Current orthopedic implant technologies used suffer from slow rates of osseointegration, short lifetime, and lack of mechanical integrity as a result of poorly controlled cell-surface interactions. Recent biologically-inspired surface strategies (biomimetic) have focused on mimicking the biofunctionality of the extracellular matrix (ECM) by using short, adhesive oligopeptides, such as arginine-glycine-aspartic acid (RGD) present in numerous ECM components. However, these strategies have yielded mixed results in vivo and marginal bone healing responses. The central goal of this dissertation project was to engineer bioactive surfaces that specifically target integrin receptors important for osteogenic functions in order to improve bone tissue repair. In order to create integrin-specific interfaces, integrin-specific ligands reconstituting the fibronectin (FN) secondary/tertiary structure were first engineered and functionalized on material surfaces using several robust presentation schemes. We demonstrated that FN-mimetic-functionalized surfaces that directed α5β1 binding enhanced osteoblast and stromal cell integrin binding and adhesion, osteogenic signaling, and osteoblastic differentiation compared to various other RGD-based ligand-functionalized surfaces. Next, we investigated the effect of integrin-specific biointerfaces to modulate bone healing in a rat tibia implant bone model. We demonstrated, using a robust polymer brush system, that bioactive coatings on titanium implants that conferred high α5β1 integrin specificity in vitro enhanced bone formation and implant integration in vivo. Moreover, we showed that integrin specificity can be engineered using different immobilization schemes, including clinically-relevant ligand dip-coating, and promote the same robust in vivo effect. Furthermore, we investigate the synergistic roles of integrin specificity and ligand clustering on cell response by engineering biointerfaces presenting trimeric and pentameric "heads" of FNIII7-10 with nanoscale spacing. Integrin-specific ligand clustering supported α5β1-specific binding and cell adhesion and enhanced implant osseointegration in vivo compared to monovalent FNIII7-10 or non-functionalized biointerfaces. In summary, the FN-mimetic integrin-specific biointerfaces engineered in this thesis provide a clinically-relevant material surface strategy to modulate tissue healing responses. In addition, these results contribute to our greater understanding of how two specific material design parameters, integrin binding specificity and clustered ligand presentation, contribute individually and synergistically toward directing cell and tissue function.

Toll-like receptor (TLR)-4 is a pattern recognition receptor (PRR) that recognises the pathogen-associated molecular pattern (PAMP) lipopolysaccharide (LPS) produced by Gram-negative bacteria. LPS binds to Myeloid differentiation 2 (MD-2)/TLR-4 heterodimers, driving their dimerisation and inducing a conformational change of the intracellular TLR-4 toll/interleukin-1 receptor (TIR) domains. The adaptor protein Myeloid differentiation primary response gene 88 (MyD88)-adaptor-like (Mal)/TIR domain-containing adaptor protein (TIRAP) then binds to the TIR domains of TLR-4 and acts as a bridge for MyD88 which goes on to form the myddosome, a large protein complex of six to eight MyD88 molecules and four Interleukin-1 receptor- associated kinase (IRAK) 4 and four IRAK1/2 molecules. This triggers a signalling cascade which results in nuclear factor (NF)-κB transcription factor activation and production of pro-inflammatory effector molecules such as the cytokine Tumour Necrosis Factor (TNF)-α. Upon activation TLR-4 is also endocytosed where it interacts with a second set of adaptor proteins TIR-domain-containing adaptor- inducing interferon (IFN)-β (TRIF)-related adaptor molecule (TRAM) and TRIF to initiate the type I IFN response. How TLR-4 dimerisation results in the formation of the oligomeric myddosome is not fully understood, but it is possible that the stoichiometry of Mal/TIRAP may be important in the formation of this protein complex. The aim of my thesis was to determine the stoichiometry of Mal/TIRAP at the plasma membrane of immortalised bone marrow derived macrophages (iBMDMs) and whether this stoichiometry changes upon stimulation with different TLR-4 ligands. To investigate Mal/TIRAP stoichiometry I first developed a viral transduction experimental cell model to visualise fluorescently labelled Mal/TIRAP. Mal/TIRAP-/- iBMDMs were lentivirally transduced with a Mal/TIRAPHALO construct. The halotag was fluorescently labelled then the cells were stimulated with TLR-4 ligands, such as LPS, fixed at different time points, then imaged. Total internal reflection fluorescence (TIRF) microscopy was used to image the plasma membrane and photobleaching experiments performed to determine Mal/TIRAP stoichiometry. I developed a computer-based analysis pipeline to analyse the resulting photobleaching data. Under resting conditions, Mal/TIRAP is present at the plasma membrane in clusters of approximately ten Mal/TIRAP molecules per cluster. After five minutes of stimulation with 10 ng/ml LPS Mal/TIRAP redistributes into cluster sizes of approximately six, twelve and much larger. After ten and fifteen minutes stimulation with 10 ng/ml LPS the clusters return to the resting size of approximately ten Mal/TIRAP molecules per cluster with a few much larger clusters remaining present. This confirms the rapid time frame within which TLR-4 signalling occurs at the plasma membrane and is consistent with myddosome stoichiometry of six MyD88 molecules or proposed super myddosomes of twelve MyD88 molecules. The computer-based analysis pipeline developed can be used to analyse any protein of interest at the plasma membrane. Protein ligands have also been found to activate TLR-4; for example allergens, such as Fel d 1 and Der p 2, as well as endogenous damage associated molecular patterns (DAMPs), such as extracellular matrix (ECM) proteins, for example fragments of fibronectin and tenascin-C. The mechanism by which these proteins interact with TLR-4 and induce signalling is unclear. Proteins from the ECM (fragments FNIII1c, FNIII13-14, FNIII9-E and FNIII9-E-14 from fibronectin and the fibrinogen-like globe (FBG) domain of tenascin-C) were tested using a transient transfection assay in HEK293 cells and shown to activate TLR-4. In conclusion, I have developed new tools and methodology to investigate how TLR-4 signals in response to LPS and DAMPs in living cells. Whether DAMP- activated TLR-4 forms similar signalling complexes to those induced by LPS will form part of a future study.

Made available in DSpace on 2016-06-02T20:19:17Z (GMT). No. of bitstreams: 1 4141.pdf: 1483003 bytes, checksum: 97d4909d05c961c3bb73e05056aee7d3 (MD5) Previous issue date: 2012-02-29 / Financiadora de Estudos e Projetos / The aim of this study was to evaluate the effect of a strength exercise protocol on articular cartilage in animal model of osteoarthritis (OA). Thirty-six rats were divided in 6 groups: Control (C, n = 6), Osteoarthritis (OA, n = 6), Sham (S, n = 6), Control with exercise (E n = 6), Osteoarthritis with exercise (OAE, n = 6) and Sham with exercise (SE, n = 6). The animal model of osteoarthritis was anterior cruciate ligament transection (ACLT) in rats. After 2 weeks of ACLT, groups E, OAE and SE started the strength exercise protocol, three times a week for 8 weeks. We used the Mankin Histologic Grading System, measured the density of chondrocytes, the density of collagen fibers and the expression of collagen type II, chondroitin sulfate and fibronectin. The groups subjected to strength exercise protocol, OAE and SE, had lower values for the Mankin score, chondrocyte density and fibronectin expression and higher values for collagen fibers density, type II collagen expression and chondroitin sulfate expression when compared with OA and S, respectively. Group E was different from group C only in chondrocyte density and fibronectin expression. Therefore, strength exercise changes the content and expression of different articular cartilage constituent, having influence on our animal model of osteoarthritis and provides benefits to the cartilaginous tissue. / O objetivo desse estudo foi avaliar o efeito de um protocolo de exercício resistido na cartilagem articular de modelo animal de osteoartrite (OA). Trinta e seis ratos foram divididos em 6 grupos: Controle (C), Osteoartrite (OA); Sham (S), Exercício (E), Osteoartrite e Exercício (OAE), Sham e Exercício (SE). Os grupos OA, OAE, S e SE foram submetidos à transecção cirúrgica do ligamento cruzado anterior (LCA) do joelho esquerdo, sendo que somente os grupos OA e OAE tiveram o LCA seccionado. Após 2 semanas da cirurgia, os grupos E, OAE e SE iniciaram o protocolo de exercício resistido, 3 vezes por semana, durante 8 semanas. Foi aplicado o sistema de graduação histológica de Mankin, mensurado a densidade de condrócitos, a densidade de fibras colágenas e a expressão de colágeno tipo II, sulfato de condroitina e fibronectina. Os grupos submetidos ao protocolo de exercício resistido, OAE e SE, apresentaram menores valores para o sistema de graduação de Mankin, densidade de condrócitos e expressão de fibronectina e maiores valores para densidade de fibras colágenas, expressão de colágeno tipo II e sulfato de condroitina quando comparados aos grupos OA e S, respectivamente. O Grupo E apresentou diferença do grupo C somente na avaliação da densidade de condrócitos e na expressão de fibronectina. Portanto, o exercício resistido promove modificações no conteúdo e na expressão de diferentes constituintes da cartilagem articular, exercendo influência em nosso modelo de osteoartrite e trazendo benefícios para o tecido cartilaginoso.

Estudamos a ação das proteases bothropasina e Bothrops protease A, do veneno da serpente Bothrops jararaca, sobre o fibrinogênio (FBG), fibronectina (FN) e cininogênio (HK), como ferramenta para geração de peptídeos bioativos. As sequências primárias dos produtos de digestão foram identificadas por espectrometria de massas, com as buscas direcionadas por peptídeos em comum gerados pelas duas proteases. Foram encontradas oito sequências em comum provenientes do FBG e onze, da FN. Apenas a bothropasina clivou o HK, liberando desArg9BK. Foram sintetizados peptídeos derivados do FBG (FBG1-6) e da FN (FN1-4), além de des-Arg9-BK. Oito peptídeos apresentam potencial atividade antiangiogênica predita in silico. Observamos a inibição da elastase (28-20%) causada por FBG1-2-5-6. A melhor inibição da trombina foi de 17%, por FBG1. Contudo, a maioria dos peptídeos intensificou sua atividade. Por fim, este trabalho sugere que as proteases de veneno de serpentes podem ser usadas como ferramentas para processar componentes do plasma, visando à busca por peptídeos bioativos. / We studied the action of the proteases bothropasin and Bothrops protease A purified from the venom of snake Bothrops jararaca upon fibrinogen (FBG), fibronectin (FN) and kininogen (HK), as a tool to generate bioactive peptides. The primary sequences of the digestion products were identified by mass spectrometry and we focused the search for common peptides released by both proteases simultaneously. Sequences in common released by both proteases were found, being eight peptides from FBG, and 11 from FN. Only bothropasin was able to cleave HK releasing des-Arg9-BK. Peptides from fibrinogen (FBG1-6) and from fibronectin (FN1-4), as well as the des-Arg9-BK were synthetized. Eight peptides have potential antiangiogenic predicted in silico. We observed the inhibition of elastase (28-20%) caused by FBG1-2-5-6. The best inhibition of thrombin was 17% by FBG1. However, most of the peptides intensified its activity. Finally, this work suggests that the snake venom protease can be used as tools to process plasma components in order to search for bioactive peptides.

Les carcinomes sont des cancers touchant plusieurs organes du corps humain, notamment les seins, le pancréas, les poumons, l'intestin… et sont issus de la transformation de cellules épithéliales en cellules tumorales. Au cours du développement d'une tumeur, les cellules cancéreuses, contrairement aux cellules normales, acquièrent la capacité de se déplacer dans le corps humain, jusqu'à coloniser des organes voisins. Ces colonies sont appelées métastases. Le processus métastatique est responsable de 90% des décès dans le cadre des carcinomes. Ce processus n'est pas dû à l'action isolée des cellules cancéreuses mais est aussi le résultat d'une coopération entre la tumeur et son voisinage – le microenvironnement tumoral – favorisant la survie et la migration des cellules cancéreuses. Les fibroblastes sont une population cellulaire du microenvironnement tumoral. Il a été démontré que les fibroblastes sont activés à proximité des cellules cancéreuses ; on les qualifie de fibroblastes associés au cancer ou CAFs. Dans des tissus de patients, les tumeurs les plus agressives corrèlent avec un enrichissement en fibroblastes et une matrice plus dense. Mon projet de thèse illustre un nouveau mécanisme de coopération entre CAFs et cellules cancéreuses. Cibler l’action des fibroblastes pourrait ralentir la progression tumorale, voire bloquer la formation de métastases. / Cancer-associated fibroblasts (CAFs) are the most abundant cells of the tumor stroma. Their capacity to contract the matrix and induce invasion of cancer cells has been well-documented. However, it is not clear if CAFs remodel the matrix by other means (degradation, matrix deposition or stiffening). This project demonstrates that CAFs induce cancer cell invasion through assembly of FN into the matrix. CAFs assembled fibronectin (FN) mainly via integrin α5 but integrin αvβ3 was necessary for initial mechanosensing and fibrillar adhesion formation. In the absence of FN, contractility of the matrix by CAFs is preserved. When degradation is impaired, CAFs retain the capacity to induce invasion in a FN-dependent manner. In all cases, the levels of expression of integrin β3 and the amount of assembled FN was directly proportional to the invasion induced by fibroblast populations. Our results highlight FN assembly and integrin β3 as new hallmarks of CAFs. We also noticed that cancer cells migrate towards CAFs suggesting a possible chemotactic response. Using Dunn’s chemotaxis chamber, we found that cancer cells migrate along a gradient of CAF-conditioned media and a gradient of fibronectin. Finally, orthotopic injections of cancer cells and CAFs in the colon wall of mice revealed that CAFs stimulate metastasis of cancer cells to the liver. In conclusion, our data show that CAFs promote cancer cell invasion by depositing fibronectin that can guide cancer cells favoring metastasis formation.

Biomaterials have evolved over the years from the passive role of mere biocompatibility to an increasingly active role of presenting instructive cues to elicit precise responses at the molecular and cellular levels. Various characteristics common to synthetic biomaterials in vitro and extracellular matrices in vivo, such as immobilized functional or peptide groups, mechanical stiffness, bulk physical properties and topographical features, are key players that regulate cell response. The dynamics in the cell microenvironment and at the cell adhesive interface trigger a web of cell-material and cell-cell information exchanges that have a profound impact in directing the ultimate cell fate decision. Therefore, comprehension of cell substrate interactions is crucial to propel forward the evolution of new instructive biomaterials. Combinatorial biomaterials that encompass a wide range of properties can help to recapitulate the complexity of a cell microenvironment. The objective of this research was to fabricate combinatorial biomaterials with properties that span wide ranges in both surface chemistries and mechanical moduli. These materials were based on polydimethyl siloxane (PDMS), an elastomeric silicone biomaterial with physiologically relevant stiffness. After developing these mechano-chemical gradient biomaterials, we conducted high throughput screening of cell responses on them to elucidate cell substrate interactions and material directed behaviors. Our central hypothesis was that materials encompassing monotonic gradients in mechanical elastic modulus and orthogonal surface chemistry gradients could be engineered using the soft biomaterial, polydimethyl siloxane (PDMS) and that these gradient biomaterials would evoke a varied cell response. Furthermore, we expected high throughput screening of cell-material interactions using these materials would elucidate patterns and thresholds of synergy or antagonism in the overall cell response to the increased complexity presented by combinatorial materials. First, reproducible gradients in surface chemistry were generated on PDMS through surface modification techniques. Cell response to PDMS surface chemistry gradients was then screened in a rapid high throughput manner. Additionally, characteristics of the adhesive interface were probed to understand its role in cell response. Finally, a 2D combinatorial gradient with a gradient in mechanical elastic modulus and an orthogonal gradient in surface chemistry was fabricated with PDMS. High throughput screening of the synergistic influence of the varied mechanical and biochemical extracellular signals presented by the combinatorial biomaterial on cell response was conducted in a systematic manner. This research demonstrates the fabrication of combinatorial biomaterials with a wide range of mechanochemical properties for rapid screening of cell response; a technique that will facilitate the development of biomaterial design criteria for numerous biomedical engineering applications including in vitro cell culture platforms and tissue engineering.

Abstract This study was undertaken to identify mechanisms of adhesion of intraocular lenses (IOLs) to the capsular bag after cataract surgery and IOL implantation. It was also done to challenge the sandwich theory presented for posterior capsular opacification (PCO): If the IOL is made of a bioactive material it would allow a single lens epithelial cell layer to bond both to the IOL and the posterior capsule at the same time. This would produce a sandwich pattern including the IOL, the cell monolayer and the posterior capsule. The sealed sandwich structure would prevent further epithelial ingrowth. The degree of bioactivity of the IOL could explain the basic difference in the incidence of PCO and capsulotomy rates with different IOL materials. The sandwich theory was put forward on the basis of a search for a keratoprosthesis material, which would allow maximal adhesion of the prosthesis to corneal tissue. Titanium and glass-ceramic coated titanium were found to develop better adhesion than poly (methyl methacrylate) (PMMA). The adhesion of PMMA to the corneal stromal tissue was loose, and down growth of corneal epithelial cells was seen around the prosthesis. The differences between various IOL materials were first tested with rabbit corneal tissue cultures. There was better adhesion of corneal tissue to soft, hydrophobic acrylate than to PMMA, heparin surface modified (HSM)-PMMA, silicone or hydrogel IOLs. To assess differences in protein adhesion to IOL surfaces, different IOLs were incubated for 24 hours with radioactive iodine labeled fibronectin. Soft hydrophobic acrylate (AcrySof®) showed the highest binding of fibronectin, and the differences relative to all the other materials were significant (p < 0.01-0.001), except to PMMA (p = 0.31). The sandwich theory and the results with rabbit corneal tissue cultures and the protein adhesion study in vitro were evaluated against the results found in pseudophakic autopsy eyes. Altogether, 70 autopsy eyes were analyzed. From 38 autopsy eyes containing PMMA, silicone, soft hydrophobic acrylate or hydrogel IOLs histological sections were prepared from the capsular bag and immunohistochemical analyses were performed for fibronectin, vitronectin, laminin and collagen type IV. A total of 152 specimens were analyzed. From 32 autopsy eyes containing IOLs made of PMMA, silicone, acrylate or hydrogel, IOLs were explanted from the capsular bag and immunohistochemical analysis was done on both sides of the IOLs for fibronectin, vitronectin, laminin or collagen type IV. Soft hydrophobic acrylate IOLs had significantly more adhesion of fibronectin to their surfaces than PMMA or silicone IOLs. Also, more vitronectin was attached to acrylate IOLs than to the other IOL materials. Silicone IOLs had more collagen type IV adhesion in comparison to the other IOL materials studied. In histologic sections a sandwich-like structure (anterior or posterior capsule-fibronectin-one cell layer-fibronectin-IOL surface) was seen significantly more often in eyes with acrylate IOLs than in PMMA, silicone or hydrogel IOL eyes. These studies support the sandwich theory for posterior capsule opacification after cataract surgery with IOLs. The results suggest that fibronectin may be the major extracellular protein responsible for the attachment of acrylate IOLs to the capsular bag. This may represent a true bioactive bond between the IOL and the lens epithelial cells, and between the IOL and the capsular bag. This may explain the reason for clinical observations of less posterior capsular opacification and lower capsulotomy rates with the soft hydrophobic acrylate material of AcrySof® IOLs compared to the other IOL materials studied.

Streptococcus pyogenes is an important human pathogen. One of the most prominent virulence factors produced by S. pyogenes is SfbI, a surface adhesin composed of three domains: thioester domain (TED), proline-rich repeat domain (PRR) and fibronectin-binding repeat domain (FnBD). The structures and functions of TED and PRR and their contributions to the pathogenesis of streptococcal diseases are unknown. The interaction between PRR and its putative target, the intracellular actin cytoskeleton regulator Arp2/3, was investigated by both in vitro and in vivo studies. PRR was shown to inhibit Arp2/3-dependent actin polymerisation. The expression of PRR in HeLa cells caused disruption to the cytoskeleton of the cells. All data point towards a role of PRR in inhibiting the Arp2/3 complex but more evidence is needed to support this. The N-terminal domain of SfbI (TED) and four homologous domains from S. pyogenes, group G streptococci and Streptococcus pneumoniae were characterised by mass spectrometry, NMR spectroscopy and biochemical assays. All were shown to possess intramolecular thioester bonds, spontaneously formed between sides chains of Cys and Gln residues. Fibrinogen (Fg) was identified as the first binding target of bacterial TEDs with direct evidence that the thioester bond was involved in the interaction with Fg. A pull-down experiment using human plasma showed Fg is a specific binding partner of SfbI-TED. The binding sites were narrowed down to the thioester-forming Gln of SfbI-TED and Lys residues in the Fg-Aα chain, and binding potentially occurred via covalent isopeptide linkage. The data presented here suggest two new roles for SfbI, previously unknown in bacterial pathogenesis. The PRR may be the first bacterial inhibitor of the actin cytoskeleton acting by inhibiting the Arp2/3 complex. Thioester domains appear to be a shared common feature of surface proteins of many Gram-positive pathogens. They may form covalent crosslinks between bacteria and host tissue.

Les cancers de l’ovaire représentent la première cause de mortalité due aux cancers gynécologiques dans les pays développés. La plupart de ces cancers sont diagnostiqués à des stades tardifs quand les cellules cancéreuses ovariennes ont disséminé et colonisé les parois de la cavité abdominale. En conséquence, ces cancers sont associés à un sombre pronostic.Plus d’un tiers des patientes présentent une accumulation de liquide d’ascite au moment du diagnostic. L’ascite est un fluide exsudatif ayant une composante cellulaire et également une composante liquide acellulaire, constituant un réservoir dynamique de molécules bioactives. Malgré une réponse efficace aux traitements standards, les taux de récidive de cette pathologie restent élevés. L’acquisition d’une chimiorésistance et le mode de propagation atypique des cellules cancéreuses sont deux éléments importants impliqués dans la récidive des cancers ovariens.Le rôle de l’ascite et plus particulièrement de la composante matricielle ascitique, dans le processus de dissémination et dans la chimiorésistance des cellules cancéreuses ovariennes reste peu étudié et constitue le cœur de ce travail de thèse.L’objectif des travaux a été d’étudier l’influence de l’ascite et de deux glycoprotéines matricielles purifiées à partir d’échantillons d’ascites sur les comportements cellulaires propices à la dissémination et à la récidive des cancers ovariens. De plus, l’influence de l’ascite sur la réponse des cellules aux traitements à base de platine a également été recherchée et estimée.L’étude démontre que l’ascite est un microenvironnement propice à la dissémination des cellules cancéreuses ovariennes et que la fibronectine et la vitronectine ascitiques ainsi que leurs récepteurs d’adhérence spécifiques sont des éléments qui participent à la régulation de cette dissémination. L’ascite exerce également un effet protecteur contre l’effet cytotoxique des agents chimiothérapeutiques.Ces résultats mettent en perspectives l’intérêt potentiel de la fibronectine et de la vitronectine ascitiques comme outils diagnostiques et/ou cibles thérapeutiques des cancers ovariens et nous engagent à approfondir l’étude de ces deux molécules et de leurs conséquences sur la progression de la maladie. / Ovarian cancers are the leading cause of death among gynaecological cancers in western countries. Most of these cancers are diagnosed at a late stage, when ovarian cancer cells have spread and colonized the walls of the abdominal cavity. Therefore, these cancers are associated to a poor prognosis.More than one third of the patients show an accumulation of ascites at the time of the diagnosis. Ascites are exudative fluids composed of a cellular and also an acellular fraction. In fact, ascites constitute a dynamic reservoir of bioactive molecules. Even if the response of ovarian cancers to the current first-line therapy, that consit in debulking surgery followed by chemotherapy, is satisfactory, the rate of recurrence remains important. The gain of a chemoresistance and the atypical widespread of cancer cells are two important factors involved in the recurence of the ovarian cancers.The role of ascites and more particularly, of the ascitic matrix componants on the dissemination process and on the chemoresitance of the ovarian cancer cells remain poorly studied and is the aim of this work.The objective of this study was to investiguate the influence of ascites and of two matrix glycoproteins purified from samples of ascites i) on the cells behavior convenient to the dissemination and the recurrence of the ovarian cancers and ii) on the response of cells to therapeutics treatments with platinum.This study suggests that ascites are a permissive microenvironment to the dissemination of ovarian cancer cells and that ascitic fibronectin and vitronectin as well as their specific receptors are actors which participate to the regulation of this dissemination. Ascites also protect ovarian cancer cells against the cytotoxicity of chemotherapeutic drugs.These results illustrate the potential interest of ascites derived fibronectin and vitronectin as diagnosis tools and/or therapeutic targets for ovarian cancers and encourage us to deepen the study of these two molecules and their consequences in the progress of the disease.

O estudo de macrointeraçães na matriz extracelular (ECM) foi abordado em três modelos; a interação colágeno/colagenase, a interação colágeno/fibronectina e finalmente, a evolução da família enzimática das metaloproteinases de matriz (MMP). As MMP se caracterizam por sua notável especificidade contra componentes da ECM. As colagenases intersticiais, os membros mais estudados, clivam os colágenos intersticiais em um único ponto da molécula produzindo dois fragmentos característicos. Usando a hipótese da hidropaticidade complementar, a qual estipula que peptídeos codificados por sequências nucleotídicas complementares podem interagir entre si, foi possível caracterizar a sequência SQNPVQP em colagenase de fibroblasto ou SSNPIQP em colagenase de neutrófilo como importantes na interação das respectivas enzimas com o colágeno nativo. O fato da fibronectina, outro componente da ECM, ligar-se no mesmo domínio do colágeno clivado pelas colagenases possibilitou a utilização da mesma abordagem acima (hidropaticidade complementar) no estudo da interação colágeno/fibronectina. Sendo assim, foi possível caracterizar a sequência TNEGVMY da fibronectina como importante na interação com o colágeno. Adjacente a este sítio na fibronectina, existe uma sequência (AAHEEIC) que é homóloga ao sítio de ligação ao zinco presente nas MMP. Isto originou a possibilidade de que zinco pudesse modular a interação colágeno/fibronectina. De fato, zinco aumentou especificamente a ligação entre colágeno e fibronectina. Finalmente, abordou-se alguns aspectos filogenéticos da família das MMP. Foi caracterizada uma relação filogenética entre o núcleo enzimático das MMP e o domínio correspondente tanto na protease de Serratia como na protease B de Erwinia chrysanthemi, membros da mesma família de metaloproteinases bacterianas. Sendo assim, a atividade catalítica das MMP pode ter sido herdada das metaloproteinases bacterianas, enquanto a especificidade ao substrato talvez se constitua em uma aquisição das MMP eucarióticas. / The study of extracellular matrix (ECM) macrointeractions was approached in three models: the collagen/collagenase interaction, the collagen/fibronectin interaction and the molecular evolution of the matrix metalloproteinase (MMP) family of enzymes. MMP is characterized by its remarkable specificity against ECM components. Interstitial collagenases, the best studied members, attack interstitial collagens at a unique site producing two fragments. Using the complementary hydropathy hypothesis that states that peptides encoded by complementary nucleotide sequence can interact one to another, it was possible to characterize the sequence SQNPVQP (in fibroblast collagenase) or SSNPIQP (in neutrophil collagenase) as important in the interaction of the respective enzymes with collagen. The fact that fibronectin, another ECM component, binds at the same domain on collagen that is cleaved by collagenases arose the possibility of using the same approach (complementary hydropathy) in the study of collagen/fibronectin interaction. It was possible to characterize the fibronectin sequence TNEGVMY as important in the interaction with collagen. Adjacent to this site, there is a sequence (AAHEEIC) that shows an homology to the zinc-binding site present in several MMP. Therefore, zinc could be a modulator the collagen/fibronectin interaction. Finnaly, some phylogenetic aspects of the MMP family were studied. It was characterized a phylogenetic relationship between the catalytic core of MMP and the corresponding domain in Serratia protease and protease B from E. chrysanthemi, members of the same family of bacterial metalloproteinases. The catalytic activity of MMP can have evolved from the bacterial metaloproteinases whereas the substrate specificity is an acquisition of eukaryotic MMP