No description available.

[ES] El mieloma múltiple es una neoplasia hematológica caracterizada por una expansión descontrolada de células plasmáticas monoclonales (mPCs) en medula ósea que producen, en la mayoría de los casos, un componente monoclonal secretado en el suero y/o en orina. En la actualidad, se sigue considerando una enfermedad incurable con la constante aparición de recaídas en los pacientes. Una de las causas que condicionan esta situación, radica en la generación de resistencia frente a fármacos por parte de las mPCs. Este mecanismo de resistencia a fármacos (DR) se ha visto que no solo depende de factores intracelulares, sino que la propia interacción de las mPCs con el microambiente medular juega un papel fundamental para su supervivencia, crecimiento y desarrollo de DR. Entre los componentes del microambiente tumoral, destaca la adhesión de las mPCs a componentes de la matriz extracelular (ECM) que se ha visto relacionada con la generación de DR. Por este motivo el desarrollo de esta tesis doctoral consistió en la elaboración y validación de una plataforma de cultivo 3D basado en la síntesis de un microgel. Este sistema estará constituido por microesferas funcionalizadas con componentes de la ECM como son la fibronectina (FN), colágeno tipo I (COL), heparina (Hep), heparan sulfato (HS) y ácido hialurónico (HA), generando un entorno 3D biomimético con la capacidad de poder analizar la respuesta celular desencadenada por la interacción de las mPCs con los componentes de la ECM, así como la DR generada por la adhesión de las mPCs a estas biomoléculas. El primer estudio consistió en la realización y puesta a punto de varios protocolos para la síntesis de distintos microgeles; un primer sistema se produjo mediante la polimerización por vía radical en bloque de co-polímeros de poliacrilato de etilo (EA) y polimetacrilato de etilo (EMA) o bien por EA, EMA y ácido acrílico (AAc). Mediante una emulsión del tipo aceite en agua se consiguió producir con estos copolímeros, microesferas de un tamaño próximo al de las mPCs. Un segundo sistema se basó en microesferas de alginato. Estas microesferas se obtuvieron en un dispositivo de microfluidica produciéndose la gelificación externa de las micro-gotas con la incorporación de iones de calcio consiguiendo microesferas de un tamaño medio de 177 µm. Debido a la gran variedad de microesferas sintetizadas con diferentes grupos químicos en sus superficies, se consiguió establecer protocolos de funcionalización similares a los establecidos en la literatura, teniendo en cuenta la estabilidad de la biomolécula a lo largo del tiempo del cultivo celular. Este enfoque, permitió la funcionalización con una gran variedad de biomoléculas disponiendo así de microgeles funcionalizados con FN, COL, Hep, HS y HA. Una vez desarrollados los microgeles, en un segundo estudio se procedió a evaluar la respuesta celular en un entorno 3D basado en microgel, valorando la interacción con los componentes de la ECM. Entre los resultados observados se pudo determinar como el tamaño de las microesferas afecta al crecimiento celular incluso en ausencia de cualquier funcionalización. Con los microgeles constituidos por microesferas de un tamaño próximo al de las mPCs se obtuvo un mayor crecimiento celular que con los microgeles formados por partículas de mayor tamaño, y en ambos el crecimiento fue superior al del cultivo en suspensión. Se plantea la hipótesis de que la presencia de las microesferas favorece en gran medida que se produzca un mayor contacto célula-célula que se ve incrementado cuanto mayor es la superficie específica del microgel. Entre los componentes de la ECM estudiados, mientras que el COL no genera ninguna respuesta celular diferente al control (microgel no funcionalizado), el HA favorece la proliferación celular. La adhesión de las mPCs a la FN condiciona el bloqueo de las células en la fase G0-G1 del ciclo celular. Esta adhesión está mediada / [CA] El mieloma múltiple és una neoplàsia hematològica caracteritzada per una expansió descontrolada de cèl·lules plasmàtiques monoclonals (mPCs) en medul·la òssia que produeixen, en la majoria dels casos, un component monoclonal secretat en el sèrum i/o en orina. En l'actualitat, es continua considerant una malaltia incurable, amb la constant aparició de recaigudes en els pacients. Una de les causes que condicionen aquesta situació, radica en la generació de resistència enfront de fàrmacs per part de les mPCs. Aquest mecanisme de resistència a fàrmacs (DR) s'ha vist que no sols depèn de factors intracel·lulars, sinó que la mateixa interacció de les mPCs amb el microambient medul·lar juga un paper fonamental per a la seua supervivència, creixement i desenvolupament de DR. Entre els components del microambient tumoral, destaca l'adhesió de les mPCs a components de la matriu extracel·lular (ECM) que s'ha vist relacionada amb la generació de DR. Per aquest motiu, el desenvolupament d'aquesta tesi doctoral va consistir en l'elaboració i validació d'una plataforma de cultiu 3D basada en la síntesi d'un microgel. Aquest sistema estarà constituït per microesferes funcionalitzades amb components de l'ECM com són la fibronectina (FN), col·lagen tipus I (COL), heparina (Hep), heparan sulfat (HS) i àcid hialurònic (HA), generant un entorn 3D biomimètic amb la capacitat de poder analitzar la resposta cel·lular desencadenada per la interacció de les mPCs amb els components de la ECM, així com la DR generada per l'adhesió de les mPCs a aquestes biomolècules. El primer estudi va consistir en la realització i posada a punt de diversos protocols per a la síntesi de diferents microgels; un primer sistema es va produir mitjançant la polimerització per via radical en bloc de copolímers de poliacrilat d'etil (EA) i polimetacrilat d'etil (EMA), o bé per EA, EMA i àcid acrílic (AAc). Mitjançant una emulsió del tipus oli en aigua es va aconseguir produir amb aquests copolímers, microesferes d'una grandària pròxima al de les mPCs. Un segon sistema es va basar en microesferes d'alginat. Aquestes microesferes es van obtenir en un dispositiu de microfluidica produint-se la gelificació externa de les microgotes amb la incorporació d'ions de calci aconseguint microesferes d'una grandària mitjana de 177 ¿m. A causa de la gran varietat de microesferes sintetitzades amb diferents grups químics en les seues superfícies, es va aconseguir establir protocols de funcionalització similars als establerts en la literatura, tenint en compte l'estabilitat de la biomolècula al llarg del temps del cultiu cel·lular. Aquest enfocament va permetre la funcionalització amb una gran varietat de biomolècules disposant així de microgels funcionalitzats amb FN, COL, Hep, HS y HA. Una vegada desenvolupats els microgels, en un segon estudi es va procedir a avaluar la resposta cel·lular en un entorn 3D basat en microgel, valorant la interacció amb els components de l'ECM. Entre els resultats observats es va poder determinar com la grandària de les microesferes afecta el creixement cel·lular fins i tot en absència de qualsevol funcionalització. Amb els microgels constituïts per microesferes d'una grandària pròxima al de les mPCs es va obtenir un major creixement cel·lular que amb els microgels formats per partícules de major grandària, i en tots dos el creixement va ser superior al del cultiu en suspensió. Es planteja la hipòtesi que la presència de les microesferes afavoreix en gran manera que es produïsca un major contacte cèl·lula-cèl·lula que es veu incrementat com més gran és la superfície específica del microgel. Entre els components de l'ECM estudiats, mentre que el COL no genera cap resposta cel·lular diferent del control (microgel no funcionalitzat), l'HA afavoreix la proliferació cel·lular. L'adhesió de les mPCs a la FN condiciona el bloqueig de les cèl·lules en la fase G0-G1 del cic / [EN] Multiple myeloma is a haematological neoplasm characterized by an uncontrolled expansion of monoclonal plasma cells (mPCs) in bone marrow that produce, in most cases, a monoclonal component secreted in serum and/or urine. At present, it is still considered an incurable disease with the constant appearance of relapses in patients. One of the causes that condition this situation lies in the generation of drug resistance by the mPCs. This mechanism of drug resistance (DR) has been seen to depend not only on intracellular factors, but the very interaction of mPCs with the medullary microenvironment plays a fundamental role in their survival, growth and development of DR. Among the components of the tumor microenvironment, the adhesion of the mPCs to components of the extracellular matrix (ECM) stands out, which has been related to the generation of DR. For this reason, the development of this doctoral thesis consisted in the elaboration and validation of a 3D culture platform based on the synthesis of a microgel. This system will be made up of micropsheres functionalized with the components of the ECM such as fibronectin (FN), collagen type I (COL), heparin (Hep), heparan sulphate (HS) and hyaluronic acid (HA), generating a 3D biomimetic environment with the ability to analyse the cellular response triggered by the interaction of mPCs with the ECM components, as well as the DR generated by the adhesion of the mPCs to these biomolecules. The first study consisted in the realization and development of several protocols for the synthesis of different microgels. A first system was produced by the radical block polymerization of polyethylene acrylate (EA) and polymethacrylate (EMA) co-polymers or by EA, EMA and acrylic acid (AAc). By means of an oil-in-water emulsion technique, it was possible to produce, with these copolymers, microspheres of a size close to that of the mPCs. A second system was based on alginate microspheres. These microspheres were obtained in a microfluidic device producing the external gelification of the micro-drops with the incorporation of calcium ions, obtaining microspheres with an average size of 177 µm. Due to the great variety of microspheres synthesized with different chemical groups on their surfaces, it was possible to establish functionalization protocols similar to those established in the literature, taking into account the stability of the biomolecule along with the time of cell culture. This approach allowed for functionalization with a great variety of biomolecules, having in this way functionalized microgels with FN, COL, Hep, HS and HA. Once the microgels were developed, a second study was carried out to evaluate the cell response in a 3D microgel-based environment, assessing the interaction with the components of the ECM. Among the results observed, it was possible to determine how the size of the microspheres affects cell growth even in the absence of any functionalization. With the microgels constituted by microspheres close to the size of the mPCs, a greater cellular growth was obtained than with the microgels formed by larger particles, and in both the growth was higher than in suspended culture. It is hypothesized that the presence of microspheres greatly favours a greater cell-cell contact, which is increased the larger the specific surface area of the microgel. Among the components of the ECM studied, while the COL does not generate any cellular response different from the control (non-functionalized microgel), HA favours cell proliferation. The adhesion of mPCs to FN conditions the blocking of cells in the G0-G1 phase of the cell cycle. This adhesion is mediated by the integrin ¿4ß1. / La presente tesis doctoral no se podría haber realizado sin la financiación del proyecto PROMETEO/2016/063, trabajo que también estuvo parcialmente financiado con fondos FEDER (CIBERONC (CB16/12/00284)). La iniciativa CIBER-BBN está financiada por el proyecto VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. Las acciones CIBER están financiadas por el Instituto de Salud Carlos III con ayuda del Fondo Europeo de Desarrollo Regional. / Marin Paya, JC. (2021). 3D Culture o Multiple Myeloma Cell Line Using Microgel Environments [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/167427 / TESIS

No description available.

Yes / Studies have been conducted to highlight the anti-inflammatory and immunosuppressive properties of synthetic cannabinoids as well as their potential for cartilage repair. Various wound healing techniques can be used to investigate the mechanisms of chondrocyte repair in monolayers or three dimensional tissues constructs. In this work the effect of WIN55, 212-2 (WIN-2) on nitric oxide (NO) and matrix metalloproteinase-2 (MMP-2) expressed by wounded chondrocyte monolayers was investigated. Moreover, expression of collagen type-I and type-II, fibronectin and S100 proteins were detected using immunofluorescence and quantitatively verified using ELISA based techniques following treatment with 1 μM and 2 μM of WIN-2. Treating chondrocytes with 1 μM of WIN-2 significantly increased expression of collagen type-II, fibronectin and S100, and significantly reduced collagen type-I expressions as compared to the control groups. On the other hand, both concentrations of WIN-2 significantly reduced the expression of the inflammation markers NO and MMP-2 in a dose dependent manner. These findings highlight the potential use of the synthetic cannabinoids for improving cartilage healing properties as well as acting as an anti-inflammatory agent which could be used to enhance tissue engineering protocols aimed at cartilage repair.

Receptor proteins on the surfaces of cells equip them to communicate with each other and to sense and interact with their environment. One receptor family, the αβ T-cell receptors (TCRs), allow T lymphocytes to detect and respond to pathogens via interactions with antigen-presenting major histocompatibility complex (MHC) molecules on target cells. A degree of TCR cross-reactivity (e.g. through structural similarity between peptide-MHC (pMHC) complexes) is essential to account for all possible pathogens, but can also lead to the misinterpretation of self antigens as foreign, and thereby elicit an autoimmune response, resulting in diseases such as multiple sclerosis (MS). Structural studies of pMHC and TCR-pMHC complexes have been key to developing of an understanding of the molecular basis of TCR cross reactivity, and the first strand of this thesis describes attempts to express and purify a highly cross-reactive MS patient-derived TCR for structural characterisation. The formation, purification and crystallisation of a TCR-self pMHC complex including another autoreactive TCR is also described. Another family of receptors, the fibronectin leucine-rich transmembrane proteins (FLRTs), has been implicated in roles in embryonic development including cell sorting and adhesion. In the second strand of this thesis, the nature of homotypic interactions between FLRTs, which may underlie adhesion between FLRT transfected cells, is investigated. Biophysical analyses demonstrate that these interactions may be mediated by the extracellular leucine-rich repeat (LRR) domain, and crystal structures of all three FLRT LRR domains suggest how interactions between them may underlie FLRT self-association at the cell surface. Residues which contribute to these interactions are conserved across different members of the FLRT family and different species. These findings confirm that FLRTs induce homotypic cell-cell adhesion, and suggest that this behaviour is mediated by self association at the cell surface via the LRR domain.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

No description available.

As células sangüíneas originam-se da medula óssea através da célula tronco que sofre processo de proliferação, diferenciação e maturação no microambiente hematopoiético. O microambiente hematopoiético é uma estrutura altamente organizada composta de células estromais, moléculas da matriz extracelular (MEC) e citocinas. A desnutrição protéico-energética diminui a produção de células sangüíneas e interfere na defesa do organismo. Neste trabalho estudamos os efeitos da desnutrição protéica (dieta contendo 4% de caseína) sobre a MEC da medula óssea em camundongos. Avaliamos a composição da MEC através de SDS-PAGE 7,5% e Western blot para Fibronectina (FN), laminina (LN) e trombospondina (TSP). Verificamos a capacidade da MEC aderir e sustentar proliferação da célula mielóide FDC-P1, na ausência e na presença de citocinas (GM-CSF e IL3). Avaliamos também a capacidade de ligação destas citocinas na MEC. O perfil eletroforético mostrou diferenças nas proteínas da MEC do animal desnutrido em relação ao controle. Através da densitometria dos géis observamos nas amostras obtidas do animal desnutrido, maior intensidade nas bandas de peso molecular 220, 182, 108 e 56 KDa em relação ao controle. Em 72 KDa a banda foi mais intensa nas amostras dos animais controles. A banda de 60 KDa foi evidenciada apenas nas amostras obtidas dos animais desnutridos. As bandas de 123 e 49 KDa foram evidenciadas apenas nas amostras dos animais controles. A expressão de FN, LN e TSP foi maior nas amostras obtidas dos animais desnutridos. Os ensaios de adesão e proliferação na presença e ausência de citocinas não apresentaram diferenças significativas entre as amostras. Quando avaliamos a capacidade da MEC ligar-se ao GM-CSF, houve maior interação com a MEC proveniente do animal desnutrido do que a MEC do animal controle. O teste de ligação para o IL3 não mostrou diferenças entre as amostras. Esses achados sugerem que a desnutrição protéica induz modificações na MEC, alterando o microambiente hematopoiético. / Blood cells have their origin at the bone marrow through the stem cell which undergoes a proliferation, differentiation and maturation process in the hematopoietic microenvironment. The hematopoietic environment is a highly organized structure formed by stromal cells, extracellular matrix (ECM) molecules, and cytokines. Protein-energy malnutrition reduces the production of blood cells, interfering with the defense of the organism. In the present work we have studied the effects protein malnutrition has on the ECM of bone marrow in mice. We have evaluated ECM composition by means of SDS PAGE 7,5% and Western blot for fibronectin (FN), laminin (LN) and thrombospondin (TSP). We assessed the capacity ECM has in adhesion and support of proliferation of the FDC-P1 myeloid cell both in the absence and in the presence of GM-CSF and IL3 cytokines. We have also measured the binding capacity of these cytokines in the ECM. The electrophoresis profile showed the existence of differences between the ECM proteins in the undernourished animal and the control. Using gel densitometry, we observed in samples from the undernourished animal a greater intensity of bands of 220, 182, 108, 60 and 56 KDa molecular weight as compared to control. At 72 KDa the band was more intense on samples from control animals. The 60 KDa band was evident only on samples taken from undernourished animals. The 123 and 49 KDa bands were evident on control animals only. Expression of FN, LN, and TSP was greater on samples from undernourished animals. Adhesion and proliferation assays, both in the presence and in the absence of cytokines, did not show significant differences among samples. When we evaluated the capacity ECM has to bind to GM-CSF, a greater interaction was seen with the ECM from the undernourished animal than the ECM from the control. Binding test for IL3 showed no differences existed among samples. Such findings suggest protein malnutrition causes alterations of the ECM, modifying the hematopoietic microenvironment.

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

Le cancer du rein compte parmi les 10 types de cancers les plus fréquents chez l’Homme. Il n’existe aujourd’hui aucun marqueur biomoléculaire dans ce type de cancer, et dans le cas d’un cancer métastatique, l’arsenal thérapeutique aujourd’hui disponible manque d’efficacité. Les différents processus mis en jeu lors de la progression tumorale sont encore mal connus. La connaissance de ces processus pourrait permettre de mettre en évidence de nouvelles cibles thérapeutiques, ainsi que des marqueurs biomoléculaires pronostiques ou diagnostiques de la maladie. Dans un premier projet, et afin de mieux comprendre les mécanismes de la progression tumorale et d’identifier de nouvelles cibles thérapeutiques potentielles et de nouveaux marqueurs biomoléculaires dans le cancer du rein, un nouveau modèle innovant a été généré à partir d’une lignée tumorale de RCC murine. Ce modèle de réimplantations successives de cellules tumorales issues de tumeur primaire ou de métastases a permis de générer différentes lignées cellulaires montrant une agressivité accrue au cours des passages. En utilisant une stratégie de biologie des systèmes, ce modèle pourra permettre de mettre en évidence des cibles d’études prometteuses qui pourraient être de nouvelles cibles thérapeutiques ou de nouveaux marqueurs biomoléculaires dans le RCC. L’interleukine-34 est l’exemple d’une cible d’étude d’ores et déjà été sélectionnée, mettant en évidence la puissance du modèle généré. Dans un second projet, les rôles de certains membres de la matrice extracellulaire tumorale ont été évalués en utilisant cette même lignée de RCC murine (collagène de type I, fibronectine, matrigel). Cette étude a permis de mettre en évidence le potentiel pro-invasif et pro-métastatique du dépôt de collagène de type I dans les tumeurs. Des récepteurs activés par le collagène sont proposés comme potentiellement impliqués dans les effets induits par le collagène de type I dans le modèle. Ces deux projets permettent et permettront de mieux comprendre certains mécanismes de la progression tumorale, ainsi que de mettre en évidence des marqueurs biomoléculaires et de nouvelles cibles thérapeutiques. / Kidney cancer is one of the 10 commonest human cancers. To date, no biomolecular markers are available in this type of cancer, and in the case of metastatic cancer, the therapeutic arsenal is still inefficient. The different processes involved in cancer progression are still poorly understood. Understanding those processes could highlight new therapeutic targets, and new prognostic or diagnostic biomolecular markers of this disease. For a first project, a new innovative model has been generated from a murine RCC cell line as a tool to understand cancer progression mechanisms and to identify new therapeutic target and new biomolecular markers in kidney cancer. This model of sequential reimplantation of cancer cells isolated from primary tumours or metastases allowed us to generate different cell lines showing increased aggressiveness after passages. Using a systems biology strategy, this model will allow us to identify new potential therapeutic targets and new biomolecular markers in RCC. Interleukin-34 is an example of an already selected target, showing the power of the model generated. For a second project, the role of some members of extracellular matrix (collagen type I, fibronectin, matrigel).was studied using this same murine RCC cell line. This study demonstrated the potential pro-invasive and pro-metastatic roles of collagen type I deposition in tumors. Collagen-activated receptors are proposed as mediators of the effect induced by collagen type I in this model. Those two projects have and will continue to contribute to a better understanding of cancer progression mechanisms, and will bring out new biomolecular markers and new therapeutic targets.