RésuméLes lésions du cerveau sont un problème médical majeur, celui-ci possédant des ressources limitées pour la guérison. Les patients souffrent souvent des déficiences graves et durables, dégradant leur qualité de vie et imposant des couts importants. Des thérapies qui visent l'implantation des cellules souches neurales supportées par un biomatériau qui imite la matrice extracellulaire du cerveau sont en développement. L’ECM du cerveau a une teneur élevée en acide hyaluronique (HA). Ce glycosaminoglycane possède la biocompatibilité et l'activité biologique requises par les applications avec des cellules souches neurales.Nous avons développé des hydrogels à base de HA, possédant des propriétés mécaniques et des densités en peptide d’adhésion cellulaire (GRGDS) contrôlées, pour l'étude in vitro de la différenciation de cellules souches neurales en neurones. L'analyse de neurites en 3-D par microscopie biphotonique a montré une excroissance accrue et une densité élevée des neurites dans les hydrogels les plus élastiques (G '= 400 Pa), combinées avec l'existence d'un optimum dans l'extension des neurites en fonction de la densité des ligands dans le cas des hydrogels contenant des GRGDS. La croissance des neurites relève vraisemblablement d’une combinaison d’interactions adhésives cellule-HA, cellule-GRGDS, et cellule-molécules extracellulaires secrétées.Par la suite la dégradabilité enzymatique des hydrogels de HA a été étudiée. Les hydrogels de HA se dégradent sous l'effet de l'enzyme hyaluronidase suivant un modèle mono-exponentiel, ce qui correspond à une population homogène de chaînes de HA clivables. Les hydrogels avec des modules d'élasticité plus élevés, montrent des vitesses de dégradation enzymatique plus faibles. Le remplacement de l'agent de réticulation PEG-bis(thiol) pour un polymère HA-(SH)3 clivable par voie enzymatique conduit à une réduction du temps nécessaire à la dégradation complète des hydrogels.Dans un troisième temps, nous avons développé des gels de héparosane sans activité biologique qui pourraient révéler une meilleure compréhension du rôle joué par le HA dans la différentiation des NSCs et dans l’extension des neurites. Nous avons montré que le CD44 joue un rôle mesurable dans le processus d'adhésion des cellules MEF. Il existe d'autres procédés par lesquels ces cellules peuvent adhérer sur les hydrogels d’héparosane, cependant la force de ces interactions est plus faible. / AbstractDamage caused to the central nervous system (CNS) is a major medical concern. As the CNS has limited ability to regenerate its damaged cells, patients can suffer from serious and long-term disabilities and impairments, which put strains on public healthcare systems. Therapies that aim to implant neural stem cells together scaffolds that mimic the extracellular matrix of the brain are being developed. Hyaluronic acid is an important component of the brain ECM. This glycosaminoglycan possesses the required biocompatibility and bioactivity for use in neural stem cells applications.We have developed HA-based hydrogels with controlled mechanical properties and cell adhesion peptide (GRGDS) densities for the in vitro study of neural precursor cells’ differentiation into neurons. The analysis of neurite outgrowth in 3-D by two-photon microscopy showed an increased outgrowth and density of neurites in the softest hydrogels (G’ = 400 Pa), combined with the existence of an optimum in neurite outgrowth as a function of ligand density in the case of hydrogels containing GRGDS. Neurite outgrowth in these hydrogels most likely involves a combination of adhesive interactions between cell-HA, cell-GRGDS moieties, and cell-secreted extracellular molecules.The enzymatic degradability of HA hydrogels was then investigated. The HA hydrogels degrade under the effect of the Hyaluronidase enzyme following a mono-exponential model, corresponding to a homogenous population of cleavable HA polymer chains. Hydrogels with higher elastic moduli have progressively lower enzymatic degradation rates. The substitution of the PEG-bis(thiol) crosslinker by an enzymatically cleavable HA-(SH)3 polymer led to a reduction in the time required for the complete degradation of the hydrogels.Finally we developed heparosan hydrogels that are devoid of biological functions and thus provide better insight into the role of HA in NSCs differentiation and neurite outgrowth. We showed that CD44 plays a measurable role in the adhesion process of MEF cells. There are alternative processes through which cells can attach to the heparosan hydrogels however the strength of these adhesions is weaker. Heparosan is a viable biomaterial for hydrogel synthesis that does not interact with the CD44 receptor, resulting in lower cellular adhesions.

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

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

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

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

Au laboratoire, une méthode originale d'encapsulation par transacylation entre l'alginate de propylène-glycol (PGA) et une protéine a été mise au point. Cette méthode est basée sur la création de liaisons amides entre les fonctions amines libres de la protéine et les groupes esters du PGA dans une phase aqueuse émulsionnée (E/H) après alcalinisation. Les microparticules obtenues, stables, biocompatibles et biodégradables, sont potentiellement intéressantes pour la délivrance de substances actives en thérapeutique ou en cosmétique.Le premier objectif de ce travail est d'étudier l'influence des propriétés physicochimiques des deux biopolymères (protéine et PGA) et de leurs solutions, ainsi que l'effet des paramètres de préparation sur la réaction de transacylation et sur les propriétés des microparticules obtenues. Pour cela, la sérumalbumine humaine (HSA) a servi de protéine modèle et les microparticules ont été préparées dans différentes conditions physicochimiques puis caractérisées. Différents liens ont été établis entre les propriétés physicochimiques des solutions initiales des deux polymères et les propriétés fonctionnelles des microparticules obtenues.Le deuxième objectif est de remplacer le PGA, seul polysaccharide utilisable jusqu'à présent pour la microencapsulation par transacylation, par d'autres polysaccharides naturels, dans la préparation de microparticules. Etant donné ses propriétés intrinsèques limitantes, le remplacement du PGA par d'autres esters polysaccharidiques parait avantageux dans le domaine d'application des microparticules.Dans ce travail, le PGA a été remplacé par une série d'esters semi-synthétiques d'alginate puis par d'autres polysaccharides estérifiés naturels (pectines) ou semi-synthétiques (esters polypectiques et esters de l'acide hyaluronique). Les conditions optimales pour l'utilisation de chaque ester ont été alors déterminées. / In our laboratory, an original method of microencapsulation was developed, based on the use of a transacylation reaction, creating covalent bonds between proteins and propylene glycol alginate (PGA). The covalent bonds are created after alkalization of the aqueous phase of a W/O emulsion, without using bifunctional crosslinking reagent.The resulting microparticles, which are stable, biocompatible and biodegradable, have potential applications for the delivery of active compounds for therapeutics or cosmetics.The first aim of this work is to study the influence of the physicochemical properties of the two polymers (protein and PGA) and of their solutions, as well as the effect of the preparation parameters on the transacylation reaction and on microparticle characteristics. For this purpose, human serum albumin (HSA) was picked as a model protein and microparticles were prepared using several physicochemical conditions then characterized. Several relationships were established between the physicochemical properties of the initial solutions of the two polymers and the functional properties of the resulting microparticles.The second purpose is to replace the PGA, only polysaccharide used for microencapsulation by transacylation so far, by other natural polysaccharides in the preparation of microparticles. Given its limiting intrinsic properties, the replacement of PGA by other polysaccharidic esters seems advantageous in the field of microparticle applications.In this work, the PGA was successfully replaced by a series of semisynthetic alginate esters, and then by other polysaccharidic esters, either natural esters (pectin) or semisynthetic esters (polypectate esters and hyaluronate esters). The optimal conditions for the use of each ester were then determined.

La métastase et la chimiorésistance sont les principales causes de mortalité chez les patients atteints d’un cancer. La compréhension des mécanismes moléculaires régissant ces deux processus devient donc un domaine de recherche important pour la conception de nouvelles stratégies thérapeutiques. Dans ma thèse, je me concentre sur la compréhension du rôle du facteur d’initiation de la traduction chez les eucaryotes 4E (eIF4E) dans l’invasion du cancer, et je décris un nouveau mécanisme de résistance que nous avons découvert en étudiant le développement de la résistance à un inhibiteur connu d’eIF4E, la ribavirine. eIF4E est un puissant oncogène qui est connu pour être élevé dans une multitude de cancers comprenant entre autres les sous-types M4 / M5 de la leucémie myéloïde aiguë (AML). Il fonctionne dans la traduction et l'exportation nucléocytoplasmique d'ARNm en se liant à la coiffe m7G des ARNm possédant des codes USER spécifiques dans leur région UTR 5' et/ou 3'. En reconnaissant ces codes USER, le complexe dans lequel se trouve eIF4E régule de manière coordonnée l'expression de gènes essentiels à la croissance, à la prolifération et à la survie, et ainsi, eIF4E a été placée en tant que nœud central d'un régulon d'ARN régissant la prolifération. En analysant les voies dans lesquelles l’export est régulé de façon coordonnée par eIF4E et les effets physiologiques qui en découlent, j'ai trouvé un enrichissement de la voie biosynthétique de l'acide hyaluronique (HA) et de son principal récepteur CD44 qui sont des médiateurs clés connus des métastases cancéreuses. J’ai également démontré que l'élévation d’eIF4E modifie la surface des cellules cancéreuses en les recouvrant de protrusions riches en HA de type microvillus et enrichies d'armes de destruction métastatique. Heureusement, en dégradant le manteau HA ou en utilisant des inhibiteurs de CD44 en combinaison avec la ribavirine, nous pouvons alors nous défendre. Compte tenu de l'avantage prolifératif que confère la surexpression d’eIF4E, il est devenu un talon d'Achille attrayant pour le traitement de cancers ayant un niveau élevé d'eIF4E. En effet, lors d'un essai clinique de phase II parmi des patients atteints de leucémie myéloïde aiguë M4 / M5 réfractaire et récidivante, la ribavirine a conduit au ciblage d'eIF4E et a donné lieu à des réponses cliniques significatives, incluant des réponses complètes ou partielles. Cependant, tel qu’attendu lors d’un traitement monothérapique, les patients ayant répondu finissent par développer une résistance au médicament. Mon analyse a révélé que cette résistance est due à un mécanisme nouveau caractérisé par l'élévation du facteur de transcription Sonic Hedgehog GLI1 qui conduit à la glucuronidation du médicament et donc à la perte de l'interaction entre la drogue et sa cible. Heureusement, ce mécanisme peut être inversé en utilisant des inhibiteurs de la voie Hedgehog. En conclusion, ces découvertes fournissent de nouvelles cibles thérapeutiques pour le traitement des cellules cancéreuses agressives et résistantes. / Metastasis and chemoresistance are the leading cause of mortality among cancer patients. The discovery of molecular mechanisms governing these two processes is becoming an important area of research for the design of novel therapeutic strategies. In my thesis, I focus on understanding the role of the eukaryotic translation initiation factor 4E (eIF4E) in cancer invasion and describe a novel mechanism of resistance that we discovered while studying the development of resistance to a known eIF4E inhibitor, ribavirin. eIF4E is a potent oncogene that is known to be elevated in a multitude of cancers including M4/M5 subtypes of acute myeloid leukemia (AML). It functions in mRNA translation and nucleocytoplasmic export by binding to the m7G cap of mRNAs possessing specific USER codes in their 5’ and/or 3’ UTRs. By recognizing these USER codes, eIF4E complex coordinately regulates the expression of genes essential for growth, proliferation and survival and as such has been placed as a central node of an RNA regulon governing proliferation. When analyzing which pathways have their export coordinately regulated by eIF4E and what physiological effects arise from it, I found an enrichment in the hyaluronic acid (HA) biosynthetic pathway as well as its major receptor CD44 which are known key mediators of cancer metastasis. I demonstrate that eIF4E elevation changes the surface of cancer cells sugar-coating them with HA-rich microvillus-like protrusions that are enriched with weapons of metastatic destruction. Luckily, through degrading the HA-coat or using inhibitors of CD44 in combination with ribavirin we can strike back. Given the proliferative advantage that eIF4E overexpression conveys, this rendered it as an attractive Achilles heel for the treatment of cancers where eIF4E levels are high. Indeed, in a phase II clinical trial in refractory and relapsed poor prognosis M4/M5 AML patients, ribavirin led to eIF4E targeting and resulted in significant clinical responses including complete and partial remissions. However, as it is expected for monotherapy treatment, all responding patients eventually developed resistance to the drug. My analysis revealed that resistance is due to a novel mechanism characterized by elevation of the Sonic Hedgehog transcription factor GLI1 which leads to drug glucuronidation and the subsequent loss of drug-to-target interaction. Fortunately, this mechanism can be reversed using Hedgehog pathway inhibitors. Taken together, these findings provide novel therapeutic venues for the treatment of aggressive and resistant cancer cells.

Les traitements des pathologies de l’oreille interne par voie locale se développent en alternative aux traitements par voie générale peu efficaces et responsables de nombreux effets secondaires. Dans ce travail, nous avons développé une formulation originale constituée de liposomes dispersés au sein d’un gel d’acide hyaluronique pour la libération prolongée d’un corticoïde dans l’oreille interne après administration locale dans l’oreille moyenne. D'abord, une étude physicochimique approfondie a permis d'identifier les paramètres clés de formulation qui ont un impact sur les propriétés du gel de liposomes en termes de caractéristiques rhéologiques, de seringabilité, de stabilité, de microstructure et enfin de diffusion des liposomes dans le gel. Ensuite, le gel de liposomes contenant la dexamethsone phosphate a été évalué in vivo chez le cobaye. L'administration locale du gel de liposomes a abouti à une libération prolongée du corticoïde dans l'oreille interne, sans aucun effet négatif sur la fonction auditive. L’évaluation de l’efficacité thérapeutique de la formulation pour le traitement de la surdité secondaire à un traumatisme sonore et pour la préservation de l’audition durant l’implantation cochléaire a montré des résultats préliminaires très prometteurs. / Local, rather than systemic drug delivery is being developed to treat inner ear diseases. In this work, we developed an original drug delivery system based on liposomes dispersed within a hyaluronic acid gel for the sustained delivery of a corticoid to the inner ear after local administration in the middle ear. First, a thorough physicochemical study allowed to identify the key formulation parameters that impact the liposomal gel properties in terms of rheological behavior, syringeability, stability, microstructure and diffusion of liposomes within the gel. Then, the liposomal gel containing dexamethsone phosphate was evaluated in vivo in guinea pig. The local administration of the liposomal gel in the middle ear resulted in a sustained release of the corticoid in the inner ear without any negative effect on the hearing function. Promising preliminary data were obtained regarding the therapeutic efficacy of the formulation for hearing recovery after acoustic trauma and for hearing preservation during cochlear implantation.

Ziel dieser Studie war es, Auswirkungen von subgingival applizierter Hyaluronsäure (Gengigel®) nach scaling and root planing (SRP) auf mikrobiologische Variablen bei Parodontitispatienten nachzuweisen. Dabei wurden die möglichen Zusammenhänge zwischen Ergebnissen der Bakterienspezies und zuvor ermittelten Ergebnissen klinischer Variablen untersucht. An der Untersuchung nahmen 20 Männer und 29 Frauen teil. Es erfolgte eine Randomisierung in zwei Gruppen, bestehend aus einer Testgruppe mit 23 und einer Kontrollgruppe mit 26 Probanden. Bei den Versuchteilnehmern wurde in zwei Sitzungen in einem 24-stündigen Abstand ein SRP mittels Hand- und Ultraschallinstrumenten durchgeführt. Am Ende des SRP wurde in der Testgruppe Gengigel prof® (mit 0,8% Hyaluronsäure) in die parodontalen Taschen eingebracht. Zusätzlich trugen die Probanden der Testgruppe während der folgenden 14 Tage zweimal täglich morgens und abends Gengigel® (0,2%) auf den Gingivarand auf. In der Kontrollgruppe erfolgte das übliche SRP ohne Verwendung von Gengigel®. Alle Probanden wurden zu Beginn der Untersuchung, nach drei und sechs Monaten aus parodontologischer Sicht untersucht. Des Weiteren wurden Proben der Sulkusflüssigkeit für eine spätere Analyse von zehn parodontopathogenen Keimen sowie der Peroxidase- und Granulozytenaktivität gewonnen. Im Gegensatz zur kontinuierlichen Zunahme in der Kontrollgruppe (p=0,035) konnte beim Verlauf der Gesamtbakterienzahl für die Testgruppe keine Veränderung der Keimzahlen (p=0,737) beobachtet werden. In der Testgruppe wurde nach sechs Monaten für Campylobacter rectus, Treponema denticola und Aggregatibacter actinomycetemcomitans eine Reduktion der Keimbelastung festgestellt (p=0,05; p=0,043; p=0,066). Am Ende der Untersuchung waren in der Testgruppe keine Unterschiede in der bakteriellen Besiedlung unterschiedlich tiefer Taschen mehr nachweisbar (p=1). In der Testgruppe bestand eine stark signifikante Korrelation der Granulozytenaktivität mit der Zeit (r=0,443; p<0,0001) und mit der Gesamtbakterienzahl (r=0,336; p=0,009). Die Ergebnisse der Studie weisen auf einen wachstumshemmenden Effekt der Hyaluronsäure auf parodontopathogene Bakterien hin, welcher womöglich auf einer indirekten Interaktion mit dem Immunsystem basiert.