Les ligaments jouent un rôle important dans le mouvement et la stabilité des articulations. Les accidents et la fatigue chronique sont les principales raisons des ruptures de ligaments qui n’ont généralement pas de capacité de guérison, ce qui conduit à de graves dysfonctionnements du ligament et des articulations. La construction des ligaments en bioingénierie donne un nouvel espoir thérapeutique. Pour construire un tel tissu, les cellules sont très importantes dans la mise en oeuvre de la construction d’un biotissu ayant de bonnes propriétés tant biologiques que mécaniques. Quelle source cellulaire et quel microenvironnement doivent être utilisés pour la reconstruction des ligaments ? L’objectif de ce travail était d’étudier la différenciation des cellules souches mésenchymateuses médullaires (CSMM) en fibroblaste. Pour ce faire, d’une part, nous avons co-cultivé des CSMM du rat sans contact avec des fibroblastes ligamentaires, et d’autre part, nous avons stimulé mécaniquement les CSMM. Un suivi des ARNm et des protéines associées caractéristiques des ligaments (collagènes I et III et ténascine-C) a été analysés. Nos résultats expérimentaux ont montré que la culture des CSMM dans un microenvironnement fibroblastique de ligaments ou l’étirement favorisent les synthèses de collagènes I et III et de ténascine-C dans les proportions proches des ligaments. L’ensemble de cette étude suggère qu’il est envisageable d’utiliser les CSMM comme source cellulaire, pour une application clinique, en ingénierie tissulaire du ligaments / Ligaments play an important role in the movement and stability of joints. Accidents and chronic fatigues are the main reasons for ligament lesion which usually is difficult for self healing and leads to serious dysfunction of ligaments and joints. The construction of bioengineering ligaments gives a new way to overcome this problem. Cells are very important in the construction of a biotissue with appropriate biological as well as mechanical properties. Which cellular source and microenvironment should be used for the reconstruction of ligaments? The objective of this work is to study the differentiation of bone marrow mesenchymal stem cell (BMSC) into fibroblast. We co-cultured indirectly rat BMSC with ligament fibroblasts or stimulated them by mechanical stretching. After that, the expressions of characteristics mRNA and protein of ligaments (collagen I, III and tenascin-C) have been analyzed. Our experimental results showed that the culture of BMSC in a microenvironment of ligament fibroblast or under stretching favored the syntheses of collagen I, III and tenascin-C in the proportions close to ligaments. In summary, these studies suggest it is feasible to use BMSC as cellular source for a clinical application in tissue engineering of ligaments

The extracellular matrix (ECM) is a complex network of molecules secreted by cells, which is essential for providing structural support and facilitating cell processes including adhesion, migration and survival. Tenascin-C is an immunomodulatory ECM protein that exhibits limited expression in healthy tissues, but is transiently elevated at sites of tissue injury, and is persistently expressed in chronic inflammatory diseases and tumours. Alternative splicing of 9 of tenascin-C's fibronectin type III-like domains (FnIII- A1, A2, A3, A4, B, AD2, AD1, C and D) generates enormous diversity in form; yielding 511 possible isoforms. Post-transcriptional modification of tenascin-C has been studied in cancer and during development where disease and tissue specific isoforms exhibit distinct adhesive, migratory and proliferative effects. However, little is known of how tenascin-C is expressed or alternatively spliced during inflammation. This study characterises inflammation and disease specific tenascin-C isoforms made by immune cells and fibroblasts, and investigates their functional relevance. Biosynthesis and alternative splicing of tenascin-C was examined using standard curve qPCR, ELISA, Western blot and confocal immunocytochemistry in resting and activated primary human immune cells, dermal fibroblasts, and in synovial fibroblasts isolated from healthy controls and from osteoarthritis (OA) and rheumatoid arthritis (RA) patients. Based on these data, three recombinant proteins comprising FnIII domains AD2-AD1, B-C-D and B-AD2-AD1-C-D were cloned, expressed and purified, and their impact on cell behaviour including adhesion, morphology and migration was assessed. Basal tenascin-C expression was lower in myeloid and lymphoid cells than fibroblasts, and was induced in all following inflammatory stimulation. Tenascin-C expression was elevated in disease with RA and OA synovial fibroblasts containing higher levels than healthy controls. Alternative splicing following cell activation was cell-type specific: all FnIII except AD2 and AD1 were upregulated in dendritic cells and macrophages, in T-cells all FnIII remained unchanged with FnIII A1 absent; and no change in splicing was observed in activated dermal fibroblasts. Normal and OA synovial fibroblasts exhibited similar tenascin-C splicing patterns, but FnIII B and D were specifically elevated in RA. Functional analysis revealed differences in the adhesion, morphology and migration of myeloid cells and dermal fibroblasts cultured on FnIII AD2-AD1, B-C-D, B-AD2-AD1-C-D and full length tenascin-C substrates; FnIII B-C-D promoted MDDC migration while B-AD2-AD1-C-D promoted fibroblast adhesion, compared to full length tenascin-C. For the first time, this study reveals differences in tenascin-C biosynthesis and alternative splicing by immune cells and fibroblasts following activation with inflammatory stimuli; and starts to reveal how alternative splicing of tenascin-C may influence the behaviours of both stromal and immune cells types during inflammation and in inflammatory diseases.

Made available in DSpace on 2014-08-20T14:30:18Z (GMT). No. of bitstreams: 1 Elaine Baldissera_TESE.pdf: 2456356 bytes, checksum: eaccf33a07294f70b9babaa3140cc017 (MD5) Previous issue date: 2006-07-06 / Aim Based on the great importance of the extracellular matrix (EM) in tissue development and repair, the aim of this study was to investigate the expression of their major glycoproteins, tenascin (TN) and fibronectin(FN) in the human pulp repair. Methodology Using immunohistochemistry, the expression of TN and FN was analyzed in forty-two human teeth, which were taken from a previous study. TN and FN profiles were evaluated after 7, 30, and 90 days after pulp capping with calcium hydroxide, being used three different haemostatic agents (0.9% salin solution, 5.25% sodium hypoclorite and 2% chlorhexidine digluconate) before pulp capping. Results There was no difference in the expression of TN and FN among the distinct haemostatic agents being all the time intervals taken into consideration. Both glycoproteins were found in all the pulp tissue, accomplishing the collagen fiber, and they were absent in all the mineralized tissues. Within 7 days post-treatment, it was observed a slightly more pronounced immunostaining on the exposure pulp site. Within 30 days, TN and FN demonstrated a stronger expression around the dentin barrier. TN also showed focal staining inside the reparative dentin, which was in mineralization. Within 90 days, it was observed a thin and linear expression of TN and FN delimitating the reparative dentin. In the predentin, TN showed strong immunostaining, and FN had a variable expression. Conclusions Based on the results, it may be concluded that there was no difference in the expression of TN and FN among the distinct haemostatic agents being all the time intervals taken into account. Moreover, TN and FN were present in pulp repair,confirming their active participation in this event. It might also be suggested that both glycoproteins are responsible for the odontoblastic differentiation and for the maintenance of the cell shape. It can also be concluded that TN and FN are important factors in the mineralization of the newly-formed dentin matrix. / O controle do sangramento frente a uma exposição pulpar é de fundamental importância no sucesso do capeamento direto. As soluções de hipoclorito de sódio e de gluconato de clorexidina têm sido utilizadas como agentes de limpeza e hemostasia na terapia pulpar conservadora. Alguns estudos têm indicado para o controle da hemorragia e sucesso do capeamento pulpar adesivo o hipoclorito de sódio (COX et al, 1998; COX et al., 1999). Além de ser bom agente antimicrobiano, o hipoclorito possui elevado Ph, o que implicaria na solubilização de fatores de crescimento da dentina, e conseqüentemente, na estimulação à formação de dentina (SMITH et al., 2002). No entanto, pesquisas também demonstram a atividade de dissolução tecidual do hipoclorito quando empregado a 5.25%. Porém esta é limitada às células superficiais pulpares sem efeitos adversos sobre o tecido pulpar subjacente (SENIA et al., 1971; ROSENFELD et al., 1978). Pouco se sabe a respeito da biocompatibilidade da clorexidina (THOMAS et al., 1995). Cox et al. (1998) sugeriram que esta substância poderia ser a causa de desastrosos resultados encontrados no estudo de Pameijer & Stanley (1998). Em contrapartida, a clorexidina a 0,2% utilizada no capeamento pulpar direto como agente hemostático e de limpeza, tem demonstrado boa performance em estudos em humanos (HORSTED et al., 2003) e em macacos (MURRAY et al. 2004) Diante das evidências apresentadas, pode-se observar que não há unanimidade entre os pesquisadores a respeito de qual dessas substâncias e suas respectivas concentrações seria mais efetiva na realização das terapias conservadoras vitais, especialmente no capeamento pulpar direto. Adicionalmente, as alterações na distribuição de componentes da matriz extracelular (ME) têm sido estudadas durante o desenvolvimento dentário e nos processos de reparo pulpar. As duas maiores glicoproteínas da ME, Fibronectina (FNC) e Tenascina (TNC) têm sido descritas como importantes para o estímulo e mobilidade celulares, durante a diferenciação de células odontoblastóides a partir de células multipotentes da polpa. No entanto, a participação da ME e sua interação com as reações celulares têm sido pouco exploradas e compreendidas. Desta forma, tornam-se fundamentais as investigações sobre a expressão dos componentes da 15 ME durante o processo de reparo pulpar, na tentativa de buscar melhor entendimento dos eventos de resposta deste tecido após o capeamento direto. O objetivo deste estudo é avaliar a biocompatibilidade do hipoclorito de sódio e do gluconato de clorexidina em diferentes concentrações, através de estudo in vitro de cultivo celular; a genotoxicidade dessas duas soluções, utilizando a reação de Feulgen para detecção e análise de micronúcleos e a expressão de componentes da matriz extracelular, através de estudo imunoistoquímico (técnica da estreptoavidina-biotina) em polpas humanas expostas, tratadas com os agentes hemostáticos hipoclorito de sódio a 5,25% e gluconato de clorexidina a 2%, empregados previamente ao capeamento pulpar direto. Para o ensaio de citotoxicidade do MTT será utilizada a linhagem celular NIH/3T3 (fibroblastos de rato). Este ensaio foi escolhido porque estudos prévios (COSTA, 2001; ZHANG et al., 2003) têm mostrado ser o mesmo apropriado para a avaliação da viabilidade ou citotoxicidade celulares, frente a materiais de uso odontológico. Serão testadas em diferentes tempos de exposição, as soluções de hipoclorito de sódio a 0,5%, 1%, 2,5% e 5,25% e de gluconato de clorexidina a 0,12%, 0,2%, 1% e 2%, bem como a solução salina 0,9%, que funcionará como controle negativo dos testes. Para o ensaio de genotoxicidade, será feita a análise semi-quantitativa dos micronúcleos de amostras previamente emblocadas, oriundas de 45 espécimens com polpas humanas tratadas com hipoclorito de sódio a 5,25% (n=16), gluconato de clorexidina (n=15) e

Abstract Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral cavity. The 5-year mortality of OTSCC remains at about 50%. The tumor microenvironment (TME) is now recognized as an important factor in cancer progression and metastasis, as well as a tool for prognostication. The aim of this study was to elucidate the roles of TME hypoxia and soluble factors on cancer cell migration and invasion, and the prognostic value of two extracellular matrix (ECM) molecules: tenascin-C (TNC) and fibronectin (FN). Hypoxia was studied using oral squamous cell carcinoma cells in migration and invasion assays. Invasion assays were carried out using a 3D-myoma invasion method. Similarly, the effect of soluble factors as well as ECM alterations were studied using the myoma model: the effect of soluble factors was studied by rinsing the myoma discs prior to experiments, and ECM alterations by lyophilizing and rehydrating. ECM was further studied by analyzing the prognostic value of TNC and FN from OTSCC samples. The effect of hypoxia was shown to be OTSCC cell line dependent: the effect of hypoxia on migration and invasion was increased in aggressive cell lines. Additionally, the response to hypoxia was altered in rinsed tissue. Tissue rinsing media were analyzed and factors affecting cell motility were found. The TME was found to be pivotal for cancer invasion: invasion was impaired in non-neoplastic tissue. Furthermore, changes in the ECM by lyophilization and rehydration led to a change in the invasion mechanism. High expression of stromal TNC and FN were excellent prognosticators in early-stage OTSCC. In conclusion, the present study highlighted the role of various TME components in cancer cell invasion as well as prognostication in OTSCC. Additionally, this study provided feasible tools for more precise diagnosis of early-stage OTSCC. / Tiivistelmä Liikkuvan kielen levyepiteelikarsinooma (OTSCC) on suuontelon yleisin syöpä. Viiden vuoden kuolleisuus OTSCC:an on edelleen noin 50 %. Kasvaimen mikroympäristön (TME) tiedetään nykyään olevan tärkeässä roolissa syövän kehityksessä ja etäpesäkkeiden muodostuksessa, sekä tarjoavan työkaluja ennusteiden laadintaan. Tämän tutkimuksen tarkoituksena oli selvittää TME:n hypoksian ja liukoisten tekijöiden vaikutusta syöpäsolujen liikkumiseen ja invaasioon ympäröivään kudokseen, sekä tutkia kahden solunulkoisen matriksin (ECM) proteiinin, tenaskiini-C:n (TNC) ja fibronektiinin (FN), vaikutusta OTSCC:n ennusteeseen. Hypoksian vaikutusta tutkittiin käyttäen suun levyepiteelikarsinoomasoluja liikkuvuus- ja invaasiokokeissa. Invaasiokokeissa hyödynnettiin kolmiulotteista ihmisen myoomaan perustuvaa invaasiomallia. Myös liukoisten tekijöiden ja ECM:n muutosten vaikutusten tutkimisessa käytettiin myoomamallia: liukoisten tekijöiden vaikutusta tutkittiin huuhtomalla myoomakiekot ennen niiden käyttämistä, ja ECM:n muutosten vaikutusta kylmäkuivaamalla ja uudelleen nesteyttämällä myoomakiekot. ECM:ia tutkittiin myös analysoimalla TNC:n ja FN:n värjäytyvyyden merkitystä OTSCC:n ennusteessa. Hypoksian vaikutus osoittautui solulinjariippuvaiseksi: hypoksia lisäsi kielisyöpäsolujen liikkuvuutta ja invaasiota eniten aggressiivisimmilla solulinjoilla. Lisäksi solujen vaste hypoksialle oli erilainen huuhdotussa kudoksessa. Huuhteluliuos analysoitiin ja siitä löydettiin solujen liikkumiseen vaikuttavia tekijöitä. TME:n havaittiin olevan ratkaisevassa roolissa syöpäsolujen invaasiossa: syöpäsolut eivät kyenneet invasoitumaan lainkaan ei-neoplastiseen kudokseen. Lisäksi muutosten ECM:ssä havaittiin johtavan muutoksiin solujen käyttämässä invaasion mekanismissa. Strooman TNC:n ja FN:n värjäytyvyyden todettiin olevan erinomaisia ennustekijöitä aikaisen vaiheen OTSCC:ssa. Tiivistettynä voidaan todeta, että tämä tutkimus alleviivasi useiden TME:n komponenttien vaikutusta syövän invaasiolle ja ennusteelle OTSCC:ssä. Lisäksi se tarjoaa käyttökelpoiset työkalut (TNC ja FN) tarkemmalle diagnostiikalle aikaisen vaiheen OTSCC:ssä.

Les métastases sont la principale cause de décès chez les patients atteints d’un cancer. Lors du développement métastatique, les cellules tumorales disséminées (CTD) doivent franchir certaines étapes clés avant de coloniser des organes distants de la tumeur primaire. Notre hypothèse est que la TNC pourrait jouer différents rôles dans la migration des cellules cancéreuses et par conséquent dans le développement métastatique. Considérant l’actine comme un réservoir de facteurs de croissance, la TNC pourrait induire la TEM ainsi que la survie et l’extravasation des cellules tumorales. Cependant, des cellules cancéreuses individualisées localement pourraient répondre à la TNC en initiant des changements rapides menant à un phénotype migratoire de type amiboïde. L’objectif de cette thèse a été d’étudier comment la TNC stimule le développement métastatique dans le cancer du sein au niveau cellulaire et moléculaire en utilisant des modèles tumoraux et cellulaires. / A high TNC expression correlates with lung metastagenicity and was shown to promote experimental lung metastasis, but the underlying mechanisms are poorly understood. The results of my thesis have provided insight into the roles of TNC in metastasis suggesting that TNC contributes to extravasation by impacting on survival, endothelialization, EMT and migration. Moreover, I have identified TGF-β signaling and integrin α9β1 as important pathway and molecule, respectively to be employed by TNC. Whether both molecule/pathway play a similar role in the investigated models of breast cancer, osteosarcoma and glioblastoma remains to be seen.