Made available in DSpace on 2014-08-20T14:30:10Z (GMT). No. of bitstreams: 1 tese_anelise_fernandes_montagner.pdf: 2069040 bytes, checksum: f8827dc228fd6085f72280a7c6d30b98 (MD5) Previous issue date: 2013-08-29 / The aim of this study was to evaluate the influence of chlorhexidine digluconate and sodium hypoclorite application on dentin after acid ecthing on the performance of adhesive restorations subjected to aging of the adhesive interface. For this, three studies were performed (in vitro, in situ and a systematic review). The in vitro study aimed to evaluate the effect of chlorhexidine digluconate (CHX) and sodium hypochlorite (NaOCl) on the microtensile bond strength values (μTBS) in dentin after cariogenic challenge and aging. Thirty sound human molars were selected and randomly assigned into 3 dentin pre-treatments (control, 2% chlorhexidine and 10% NaOCl) and 4 different types of aging (control -C-, biofilm without cariogenic challenge -NCC-, biofilm with cariogenic challenge -CC- and 18-month water storage -WS-). The same etch-and-rinse adhesive system (Adper Single Bond 2, 3M ESPE) and composite resin (Z250, 3M ESPE) were used for all the groups (n=30). For NCC and CC groups, dental biofilm in microcosm derived from saliva of a healthy donor was added on the samples. The biofilm grew up in a defined medium enriched with mucin (DMM), with or without 10% sucrose, according to the group. After the experimental period, all the samples were cleaned up and microtensile test was carried out. The data, in MPa, were subjected to ANOVA and a post-hoc Tukey Test (p < 0.05). The pre-treatment did not influence the μTBS, for all the aging conditions (p = 0.188); however the type of aging statistically affected the bond strength (p = 0.000). The bond strength values showed the following trend: C = NCC < CC = WS. Cariogenic challenge and water storage aging affected the bond strength stability and the former seems to be a suitable short-term methodology to assess the degradation of the resin-dentin. The in vivo study aimed to evaluate the effect of chlorhexidine (CHX) application in etched dentin on the 6-month retention of restorations of noncarious cervical lesions (NCCLs). A randomized controlled splitmouth and blind trial was carried out. Patients (n = 42) with at least two noncarious cervical lesions were included. NCCLs were randomly assigned according to the type of tooth into two groups: control (application of a placebo solution) or experimental group (application of 2% CHX for 60s after acid etching). Class V restorations (n = 169) were performed with an etch-and-rinse adhesive system (Adper Single Bond 2) and a composite resin (Filtek Z350) according to the manufacturer instructions, by 10 previously trained operators. The restorations were evaluated by a calibrated examiner at 1 week (baseline) and 6-month using the FDI criteria. The outcome was retention of the restoration. The analysis of factors associated to the failure of restoration was carried out through Fisher s Exact Test (α = 0.05). After 6 months of follow-up, 3.4% (CI 95% 1.3 - 7.3) of the Class V restorations failed. There was no significant difference between control and CHX (p = 0.920). Regarding the cavity s variables, deeper (p = 0.024) and wider cavities (p = 0.004) showed more failures, and wedge-shaped cavities failed more than saucer-shaped ones (p = 0.033). Regarding the patient s variables, restorations in patients with lower salivation 8 showed a higher percentage of failure (p = 0.034). Both treatments provided acceptable clinical performance of the restorations. The use of CHX as a coadjuvant in dentin adhesion did not influence the retention of Class V restorations after 6 months of follow-up. The systematic review aimed to systematically review the literature for in vitro and ex vivo studies that evaluated the resin-dentin bond strength immediately and longitudinal with the use of MMP inhibitor on the adhesive procedure. The systematic review was conducted sourcing the following databases (Pubmed, TRIP, LILACS, Scielo, Cochrane and ISI web Science) with no publication year or language limit. From 1.336 potentially eligible studies, 48 were selected for full text analysis and 30 were included for review. Two independently reviewers selected the studies, abstracted information and assessed the risk of bias on standardize scales. The results showed high heterogeneity in some comparisons and showed no difference for CHX 2% and control at baseline, however after 6-month of aging the CHX 2% and 0.2% presented a trend showing higher bond strength values than control groups (p < 0.05). The literature review was possible to observe the trend towards the decreased of the bond strength through time most for the control groups than for the experimental one. From of the 30 studies included, only 5 studies (16.6%) presented medium risk of bias while 25 (83.4%) showed high risk of bias and none showed low risk of bias. The use of MMPs inhibitor did not negatively affect the immediate bond strength, and it influenced the bond strength values long-time regarding the dentin adhesion stability. / O objetivo deste estudo foi avaliar a influência da aplicação do digluconato de clorexidina (CRX) e hipoclorito de sódio (NaOCl) em dentina, após o condicionamento ácido, na performance da interface adesiva submetida a envelhecimento da interface adesiva. Para isto, foram realizados 3 estudos (in vitro, in vivo e revisão sistemática). O estudo in vitro objetivou avaliar o efeito do prétratamento da dentina nos valores de resistência de união (RU) após diferentes tipos de envelhecimentos da interface adesiva. A dentina de todos os espécimes foi submetida ao condicionamento com ácido fosfórico por 15s e em seguida a 3 tipos de pré-tratamento: água destilada (controle - soluçao placebo - 60s), digluconato de clorexidina 2% - 60s, ou hipoclorito de sódio 10% - 60s. O sistema desivo (Single Bond 2 3M/ESPE) foi aplicado na superfície dentinária conforme indicação do fabricante. Palitos resina-dentina foram obtidos e submetidos a 4 níveis de envelhecimento: controle (CO), biofilme sem desafio cariogênico (BSD), biofilme com desafio cariogênico (BCD) e armazenamento em água por 18 meses (AGU). Nos grupos com biofilme, modelo de microcosmo foi utilizado e o biofilme foi crescido em meio definido enriquecido com mucina, com (BCD) ou sem (BSD) sacarose por 14 dias. Após o período experimental os espécimes foram limpos e preparados para o teste de microtração e o padrão de fratura foi analisado. Os valores de RU, em MPa, foram submetidos a ANOVA e teste post hoc Tukey (p < 0,05). O pré-tratamento não influenciou nos valores de RU, para todos os envelhecimentos avaliados (p = 0,188), entretanto o tipo de envelhecimento estatisticamente influenciou os valores de RU (p = 0,000). O envelhecimento CO foi similar ao BSD, com valores RU maiores que o BCD e AGU. A interface adesiva foi negativamente afetada pelo desafio cariogênico e pela degradação em água. No estudo in vivo, com objetivo de avaliar a retenção de restaurações classe V em dentina tratada ou não com CRX, um ensaio clínico controlado randomizado, boca dividida e triplo-cego foi conduzido. Pacientes (n = 42) com no mínimo 2 lesões cervicais não cariosas (LCNC) foram selecionados. As LCNC foram randomizadas em 2 grupos: tratamento controle e tratamento experimental (CRX 2% - 60s). Restaurações (n = 169) foram realizadas com Single Bond 2 (3M EPSE) e Filtek Z350 por 10 operadores previamente treinados. As restaurações foram avaliadas por um examinador calibrado em 1 semana e 6 meses após, usando os critérios da FDI. A análise dos fatores associados às falhas nas restaurações foi conduzida através do teste exato de Fisher (α = 0,05). Após 6 meses de acompanhamento, 3,4% (IC95% 1,3 - 7,3) das restaurações falharam devido a retenção. Não houve diferença significante entre os tratamentos controle e CRX para retenção (p = 0,920) e para manchamento marginal (p = 0,734). Em relação às variáveis relativas à cavidade, cavidades com maior profundidade (p = 0,024) e com maior altura (p = 0,004) 6 apresentaram maior ocorrência de falhas. Ainda, cavidades em formato de V apresentaram mais falhas que aquelas em formato de U (p = 0,033). Quanto as variáveis relativas aos pacientes, restaurações em pacientes que apresentavam boca seca falharam em maior porcentagem (p = 0,034). O uso de clorexidina como um coadjuvante na adesão a dentina não influenciou na retenção das restaurações após 6 meses de acompanhamento. E a revisão sistemática teve o objetivo de revisar a literatura acerca de estudos in vitro que avaliaram a RU resina-dentina imediata e após envelhecimento com o uso de inibidores de MMPs no procedimento adesivo. A revisão sistemática foi conduzida em 6 databases (Pubmed, TRIP, LILACS, Scielo, Cochrane and ISI web Science) sem limite de ano ou língua. De 1.336 estudos potencialmente elegíveis, 48 foram selecionados para análise de todo texto e 30 estudos foram incluiídos na revisāo. Dois revisores selecionaram os estudos, extraíram as informações e dados e verificaram o risco de viés em escalas padronizadas. Os resultados demostraram uma grande heterogeneidade em algumas comparações e nāo mostraram diferenças entre clorexidina 2% e o grupo controle sem envelhecimento, porém após 6 meses de envelhecimento observou-se uma tendência a favor dos grupos controle (p < 0,05). Todavia, foi possível observar na revisāo de literatura descritiva a tendência da diminuiçāo dos valores de RU (porcentagem) ao longo do tempo, mais para o grupo controle do que para o grupo experimental. De 30 estudos incluídos, apenas 5 estudos (16,6%) apresentaram médio risco de viés, enquanto 25 estudos (83,4%) mostraram alto risco de vieés e nenhum mostrou baixo risco. Os inibidores de MMPs nāo afetaram negativamente a RU da resina a dentina sem envelhecimento, e nāo influenciaram os valores de RU após envelheciemento.

Made available in DSpace on 2014-08-20T14:30:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-01-30 / The aim of this study was to evaluate the effect of different concentrations of 2-hydroxyethyl methacrylate (HEMA) triethyleneglycol dimethacrylate (TEGDMA) on the inhibition of matrix metalloproteinase 2 (MMP-2). Mouse gingival explants were cultured overnight in DMEM and the expression of secreted enzymes was analyzed by gelatin zymography in buffers containing 5 mM CaCl2 (Tris-CaCl2) in 50 mM Tris-HCl buffer with the addition of HEMA and TEGDMA at different concentrations (0.62, 1.25, 2.5 or 5.0% (v/v)). The gelatinolytic proteinase present in the conditioned media was characterized as matrix metalloproteinase by means of specific chemical inhibition with 0.5 mM of EDTA and 0.5 mM of NEM. The matrix metalloproteinases present in the conditioned media were characterized as MMP-2 by immunoprecipitation.The eletrophoretic bands were scanned and the transmittance values were analyzed with ImageJ software. Data was plotted and submitted to linear regression to investigate MMP-2 inhibition as a function of HEMA and TEGDMA concentration. Three major bands were detected in the zymographic assays. Those bands were characterized as MMP-2. Zymogene (72 kDa), intermediate (66 kDa) and active forms of MMP-2 (62 kDa) were inhibited by HEMA and TEGDMA in a dose-dependent way. These findings suggest that HEMA and TEGDMA could inhibit MMP-2 expression even at small concentrations. / O objetivo do presente estudo foi avaliar o efeito de diferentes concentrações de 2-hidroxietil metacrilato (HEMA) e trietilenoglicol dimetacrilato (TEGDMA) na inibição da metaloproteinase da matriz 2 (MMP-2). Tecidos gengivais de ratos foram incubados em DMEM e a expressão das enzimas secretadas foi analisada por zimografia em tampões de incubação contendo 5 mM de CaCl2 (Tris-CaCl2) em 50 mM de Tris-HCl com a adição de diferentes concentrações de HEMA e TEGDMA (0,62; 1,25; 2,5 e 5%) em volume. Para caracterizar as enzimas como MMPs foi realizado ensaio de inibição química específica com 0,5 mM de EDTA (um conhecido inibidor de MMPs) e 0,5 de NEM (um conhecido inibidor de proteinases serinas).Para caracterizar as enzimas como MMP-2 foi realizado o ensaio de imunoprecipitação. As bandas produzidas no gel foram escaneadas e os valores de transmitância foram analizados com o auxílio do programa ImageJ. Os resultados foram submetidos a regressão linear em função da concentração de HEMA e TEGDMA. Três bandas foram identificadas após a zimografia. Essas bandas foram caracterizadas como MMP-2. O zimogênio (72 kDa), a forma intermediária (66 kDa) e a forma ativa da MMP-2 (62 kDa) foram inibidas pelo HEMA e pelo TEGDMA de forma dose-dependente. Nossos resultados sugerem que o HEMA e o TEGDMA podem inibir a expressão da MMP-2 mesmo em pequenas concentrações.

Les travaux réalisés portent sur la conception de ligands protéiques capables de cibler le site catalytique des métalloprotéases matricielles (MMPs) grâce à une méthode d’ingénierie développée au laboratoire qui repose sur le greffage de motifs fonctionnels. Le motif fonctionnel choisi correspond aux 4 résidus N-terminaux du TIMP-2, un inhibiteur naturel des MMPs. Des plates-formes protéiques possédant des motifs d’acides aminés dans une topologie similaire à celle du motif de référence dans le complexe TIMP-2/MMP-14 ont été identifiées par criblage systématique de la PDB à l’aide du logiciel STAMPS (Search for Three-dimensional Atom Motif in Protein Structure). Dix candidats ligands satisfaisant les contraintes topologiques, stériques et de similarité électrostatique avec le ligand naturel TIMP-2 ont été sélectionnés. Ces ligands ont été produits par synthèse chimique ou par voie recombinante puis leur capacité à inhiber une série de 6 MMPs a été évaluée. Les résultats indiquent que tous les ligands protéiques conçus in silico sont capables de lier les sites catalytiques des MMPs avec des constantes d’association allant de 450 nM à 590 mM, sans optimisation supplémentaire. La caractérisation structurale par diffraction X de 2 variants d’un de ces ligands protéiques a permis de montrer que les interactions établies par le motif 1-4 dans ces ligands étaient similaires à celles observées dans le complexe TIMP-2/MMP-14, avec cependant des différences dans la géométrie de certaines d’entre elles. Des études de simulation par dynamique moléculaire ont également permis de mettre en évidence de possibles différences dans la géométrie et la stabilité de certaines des interactions reproduites dans les 10 plates-formes, pouvant contribuer aux affinités modestes observées pour ces ligands. Cependant, les résultats obtenus montrent que la méthode de conception in silico utilisée est capable de fournir une série de ligands protéiques de 1ère génération ciblant de manière spécifique un site catalytique d’intérêt avec un bon rendement. Cette méthode pourrait constituer la 1ère étape d’une approche hybride de conception in silico de ligands combinée à des techniques de sélection expérimentales. / Artificial mini-proteins able to target catalytic sites of matrix metalloproteinases (MMPs) were designed using a functional motif grafting approach. The motif corresponded to the 4 N-terminal residues of TIMP-2, a broad-spectrum natural protein inhibitor of MMPs. Scaffolds able to reproduce the functional topology of this motif as described in the TIMP-2/MMP-14 complex were obtained by exhaustive screening of the Protein Data Bank (PDB) using the STAMPS software (Search for Three-dimensional Atom Motif in Protein Structure). Ten artificial protein binders satisfying all topologic, steric and electrostatic criteria applied for selection were produced for experimental evaluation. These binders targeted catalytic sites of MMPs with affinities ranging from 450 nM and 590 μM prior to optimization. The crystal structures of two artificial binders in complex with the catalytic domain of MMP-12 showed that the intermolecular interactions established by the functional motif in these artificial binders corresponded to those found in the TIMP-2/MMP-14 complex, albeit with some differences in their geometry. Molecular dynamics simulations of the 10 binders in complex with MMP-14 suggested that these scaffolds could allow reproducing in part the native intermolecular interactions, but some differences in geometry and stability could contribute to the lower affinity of the artificial protein binders as compared to the natural one. Nevertheless, these results show that the in silico design method used can provide sets of starting protein binders targeting a specific binding site with a good rate of success. This approach could constitute the first step of an efficient hybrid computational-experimental protein binder design approach.

Abstract Cancers of the alimentary tract include many common cancer types, some of which have well-established treatment protocols and relatively good prognosis, such as colorectal cancer, and others, which have generally very poor prognosis. The gastrointestinal canal is colonized by a multitude of bacteria, the effects of which are currently poorly understood. Toll-like receptor 9 (TLR9) in cells of the alimentary tract recognizes the bacterial DNA-fragments and regulates immune functions in the host and the cancer. This thesis examines the function and prognostic significance of Toll-like receptor 9 in oral and esophageal squamous cell carcinoma as well as in esophageal, gastric and colorectal adenocarcinoma. The studies were made using tissue samples from patient cohorts and various cell culture techniques. Our data indicate that high expression of Toll-like receptor 9 in cancer cells associates with metastatic properties in oral and esophageal cancers and poor prognosis in esophageal adenocarcinoma and oral squamous cell carcinoma. Cell culture studies further suggest that TLR9 is functional in alimentary tract cancers and mediates cellular invasion when activated. Based on the results, TLR9 is active in alimentary tract cancers and its expression is related to poor cancer prognosis. Thus, TLR9 may represent a novel therapeutic target in alimentary tract cancers and might provide a link between bacteria and oral and gastrointestinal cancer. / Tiivistelmä Ruuansulatuskanavan syöpiin lukeutuu useita yleisiä syöpätyyppejä, kuten kohtalaisen hyväennusteinen paksusuolen syöpä, jonka hoitokäytäntö on vakiintunut. Toisissa ruuansulatuskanavan syövissä puolestaan ennuste on hyvin huono. Mahasuolikanavaa asuttavat moninaiset bakteerikannat, joiden vaikutuksia ymmärretään vielä kehnosti. Tollinkaltainen reseptori 9 (TLR9) tunnistaa näiden bakteerien DNA-rakenteita ja vaikuttaa yksilön ja syövän immuunivasteeseen. Tämä väitöstutkimus selvittää TLR9:n toimintaa ja ennustevaikutusta suun ja ruokatorven levyepiteelisyövissä, sekä ruokatorven, mahalaukun ja paksusuolen adenokarsinoomassa. Tutkimus toteutettiin käyttäen syöpäpotilaiden kudosnäytteitä sekä soluviljelytekniikoita. Tuloksemme osoittavat, että TLR9:n lisääntynyt ilmentyminen syöpäsoluissa yhdistyy metastasointiin suu- ja ruokatorvisyövissä, sekä korkeaan kuolleisuuteen suun levyepiteelisyövässä ja ruokatorven adenokarsinoomassa. Soluviljelykokeidemme tuloksiin nojaten TLR9 toimii ruuansulatuskanavan syövissä ja sen aktivaatio saa aikaan solujen invasoitumisen. Tutkimustuloksiimme vedoten TLR9 on aktiivinen ja toimiva ruuansulatuskanavan syövissä ja sen ilmentyminen liittyy huonoon ennusteeseen. TLR9 saattaa osoittautua uudeksi syöpähoitojen kohteeksi tai yhdistäväksi tekijäksi syövän ja bakteerien välillä ruuansulatuskanavan syövissä tulevaisuudessa.

Abstract Cancer progression is a multistep process dependent on tumour-stroma interactions. Various cell types, such as fibroblasts, endothelial, inflammatory and stem cells, as well as extracellular matrix (ECM) proteins, such as collagens, contribute to the tumour outcome. Tumour growth and invasion is accompanied by the proteolysis of ECM components mediated by various enzymes, such as matrix metalloproteases (MMPs). Proteolytic fragments released into the circulation may reflect cancer progression. The aim of this study was to develop novel in vitro methods for investigating cell invasion and for measuring cancer-associated collagen degradation. Human carcinoma cell invasion studies in vitro are often performed in organotypic cell culture models, mainly composed of rat or mouse ECM proteins. To create a human microenvironment for invasion studies, a novel organotypic model based on human uterine myoma (benign tumour) tissue was developed. Compared to the conventional collagen-based organotypic model, in the myoma model the carcinoma cell invasion depth was about eightfold and the invasion resembled, to a greater degree, the invasion pattern of dissected tissue samples of cancer patients. In addition, the invasion was easily quantified with a novel radioimmunoassay measuring type III collagen degradation products from the organotypic culture media. As human mesenchymal stem cells (MSCs) are one of the stromal cell types that may affect tumour progression, the mechanisms of stem cell invasion were also studied. On the surface of MSCs, Toll-like receptor 9 (TLR9) functions in immune defence against microbes. The activation of TLR9 with microbial DNA-resembling molecules induced human MSC invasion into the myoma tissue in a MMP-13-mediated fashion. To analyse cancer-associated soft tissue degradation, a novel enzyme immunoassay was developed. This novel assay enabled, for the first time, the measurement of type III collagen degradation products from human serum samples. In head and neck cancer patient sera, high levels of type III and type I collagen degradation products were shown to predict poor survival. In conclusion, the novel myoma model showed that the tumour microenvironment crucially affects carcinoma cell invasion. In addition, cancer-associated type III collagen degradation was successfully measured in cell cultures and in human sera by novel immunoassays. / Tiivistelmä Syövän eteneminen on monivaiheinen tapahtuma, jossa syöpäsolut ovat vuorovaikutuksessa lähiympäristönsä kanssa. Ympäristön eri solutyypit, kuten kantasolut ja sidekudoksen fibroblastit sekä soluväliaineen proteiinit kuten kollageenit, vaikuttavat syöpäsolujen invaasioon eli tunkeutumiseen ympäröivään kudokseen. Syövän invaasiossa useat entsyymit, mm. matriksin metalloproteaasit (MMP:t), hajottavat soluväliainetta. Kasvaimen kehittymisen aikana verenkiertoon vapautuu soluväliaineen hajoamistuotteita, joiden määrä voi kuvastaa sairauden etenemistä. Väitöstutkimuksen tarkoituksena oli kehittää uusia menetelmiä solujen invaasion ja syöpään liittyvän kollageenin hajoamisen tutkimiseen. Ihmisen karsinoomasolujen invaasion tutkimuksessa on perinteisesti käytetty kolmiulotteisia soluviljelymalleja, jotka koostuvat pääasiassa rotan tai hiiren soluväliaineproteiineista. Työssä kehitettiin uusi viljelymalli, jossa soluja kasvatettiin ihmisen hyvälaatuisen kohtukasvainkudospalan eli myooman päällä. Perinteiseen kollageenimalliin verrattuna myoomamallissa karsinoomasolut tunkeutuivat noin kahdeksan kertaa syvemmälle, ja solujen kasvu muistutti enemmän potilaiden syöpäkudosnäytteissä havaittua kasvutapaa. Invaasion voimakkuuden määrittämiseen kehitettiin vasta-aineisiin perustuva menetelmä, jolla mitattiin soluviljelmän kasvatusliuokseen myoomakudoksesta vapautuneiden tyypin III kollageenin hajoamistuotteiden määrää. Koska kantasolujen tiedetään voivan vaikuttaa syöpäkasvaimen leviämiseen, tutkimme myös ihmisen luuytimen kantasolujen invaasiota. Kantasolujen pinnalla TLR9-reseptori osallistuu immuunipuolustukseen mikrobeja vastaan. Kun reseptoria aktivoitiin mikrobi-DNA:ta muistuttavilla molekyyleillä, kantasolut alkoivat invasoitua myoomakudokseen ja MMP-13:n aktiivisuus soluissa lisääntyi. Syöpään liittyvän pehmytkudoksen hajoamisen tutkimiseksi kehitettiin vasta-ainemenetelmä, jolla onnistuttiin ensi kertaa mittaamaan potilaiden seeruminäytteistä tyypin III kollageenin hajoamistuotteita. Pään ja kaulan alueen syöpäpotilailla korkean tyypin III kollageenin hajoamistuotepitoisuuden todettiin liittyvän huonoon ennusteeseen. Tutkimus osoitti, että kasvaimen ympäristö vaikuttaa olennaisesti syöpäsolujen leviämiseen. Syöpään liittyvää tyypin III kollageenin hajoamista pystyttiin työssä kehitetyillä menetelmillä mittaamaan sekä soluviljelmistä että potilaiden seeruminäytteistä.

Lorsque le système nerveux central des mammifères est lésé, un ensemble de réactions secondaires impliquant l’inflammation et une gliose réactive conduit à la formation d’une cicatrice gliale qui inhibe la régénération axonale. Dans le cas d’une lésion de la moelle épinière l’absence de réparation efficace des réseaux axonaux lésés peut conduire à la paraplégie ou à la tétraplégie. Aujourd’hui on estime à plus de 2,5 millions le nombre d’individus dans le monde souffrant de ces handicaps et il n’existe à ce jour aucun traitement validé pour améliorer la situation des patients. Cependant, certaines approches de thérapie moléculaire, cellulaire, et de réadaptation semblent toutefois prometteuses sur modèle animal. La dégradation des chondroitines sulfates protéoglycanes (CSPGs), principales protéines inhibitrices de la cicatrice gliale, par clivage des coeurs protéiques et ou des chaînes latérales glycosaminoglycanes favorise la régénération axonale et entraîne une récupération fonctionnelle. Des études ont montré que la métalloprotéase matricielle MMP&#8208;2 est capable de dégrader le coeur protéique de ces CSPGs. Par ailleurs, les cellules engainantes de la muqueuse olfactive (CEOs) occupent une place privilégiée parmi les types cellulaires proposés dans la thérapie cellulaire en favorisant la croissance axonale et la récupérationfonctionnelle après lésion de la moelle épinière. Cependant, les mécanismes qui sous&#8208;tendent les propriétés régénératrices des CEOs restent essentiellement inconnus. Dans notre Thèse, nous présentons nos travaux en trois parties. Dans la première, nous montrons in vitro que : i) les CEOs en culture primaire secrètent des taux élevés de MMP&#8208;2, au moins en partie active ; ii) les gélatinases MMP&#8208;2 et MMP&#8208;9 présentent une sécrétion vésiculaire golgi&#8208;dépendante; iii) la distribution des vésicules contenant les MMPs est liée à celle du cytosquelette et des moteurs moléculaires qui participent probablement à une sécrétion focalisée de ces molécules en fonction d’interactions entre le milieu extracellulaire et le cytosquelette ; iv) les MMPs peuvent avoir une distribution nucléaire dans les CEOs ; v) MMP&#8208;2 jouerait un rôle dans la migration des CEOs, un processus important dans leurs capacités à réparer le tissu nerveux. Dans la seconde partie de notre thèse, nous avons développé un modèle de cicatrice gliale in vitro et nous montrons que : i) la migration des cellules astrocytaires de la cicatrice gliale in vitro est sensible aux effets des inhibiteurs des MMPs, contrairement aux cellules microgliales ; ii) les CEOs lèvent l’inhibition de croissance axonale due aux cellules astro&#8208;microgiales ; iii) le potentiel des CEOs à créer un environnement permissif à la croissance axonale serait lié aux gélatinases sécrétées par ces cellules, en particulier MMP&#8208;2. Dans la troisième partie de notre Thèse, nous avons évalué in vivo si MMP&#8208;2 contribuait aux effets bénéfiques des CEOs. Nous montrons pour la première fois, dans un model animal d’hémisection de la moelle épinière, et en utilisant des approches anatomiques, électrophysiologiques et d’analyse de la locomotion, qu’une administration chronique de MMP&#8208;2 recombinante : i) augmente le nombre et le diamètre des axones du coté distal du site de lésion ; ii) restaure la réponse évoquée du reflexe&#8208;H distal au site de lésion ; iii) améliore la réponse respiratoire à la fatigue musculaire induite électriquement et, iv) le plus important, améliore la récupération de la locomotion. L’ensemble de notre travail suggère que MMP&#8208;2 sécrétée par les CEOs jouerait un rôle important des les propriétés bénéfiques de ces cellules lorsqu’elles sont transplantées dans des sites de lésions de la ME, et que cette MMP présente un réel potentiel thérapeutique qui reste à explorer. / When the mammalian central nervous system is injured, a set of secondary reactions involving inflammation and reactive gliosis leads to the formation of a glial scar that inhibits axonal regeneration. In the case of a spinal cord lesion, the lack of effective repair of injured axonal networks can lead to paraplegia or quadriplegia. Today it is estimated that more than 2.5 million people are suffering from these handicaps worldwide, and there is as yet no validated treatment to improve the situation of patients. However, based on animal models, some molecular, cellular, and rehabilitation therapy approaches seem promising. Degradation of chondroitin sulfate proteoglycan (CSPG), the main inhibitory protein of the glial scar, by cleavage of either the protein core or side chains glycosaminoglycans, promotes axonal regeneration and leads to functional recovery. Studies have shown that the matrix metalloproteinase MMP-2 is capable of degrading the core protein of the CSPG. In addition, olfactory mucosa ensheathing cells (OECs) represent the most promising cell type for promoting axonal growth and functional recovery after spinal cord injury. However, the mechanisms underlying the regenerative properties of OECs remain essentially unknown. Here, we present our work in 2 parts. First, we show in vitro that: i) OECs in primary culture secrete high levels of active MMP-2; ii) both gelatinases, MMP-2 and MMP-9, have a vesicular Golgi-dependent secretion; iii) the distribution of vesicles containing the MMPs is linked to cytoskeleton and molecular motors distribution, which are probably involved in focused secretion of these molecules; iv) MMPs may have a nuclear distribution in OECs; v) MMP-2 plays a role in the migration of EOCs, an important process in their ability to repair nerve tissue. In the second part of my work, we evaluated whether the MMP-2 contributed to the beneficial effects of EOCs. We used an in vivo approach and we show for the first time, in an animal model of hemisection of the spinal cord, and using anatomical, electrophysiological analysis of locomotion approaches, that a chronic administration of recombinant MMP-2: i) increases the number and diameter of axons in the distal side of the site of injury; ii) restores the response-evoked H-reflex distal to the lesion site, iii) enhances the respiratory response to electrically-induced muscle fatigue, and iv) most importantly, improves the recovery of locomotion. All our work suggests that MMP-2, secreted by the EOCs, plays an important role in the recovery properties of these cells, when transplanted into spinal cord lesions, and that this MMP has a real therapeutic potential that remains to be explored.

No description available.

No description available.

INTRODUÇÃO: A propensão do CaP em gerar metástases decorre de mecanismos moleculares específicos em um processo composto por múltiplas etapas, sendo que o remodelamento do meio extracelular através de ações de enzimas proteolíticas denominadas metaloproteinases da matriz (MMP) é uma etapa fundamental. As MMP degradam vários componentes da matriz extracelular, sendo que seu controle pode ser exercido por outras proteínas denominadas TIMPs. Em nível gênico, outro controle pode ser exercido por moléculas chamadas microRNAs. OBJETIVO: O objetivo deste estudo é avaliar a regulação da MMP-9 por miRNAs. A partir de dados da literatura identificamos que a MMP-9 pode sofrer influência do miR-21 e 338-3p. MÉTODOS: Para os experimentos in vitro, linhagens celulares de CaP (DU145, PC3 e LNCaP) foram transfectadas com os miRNAs de interesse e a expressão de MMP-9 foi avaliada por reação em cadeia de polimerase quantitativa com transcriptase reversa (qRT-PCR). O sobrenadante da transfecção foi usado para ensaios de invasão com matrigel, e ELISA. Nos experimentos in vivo, células da linhagem PC-3-luc foram implantadas no subcutâneo de camundongos Balb-c nude e tratadas com injeções de anti-miR-21, miR-338-3p ou a combinação de ambos. RESULTADOS: O miR-21 aumentou expressão de MMP-9 em 72% na PC3. Houve maior invasão celular tanto na PC3 como DU145. In vivo, o bloqueio do miR-21 reduziu em 10% a expressão de MMP-9 nos tumores implantados (p=0,04). O miR-338-3p reduziu a expressão de MMP-9 em 53% na PC3 (p=0,001), 31% na LnCaP (p=0,23) e 24% na DU145 (p=0,16). No ensaio de invasão, menor número de células e colônias foram capazes de invadir a membrana de matrigel. In vivo, houve redução de 27% na expressão de MMP-9 nos camundongos tratados com o miR-338-3p (p=0,07). A combinação anti-miR-21+miR-338-3p reduz a expressão de MMP-9 em maior intensidade tanto in vitro como in vivo. CONCLUSÕES: A expressão de MMP-9 pode ser regulada pelo miR-21 e miR-338-3p. O primeiro se comporta como um oncomiR ao passo que o segundo como um supressor tumoral. A combinação de miRNAs é uma estratégia plausível para ampliar o efeito sobre expressão de genes de interesse / INTRODUCTION: The propensity of CaP to generate metastases results from specific molecular mechanisms in a multiphase process and the remodeling of the extracellular medium through the actions of proteolytic enzymes called matrix metalloproteinases (MMP) is a fundamental step. MMPs degrade several components of the extracellular matrix, and their control can be exerted by other proteins called TIMPs. At the gene level, another control can be exerted by molecules called microRNAs. OBJECTIVE: The objective of this study is to evaluate the regulation of MMP-9 by miRNAs. From literature data we have identified that MMP-9 may be influenced by miR-21 and 338-3p. METHODS: For in vitro experiments, CaP cell lines (DU145, PC3 and LNCaP) were transfected with the miRNAs of interest and the expression of MMP-9 was assessed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The transfection supernatant was used for matrigel and ELISA invasion assays. For the in vivo experiments, PC3-luc cells were implanted into the subcutaneous Balb-c nude mice and treated with anti-miR-21, miR-338-3p injections or the combination of both. RESULTS: The miR-21 increased MMP-9 expression by 72% in PC3. There was greater cell invasion in both PC3 and DU145. In vivo, miR-21 blockade reduced MMP-9 expression by 10% in implanted tumors (p = 0.04). MiR-338-3p reduced MMP-9 expression by 53% in PC3 (p = 0.001), 31% in LNCaP (p = 0.23), and 24% in DU145 (p = 0.16). In the invasion assay, fewer cells and colonies were able to invade the matrigel membrane. In vivo, there was a 27% reduction in MMP-9 expression in mice treated with miR-338-3p (p = 0.07). The combination of anti-miR-21 + miR-338-3p reduces MMP-9 expression in greater intensity both in vitro and in vivo. CONCLUSIONS: MMP-9 expression can be regulated by miR-21 and miR-338-3p. The former behaves as an oncomyR while the second as a tumor suppressor. The combination of miRNAs is a plausible strategy to extend the effect on gene expression of interest

O câncer de mama é o tipo de câncer mais comumente detectado em mulheres de todo o mundo. Na maioria das pacientes, a causa de morte se deve, principalmente, à doença metastática que pode se desenvolver a partir do tumor primário. O processo metastático envolve uma complexa cascata de eventos, incluindo a quebra organizada dos componentes da matriz extracelular por metaloproteinases de matriz (MMPs). A atividade das MMPs é precisamente regulada por inibidores específicos, os inibidores teciduais das MMPs (TIMPs). Dado seu papel na progressão tumoral, níveis elevados de MMPs têm sido associados com prognóstico desfavorável para pacientes com câncer. Por outro lado, sendo os TIMPs proteínas multifuncionais, níveis elevados de TlMP-1 e de TIMP-2 correlacionam com agressividade do tumor e prognóstico ruim em diferentes tipos de câncer, incluindo o câncer de mama. O gene supressor de metástase RECK codifica uma glicoproteína de membrana capaz de inibir a invasão e a metástase tumoral através da regulação negativa da atividade de MMPs envolvidas em carcinogênese: MMP-2, MMP-9 e MMP-14 (MT1-MMP). A fim de analisar o papel das MMPs e de seus inibidores (TIMPs e RECK) na progressão tumoral do câncer de mama, o perfil de expressão destes genes foi detectado, através de ensaios de Real-Time PCR, em um painel de cinco linhagens celulares de carcinoma de mama humano com diferentes potenciais invasivos e metastáticos e em 72 amostras teciduais de tumores primários de mama e 30 amostras teciduais de borda normal adjacente ao tumor. O perfil de expressão protéica de RECK foi avaliado em 236 amostras de tumores primários de mama através de ensaios de Tissue Microarray. Além disso, a atividade proteolítica das MMPs foi detectada em ensaios de Zimografia. Os resultados obtidos indicam que a progressão do câncer de mama humano está relacionada com um aumento dos níveis de expressão das MMPs e de seus inibidores específicos. O aumento dos níveis de expressão dos TIMPs parece estar relacionado ao seu papel como proteína multifuncional que pode estar funcionando de maneira a promover, mais do que suprimir, a progressão tumoral. Níveis elevados da expressão protéica de RECK estão associados com pior prognóstico. No entanto, para pacientes em estádios clínicos avançados, altos níveis de expressão de RECK podem estar correlacionados com melhor prognóstico, dependendo do balanço MMP/inibidor. Os níveis de expressão das MMPs apresentaram correlação positiva em relação aos níveis de expressão de seus inibidores específicos, sugerindo a existência de fatores e vias de sinalização comuns envolvidas na regulação coordenada destes genes. Além disso, a síntese do inibidor pode estar relacionada a uma resposta celular ao aumento da expressão e atividade de proteases. O balanço transcricional enzima/inibidor favorece a enzima nas amostras tumorais e, de modo contrário, o inibidor específico nas amostras de borda normal, sugerindo o balanço como o principal fator na determinação da degradação da MEC em processos invasivos e metastáticos. Os resultados obtidos podem contribuir para um melhor entendimento da complexidade dos mecanismos envolvidos na metástase do câncer de mama. / Breast cancer is among the most common tumors affecting women. Like most solid tumors, metastatic disease rather than the primary tumor itself is responsible for death. The metastatic process involves a complex cascade of events, including the organized breakdown of the extracellular matrix by matrix metalloproteinases (MMPs). The activity of these proteases is tightly regulated by specific inhibitors, known as tissue inhibitors of MMPs (TIMPs). Consistent with their role in tumor progression, high levels of a number of MMPs have been shown to correlate with poor prognosis in human cancers. On the other hand, TIMPs are multifunctional molecules with high levels of TIMP-1 and TIMP-2 having been shown to predict adverse prognosis and correlate with tumor aggressiveness in several different human cancers, including breast cancer. The RECK metastasis suppressor gene encodes a membrane-associated MMP regulator protein that is able to suppress tumor invasion and metastasis by negatively regulating MMPs involved in carcinogenesis, namely: MMP-2, MMP-9 and MMP-14 (MT1-MMP). In order to analyse the role of these genes in breast cancer progression, the expression levels of MMPs and theirs inhibitors were detected by Real Time PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential and in 72 primary breast cancer and 30 adjacent normal tissue specimens. The RECK protein expression profile was also examined in 236 primary breast cancer tissue specimens by Tissue Microarray technology. The proteolytic activity of MMPs was examined by Zymography. The results suggest that high expression levels of MMPs and their inhibitors are correlated with breast cancer progression. High levels of TIMP transcript may be involved in tumor-promoting activity as a result of their multifunctional role. Increased levels of the RECK protein are correlated with poor prognosis for the patient. However, high levels of RECK would be expected to confer a favorable prognosis to patients with advanced disease. The expression levels of MMPs significantly correlated with the levels of TIMPs and may be explained by coordinate correlation of these molecules or, alternatively, the synthesis of an inhibitor may be a cellular reaction to the presence of the protease. The enzyme/inhibitor balance at the transcriptional level favors the enzyme in tumor tissue and the inhibitor in adjacent normal tissue. It is probably the parameter that will determine the matrix degradation at invasion and metastatic process. Our results are likely to contribute for better understanding of the complex mechanisms involved in breast cancer metastasis.