A complacência da bexiga depende de músculos lisos, fibras colágenas, fibras elásiticas e suas relações. O objetivo deste trabalho é determinar a composição da matriz extracelular em amostras de bexigas normais através de análise bioquímica de colágeno e glicosaminoglicanos em amostras obtidas de mulheres em diferentes grupos de idade, analisando separadamente as camadas urotelial e muscular. Avaliamos 17 amostras de bexiga divididas em três grupos: infância (N=5), menacme (N=6) e pós-menopausa (N=6). As bexigas foram analisadas para concentração de GAG total e colágeno e para análise qualitativa de GAG por eletroforese em gel de agarose. Na camada muscular, não houve diferença entre os grupos tanto para GAG quanto para colágeno. Na camada urotelial, a análise da concentração de colágeno não mostrou diferença entre os grupos, mas a concentração de GAG no grupo da pós-menopausa (0.21 0.12 μg de ácido hexurônico/mg de tecido seco) apresentou diferença em relação aos grupos do menacme (1.78 1.62 μg de ácido hexurônico/mg de tecido seco) e da infância ( 2.29 1.32 μg de ácido hexurônico/mg de tecido seco).Nosso trabalho concluiu que a concentração de GAG está substancialmente diminuída na camada urotelial da bexiga de mulheres na pós-menopausa. / Bladder compliance is dependent on smooth muscle, collagen fibers, elastic fiber and their ratios. The luminal surface of the urothelium is covered by an adhering glycosaminoglycan (GAG) layer. The aim of this study was to determine the composition of the extracellular matrix (ECM) in normal samples of women bladders through biochemistry analysis of collagen and GAG on samples obtained from individuals from different age groups, analyzing separately the urothelial and muscular layers. We studied samples taken from bladders of 17 patients divided in three different groups: childhood (N=5), menacme (N=6) and menopause (N=6). Bladders were analyzed for total GAG and collagen concentration per mg dry tissue and for the contents of GAG species, as determined by agarose electrophoresis and reported as the percent of total sulfated GAG. In muscular layer, collagen and GAG concentration showed no difference between groups. In urothelial layer, collagen concentration showed no difference between groups but GAG concentration in menopause ( 0.21 0.12 μg hexuronic acid /mg dry tissue) was different from menacme (1.78 1.62 μg hexuronic acid /mg dry tissue) and childhood ( 2.29 1.32 μg hexuronic acid /mg dry tissue). There was no difference between sulfated GAG in three groups.In conclusion, GAG concentration in urothelial layer was substantially lower in menopause women.

O prolapso uterino tem sua incidência aumentada na pós-menopausa. O objetivo deste estudo é identificar as alterações na matriz extracelular do ligamento cardinal associadas à menopausa e ao prolapso uterino. Ligamento cardinal de três diferentes grupos de mulheres, pré-menopausa, prolapso uterino e pós-menopausa, foram identificados e biopsiados durante 57 histerectomias abdominais ou vaginais. As amostras foram processadas por métodos bioquímicos para caracterização e quantificação de glicosaminoglicanos sulfatados e colágeno. As concentrações relativas de glicosaminoglicanos foram obtidas por eletroforese. Procedimentos histológicos foram feitos para identificar fibras elásticas (Weigert), distribuição de colágeno (Picro Sirius) e decorin (imunohistoquímica). Nossos resultados mostraram aumento na concentração de GAG de 72,2%, redução na concentração de colágeno de 37% e diminuição de 22% de fibras elásticas no grupo de prolapso uterino quando comparado ao grupo da pós-menopausa (p<0,05, p<0,04 e p<0,05 respectivamente). As concentrações relativas de glicosaminoglicanos sulfatados para condroitin sulfato, heparan sulfato e dermatan sulfato não mostraram diferenças entre os três grupos. A organização do colágeno foi similar entre os três grupos e a marcação do decorin pareceu estar diminuída no grupo de prolapso uterino. Nossos resultados indicam alterações no metabolismo do tecido conjuntivo. O ligamento cardinal da mulher na pós-menopausa possui uma matriz extracelular mais densa. Esta alteração não ocorre na mulher com prolapso uterino. / Uterine prolapse has increase of incidence after menopause. The aim of this study was to identify the changes in extracellular matrix of cardinal ligaments associated to menopause and uterine prolapse. Cardinal ligament of 3 different groups (pre-menopause, menopause and uterine prolapse) are identified and biopsied during 57 womens abdominal or vaginal hysterectomy. Biopsy specimens were assessed by biochemical methods to characterize and quantify sulfated glycosaminoglycans and collagen. Relative concentrations of GAG were obtained by electrophoresis. Histological procedures are made to identify elastic fibers (Weigert) collagen distribution (Picro sirius) and decorin (immunohistochemistry). Our results showed increase in GAG concentration 72.2% in uterine prolapse group compared to menopause group (p<0.05). Collagen concentration was 37% lower in uterine prolapse group compared to menopause group (p<0.04). Relative concentration of GAG: heparan sulfate, chondroitin sulfate and dermatan sulfate showed no differences among three groups. Elastic fibers showed a significant reduction of approximately 22% uterine prolapse group compared to menopause group (p<0.05). Collagen organization was similar in three groups and the staining pattern of decorin seemed to be decreased in uterine prolapse group. Our results indicate changes in connective tissue metabolism. Cardinal ligament in postmenopausal women has a denser extracellular matrix. This change is not observed in women with uterine prolapse.

Introdução: As mucopolissacaridoses (MPS) são um conjunto de sete doenças genéticas incluídas dentro das Doenças Lisossômicas que por sua vez fazem parte dos Erros Inatos do Metabolismo (EIM). São doenças multissistêmicas que afetam todo o organismo, com variações conforme o tipo de MPS, sendo que algumas delas possuem tratamento específico. Quase todas comprometem, em graus variados, o sistema osteoarticular, e praticamente todos os pacientes apresentam excreção alterada de glicosaminoglicanos (GAGs) na urina. As MPS são doenças raras que podem ser subdiagnosticadas em função do pouco conhecimento dos profissionais de saúde sobre elas, do pouco acesso aos métodos de triagem e diagnóstico e da sua ampla heterogeneidade clínica, podendo ocorrer formas atenuadas nas quais pode ser difícil de levantar a suspeita clínica de MPS. Material e métodos: o presente estudo foi realizado no período de março de 2012 à janeiro de 2014, tendo incluído 55 pacientes atendidos em serviços de Reumatologia e/ou Ortopedia de Porto Alegre, RS, Brasil e que, apresentavam como principal queixa, manifestações articulares sem etiologia definida. Esses pacientes foram inicialmente investigados através da avaliação quantitativa e qualitativa dos GAGs urinários. Resultados e Discussão: entre os 55 casos investigados, em 1 paciente de 15 anos de idade foi observada na análise dos GAGs urinários excreção aumentada e alteração do padrão qualitativo, sendo posteriormente confirmado o diagnóstico de uma forma atenuada de MPS II, a qual não havia sido suspeitada anteriormente. Conclusão: embora a proporção de pacientes identificados com MPS na amostra estudada tenha sido pequena (1/55), este estudo mostra que ocorre subdiagnóstico dessas doenças e que a triagem sistemática pode contribuir para a identificação de pacientes, os quais podem se beneficiar das medidas de tratamento disponíveis. / Introduction: Mucopolysaccharidoses (MPS) are a set of 7genetic diseases including Lysosomal Diseases , which in turn are part of Inborn Errors of Metabolism (IEM). The MPS are multisystemic conditions that affect the entire body, with variations depending on the type, some of which have specific treatment. Almost all affect, in variable degrees, the osteo-articular system, and virtually all patients have abnormal excretion of glycosaminoglycans (GAGs) in urine. The MPS are rare diseases that are being underdiagnosed due to the little knowledge of health professionals about them, the poor access to screening and diagnostic methods and their extensive clinical heterogeneity. Also, attenuated forms may occur in which it may be difficult to raise the clinical suspicion of MPS. Material and Methods: The present study was conducted from March 2012 to January 2014 and included 55 patients from Rheumatology and/or Orthopedics services of Porto Alegre, RS, Brazil and which had, as main complaint, articular manifestations without defined etiology. These patients were screened by quantitative and qualitative assessment of urinary GAGs. Results and Discussion: Among the 55 cases investigated, in 1 patient 15 years of age was observed in the analysis of urinary GAG excretion and increased change in qualitative standard and subsequently confirmed the diagnosis of an attenuated form of MPS II, which had not previously been suspected. Conclusion: Although the proportion of patients with MPS identified in the study sample was small (1/55), this study shows that occurs underdiagnosis of these diseases and that systematic screening can help to identify patients who may benefit from measures treatment available.

INTRODUÇÃO: As mucopolissacaridoses (MPS) são doenças de depósito lisossômico, caracterizadas pela deficiência de enzimas lisossômicas envolvidas na degradação dos glicosaminoglicanos (GAGs). O acúmulo anormal dessas macromoléculas no interior dos lisossomos provoca alterações estruturais e funcionais, de caráter multissistêmico e progressivo. Os GAGs acumulados também são excretados na urina, onde podem ser identificados através de diversos métodos bioquímicos. Estas doenças estão presentes em todos os grupos étnicos e a incidência conjunta das MPS, é estimada entre 1:10.000 a 1:25.000 nascidos vivos. (Baehner, 2005). A causa das MPS é a deficiência de uma enzima específica na rota de degradação dos GAGs. As MPS são classificadas segundo o tipo de substrato (GAGs) acumulado e a enzima específica deficiente. Na síndrome de Morquio A, ou mucopolissacaridose tipo IVA (MPS IVA), o substrato acumulado é o queratan sulfato e a enzima deficiente é a N-acetilgalactosamina-6-sulfatase. (GALNS). Os pacientes afetados por MPS IVA apresentam baixa estatura, disostose múltipla, opacidade de córnea, entre outros sinais e sintomas. O desenvolvimento psicomotor e mental é normal. O método de detecção inicial das MPS baseia-se na identificação dos GAGs, que são excretados em excesso na urina destes pacientes. A presença de queratan sulfato na eletroforese ou a detecção de níveis aumentados na dosagem quantitativa, direciona a investigação laboratorial para a MPS IV. O diagnóstico definitivo se estabelece através da medida da atividade enzimática em leucócitos ou fibroblastos, onde se constata a deficiência enzimática. OBJETIVOS: Este estudo teve como objetivo principal, tornar disponível um novo método, mais simples, rápido e acessível, para o diagnóstico bioquímico de mucopolissacaridose tipo IVA, utilizando amostras de sangue impregnadas em papel filtro (SIPF). MATERIAIS E MÉTODOS: Amostras de SIPF e leucócitos de 35 pacientes de ambos os sexos, com idade entre 3 e 47 anos, com diagnóstico previamente estabelecido de MPS IVA, pelo método convencional, em leucócitos e /ou fibroblastos, foram analisadas. Para o estabelecimento dos valores de referência, foram estudadas amostras de leucócitos e de SIPF de 54 indivíduos saudáveis (18-50 anos), de ambos os sexos. Após assinatura do termo de consentimento, amostras de sangue periférico de pacientes e controles, foram coletadas, para a obtenção de leucócitos e sangue impregnado em papel filtro.(SIPF). Os ensaios enzimáticos foram realizados nas amostras de leucócitos e SIPF, simultaneamente, para comparar os resultados. RESULTADOS: Os resultados obtidos nos ensaios enzimáticos de todos os pacientes apresentando MPS IVA, confirmaram a deficiência da atividade enzimática em ambos materiais (leucócitos e SIPF) com uma diferença estatisticamente significativa em relação ao grupo controle. (Mann-Witney U test, p< 0,001). Neste estudo, a medida de GALNS em amostras de SIPF permitiu a identificação dos pacientes com MPS IVA, com sensibilidade de 100 %. Os testes de estabilidade realizados nas amostras de SIPF indicaram que amostras coletadas para a medida da atividade de GALNS devem ser mantidas a 4ºC sempre que possível, sendo estáveis nesta temperatura por mais de 30 dias. CONCLUSÕES: Nas condições utilizadas, amostras de SIPF se mostraram adequadas para a identificação segura de pacientes com MPS tipo IVA. O método que utiliza amostras de SIPF é mais acessível e rápido, simplificando a etapa de coleta e transporte, podendo ser utilizado para detectar pacientes afetados, especialmente em áreas de difícil acesso para a coleta e transporte de amostras líquidas. / INTRODUCTION: Mucopolysaccharidosis (MPS) are lysosomal deposit diseases characterized by lysosomal enzymes deficiency involved in the degradation of glycosaminoglycans (GAGs). The abnormal accumulation of these macromolecules inside the lysosomes provokes structural and functional alterations multi-systemically and progressively. The accumulated GAGs are also excreted in the urine, where they may be identified through many different biochemical methods. These diseases occur among all ethnical groups and the combined incidence of MPS is estimated at 1:10.000 to 1:25.000 live births. (Baehner, 2005). The MPS’ cause is the deficiency of a specific enzyme in the GAGs degradation route. The MPS are classified according to a type of substrate accumulated (GAGs) and the deficiency of a specific enzyme. In Morquio syndrome A or Mucopolysaccharidosis type IVA (MPS IVA), the accumulated substrate is the keratan sulfate and the deficient enzyme is the N-acetylgalactosamine-6-sulfatase (GALNS). The patients affected by MPS IVA present short stature, dysostosis multiplex, corneal opacity, among others signs and symptoms. The cognitive and mental developments are normal. The MPS initial detection method is based on the identification of the GAGs which are excreted in the patients’ urine. The presence of the keratan sulphate in the electrophoresis or the detection of the increased levels in the quantitative dosage directs the laboratory investigation to MPS IV. The definitive diagnosis is established through measuring the enzymatic activity in leukocytes or fibroblasts, in which the enzymatic deficiency is proved. OBJECTIVE: This study’s main purpose is to offer an original, simpler, faster and more accessible method for biochemical diagnosis of Mucopolysaccharidosis type IVA using dried blood samples (DBS). MATERIALS AND METHODS: DBS and leukocytes from 35 patients from both sexes between 3 and 47 years of age with previously established diagnosis of MPS IVA through the conventional method in leukocytes and/or fibroblasts were analyzed. In order to establish reference values DBS and leukocytes samples from 54 healthy people (18-50 years of age) from both sexes were studied. After signing a paper consent form, peripheral blood samples from patients and controls were collected for obtaining leukocytes and dried blood samples (DBS). To validate the method, we made a simultaneous GALNS assay in leukocytes and DBS. RESULTS: The results obtained in the enzymatic assays from all patients presenting MPS IVA confirmed the deficiency of enzymatic activity in both materials (leukocytes and DBS) with a significant statistical difference in relation to the control group. (Mann-Witney U tes, p< 0,001). In this study, the quantity of GALNS in DBS allowed the identification of patients with MPS IVA with sensibility of 100%. The stability tests indicate that DBS samples collected for measuring the activity of GALNS must be kept at 4ºC whenever possible, being stable in this temperature for more than 30 days. CONCLUSION: In the used conditions, DBS were adequate for a safe identification of patients with MPS type IVA. The method which utilizes DBS is cheaper and faster, what simplifies the collection and transportation stage and can be used to detect affected patients especially in difficult access areas for the collection and transportation of liquid samples.

Orientador: Cristina Pontes Vicente / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T00:19:46Z (GMT). No. of bitstreams: 1 Godoy_JulianaAparecidaPretode_D.pdf: 5686269 bytes, checksum: 95147a9fe0fec8d741716bf7ed2bd30e (MD5) Previous issue date: 2014 / Resumo: A aterosclerose é uma doença que acomete os vasos sanguíneos através da formação exacerbada de placas de gordura, interrompendo o fluxo de sanguíneo. A intervenção cirúrgica mais utilizada nesses casos é a angioplastia coronária percutânea, entretanto, esse procedimento pode ocasionar uma lesão ao endotélio, onde os processos trombótico e inflamatório são iniciados. Neste processo ocorre o recrutamento de células inflamatórias e a migração e adesão das células progenitoras endoteliais (CPE) para o local da lesão de modo a recuperar o dano endotelial. As CPE são células existentes na população de células mononucleares (MNC) da medula óssea e podem estar envolvidas no processo de reparo endotelial. Os glicosaminoglicanos, como o dermatan sulfato (DS) e o condroitin sulfato fucosilado (CSF), são moléculas que atuam como agentes antitrombóticos, anticoagulantes e anti-inflamatórios. Neste estudo, analisamos a influência do DS e CSF e da terapia com CPE na inflamação e trombose após lesão arterial em camundongos selvagens (C57BL06) e deficientes em apolipoproteína E (ApoE-/-). Todos os animais foram submetidos a um procedimento cirúrgico na artéria carótida comum esquerda, mimetizando a lesão causada por angioplastia em humanos. Proteínas/fatores envolvidos nos processos iniciados logo após a lesão endotelial foram analisados por western blotting (ICAM-1, P-selectina, eNOS e SDF-1) e por Elisa (TNF-?, IL-10, SDF-1 e TGF-?); as artérias carótidas lesionadas foram analisadas histologicamente para se verificar a formação de trombo e a atividade de gelatinases por zimografia `in situ¿. Todas as análises foram realizadas 1 e 3 dias após a lesão arterial. Observamos nos camundongos C57BL/6 que o tratamento com DS foi capaz de diminuir a reação inflamatória inicial e, ao mesmo tempo, estimular a migração de células progenitoras para o local da lesão; tal resultado foi observado em relação ao uso de CSF, apesar deste não prevenir a formação de trombo. A terapia utilizando MNC isoladas ou em conjunto com DS ou CSF aumentou a resposta inflamatória inicial. O uso de CPE também ocasiona essa resposta, mas quando utilizamos a EPC junto com DS obtemos uma menor resposta inflamatória e, ao mesmo tempo, aumento da expressão de eNOS, melhorando o tônus vascular. Nos camundongos ApoE-/-, os tratamentos não foram capazes de melhorar a resposta inflamatória; o tratamento com CPE e CSF foi o mais eficaz, diminuindo a resposta inflamatória e melhorando o tônus vascular. Concluímos que as terapias com os glicosaminoglicanos (DS ou CSF) foram capazes de diminuir as respostas trombóticas e inflamatórias iniciais. Além disso, que a injeção de MNC leva a um aumento da resposta inflamatória provavelmente devido ao caráter inflamatório destas células. Concluímos, que os GAGs podem estar atuando como moléculas de quimioatração das células ao mesmo tempo em que evitam a trombose e inflamação iniciais exacerbadas e que podem ser utilizados como agentes coadjuvantes na terapia celular para a recuperação de dano endotelial / Abstract: Atherosclerosis is a disease that undertakes blood vessels by exacerbated atheroma plaque formation, and blocking blood flow. The most used surgical intervention in these cases is the angioplasty, however, this procedure can cause a lesion to the endothelium, where thrombotic and inflammatory processes are initiated, recruiting inflammatory cells to the lesion site and also promoting migration and adhesion of endothelial progenitor cells (EPC) in order to recovery endothelial damage. Glycosaminoglycans (GAGs), like dermatan sulfate (DS) and fucosilated chondroitin sulfate (FCS), are molecules that act as antithrombotic, anticoagulant and anti-inflammatory agents. EPC are cells that exist in bone marrow mononuclear (MNC) cells fraction population and can be involved in vascular repair. In this study, we analyzed the influence of DS and FCS and cellular therapy using EPC in inflammation and thrombosis after arterial lesion in wild-type (C57BL6) and apolipoprotein E deficient (ApoE-/-) mice. All animals were submitted to a surgical procedure in the left common carotid artery, mimicking the lesion caused by angioplasty in humans. Proteins/factors involved in the initial process after an endothelial lesion were analyzed by western blotting (ICAM-1, P-selectina, eNOS and SDF-1) and by Elisa (TNF-?, IL-10, SDF-1 and TGF-?); lesioned carotid artery were analyzed histologically to verify thrombus formation and gelatinase activity by zimography `in situ¿. All analyzes were done 1 and 3 days after arterial lesion. We observed that C57BL6 mice treatment with DS was able in decrease the initial inflammatory reaction and, at the same time, stimulate progenitor cells migration to the lesion site; this result was also observed in relation to FCS use, despite its did not prevent thrombus formation. Therapy using MNC alone or with DS or FCS increased the initial inflammatory response. Use of EPC also promoted this response but when used together with DS, we can obtain a decreased inflammatory response and, at the same time, an increased in the eNOS expression, improving vascular tonus. In ApoE-/- mice, treatments were not able in improving inflammatory response; treatment with EPC and FCS was the one with best action, decreasing inflammatory response and improving the vascular tonus. GAGs (DS or FCS) therapy was able to decrease the initial inflammation. Besides, MNC injection leads to an increase in inflammation probably due to its inflammatory characteristics. We conclude that GAGs can be acting quimio-atracting molecules to the cells and, at the same time, avoiding the initial thrombosis and inflammation after injury and they can be used as supporting agents in cellular therapy use to help in vascular recovery after injury / Doutorado / Biologia Celular / Doutora em Biologia Celular e Estrutural

L’arthrose du genou est une maladie rhumatismale dégénérative touchant tous les tissus de l’articulation. Elle est classée parmi les dix affections les plus invalidantes dans le monde et l’absence de traitement curatif efficace en fait un véritable problème de santé publique. Dans le cas d’arthrose post-traumatique, les lésions méniscales sont fréquentes et entravent leurs fonctions de répartition des efforts, d’absorption des chocs et de stabilité. Les objectifs de ce travail de thèse sont (1) d’analyser les changements de propriétés mécaniques des ménisques à un stade précoce de développement de l’arthrose et de les corréler avec les modifications morphologiques, biochimiques et microstructurales, et (2) d’évaluer l’effet d’une thérapie de viscosupplémentation commerciale. Le modèle d’arthrose post-traumatique choisi dans cette étude est la rupture du ligament croisé antérieur chez le lapin. Les ménisques de lapins sains, arthrosiques non traités et arthrosiques traités ont été caractérisés six semaines après l’opération par un score macroscopique, puis par indentation – relaxation grâce au développement d’une méthode de test adaptée, ainsi que par microspectroscopie Raman, microscopie confocale biphotonique et histologie. Cette étude a révélé la formation de lésions à la surface des ménisques arthrosiques et une diminution de leurs propriétés viscoélastiques, s’expliquant par une désorganisation de leurs fibres de collagène et une diminution de leur quantité de glycosaminoglycanes. Bien que le traitement améliore significativement l’état de surface des ménisques et joue un rôle dans la régulation des glycosaminoglycanes, il a peu d’effet sur l’organisation des fibres de collagène et sur les propriétés viscoélastiques. Ce travail de thèse apporte une meilleure compréhension des modifications affectant le ménisque, dans l’optique de développer de nouveaux traitements. / Knee osteoarthritis (OA) is a rheumatic degenerative disease affecting all tissues of the joint. It is classified among the ten most disabling affections in the world and the lack of efficient curative treatment makes it a real public health issue. In case of post-traumatic OA, meniscal tears are frequent and interfere with their functions of load distribution, shock absorption and stability. The aims of this work are (1) to analyze the changes of mechanical properties of the menisci at an early stage of OA development and to correlate them with morphological, biochemical and microstructural modifications, and (2) to assess the effect of a commercial viscosupplementation therapy. For this study, an anterior cruciate ligament transection model of post-traumatic OA was used in rabbits. Menisci from healthy, non-treated OA and treated OA rabbits were characterized six weeks after surgery through macroscopic grading, indentation-relaxation with the development of an adapted test method, Raman microspectroscopy, biphotonic confocal microscopy and histology. This study revealed the formation of tears at the surface of OA menisci and a decrease in their viscoelastic properties, which was explained by a disorganization of their collagen fibers and a decrease in their glycosaminoglycan content. Although the treatment significantly improves the surface integrity of the menisci and plays a role in the glycosaminoglycan regulation, it has little effect on the organization of their collagen fibers and on their viscoelastic properties. This work brings a better understanding of the disease processes affecting the meniscus, for the purposes of developing new treatments.

La bioraffinerie, avec l’utilisation de la biomasse végétale comme matière première renouvelable, est un moyen alternatif aux ressources fossiles qui s’amenuisent pour l’obtention de molécules d’intérêt, de biomatériaux ou de biocarburant nécessaires à notre vie quotidienne. Les hémicelluloses constituent 20 à 45% de la biomasse végétale et sont riches en pentoses (D-xylose en particulier) dans le cas des xylanes. Dans le cadre du projet multi-partenaires XYLOCOS, financé par la Région Champagne-Ardenne et le FEDER, l’objectif de cette thèse consiste en l’utilisation d’une xylanase pour mettre au point la préparation par voie enzymatique ou chimio-enzymatique de nouveaux β-xylosides et β-xylobiosides présentant des aglycones de différentes natures et d’évaluer leurs propriétés biologiques. L’étude d’une xylanase GH11 a d’abord été réalisée pour améliorer son activité de transglycosylation en présence de divers accepteurs. Une étude de modélisation in silico a été effectuée et a conduit à cibler la mutation du résidu W126. Les paramètres cinétiques et les propriétés de transglycosylation de la xylanase sauvage et du mutant ont été comparés et ont montré que le mutant a une capacité de transglycosylation améliorée. La synthèse de séries de xylosides et xylobiosides comportant une partie aglycone aromatique par voie enzymatique ou présentant des hétérocycles triazoles diversement fonctionnalisés par voie chimio-enzymatique a été effectuée. L’influence de la nature de la partie aglycone mais également du degré de polymérisation des différents xylosides sur leur capacité à amorcer la biosynthèse de glycosaminoglycanes a été étudiée en présence d’un modèle cellulaire. L’ensemble des xylosides et xylobiosides synthétisés amorce la biosynthèse des GAGs. Les résultats obtenus montrent que les xylosides sont plus efficaces que leurs analogues xylobiosides. Certains xylosides à aglycones hydrophobes représentent de bons candidats pour des applications cosmétiques. / Biorefinery, with the use of plant biomass as a renewable raw material, is an alternative means to fossil resources for obtaining molecules of interest, biomaterials or biofuel. Hemicelluloses represent about 20 to 45% of the plant biomass and are rich in pentoses (D-xylose in particular) in the case of xylans. XYLOCOS is a multi-partner project funded by the Champagne-Ardenne Region and the FEDER. In this context the objective of this work consists in the use of a xylanase to develop the enzymatic or chemo-enzymatic synthesis of new β-xylosides and β-xylobiosides featuring different kinds of aglycone moieties and to assess their biological properties.First, the study of a GH11 xylanase was carried out to improve its transglycosylation activity in the presence of various acceptors. A in silico study was performed and led to target the mutation of W126 residue. Kinetic parameters and transglycosylation properties of the wild and mutant xylanases were compared and showed that the mutant has an improved capacity for transglycosylation.The synthesis of xylosides and xylobiosides bearing an aromatic aglycone part by an enzymatic transformation or functionalized triazole heterocycles by a chemo-enzymatic pathway was performed. The influence of the nature of the aglycone part but also the degree of polymerization of different xylosides was studied through their ability to initiate the biosynthesis of glycosaminoglycans in the presence of a cell model. All the xylosides and xylobiosides prepared act as GAGs primers. The obtained results show that xylosides are more efficient primers than the corresponding xylobiosides. Xylosides carrying hydrophobic aglycones represent good candidates for cosmetic applications.

Glycosaminoglycans (GAGs) are complex linear chain carbohydrate molecules found on virtually all animal cell surfaces. Owing to their negatively charged nature, GAGs interact with a number of different proteins. Thus, although they have great potential as therapeutic agents, their apparent promiscuous interactions increase their side effect risk. GAG mimetics, including GAG oligosaccharides and non-saccharide GAG mimetics (NSGMs) are viable approaches to address this. This work discusses sulfated benzofuran thrombin inhibitors with submaximal protease inhibition, sulfated diflavonoid inhibitors of plasmin and GAG oligosaccharides with selectivity for human neutrophil elastase (HNE). Anticoagulants are very important for the treatment of thrombotic diseases. The adverse effects associated with current clinically used anticoagulants warrant the continuous search for new agents. Thrombin, being the central player in the coagulation cascade, remains a very important target for anticoagulant therapy, however drugs inhibiting its activity carry the risk of prolonged bleeding. Based on a previously identified sulfated benzofuran thrombin inhibitor, we have developed analogs with submaximal inhibition of the protease. These agents inhibit thrombin with efficacies approaching 50%, for both chromogenic and macromolecular substrates, ensuring a basal level of thrombin activity even at saturating inhibitor concentrations. The most potent of these compounds had a potency of 1.8 µM, 2-3 fold better than the lead. Additionally, these compounds utilize an allosteric mechanism for thrombin inhibition. Further, studies have revealed structural features responsible for submaximal thrombin inhibition. Fibrinolysis is an important part of hemostasis and plasmin is the most important fibrinolytic enzyme. Anti-plasmin agents are thus important for conditions such as hemophilia; however, there are no clinically used direct plasmin inhibitors. By structural modifications of a previously identified sulfated diflavonoid plasmin inhibitor, we have achieved a compound with 12-fold better potency (IC50 = 6.3 ± 0.4 µM), and a selectivity index of at least 22 over closely related serine proteases. We have shown that this compound inhibits plasmin mediated clot lysis, and further demonstrated that its activity is reversible using protamine sulfate, indicating its potential as a lead for the development of clinical anti-plasmin agents. HNE, a serine protease associated with inflammatory diseases is known to be inhibited by GAGs. However, the interactions at the molecular level have remained elusive. Using biochemical methods, and by studying the inhibitory potency of different GAGs and GAG oligosaccharides, we have shown that an octasaccharide may be the ideal GAG length for the achievement of potent HNE inhibition. Under our assay conditions, the inhibition of HNE by an octasaccharide species was only 5-fold less than that of unfractionated heparin, whereas the hexasaccharide species was 30-fold less active. The data also suggests that the inhibition of HNE by GAGs is via an allosteric mechanism and using molecular modeling, we have identified putative GAG binding sites on HNE and further identified GAG species with potential selectivity for anti-HNE activity

Le muscle strié squelettique possède de grandes capacités de régénération grâce à ses cellules souches, les cellules satellites. Après une lésion, le processus de régénération musculaire qui se met en place est finement régulé dans le temps et l’espace par le microenvironnement, constitué de cellules avoisinantes mais également par des éléments de la matrice extracellulaire (MEC). Cette dernière se compose de molécules structurales comme les collagènes et de composants possédant un rôle trophique comme les glycosaminoglycanes (GAGs). La MEC musculaire est peu étudiée à cause d’une organisation tridimensionnelle complexe rendant son exploration difficile. Lors d’une lésion avec perte de substance musculaire, la régénération est altérée, associée à une fibrose et une inflammation chronique. Ce type de lésion est fréquemment rencontré en traumatologie mais survient également chez le blessé de guerre. Malgré un traitement optimal, une invalidité fonctionnelle persiste chez ces patients. L’utilisation d’un biomatériau décellularisé, constitué de MEC pourrait fournir ce support physique et trophique faisant défaut dans ce type de lésion. Dans ce travail, nous avons entrepris l'établissement d'une MEC d’origine musculaire et nous avons établi un protocole de décellularisation permettant d’obtenir un biomatériau conservant l’architecture spécifique de la MEC musculaire avec une élimination de la majorité des antigènes cellulaires afin d'éviter une réponse immunitaire délétère après implantation. Néanmoins, le protocole retenu ne permet de conserver certaines molécules trophiques d’intérêt comme les GAGs. Les « ReGeneRaTing Agent®» (RGTA®) sont des mimétiques fonctionnels de ces GAGs, utilisés en clinique pour améliorer la cicatrisation cutanée et cornéenne. Ces mimétiques conservent une capacité de liaison aux facteurs de croissance avec une résistance aux dégradations enzymatiques. Nous avons évalué l’utilisation de ces molécules au cours de la réparation musculaire, dans un modèle in vivo chez le rongeur. Nous avons réalisé une analyse histologique précoce (8e jour de régénération) mettant en évidence une augmentation du nombre de noyaux par myofibre en faveur d’une augmentation de la fusion, validée également in vitro sur des progéniteurs musculaires. Nous avons également observé une augmentation du nombre de vaisseaux, suggérant une amélioration de l’angiogenèse. Le nombre de gouttelettes lipidiques, marqueur d’une mauvaise régénération, était en diminution. L’exploration histologique plus tardive (28e jour de régénération) n’a retrouvé que l’augmentation du nombre de vaisseaux en faveur d’un effet durable sur l’angiogenèse. Ces RGTA® peuvent être couplés aux biomatériaux et sont particulièrement résistants dans un environnement inflammatoire pouvant être rencontré dans les lésions avec perte de substance musculaire. Des chimiokines et des facteurs de croissance pourront également être ajoutés au biomatériau matriciel afin de favoriser la migration des différents progéniteurs nécessaires à une néoformation musculaire. L’efficacité thérapeutique de ces biomatériaux optimisés nécessitera d’être évaluée dans un modèle in vivo de perte de substance / Skeletal muscle exhibits high capacity for regeneration after an injury that relies on resident stem cells. Muscle regeneration is tightly regulated by both the immune response and other resident cells, as well as by cues from the local extracellular matrix (ECM), contributing to a coordinated repair process. Muscle ECM is a network of structural macromolecules with a large majority of collagens and trophic molecules such as glycosaminoglycans (GAGs). In the skeletal muscle tissue, ECM was overlooked due to its complex organization making investigations difficult. Muscle regenerative ability can be overtaken in large muscle wasting, such as in volumetric muscle loss (VML), leading to fibrosis formation and chronic inflammation. This type of injury predominantly occurs in traumatology and in war-wounded patients, with functional disability despite an optimal treatment. The use of biomaterials could provide the biochemical and physical cues that are missing in this pathologic repair. In this work we have focused on obtaining a biomaterial composed of skeletal muscle ECM. We have tested several decellularization protocols both to preserve the three-dimensional architecture of the muscle ECM and to completely remove cell components in order to avoid a deleterious immune response after implantation. However, the protocol did not allow the preservation of trophic molecules such as GAGs, in the scaffold.“ReGenerating Agents” (RGTA®) are functionally analogous of GAGs with a crucial property to resist enzymatic degradation. They function to restore a proper microenvironment for tissue healing with already a clinical application in skin and corneal repair. We have explored the effects of RGTA® in muscle regeneration using an in vivo model in mouse. At early time of regeneration (day 8), we performed histologic analysis. We showed that regenerating myofibers contained more nuclei in the treated animals, in favor of an increase of progenitor fusion, which has been validated in vitro in myogenic cultures. The number of capillaries was higher in favor of a better angiogenesis. Lipid droplets, a marker of impaired regeneration, were reduced by RGTA® administration. At later time of regeneration (day 28), capillary number was still improved in favor of a durable effect of RGTA® on angiogenesis. RGTA® could be incorporated into biomaterials and are particularly resistant in an inflammatory environment, such as that occurring after a VML injury. Chemokines and growth factors could also be added in ECM-based scaffolds to promote the migration of progenitors that are essential for myofiber neoformation. Therapeutic efficacy of these optimized biomaterials will require to be evaluated in an in vivo model of VML

L'Osteoarthrose (OA) est la maladie articulaire la plus répandue avec un impact socio-économique croissant en raison du vieillissement de la population, de l’augmentation de l'obésité et surtout de l'absence d'un traitement efficace. En effet, l’OA est caractérisée par la dégradation inéluctable du cartilage articulaire, l'apoptose des chondrocytes, un remodelage osseux sous-chondral et une inflammation de la synovie. La matrice extracellulaire (MEC) du cartilage est constituée de collagènes et de protéoglycanes (PG) eux-mêmes composés de glycosaminoglycanes (GAG) liés à un corps proteique, présents dans l'ECM ou à la surface cellulaire. Les GAG sont des chaînes polysaccharidiques linéaires sulfatées comprenant les Héparine/Héparan Sulfate (Hep/HS), Chondroitin Sulfate (CS) et Keratan Sulfate (KS). L'acide hyaluronique (AH) est un GAG non sulfaté particulier, non associé à un corps proteique. Dans le cartilage, l'un des principaux composants de la MEC est l'aggrécan, un CS/KS PG qui forme des aggrégats par interaction avec de l’AH. Au cours du vieillissement, des changements dans la qualité des PG ouvrent la voie à l’OA et les études depuis 60 ans se concentrent sur les aggrécans et le catabolisme des CS. En effets, les niveaux d'expression des CS, la taille de leurs chaînes, leurs profils de sulfatation évoluent, affectant les propriétés mécaniques de la MEC. Cependant, les traitements actuels de visco-supplémentation à base d’injections locales de CS ont démontré leur limite puisque la réparation du cartilage n'est pas induite. Même si ils sont rares dans le cartilage adulte, les HSPG sont associés aux chondrocytes et leurs rôles a été démontrée lors du développement osseux. Or les HS sont des régulateurs de l’homéostasie très importants car ils peuvent lier et réguler l'activité de protéines liant l'héparine (HBP) (facteurs de croissance, cytokines, chimiokines, morphogènes), les protégeant contre la protéolyse et potentialisant leur liaison à leurs récepteurs. Tous ces effets sont contrôlés par les profils de sulfatation complexes des chaînes d’HS.Dans ce contexte les objectifs de cette thèse sont de caractériser l'évolution de la signature chimique et de la fonctionnalité des HS au cours de l’OA. En collaboration avec les Rhumatologistes et Orthopédistes de l’Hopital Henri Mondor, une évaluation quantitative des HS dans des échantillons de cartilage humain contrôle versus OA a été corrélée à la gravité des dommages. Grace à la plateforme glycomic du CRRET, des modifications dans les profils de sulfatation des disaccharides de HS ont été observées et confirmées par des analyses de l'expression des enzymes de la biosynthèse des GAG. Ces caractéristiques structurales ont été corrélées à des changements fonctionnels de l’affinité des GAG pour des HBP, telles que FGF-2, VEGF et PTN. Enfin, les GAG OA ont des capacités différentes à moduler les propriétés (prolifération, adhésion, phénotype) de cellules souches mésenchymateuses, chondrocytes, fibroblastes et cellules endothéliales. Ces résultats démontrent que des modifications des structures et fonctions des HS pourraient être impliquées dans l'évolution de l'homéostasie du cartilage vers des processus pathologiques au cours de l’OA. Ce projet se positionne clairement comme une recherche fondamentale et translationnelle qui permettra d'acquérir des connaissances sur les mécanismes régulant les interactions cellules/matrice au cours de l'OA. De plus, les outils développés au cours de ce projet ont permis de réaliser 2 projets collaboratifs sur l'hypertension artérielle pulmonaire et une pathologie éosophagique. Enfin, ces données confirment l'intérêt d’identifier de nouvelles cibles glycaniques basées sur la chimie des HS. Cela permettra de proposer une nouvelle stratégie thérapeutique basée sur des composes à même de contrôler les profils de sulfatation de la MEC, dans le but d'améliorer les propriétés de cellules souches thérapeutiques endogènes ou exogènes, associées. / Osteoarthritis (OA) is the most prevalent joint disease with increasing socio-economic impact due to population aging, obesity , and absence of an efficient medical treatment that can repair cartilage. OA is characterized by degradation of articular cartilage, hypertrophy and apoptosis of chondrocytes, subchondral bone remodeling and joint synovial inflammation. Cartilage extracellular matrix (ECM) consists of collagens, glycoproteins and proteoglycans (PGs) that are composed of Glycosaminoglycans (GAGs) linked to core proteins, presents in the ECM or at the cell surface. GAGs are linear polysaccharidic sulfated chains including Heparine/Heparan Sulfate (Hep/HS), Chondroitin Sulfate (CS) and Keratan Sulfate (KS) families. Hyaluronic acid (HA) is a particular un-sulfated GAG no associated to core protein. In cartilage, one of the major ECM component is aggrecan, a CS/KS PG that form aggregate through HA interaction. During the aging process, changes in PGs quality pave the way for OA and studies are focus on aggrecans and CS catabolism since 60 years. CS expression levels, chain size, sulfation patterns evolved during OA, affecting the mechanical properties of ECM. However, treatments based on visco-supplementation with CS local injections have demonstrated their limit since cartilage repair is not induced. Even if rare in adult cartilage, HSPG are present associated to chondrocytes also and their relevance was demonstrated mainly during bone development. HS chains are very important homeostatic regulators because they are able to bind and regulate the activity of several heparin binding proteins (HBP) (growth factors, cytokines, chemokines, morphogens), protecting them against proteolysis and potentiating their binding to their receptors. These interactions provide a stock of regulatory factors that can be release by selective degradation of the HS chains too. All these regulatory effects are mediated through the complex sulfation/acetylation pattern of HS chains but no data are available on this aspect during OA.In this context, the goals of this Thesis were to characterize the evolution of HS chemical signature and functionality during OA. In collaboration with Rheumatology and Orthopedic clinical teams from Henri Mondor Hospital, a quantitative evaluation of HS and CS amount in control versus OA human cartilage samples was correlated to the structural damage severity. According to the tools of the CRRET’s lab glycomic platform, structural changes on HS and CS sulfated disaccharides compositions was observed using HPLC, confirmed by RQ-PCR analyzes of the expression of enzymes involved in GAG biosynthesis. These structural features were correlated to functional changes on HBP affinities, such as FGF-2, VEGF and PTN, through ELISA based competition assay. Finally, GAGs from OA have different abilities to modulate properties (adhesion proliferation, phenotype…) of Mesenchymal Stem Cells, chondrocytes, fibroblast and endothelial cells. These results clearly make the proof that modifications of HS structures and functions could be involved in the evolution of cartilage homeostasis and pave the way for altered pathological processes during OA. This project is clearly positioned as a fundamental and translational research that will permit to gain knowledge on the mechanisms regulating cartilage cells/matrix interactions during OA. All these results are summarized in 2 scientific and 1 review articles. Moreover, all the tools developed during this project have permit to realize 2 collaborative projects and associated articles on Pulmonary Hypertension and Eosophagic pathology also. Finally, all these data confirmed the interest of the team to identify new glycanic targets based on HS chemistry. This will permit to propose new therapeutic strategy based on HS compounds associated to endogenous or exogenous therapeutic stem cells, with the aim of improving cell properties according to HS ability to control sulfation panels of ECM.