The excellent lubrication present within mammalian synovial joints attracted scientific interest, and some close-to-realistic models were applied to study the mechanism in vitro. In this project, the synergistic lubrication of synovial fluid was investigated by using 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), hyaluronic acid (HA), and phosphate-buffered saline (PBS buffer) to mimic the synovial fluid. Lubrication by the model synovial fluid was studied using borosilicate glass specimens in Mini-Traction Machine (MTM). The experiments proved that the DPPC vesicle solution and mixed DPPC/HA solution had excellent lubrication ability, stemming both from adsorption of a lubricious layer at the surface of glass specimens and from presence of material reservoir available for repair of wear defects in the lubricious layer. Comparing the macroscale results obtained in this project by MTM with the results in previous studies on microscale by using AFM, we concluded that the microscale study of synergistic lubrication could predict macroscale results, even though some differences were detected due to limited possibilities for exact replication of experimental conditions at the two scales.

Preterm birth (PTB), defined as birth before 37 weeks of gestation, is the leading cause of neonatal morbidity, and survivors can face lifelong medical difficulties. PTB remains a clinical challenge worldwide, with rates of PTB rising in all countries with reliable data. A lack in understanding of the mechanisms that lead to PTB has made developing diagnostics and therapeutics challenging, and existing ones are often ineffective. For a successful pregnancy, the major reproductive organs and surrounding tissues must sustain the growing loads of pregnancy. The cervix is one of these major reproductive organs. The cervix sits at the base of the uterus and has a versatile mechanical function in pregnancy. First, it must stay closed during gestation while the fetus develops; second, the cervix must remodel sufficiently and timely to dilate and allow delivery. The proper timing and extent of remodeling are critical for a healthy pregnancy. Improper cervical remodeling is a final common pathway to PTB and is the tissue of focus in this thesis. To improve our ability to identify when a PTB birth will occur and ultimately be able to treat those at risk, this thesis will identify the mechanical role of three extracellular matrix (ECM) components at various gestational ages and evaluate the ability of two major hormones to alter cervix function. Using experimental techniques (large-deformation tensile testing, digital-image correlation, imaging, biochemical) and theoretical and computational techniques (constitutive modeling, finite element analysis), the mechanical behavior of whole mouse cervices will be characterized in wild type, genetic knockout, and hormone-treated animals. First, the loss of both Class-I small leucine rich proteoglycans (SLRPs), decorin and biglycan, is detrimental to cervix function in late gestation. When the cervix should be most compliant and extensible, cervices without decorin and biglycan cannot stretch and are as stiff as the nonpregnant cervix. The loss of these proteoglycans also slows the cervix’s stress dissipation mechanism in late gestation, which could put the cervix at increased risk for damage. The mechanism of stiffening and lost viscoelasticity indicates the fibril crosslinking associated with SLRPs is a structural mechanism of the ECM contributing to cervical remodeling. Second, the loss of hyaluronic acid diminishes the cervix’s mechanical function at every gestational age tested. For nonpregnant to mid-gestational age, the cervix is softer than normal. Though by late gestation, the loss of hyaluronic acid stiffens the cervix; this is at a point when the cervix should be at its softest. The loss of hyaluronic acid also decreases the cervix’s protective stress dissipation mechanism in late gestation. There is limited knowledge of the interaction of collagen, elastic fibers, and hyaluronic acid in the cervix. The significant mechanical role of hyaluronic acid in the cervix warrants exploration of the structural mechanisms of these functional changes. Third, the loss of endogenous hormones stiffens the tissue and increases extensibility compared to the nonpregnant cervix. The administration of estrogen recovers large amounts of extensibility (beyond the stretch level of a late gestation cervix), stiffens the tissue (such that it is stiffer than a nonpregnant cervix), and recovers a significant amount of cervix strength. Fourth, relaxin increases cervix extensibility in mid-gestation and endows the cervix with viscoelastic ability in late gestation. Altogether, understanding the correlation between these extracellular matrix components, hormones, and functional changes of the cervix is fundamental to teasing out mechanisms of cervical remodeling and developing improved PTB diagnostics and therapeutics.

There is growing interest in the development of biomimetic matrices that are simultaneously cell-friendly, allow rapid vascularization, exhibit enough mechanical integrity to be comfortably handled and resist mechanical stresses when implanted in the site of interest. Meeting all these requirements with a single component material has proved to be very challenging. The hypothesis underlying this work was that hybrid materials obtained by combining scaffolds with bioactive hydrogels would result in a synergy of their best properties: a construct with good mechanical properties, easily handled and stable thanks to the scaffold; but also, because of the gel, cell-friendly and with enhanced oxygen and nutrients diffusion, and promoter of cell colonization. Moreover, such a composite material would also be useful as a controlled release system because of the gel’s incorporation. Poly (ethyl acrylate) (PEA) scaffolds prepared with two different morphologies were envisaged to provide the mechanical integrity to the system. Both types of scaffolds were physicochemically characterized and the effect of the scaffolds production process on the PEA properties was examined. The scaffolds preparation methods affected the PEA properties; nevertheless, the modifications induced were not detrimental for the PEA biological performance. Two different bioactive gels were studied as fillers of the scaffolds’ pores: hyaluronan (HA), which is a natural polysaccharide, and a synthetic self-assembling peptide, RAD16-I. HA is ubiquitously present in the body and its degradation products have been reported to be angiogenic. RAD16-I is a synthetic polypeptide that mimics the extracellular matrix providing a favourable substrate for cell growth and proliferation. Given the hydrophobic nature of poly(ethyl acrylate), the combination of PEA scaffolds with aqueous gels raised a number of problems regarding the methods to combine such different elements, the ways to gel them inside the pores, and the procedures to seed cells in the new composite materials. Different alternatives to solve these questions were thoroughly studied and yielded protocols to reliably obtain these complex structures and their biohybrids. An extensive physico-chemical characterization of the components’ interaction and the combined systems was undertaken. As these materials were intended for cardiac tissue engineering applications, the mechanical properties and the effect of the fatigue on them were studied. The different composite systems here developed were homogeneously filled or coated with the hydrogels, were easy to manipulate, and displayed appropriate mechanical properties. Interestingly, these materials exhibited a very good performance under fatigue. The use of the composite systems as a controlled release device was based on the possibility of incorporating active soluble molecules in the hydrogel within the pores. A release study of bovine serum albumin (BSA), intended as a model protein, was performed, which served as a proof of concept. The biological performance of the hybrid scaffolds was first evaluated with fibroblasts to discard the materials cytotoxicity and to optimize the cell seeding procedure. Subsequently, human umbilical vein endothelial cells (HUVECs) cultures were performed for their interest in angiogenic and vascularization processes. Finally, co-cultures of HUVECs with adipose-tissue derived mesenchymal cells (MSCs) were carried out. These last cells are believed to play an important role for clinical regenerative medicine, and their cross-talk with the endothelial cells enhances the viability and phenotypic development of HUVECs. Through the different experiments undertaken, hybrid scaffolds exceeded the outcome achieved by bare PEA scaffolds. / Arnal Pastor, MP. (2014). New scaffolding materials for the regeneration of infarcted myocardium [Tesis doctoral]. Editorial Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/46129 / Premios Extraordinarios de tesis doctorales

[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

Os organismos extremófilos são considerados atualmente reservatórios de novas biomoléculas de interesse biotecnológico. Dentro deste grupo encontram-se os microrganismos que requerem altas concentrações de sal para crescer, denominados halófilos. Também existem os halotolerantes que são aqueles microrganismos que não precisam de sal para proliferar, mas toleram altas concentrações de NaCl. Os ambientes salinos provaram ser uma fonte rica de microrganismos halotolerantes produtores de novos compostos naturais e, portanto, a pesquisa nestes ambientes torna-se de grande importância. No Brasil, na região salina de Areia Branca no bioma caatinga, foram isoladas bactérias halotolerantes que foram estudadas com o objetivo de avaliar a produção de novos biopolímeros de interesse biotecnológico. Acredita-se que os polímeros naturais desses microrganismos extremos podem ter aplicações inovadoras ou características diferentes às tradicionais. Neste estudo, os isolados foram identificados em nível de gênero com base na análise da sequência do gene 16s rRNA. Os isolados foram principalmente bactérias Gram-positivas atribuídas às famílias Bacillaceae, Staphylococcaceae, Microbacteriaceae e uma bactéria Incertae Sedis do filo firmicutes, afiliadas aos gêneros Bacillus, Staphylococcus, Curtobacterium e Exiguobacterium, respectivamente. Apenas um isolado Gram-negativo foi identificado e atribuído como membro da família Pseudomonadaceae, incluso no gênero Pseudomonas. Avaliou-se a tolerância ao sal dos isolados em meio TSB suplementado com 5, 35, 60 e 120 g/L de NaCl. Todos os isolados apresentaram a capacidade de crescer nas quatro concentrações de NaCl avaliadas, com exceção do isolado Exiguobacterium sp. sac36 que não cresceu na concentração de 120 g/L de NaCl no meio. Realizaram-se ensaios de acúmulo de polihidroxialcanoatos (PHA) e evidenciou-se que quatro isolados do gênero Bacillus são capazes de acumular 3-hidroxibutirato (3HB) a partir de glicose, xilose, e alguns destes em glicerol. Adicionalmente, confirmou-se que quando há altas concentrações de NaCl no meio, o acúmulo de 3HB dos isolados produtores diminui. Observou-se também que doze isolados halotolerantes são produtores de exopolissacarídeos (EPS). Testes realizados indicaram que os mesmos podem ter efeitos imunoestimulantes em macrófagos. Finalmente, avaliou-se a produção de ácido hialurônico (AH) pelos isolados halotolerantes. Segundo sugere o método de Alcian blue, todos os isolados foram capazes de produzir AH, mostrando que a maior parte deles acumulou o biopolímero em concentrações maiores ou semelhantes ao controle. Os resultados obtidos evidenciam que os isolados halotolerantes avaliados são uma fonte rica em compostos com atividades promissoras para as diferentes indústrias. O presente trabalho contribui no estudo do potencial biotecnológico de microrganismos isolados no bioma caatinga, destacando sua biodiversidade, versatilidade e a necessidade de continuar explorando esses ambientes extremos pouco estudados. / Extremophile organisms are considered reservoirs of new biomolecules of biotechnological interest. In this group there are microorganisms that require high salt concentration to grow, called halophiles, and halotolerant microorganisms, that do not need salt to proliferate but can tolerate high concentrations of NaCl. Saline environments proved to be a rich source of new natural compounds by halotolerant producers and therefore, research in these environments becomes of great importance. In Brazil, in the saline region of Areia Branca in the caatinga biome, halotolerant bacteria were isolated and studied in order to evaluate the production of new biopolymers of biotechnological interest. It is believed that the natural polymers of those extreme microorganisms could have innovative applications or different characteristics from the traditional biopolymers. In this study, the isolates were identified at the genus level based on 16S rRNA gene sequence analysis. Isolates were mainly Gram-positive bacteria from Bacillaceae, Staphylococcaceae and Microbacteriaceae families, and Bacillus, Exiguobacterium, Staphylococcus and Curtobacterium genera. One of the Gram-negative isolate was identified as member of the Pseudomonadaceae family, genus Pseudomonas. The evaluation of salt tolerance of the bacterial isolates on TSB medium supplemented with 5, 35, 60 and 120 g / L NaCl was performed. All the isolates showed the ability to grow in the four concentrations evaluated, except for Exiguobacterium sp. sac36, that did not grow at 120 g / L NaCl. Polyhydroxyalkanoate (PHA) accumulation assays were performed using glucose, xylose and glycerol as carbon source. The results showed that four strains of the genus Bacillus were able to accumulate 3-hydroxybutyrate (3HB) in all conditions. Additionally, it was confirmed that the presence of high concentrations of NaCl in the medium causes a decrease in 3HB accumulation in the cells. It was observed that twelve halotolerant bacteria produced exopolysaccharides (EPS). Tests performed indicated that those EPS could have immunostimulatory effects on macrophages. Finally, hyaluronic acid (HA) production was evaluated. According to Alcian blue method, all strains were able to produce HA, showing that most of the isolates accumulated the biopolymer in higher or similar concentrations to the control. The results showed that the halotolerant isolates are a rich source of compounds with promising activities for different industries. This study contributes to the knowledge of microorganisms from the caatinga biome and their biotechnological potential, highlighting their biodiversity, versatility and the need to continue exploring these poorly studied extreme environments.

O objetivo deste estudo foi avaliar se a injeção intra-articular em altas doses de ácido hialurônico, imediatamente após o trauma, pode reduzir a apoptose de condrócitos. Para cumprir este objetivo foi desenvolvido um estudo experimental com 40 joelhos de coelhos adultos. Os animais foram anestesiados e, em seguida, cada joelho sofreu três contusões com um bloco de 1 kg, solto por meio de um cilindro, a 1 metro de altura. Logo após as contusões, foram administrados, no mesmo coelho, 2 ml de ácido hialurônico em um joelho e 2 ml de solução salina no outro. Desta forma, obteve-se uma intervenção pareada, com melhora do poder estatístico do estudo. As doses foram repetidas a cada 3 ou 4 dias por 30 dias. Os coelhos foram mantidos no mesmo ambiente sob controle de temperatura, de atividades diárias e de alimentação. Após 30 dias, os animais foram abatidos e, por meio de artrotomia, foram realizadas as coletas da cartilagem do côndilo femoral medial e da tróclea de cada joelho. As peças foram preparadas para análise em microscopia óptica e coloração por TUNEL. Os indivíduos envolvidos no preparo e análise das peças não tiveram qualquer tipo de informação a respeito do experimento. A análise estatística foi feita pelo Teste t-student para dados pareados na comparação entre o grupo ácido hialurônico (AH) e o grupo controle. Foram analisados um total de 36 joelhos e obteve-se uma redução significativa (p<0,001) na taxa de apoptose de 68,01% (+ 19,73) do grupo controle para 53,52% (+ 18,09) do grupo AH. Diante dos resultados, concluiu-se que a injeção intra-articular de altas doses de ácido hialurônico, iniciando imediatamente após o trauma, reduz as taxas de apoptose (pós-traumática) de condrócitos de coelhos / The aim of this study was to assess whether intra-articular injection of high doses of hyaluronic acid immediately after trauma, can reduce apoptosis of chondrocytes. We have developed an experimental study with forty knees of adult rabbits. Animals were anesthetized and each one had had three knee injuries with a block of 1kg, released through a cylinder, 1 meter tall. After the bruises, 2ml of hyaluronic acid were injected in one knee and 2ml saline in the other. Doses were repeated each 3 or 4 days during 30 days. Rabbits were kept in the same environment under controlled temperature, daily activities and meals. Thirty days later, animals have been sacrificed. The cartilage of the medial femoral condyle and trochlea of each knee was retrieved with a scalpel by artrothomy. Specimens were prepared for optical microscopy and TUNEL staining. No information about the experiment was given to individuals who were involved in the preparation and analysis of the slides. Statistical analysis was performed by Students t test for paired data when comparing a group of hyaluronic acid (HA) and control group. We have analyzed a total of 36 knees and have obtained a significant reduction (p <0.001) in apoptosis rate of 68.01% (+ 19.73) for the control group 53.52% (+ 18.09) in the HA group. We conclude that the intraarticular injection of high doses of hyaluronic acid starting immediately after trauma, reduces impact-induced chondrocytes apoptosis rates in rabbits

A principal reação de derivatização da quitina é a hidrólise dos grupos acetamido, que gera o polímero conhecido como quitosana. O foco do presente estudo é desenvolver um processo eficiente, reprodutivo e versátil para produção de quitosanas com características controladas. Nesse sentido, o processo de desacetilação de quitina assistida por irradiação do ultrassom de alta intensidade, denominado processo DAIUS, foi estudado. Para o desenvolvimento do estudo proposto, as seguintes etapas foram realizadas: i) extração, fracionamento e caracterização de beta-quitina extraída de gládios de lulas; ii) estudo quimiométrico visando determinar as variáveis mais importantes do processo DAIUS; iii) estudo quimiométrico visando a otimização do processo DAIUS empregando gráficos de superfícies de resposta e iv) estudo cinético da desacetilação de beta-quitina via processo DAIUS. A caracterização das quitosanas, obtidas pelo processo DAIUS com o auxílio do planejamento fatorial de experimentos revelou que a intensidade da irradiação de ultrassom é a variável menos importante durante a desacetilação da beta-quitina, e que a temperatura e o tempo de reação são as variáveis que mais afetam a despolimerização da beta-quitina. Desse estudo resultaram quitosanas com elevados <span style=\"text-decoration: overline\">GD (92%) e <span style=\"text-decoration: overline\">Mv (5,42x105g/mol), enquanto o parâmetro de acetilação (PA) apresentou valores próximos de 1,0, que corresponde ao padrão randômico ideal de distribuição de unidades GlcN e GlcNAc, sugerindo que o processo DAIUS ocorre homogeneamente. A análise dos gráficos de superfícies de resposta permitiu observar que o aumento da temperatura e do tempo de sonicação gera quitosanas mais desacetiladas, porém com menores massas molares. Esta análise também permitiu avaliar os efeitos do processo DAIUS sobre <span style=\"text-decoration: overline\">GD, <span style=\"text-decoration: overline\">Mv e PA, sendo que nesse estudo quitosanas com elevada <span style=\"text-decoration: overline\">Mv (9,83x105g/mol) foram obtidas, porém o aumento da temperatura e do tempo de sonicação resultou em quitosanas mais despolimerizadas, e também mais desacetiladas. A seleção das principais variáveis do processo DAIUS, temperatura de reação e do tempo de sonicação, permitiu uma melhor compreensão da variação do <span style=\"text-decoration: overline\">GD e da <span style=\"text-decoration: overline\">Mv, e permitiu a obtenção de quitosanas que apresentaram valores de PA&asymp;1,0, correspondente ao padrão randômico ideal de distribuição de unidades GlcN e GlcNAc. O estudo da cinética da desacetilação da beta-quitina via processo DAIUS revelou a ocorrência de duas etapas bem distintas quantos às suas velocidades, sendo a primeira, atuante nos primeiros 20 minutos, mais rápida (k=29,4 min-1 103) quando comparada com a segunda etapa (k=7,6 min-1 103). As quitosanas geradas no desenvolvimento do estudo cinético do processo DAIUS foram analisadas por difração de raios X, revelando que durante o processo DAIUS ocorre perda de água do retículo cristalino da beta-quitina, fato atribuído à cavitação gerada pela irradiação de ultrassom de alta intensidade. Assim, é proposto que o fenômeno da cavitação, que resulta em importantes alterações morfológicas, reduzindo as dimensões médias das partículas e aumentando sua rugosidade e uniformidade, também atue no interior do retículo cristalino da beta-quitina, resultando na expulsão de moléculas de água e facilitando o acesso do hidróxido de sódio aos grupamentos acetamido da beta-quitina mesmo nos domínios cristalinos. A utilização do ultrassom de alta intensidade na desacetilação de beta-quitina coloca em destaque a obtenção de quitosanas com características controladas. / The main reaction of chitin is the hydrolysis of its acetamido groups, which generates a polymer known as chitosan. The focus of the present study is the development of an efficient, reproductive and versatile process for chitosan production with controlled characteristics. In this sense, the chitin deacetylation assisted by high intensity ultrasound irradiation, called USAD process, was studied. The development of the proposed study was carried out in four steps: i) the extraction, fractionation and characterization of beta-chitin, extracted from squid pens; ii) the chemometric approach, aiming to determine the most important variables of the USAD process; iii) the chemometric approach aiming to the USAD process optimization, employing response surface and iv) the deacetylation kinetics studies of beta-chitin via USAD process. The characterization of the chitosans obtained by the USAD process, supported by factorial design, showed that the intensity of the ultrasound irradiation is the least important variable in the beta-chitin deacetylation, and the temperature and reaction time are the variables that most affect the beta-chitin depolymerization. From this study, chitosans with high <span style=\"text-decoration: overline\">DD (92%) and <span style=\"text-decoration: overline\">Mv (5.42 x105g/mol) were produced, with acetylation parameter (AP) values close to 1.0, which corresponds to an ideal random pattern of distribution of GlcNAc and GlcN units, suggesting that the USAD process occurs homogeneously. The analysis of response surfaces allowed to observe that the increase of temperature and sonication time generates more deacetylated chitosans, but with lower average molecular weights. This analysis also allowed us to evaluate the effects of USAD process in <span style=\"text-decoration: overline\">DD, <span style=\"text-decoration: overline\">Mv, and AP variations: chitosans with high <span style=\"text-decoration: overline\">Mv (9.83x105g/mol) were obtained, but the increase of temperature and sonication time resulted in more degraded and more deacetylated chitosans. The selection of the main USAD process variables, temperature and sonication time, allowed a better understanding of <span style=\"text-decoration: overline\">DD and <span style=\"text-decoration: overline\">Mv variation, and allowed to obtain chitosan with PA&asymp;1.0, which corresponds to an ideal random pattern of distribution of GlcNAc and GlcN units. The study of beta-chitin deacetylation kinetics via USAD process revealed the occurrence of two stages: the first step, active in the first 20 minutes, is faster (k = 29.4 min-1 103) when compared with the second one (k = 7.6 min-1 103). The chitosans generated in the kinetic study of the USAD process were analyzed by X-ray diffraction, which revealed some water loss in the crystalline structure during the USAD process, which is attributed to the cavitation generated by irradiation of high intensity ultrasound. Thus, it is suggested that the phenomenon of cavitation, which results in significant morphological changes by reducing average particle size and increase uniformity and roughness, also act within the crystalline structure of beta-chitin, resulting in the expulsion of water molecules and facilitating the access of sodium hydroxide to beta-chitin acetamido groups even in the crystalline domains. The use of high intensity ultrasound in deacetylation of beta-chitin highlight the production of chitosans with controlled characteristics.

Introdução. O ácido hialurônico é um glicosaminoglicano não sulfatado presente na matriz extracelular. Vários estudos têm demonstrado que uma produção ou degradação aberrante dessa molécula tem influência no comportamento do câncer de mama, próstata, bexiga e pulmão. Desta forma, a dosagem do ácido hialurônico em tecidos e fluidos corporais como sangue, urina e escarro tem despertado grande interesse como rastreador de indivíduos de alto risco e marcador diagnóstico e/ou prognóstico da doença estabelecida. Objetivos. Verificar se há diferenças nos níveis de ácido hialurônico entre espécimes tumorais e não tumorais de câncer de pulmão, bem como seu impacto na sobrevida dos pacientes; verificar se diferenças encontradas nos tecidos estão também presentes no escarro; verificar se a dosagem do ácido hialurônico no escarro permite rastrear pacientes com câncer de pulmão entre pacientes com doença pulmonar obstrutiva crônica e voluntários saudáveis. Resultados. Houve uma elevação significativa nos níveis de ácido hialurônico nos espécimes tumorais em relação aos espécimes não tumorais, mesmo quando histologicamente categorizados. Não houve associação entre as concentrações do ácido hialurônico com características clínicas dos pacientes, porém houve impacto na sobrevida dos pacientes: pacientes com tumores contendo ácido hialurônico > 364,36 g/g apresentaram menor sobrevida global que pacientes cujos tumores evidenciaram ácido hialurônico < 364,36 ug/g. Demonstramos que pacientes com câncer de pulmão apresentam elevações altamente significativas da produção de ácido hialurônico no escarro independente das características clínicas dos pacientes, porém dependente do tipo histológico. Valores de ácido hialurônico >11,13ng/mg no escarro tem sensibilidade de 87% para rastreamento de pacientes com câncer de pulmão e voluntários saudáveis. Nesse grupo valores > 31,44ng/mg tem especificidade de 100% e sensibilidade de 51%. Houve exclusão com sensibilidade de 33% e especificidade de 100% de pacientes com doença pulmonar obstrutiva crônica em relação aos pacientes com câncer de pulmão para valores > 48,36ng/mg. Conclusão. Diferentes níveis de ácido hialurônico forma observados nos tumores e tecidos normais com impacto na sobrevida dos pacientes, tornando a dosagem do ácido hialurônico como promissor marcador prognóstico no câncer de pulmão. Diferenças observadas nos tecidos foram também constatadas no escarro, despontando a dosagem do ácido hialurônico como promissora no rastreamento de indivíduos com risco para câncer de pulmão / Introduction. Hyaluronan is an extracellular matrix non-sulfated glycosaminoglycan. Some have reported that its abnormal production and degradation can influence the behaviour of different types of tumours like breast, prostate, bladder and lung cancer. Therefore, hyaluronan quantitative analysis in tissues and body fluids like blood, urine and sputum has shown promise on the high risk patients screening and as diagnostic/prognostic marker of some diseases. Objectives. Verify if there are differences in the levels of hyaluronan in tumoral and non-tumoral lung cancer specimens, as well as its impact on the patients survival; verify if sputum samples present the same differences; verify the role of hyaluronan quantitative analysis in the screening of lung cancer patients between patients with chronic obstructive pulmonary disease and healthy volunteers. Results. Lung cancer tumoral specimens showed higher levels of hyaluronan when compared to non-tumoral specimens even when the specimens were histologically categorized. There was no correlation between hyaluronan levels and the patients clinical features, however, an impact in the patients survival was observed: patients with tumoral hyaluronan levels > 364,36 g/g had a lower survival rate than patients with < 364,36 ug/g. We have shown that lung cancer patients show an elevated production of hyaluronan in the sputum. This characteristic was independent of the clinical features but dependent of the histologic type. The hyaluronan quantitative analysis for the screening of lung cancer patients between healthy volunteers showed a sensitivity of 87% for hyaluronan levels >11,13ng/mg in the sputum. In this group levels > 31,44ng/mg showed 100% specificity and 51% sensibility. On a second group (lung cancer patients vs chronic obstructive pulmonary disease patients) levels of sputum hyaluronan > 48,36ng/mg showed 100% specificity to exclude chronic obstructive pulmonary disease patients from lung cancer patients. The sensibility for this cut-off point was 33%. Conclusion. Hyaluronan quantitative analysis in the tissues is a promising lung cancer prognostic marker considering the differences between hyaluronan levels on tumoral and nontumoral tissues. Not only this, but, the differences observed in the tissues were observed in the sputum as well. Hence, the hyaluronan quantitative analysis is a promising strategy in the screening of high risk individuals that might develop lung cancer

Defeitos de cartilagem e a mais comum doença articular, a osteoartrite, são caracterizadas pela destruição da cartilagem articular, e consequentemente na perda da função articular em humanos e animais. As estratégias atuais de tratamento, conservativas e cirúrgicas, são insuficientes: não resultam em restauração total da cartilagem hialina, e, portanto trazem um prognóstico reservado a longo prazo. O presente estudo tem por objetivo avaliar os efeitos do administração conjunta de concentrado de medula óssea, plasma rico em plaqueta sobre lesões condrais experimentalmente induzidas e tratadas com microfraturas e ácido hialuronico. Foram utilizadas as articulações metacarpofalangeana de 6 éguas, as quais foram divididas em 2 grupos aleatoriamente e cego. Foram feitos defeitos condrais totais através de artroscopia e, todos foram tratados com microfraturas e ácido hialurônico no transoperatório (M 0) sendo, esse repetido após 15 dias (M 15) e 30 dias (M 30). Grupo C (controle) e grupo T (tratado). O grupo T foi tratado com aspirado concentrado de células tronco de medula óssea adicionada ao plasma rico em plaquetas (PRP), os quais foram injetados na articulação no final da cirurgia (M0). O grupo T recebeu ainda 2 aplicações articulares adicionais de PRP em 15 dias (M 15) e 30 dias (M 30). As seguintes avaliações foram realizadas: exame clínico de claudicação, ultrassonografia, estudo radiográfico, avaliações de líquido sinovial (físico, bioquímico e citológico). As avaliações foram realizadas antes da cirurgia (M 0), com 3, 5 e 7 dias. Posteriormente a cada 15 dias (M 15, M 30, M 45 e M 60) e os 3 últimos momentos foram aos 90 (M90), 120 (M120) e 210 (M210) dias. Ao final do experimento os animais foram enviados para abate comercial. Foram verificadas diferenças estatísticas (p<0,05) entre o grupo tratado e controle para avaliação de proteínas no líquido sinovial corrigido por uréia em 3, 5 e 7 dias; para PGE2 no líquido sinovial em 3 e 5 dias onde para ambas as variáveis com maiores valores para o grupo tratado. Já a concentração de ácido hialurônico apresentou maiores valores (p<0,05) em 3, 45 e 90 dias no grupo controle. Para as demais variáveis não houve diferença estatística entre o grupo tratado e controle. Porém, notou-se medianas maiores para condroitin sulfato em 3 e 5 dias no grupo controle. Notavelmente, o grupo tratado apresentou melhor escore macroscópico na avaliação do tecido de reparo. Contudo, a administração intra-articular de concentrado de medula óssea e plasma rico em plaquetas sobre lesões condrais induzidas e tratadas com microfraturas e ácido hialurônico produziu uma reação articular transitória, principalmente nos primeiros 60 dias, e foi evidenciado pelo aumento de PGE2 e proteínas no líquido sinovial, bem como, claudicação, dor a flexão passiva, diminuição da mobilidade articular e aumento de volume articular. No entanto, o tratamento produziu um efeito condroprotetor e anabólico sobre tecido de reparo formado, uma vez que o grupo tratado apresentou menor concentração de ácido hialurônico 3, 45 e 90 dias e melhor escore macroscópico ICRS aos 210 dias / Cartilage defects and the most common joint disease, osteoarthritis, are characterized by destruction of articular cartilage, and consequently in loss of joint function in humans and animals. Current strategies of conservative and surgical treatment are insufficient: they don&rsquo;t result in complete restoration of hyaline cartilage, and bring a poor prognosis on the long term. This study aims to evaluate the effects of co-administration of bone marrow concentrate, platelet rich plasma on experimentally induced chondral lesions and treated with microfractures and hyaluronic acid. The metacarpophalangeal joints of 6 mares were used, which were divided into 2 groups at random and blind. Total chondral defects were made using arthroscopy, and all were treated with microfractures and hyaluronic acid during surgery (M 0) and the hyaluronic acid was repeated after 15 days (M 15) and 30 days (M 30). Group C (control) and T group (treated). Group T was treated with concentrated aspirated bone marrow stem cells added to the platelet rich plasma (PRP), which were injected into the joint at surgery (M0). The T group had another 2 additional joint PRP applications in 15 days (M 15) and 30 days (M 30). The following evaluations were performed: clinical examination of lameness, ultrasound, radiographic studies and synovial fluid analysis (physical, biochemical and cytological). The evaluations were performed before surgery (M 0), 3, 5 and 7 days. Then, every 15 days (M 15, M 30, M 45 and M 60) and the last 3 evaluation were at 90 (M90), 120 (M120) and 210 (M210) days. At the end of the experiment the animals were sent to commercial slaughter. Statistical differences were found (p <0.05) between the treated and control group for evaluation of protein in synovial fluid corrected by urea at 3, 5 and 7 days; for PGE2 in the synovial fluid in 3 to 5 days where both variables had higher values for the treated group. The hyaluronic acid concentration was higher (p <0.05) at 3, 45 and 90 days in the control group. For the other variables there were no statistical difference between the treated and control groups. However, greater medians were noticed for chondroitin sulfate in 3 to 5 days in the control group. Notably, the treated group showed better macroscopic score in the evaluation of the repair tissue. In conclusion, intra-articular administration of bone marrow concentrate and platelet-rich plasma on induced chondral lesions and treated with microfractures and hyaluronic acid produced a transient response joint, especially during the first 60 days, and it was evidenced by the increase in PGE2 and proteins of the synovial fluid, as well as lameness, pain passive flexion, decreased joint mobility and joint swelling. Besides that, the treatment produced an anabolic chondroprotective effect on repair tissue formed once the treated group showed lower concentration of hyaluronic acid 3, 45 and 90 days, and better ICRS macroscopic scoring at 210 days

Em virtude dos pobres resultados obtidos no tratamento do Câncer de Pulmão, seja em estágios iniciais ou na doença avançada localmente, há a necessidade de se desenvolver marcadores moleculares e imunohistoquímicos que possam prever o comportamento tumoral. Ácido Hialurônico (HA) é um componente da matriz extracelular, responsável pela hidratação e manutenção do equilíbrio osmótico tecidual. Concentrações de HA estão elevadas em vários tipos de cânceres, incluindo pulmão. Hialuronidases (HAases), são uma família de enzimas relacionadas com a propagação de infecções bacterianas, toxinas de venenos e progressão tumoral. A quebra do HA em pequenos fragmentos (3-25 dissacarídeos) promovidos pela ação das HAases tipo Hyal1, Hyal2 e Hyal3, está relacionada à promoção do câncer através da indução da angiogênese e estímulo a proliferação através de ativação da via tirosina quinase. Algumas isoformas de HAases, descritas como produto de splicing alternativo, possuem atividade enzimática diversificada. A heterogeneidade de expressão das HAases foi identificada em alguns tipos de câncer e pode ser correlacionada com o comportamento diferenciado dos tumores. Em uma primeira instância, o perfil de expressão das HYAL foi avaliado em tecidos pulmonares tumorais e normais de 69 tumores ressecados de pacientes com adenocarcinomas (ADC) e carcinomas de células escamosas (CCE) oriundos do Hospital das Clinicas e Hospital do Câncer AC. Camargo. A expressão da HYAL1- selvagem (wt) e variantes 1 a 5, HYAL2-wt, HYAL3-wt e variantes 1 a 3 foi identificada por PCR e seqüenciamento direto. Diferentes proporções de HYAL3-wt e variantes foram expressas em tecidos pulmonares tumorais e controles. HYAL1-wt esteve associada com prognóstico desfavorável e HYAL3-v1 com prognóstico favorável. Diante dos resultados obtidos dos tumores de pacientes do Hospital das Clínicas e Hospital AC. Camargo, prosseguimos a investigação para estudar a imunoexpressão das Hyal 1 e 3 e HAS 1, 2 e 3 nos CCE e ADC. Observamos que a intensidade de expressão de Hyal 3 foi maior pelas células tumorais quando comparada aos controles, porém esta diferença foi marginalmente significante. Já o resultado da análise da freqüência de imunoexpressão das Hyal 1 e 3, e HAS1, 2 e 3 demonstrou expressão na maioria dos espécimes tumorais e controles. A associação entre as variáveis foi testada e evidenciou imunoexpressão concomitante de HYAL e HAS nos tumores. O modelo matemático de sobrevida , controlado para sexo, idade e estadiamento mostrou risco de morte associado com adenocarcinoma sólido e imunoreatividade para HAS2 e HAS3. Para validar os resultados obtidos, sobretudo com a imunoexpressão das Hyal e HAS nos CCE e ADC, estudamos a população de pacientes do Hospital Universitário de Coimbra. Documentamos pela primeira vez uma via pela qual a hiperexpressão de HAS3 e Hyal 3 respectivamente por células epiteliais neoplásicas e mesenquimais, podem favorecer a invasão nos ADC e CCE. Surpreendentemente, demonstramos que a imunoexpressão de HAS1 e 3 pelas células epiteliais neoplásicas confere mais agressividade aos ADC acinares e papilares, mas uma expressão negativa de HAS1 pelas células mesenquimais confere um papel protetor a MEC auxiliando-a a evitar a invasão pelas células tumorais em ambos os tipos subtipos histológicos. A interação entre a expressão das hialuronidades e sintases do àcido hialurônico foi avaliada em relação à expressão de proteínas da transição epitélio-mesênquimal nos tumores de pacientes do Hospital Universitário de Coimbra. Hyal, HAS, E-caderina e TGF- modularam uma via invasiva tumorinduzida nos ADC e CCE de pulmão, e estiveram associados a um espectro diferente de agressividade, uma vez que houve uma relação inversa entre a expressão de biomarcadores epiteliais e mesenquimais. Enquanto a hiperexpressão de HAS1 e HAS3 provê uma agressividade aos CCE e ADC, uma hiperexpressão de TGF- e E-caderina, confere um efeito protetor à MEC ao evitar a invasão por células tumorais em ambos os tipos histológicos. Comparamos os níveis de imunoexpressão das Hyal1 e 3 e HAS 1, 2 e 3 nos tumores ressecados no Hospital das Clínicas e Hospital AC. Camargo com os níveis obtidos em tumores do Hospital Universitário de Coimbra. Verificamos que a imunoexpressão das HAS 1, 2, 3 e Hyal1 foi significativamente maior nos tumores de pacientes do Hospital Universitário de Coimbra, enquanto que a imunoexpressão de Hyal 3 foi significativamente maior nos tumores de pacientes brasileiros. Por todas essas razões, nossos resultados sugerem que estratégias direcionadas à modulação dos níveis de HYAL1-wt e HYAL3-v1, da hiper imuno expressão de HAS3 e Hyal 3 respectivamente por células epiteliais neoplásicas e mesenquimais, da alta síntese de HAS3 e Hyal 1, ou a resposta local baixa de TGF- e E-caderina, poderão ter grande impacto no câncer de pulmão / Given the poor results obtained in the treatment of Lung Cancer, in early stages or locally advanced disease, there is a need to develop molecular markers and immunohistochemical studies that can predict tumor behavior. Hyaluronic Acid (HA) is a component of extracellular matrix is responsible for hydration and maintenance of tissue osmotic equilibrium. Concentrations of HA are elevated in several types of cancers, including lung. Hyaluronidases (HAases) are a family of enzymes involved in the spread of bacterial toxins, poisons and tumor progression. The breakdown of HA into small fragments (3-25 disaccharides) promoted by the action of type HAases Hyal1, Hyal 2 and Hyal 3 is related to the promotion of cancer by inducing angiogenesis and stimulate proliferation through activation of the tyrosine kinase. Some isoforms HAases, described as the product of alternative splicing, have diverse enzymatic activity. The heterogeneity of expression of HAases was identified in some cancers and can be correlated with the different behavior of tumors. In a first instance, the expression profile of Hyal spliced forms was evaluated in tumor and normal lung tissue of 69 tumors resected from patients with adenocarcinomas(ADC) and squamous cell carcinomas (SqCC) from the Hospital das Clínicas and Hospital AC. Camargo. Gene expression of HYAL1 wild-type (wt) and variants 1 to 5 HYAL2-wt, and HYAL3-wt and variants 1 to 3 was identified by PCR and direct sequencing. Different proportions of HYAL3-wt and variants were expressed in tumor and normal lung tissue. HYAL1-wt was associated with unfavorable prognosis and HYAL3-v1 with favorable prognosis. Given the genetic abnormalities found in tumors of patients from Hospital das Clinicas and Hospital AC. Camargo, we continued our research to study the expression of Hyal 1.3 and HAS 1, 2, 3 in squamous cell carcinomas and adenocarcinomas. We observed that the intensity of expression of Hyal 3 was higher in tumor cells compared to controls, but this difference was marginally significant. Since the result of frequency analysis of immunoreactivity of Hyal 1 and 3, and HAS1, 2 e 3 showed expression in the majority of tumor samples and controls. The association between variables was tested and showed concomitant immunoexpression of the HAS and HYAL in tumors. The mathematical model of survival, adjusted for sex, age and staging showed risk of death associated with adenocarcinoma and solid and HAS3 HAS2 immunoreactivity.To validate the results, especially with the immunostaining of Hyal and HAS in squamous cell carcinomas and adenocarcinomas of the lung, the patient population studied at the University Hospital of Coimbra. Documented for the first time a route by which the overexpression of HAS3 and Hyal 3 respectively by neoplastic epithelial and mesenchymal cells may favor the invasion in ADC and SqCC, respectively. Surprisingly, we demonstrated that hyper HAS1 and 3 immunoreactivity by neoplastic epithelial cells confers more aggressiveness to the ADC acinar and papillary, but a negative expression of HAS1 by mesenchymal cells confers a protective role ECM-helping to prevent the invasion by tumor cells in both types histological subtypes.The interaction between the expression of hialuronidades and hyaluronic acid synthases was evaluated for protein expression of epithelial-mesenchymal transition in tumor patients at the University Hospital of Coimbra. Hyaluronidase, hyaluronan synthase, Ecadherin, and TGF- modulated via an invasive tumor-induced in the ADC and SqCC lung, and were associated with a different spectrum of aggressiveness, since there was an inverse relationship between the expression of epithelial biomarkers and mesenchymal cells. While overexpression of HAS1 and HAS3 provides an aggressiveness to SqCC and ADC, an overexpression of TGF- and E-cadherin confers a protective effect by preventing the ECM invasion by tumor cells in both histological types. We compared the levels of immunostaining Hyal 1, 3 and HAS1, 2 and 3 in tumors resected at the Hospital AC. Camargo, and the levels obtained in tumors of the Hospital Universitário de Coimbra. We found that the immunostaining of HAS 1, 2, 3 and Hyal1 was significantly higher in tumors from patients of Coimbra, while Hyal 3 immunoreactivity was significantly. higher in tumors of patients in Brazil. For all these reasons, our results suggest that strategies directed at modulating the levels of HYAL1-wt and HYAL3-v1, the immunohistochemical expression of HAS3 and Hyal 3 respectively by neoplastic epithelial and mesenchymal cells, the synthesis of HAS3 and Hyal1 or the local response of low TGF- and E-cadherin, may have great impact on lung cancer