidade complementar. Neste trabalho isolamos e identificamos este ligante de PrPc como sendo a STI-1 (Stress Inducible Protein-1). In vitro, a STI-1interage com o PrPc de maneira específica, saturável e com alta afinidade (Kd=8x10-8M). Paralelamente, mostramos que o PrPc se liga ao domínio RNIAEIIKDI da laminina (Ln) (Kd=2x10-8M). O bloqueio de PrPc na superfície de neurônios hipocampais de embriões de ratos e camundongos, reduziu a neuritogênese induzida por Ln. Além disso, neurônios provenientes de animais PrP -/- são incapazes de estender neuritos sobre o peptídeo RNIAEIIKDI, sugerindo que o PrPc é o único receptor celular para este domínio da Ln. Estes dados indicam que a interação PrPc-Ln seja relevante nos fenômenos de adesão e diferenciação neuronais. A caracterização das interações PrPc-Ln e PrPc-STI-1 representa contribuições importantes para a elucidação do papel biológico do PrPc. / Conversion of the cellular prion protein (PrPc) to its abnormal isoform PrPsc is associated with some neurodegenerative and fatal diseases called prion diseases. Although the literature has been emphasizing the mechanism of PrPsc conversion and illness propagation, little attention has been given to the PrPc physiological role. In 1997, our group described a PrPc receptor/ligand based on the complementary hydropathy theory. Herein, we identify the PrPc receptor/ligand as STI-1, the Stress Inducible Protein-1. In vitro studies showed that STI-1 is a specific, saturable and high affinity ligand for PrPc (Kd=8x10-8M). In parallel, we demonstrated that PrPc interacts with RNIAEIIKDI domain of laminin (Ln) (Kd=2x10-8M). The blockage of PrPc, both from embryonic rats and mice hippocampal neuros, inhibited Ln-induced neurite outgrowth. In addition, neurons from PrPc null mice are unable to extend neurites on RNIAEIIKDI, suggesting that PrPc is the unique cellular receptor for this Ln domain. These data indicate that PrPc-Ln interaction is relevant for neuronal adhesion and differentiation. The characterization of PrPc-Ln and PrPc-STl-1 interactions represents important contributions for the elucidation of the PrPc physiological role.

Les glioblastomes multiformes (GBM) comptent parmi les tumeurs au pronostic le plus sombre. L’extraordinaire capacité invasive des cellules tumorales rend toutes les interventions thérapeutiques actuelles totalement impuissantes. Une sous-population de Cellules Souches de Glioblastome (CSG) hautement invasive est responsable de la récurrence tumorale. Dans le cerveau, les GBM migrent principalement le long des vaisseaux sanguins au sein de l’espace périvasculaire riche en laminine, fibronectine et collagène ainsi qu’en suivant l’alignement des fibres myélinisées du corps calleux. La Matrice Extracellulaire (MEC) de ces régions joue un rôle important dans l’invasion des GBM, mais les mécanismes mis en jeu n’ont pas été complètement dévoilés. De plus, le développement de nouvelles thérapies anti-migratrices ciblant l’interaction des GBM avec la MEC reste encore limité. Dans le but de mimer la composition biochimique et les propriétés mécaniques de la MEC cérébrale et d’étudier leur rôle(s) dans la migration des CSG, nous avons développé un nouveau support de nanofibres (NF) alignées et fonctionnalisées avec de la laminine. Mes travaux de thèse ont montré que les NF génèrent un microenvironnement tridimensionnel (3D) favorisant l’adhésion et la migration des CSG. Cette adhésion est améliorée en comparaison avec les supports planaires (SP) conventionnels (2D) et récapitule mieux les mécanismes d’interaction des CSG avec la MEC au cours de l’invasion dans le modèle murin de tumeurs xénogreffées. Dans ces conditions physiologiques plus convenables générées par les NF, la variation des composantes biochimiques et mécaniques de la MEC affecte la migration des CSG. La présence ou l’absence de laminine régule le mode migratoire et l’orientation de fibres contrôle la direction de migration des CSG. D’un autre coté, l’altération de la glycosylation des protéines de la surface cellulaire module l’interaction des cellules tumorales du cerveau avec la MEC et augmente leur invasion. La deuxième partie de mes travaux de thèse a permis de démontrer que les glycomimétiques phostines « 3.1a » réorganisent le processus de la N-glycosylation des CSG diminuant leur invasivité in vitro et in vivo en inhibant les voies de signalisation de la kinase FAK et du récepteur de TGF-β impliqués dans l’interconnexion cellule-MEC. / Glioblastoma Multiforme (GBM) is a biologically aggressive tumor with an extremely poor prognosis. The highly invasive capacity of a subpopulation of Glioblastoma Initiating Cells (GIC) makes complete surgical resection impossible. GBM dissemination occurs along preexisting brain structures such as the perivascular space rich in laminin, fibronectine and collagen as well as the aligned myelinated fibers of the corpus callosum. The Extracellular Matrix (ECM) of these cerebral regions plays an important role during GBM invasion, but the underlying mechanisms remain largely unknown. Accordingly, the development of new anti-migratory therapies targeting the cell-ECM interactions is lacking. In order to mimic the compositional and physical properties of the cerebral ECM and to investigate their role(s) in GBM invasion, we have set up a new aligned nanofibers (NF)scaffold functionalized with laminin. My work demonstrated that the NFs constitute a tridimensional (3D) microenvironment supporting GIC adhesion and migration. The cell-ECM adhesion is improved on the NF in comparison to the conventional 2D planar surfaces (PS). Furthermore, the mechanisms of GIC interaction with the ECM on the NF are similar to those observed in the human GBM xenograft murine model. In this physiologically more relevant 3D microenvironment reproduced by the NF, the variation of the different biochemical and mechanical components of the ECM affects the migration of GIC. The presence or absence of laminin on the NF regulates the mode of migration and the orientation of the fibers dictates the direction of migration of GIC. On the other hand, the glycosylation that decorates cell surface proteins modulates the interaction of GBM tumor cells with the ECM and its alteration increases their invasion. The second part of my thesis demonstrated that the glycomimetics phostines « 3.1a » remodel the N-glycosylation of GIC and decrease their invasivity in vitro and in vivo via the inhibition of FAK and TGFβ-R signaling pathways known to be implicated in the cell-ECM intercommunication.

A proteólise de componentes do plasma e da matriz extracelular é um fator importante no quadro hemorrágico desencadeado pela ação de venenos de serpentes. Neste estudo, avaliamos o papel dos domínios estruturais de metaloproteinases na interação com seus alvos. Ensaios de hidrólise in vitro mostraram que as metaloproteinases HF3 e bothropasina (classe P-III), e BJ-PI (classe P-I) do veneno da Bothrops jararaca, degradaram proteínas plasmáticas (fibrinogênio, fibronectina e vitronectina), além de colágeno VI e Matrigel, mostrando diferentes perfis de hidrólise. Experimentos de N-deglicosilação das proteinases mostraram que suas porções glicosídicas são importantes para a manutenção da integridade de suas estruturas e da atividade proteolítica. E ensaios para avaliar a interação das proteinases e da proteína DC (domínios tipo disintegrina/rico em cisteínas) com alguns substratos protéicos mostraram que além de seus domínios estruturais (catalítico/tipo disintegrina/rico em cisteínas) suas cadeias de carboidratos também são importantes no reconhecimento dos mesmos. / Proteolysis of plasma and extracellular matrix proteins is a key factor in the local hemorrhage induced by snake venom metalloproteinases (SVMPs). In this work we evaluated the role of the structural domains of SVMPs in the interaction with their targets. In vitro hydrolysis assays showed that the SVMPs HF3 and bothropasin (P-III class) and BJ-PI (P-I class) from Bothrops jararaca venom degraded plasma proteins (fibrinogen, fibronectin and vitronectin), collagen VI and Matrigel resulting in different hydrolysis profiles. N-deglycosylation of these proteinases showed that their carbohydrate moieties are important for keeping their structural integrity and proteolytic activity. The assays to analyze the interaction of the proteinases and of the DC protein (composed of the disintegrin-like and cysteine-rich domains) with some proteins confirmed that not only the structural domains (catalytic, disintegrin-like and cysteine-rich) but also the sugar moieties are important in the recognition of plasma and extracellular matrix proteins.