Les glycosaminoglycanes (GAGs) sont des polymères de sucres linéaires, présents chez tous les animaux. Certaines bactéries pathogènes synthétisent également des polysaccharides identiques ou très similaires aux GAGs humains. Cette thèse a porté en particulier sur la synthèse de la chondroïtine sulfate et de l’héparosan qui font partie de cette famille de polysaccharides. L’intérêt pour ces deux GAGs est grandissant dans l’industrie pharmaceutique du fait des nombreuses applications médicales qu’ils pourraient permettre. La chondroïtine sulfate est d’ores et déjà extraite de tissus animaux ce qui peut engendrer des problèmes sanitaires, notamment des contaminations virales ou aux prions. En revanche, le procédé de production pour l’héparosan reste à mettre en place. Il est donc nécessaire de développer des procédés de production pour ces deux molécules. La synthèse enzymatique est une voie particulièrement prometteuse pour la production de la chondroïtine sulfate et de l’héparosan, et a fait l’objet de ce travail de thèse. / Glycosaminoglycans (GAGs) are long linear polysaccharide chains, found in all animals. Some pathogenic bacteria also synthesize polysaccharides identical or similar to human GAGs. This thesis deals with chondroitin sulfate and heparosan syntheses, members of the GAGs family. There is a growing interest in these two GAGs in the pharmaceutical industry due to numerous potential applications they offer. Chondroitin sulfate is currently extracted from animal tissues which can lead to sanitary problems such as viral or prion contaminations. On the other hand, a production process still needs to be developed for heparosan. Therefore, it is necessary to develop new methods for the production of these two polymers. Enzymatic synthesis, which is a promising alternative for the production of chondroitin sulfate and heparosan, was the subject of this thesis.

Titre : Nano-plaquettes à mobilité dirigée et adhésion amplifiée pour l’administration locale: vers un nouveau traitement des maladies vésicalesAbstract : L’administration locale des médicaments, définie comme une voie d’administration où la substance active est directement administrée sur ou proche de la cible ou tissus souhaités, permet d’apporter des grandes quantités des médicaments avec moins d’effets secondaires, et permet une simplification du système nanoparticulaire du fait de la non-extravasation des médicaments. Dans ce contexte, le projet de recherche de cette thèse s’est focalisé sur la voie intra-vésicale comme voie d’administration locale car il existe un besoin clinique de la part des patients, n’étant pas encore résolu. Malgré les hypothétiques avantages fournis par l’administration locale des médicaments, la voie intra-vésicale présente certaines limitations qui diminuent l’efficacité des traitements et l’observance des patients. La plupart des médicaments pour le traitement des maladies vésicales, notamment pour le cancer de la vessie et les cystites interstitielles, sont sous forme de solutions ou suspensions administrées de manière intra-vésicale via un cathéter qui passe à travers l’urètre. Dès leur arrivée à la vessie, les substances actives sont fortement diluées par les urines et éliminées rapidement lors de la miction. Cela conduit à une diminution des concentrations des substances actives au plus proche de l’épithélium, nécessitant plusieurs instillations intra-vésicales, réalisées par des praticiens hospitaliers, pour atteindre des concentrations thérapeutiques. Il y a donc un réel besoin de développer des nouvelles formulations permettant de contrecarrer les phénomènes décrits au préalable.L’objectif de cette thèse de doctorat est de créer un nouveau système nanoparticulaire de morphologie non-sphérique qui serait susceptible d’avoir un mouvement diffèrent et dirigé ainsi qu’une adhésion amplifiée. En conséquence, nous attendons de ces systèmes qu’ils apportent des concentrations en substances actives plus importantes que les systèmes nanoparticulaires sphériques et formulations galéniques traditionnelles.Aux cours de nos travaux expérimentaux, nous avons réussi à développer un système nanoparticulaire de morphologie hexagonale et aplatie. Ces nanoparticules, appellées nano-plaquettes, sont conçues à partir de l’auto-assemblage des molécules d’α-CD et des chaines alkyles greffées sur les squelettes de polysaccharides tels que l’acide hyaluronique, la chondroïtine sulfate ou l’héparine. Ces systèmes présentent l’originalité de ne pas avoir de substance active encapsulé parce que les molécules de polymère elles mêmes agissent à la fois en tant que substance active et de véhicule. Ces nano-plaquettes ont montré un mouvement en milieu isotrope et statique très diffèrent des nano-sphères utilisées comme contrôle. En effet, la majorité d’entre elles diffuse de manière plus importante et dirigée, avec des trajectoires rectilignes. Grâce à leur mouvement et aux propriétés inhérentes liées à leur forme, ces systèmes se sont montrés particulièrement intéressants vis-à-vis des interactions avec des cellules. Ils adhèrent mieux et plus longtemps à la muqueuse vésicale, elles sont mieux internalisées par des cellules et sont éliminées plus lentement une fois adhérées à la surface de l’urothélium.Un modelé in vivo de Syndrome de la Vessie Douloureuse / Cystite Interstitielle développé chez le rat nous a permis de montrer l’efficacité thérapeutique des nano-plaquettes, notamment celle constituées d’acide hyaluronique. En effet, elles présentent une meilleure bioaccumulation dans la vessie et une meilleure activité anti-inflammatoire et de régénération de la muqueuse urothéliale.Ces systèmes nanoparticulaires, conçues lors de nos travaux de thèse, constituent une approche innovante, rationnelle et efficace pouvant ouvrir de nouvelles voies de recherche pour le traitement des maladies vésicales. / Title: Directed-mobility and enhanced-adhesion nano-platelets for local drug delivery: towards a new treatment of bladder diseases.Abstract: Local drug delivery, defined as the administration route where the drug is delivered directly or very close to its target or tissue, allows to bring large amounts of drugs with reduced side effects, in comparison with systemic administration. In this context, our research project has been focused on the intravesical drug delivery as local administration route, because there is a real need to develop new pharmaceutical formulations to thwart several limitations. Despite the advantages provided by the local drug delivery, intravesical drug delivery exhibited some issues which are decreasing the therapeutic efficacy and the patient compliance to the treatment. Most of therapies for the treatment of bladder diseases are simple drug solutions or suspensions administered intravesically by using a catheter through the urethra in order to reach easily the bladder and, consequently, the urothelium. Since the drug is administered into the bladder, drug dilution is occurring because the continuous production of urine. Furthermore, active substances are being eliminated during washout when bladder urine voiding is happening. These two processes lead to the decrease of local drug concentration close to the urothelium. Patients need repeated catheterization, performed by health care practitioners, to reach therapeutic dose of the drug. Therefor, there is a need of new drug formulations to avoid these main limitations.The main goal of this PhD thesis was to create and design a new nanoparticulate system with non-spherical shape susceptible to move in a different manner compared to spherical nanoparticles. These systems may exhibit an amplified mucoadhesion allowing to bring more important amounts of drug than classical and nanoparticle administration.During this thesis, we developed a new nanoparticulate system presenting non-spherical, hexagonal and flattened shape. The driven force for the design of these nanoparticles was the self-assembling of α-cyclodextrin molecules with alkyl chains grafted on the polymer skeleton. Polymers used belong to a polysaccharide family called glycosaminoglycans including hyaluronic acid, chondroitin sulfate or heparin. This original and innovative nanoparticulate system does not encapsulate an active drug. Our polysaccharide will act, at the same time, as the active drug and the carrier. These nanoparticles, called now nano-platelets have shown different movement behavior than the spherical ones. Indeed, they diffuse more rapidly in a straight-line way. Thanks to their oriented and directed motion and to their intrinsic properties, due to the shape, these systems have shown a better mucoadhesion on the bladder tissue, a better uptake in different cell lines and they were far less rapidly eliminated from the urothelium mucosa.An in vivo model of Bladder Painful Syndrome / Interstitial Cystitis in rats demonstrated the therapeutic efficacy of nano-platelets, especially for hyaluronic acid nanoparticles. Indeed, they demonstrated a better bioaccumulation into the bladder and a better therapeutic efficacy as anti-inflammatory and urothelium regenerating agents.These nanoparticulate systems, designed during this work, represent a new innovative, rational and effectiveness approach allowing to open new research pathways for the treatment of bladder diseases.

The mechanical function of the uterine cervix is critical for a healthy pregnancy. During pregnancy, the cervix undergoes a significant remodeling from a mechanical barrier into a compliant structure to allow for a successful delivery. A too early or too late cervical softening will lead to spontaneous preterm births (sPTB) or dystocia. PTB is a leading cause of neonatal death, affecting 15 million newly born babies each year around the world. According to CDC, the rate of PTB increases in recent years. Dystocia increases the risk to both mother and newborn babies, leading to neonatal asphyxia, neonatal infection, uterine rupture, or other dangerous sequelae. Therefore, it is significant to have a better correlation of the mechanical properties change and the biological remodeling process of the cervix during pregnancy. This thesis will focus on (1) mechanical experiments of the human cervix, and (2) the development of a material constitutive model for cervix to characterize the complex microstructure-related mechanical property of the cervix. In this thesis, a spherical indentation test was designed and conducted on human cervical samples sliced perpendicular to the axial direction, to characterize the compressive mechanical behavior of the human cervix. A uniaxial tensile was designed and conducted on the strip samples cut along and perpendicular to the preferential fiber direction from the indentation samples, to characterize the tensile mechanical behavior of the cervix. Based on the detailed experimental investigation, a nonlinear time-dependent anisotropic microstructure-inspired constitutive model has been developed. The basic idea of the model is that the mechanical behavior of the human cervix can be decomposed into an equilibrium and a time-dependent part, and the tension and compression mechanical behaviors are caused by disparate mechanisms. Specifically, the collagen fibrous network plays a major role in the tensile mechanical response, while proteoglycans (PGs), glycosaminoglycans (PGs),, and liquid cause the compressive mechanical response. The tensile time-dependent mechanical behavior of the human cervix is mostly attributed to the interactions between the collagen fiber and other components, while the compressive time-dependent mechanical behavior is mainly attributed to the porous effect. The equilibrium and time-dependent mechanical responses have been well captured using the model, and the results reveal the connection between the ECM microstructure remodeling and mechanical properties change during pregnancy.

Please note: we were unable to immediately open the spreadsheet below. We repaired the spreadsheet file with Excel, and have a copy of it in storage. If you have difficulty opening the spreadsheet, please write to us at open-help@bu.edu. / Osteoarthritis (OA) is a painful, chronic, non-inflammatory disease affecting 140 million people worldwide that alters synovial joint structure and function. OA progressively breaks down hyaline cartilage, the hydrated tissue that provides a smooth, nearly frictionless surface and distributes loads applied to articulating joint surfaces. The loss of glycosaminoglycans (GAGs) from the extracellular matrix of cartilage is an early marker of OA. Therefore, imaging methods that quantify the GAG content of cartilage are of interest. This work investigates the synthesis and development of three cationic contrast agents (CAs) for imaging articular cartilage (AC): CA4+, an iodinated small molecule, and tantalum oxide nanoparticles (Ta2O5 NPs) for x-ray Computed Tomography (CT) imaging; and Gadopentetate-dilysine (Gd(DTPA)Lys2), a gadolinium small molecule for Magnetic Resonance (MR) imaging. These cationic contrast agents are attracted to the strong negative fixed charge of extracellular GAG and, therefore, infiltrate cartilage. This work begins with an overview of CT and MR imaging basic principles, current clinical CAs and contrast enhanced imaging of AC. First, the large-scale (50 g) synthesis of CA4+ is described and the partitioning over time of CA4+ into ex vivo AC is correlated to GAG content and cartilage mechanical properties. Similar partitioning studies are applied to anionic, neutral and cationic Ta2O5 NPs, where the cationic NP exhibited substantially greater affinity for AC. Moreover, by maintaining the positive charge on the NP surface and introducing a polyethylene glycol coating, a NP formulation is described for successful in vivo cartilage imaging. Next described is the MRI CA, Gd(DTPA)Lys2, which affords an equivalent T1 signal in cartilage at 1/10th the effective dosage of anionic gadopentetate. Finally, the equilibrium partitioning of the small molecule CT and MRI CAs is directly compared to GAG content and mechanical properties in human finger AC. In summary, results show cationic CAs strongly correlate to both GAG and mechanical properties and distribute in direct proportion to GAG unlike anionic CAs. The use of cationic CAs to quantify the biochemical and mechanical changes of AC may aid drug discovery and improve clinical assessment and intervention of OA for future patients. / 2017-11-03T00:00:00Z

Neutrophil extracellular traps (NETs) are a link between infection and coagulation in sepsis. The major structural component of NETs is nucleosomes, consisting of DNA and histones. NETs not only act as a scaffold to trap platelets, but NET components also promote coagulation and impair fibrinolysis. Thus, removal of extracellular DNA by DNases may be a potential therapeutic strategy for sepsis. Since heparin is used for thromboprophylaxis in sepsis and may also be a potential anti-sepsis therapy, we investigated the mechanisms by which various forms of heparins modulate DNase function. There are two types of DNases in vivo: DNase I (produced by exocrine and endocrine glands) and DNase1L3 (secreted by immune cells). DNase I cleaves free DNA, whereas DNase1L3 preferentially cleaves DNA in complex with proteins such as histones. In this study, we investigated how DNase I and DNase1L3 activities are modulated by the following heparins: unfractionated heparin (UFH), enoxaparin (a low-molecular-weight heparin), Vasoflux (a low-molecular-weight, non-anticoagulant heparin), and fondaparinux (the pentasaccharide unit). Using agarose gel experiments, we showed that UFH, enoxaparin, and Vasoflux enhance the ability of DNase I to digest DNA-histone complexes (presumably by displacing DNA from histones), whereas fondaparinux does not. These findings are consistent with the KD values of the binding of heparin variants to histones, with fondaparinux having >1000-fold lower affinity for histones compared to the other heparins. Taken together, our data suggests that the ability of heparin to enhance DNase I-mediated digestion of DNA-histone complexes is size-dependent and independent of the pentasaccharide region of heparin. With respect to DNase1L3, we observed that it is able to digest histone-bound DNA, and that all heparins, except fondaparinux, inhibited DNase1L3-mediated digestion of histone-bound DNA. Next, we visualized the degradation of NETs by fluorescence microscopy. DNase I (± heparin variants) completely degraded NETs, presumably by digesting extracellular chromatin at histone-free linker regions, thereby releasing nucleosome units. DNase1L3 also degraded NETs, but not as effectively as DNase I, and was inhibited by all heparins except fondaparinux. Finally, we showed that DNase I levels are decreased and DNase1L3 levels are elevated in septic patients. Taken together, our findings demonstrate that heparin modulates the function of DNases, and that endogenous DNase levels are altered in sepsis pathophysiology. / Thesis / Master of Science (MSc) / Sepsis, a life-threatening condition due to hyperactivation of the immune system in response to infection, results in widespread inflammation and blood clotting. During sepsis, immune cells release sticky strands of DNA that block blood vessels and damage organs. Two different enzymes in the blood (DNase I and DNase1L3) can digest these DNA strands, and may represent a new class of anti-sepsis drugs. Our goal was to determine how heparins, commonly used blood thinners, alter the function of these enzymes. We found that (a) larger-sized heparins improved the activity of DNase I towards DNA-histone complexes and do not require any specific portion of heparin, (b) DNase I is more efficient than DNase1L3 in digesting DNA strands released from immune cells, and (c) levels of DNase I and DNase1L3 are altered in septic patients. Taken together, our studies provide new insights into how these enzymes function.

Osteoarthritis (OA) is a debilitating, degenerative joint disease that affects over 32.5 million adults in the United States and nearly 595 million people globally. OA is a major cause of pain and disability and is among the most expensive conditions to treat, carrying an annual healthcare cost of over $16.5 billion. The disease has classically been characterized by the degradation of articular cartilage and subchondral bone; however, changes to the synovium have recently garnered appreciation as synovitis has been linked to OA symptoms and progression. While the importance of the synovium in diarthrodial joint health and pathology is now widely accepted, quantitative structure-function data remains sparse. There is a need to investigate synovium structure-function relationships to better understand the synovium’s role in joint homeostasis and disease. The role of sex-based differences in OA has gained attention as epidemiological studies reveal that the incidence and prevalence of OA is higher in women than in men. Sex as a variable has rarely been considered in preclinical animal studies and in vitro laboratory experiments that explore the mechanisms of OA development and progression. Furthermore, therapeutic approaches for the treatment of OA have not adequately considered sex-based differences. As the population of those at risk for OA grows, the influence of sex-based differences in OA warrants more attention, particularly in the regenerative strategies for cartilage repair. This dissertation seeks to address persistent questions regarding OA etiology and the mechanisms underlying disease progression, as well as strategies to enhance cartilage tissue engineering therapies. The objectives of this dissertations are three-fold: (1) to further the understanding of synovium tribology (2) to develop a tissue-engineered (TE) human synovium to facilitate the study of synovium structure-function relationships and (3) to elucidate sex-based differences in cartilage regenerative medicine strategies. In Chapter 2, we assess the hypothesis that tissue glycosaminoglycan (GAG) content contributes to the low friction properties of the synovium. Bovine and human synovium tribological properties were evaluated using a custom friction testing device. Following proteoglycan depletion, synovium friction coefficients increased while GAG content decreased. In a second study, synovium samples were treated with interleukin-1 (IL) to observe inflammatory-induced structural changes. IL treatment elevated GAG concentration and decreased friction coefficients. No changes to collagen content were observed following IL treatment. For the first time, a relationship between synovium friction coefficient and GAG concentration was demonstrated. The study of synovium tribology is necessary to fully understand the mechanical environment of the healthy and diseased joint. Chapter 3 documents the development of a human TE synovium and its ability to recapitulate native tissue properties and responses to chemical stimuli. A mixed donor population of primary human fibroblast-like synoviocytes was combined with a commercially available extracellular protein mixture to fabricate TE synovium constructs. At baseline, mature TE synovium exhibited characteristics of native synovium such as the formation of an intimal lining and the expression of critical proteins like lubricin, cadherin-11, and collagen type IV. In response to IL and dexamethasone treatment, TE synovium underwent biochemical changes that mimicked the changes observed in human explants. In addition, solute transport measurements were performed to highlight the relationship between synovium extracellular matrix (ECM) composition and its functional properties, resulting in a proposed link between tissue GAG content and diffusion coefficient. A human TE synovium enables the investigation of synovium structure-function relationships in a controlled manner and can serve as a platform for disease modeling and drug screening, which may accelerate the development of new treatments for maintaining joint health that specifically target the synovium. In Chapter 4, sex-based differences in the ECM properties of canine engineered cartilage and in its degradative response to IL insult are evaluated. Isolated chondrocytes from male or female cartilage donors were encapsulated in agarose to create cylindrical cartilage constructs. Mechanical and biochemical measurements demonstrated that the sex of the donor chondrocytes did not influence intrinsic, de novo tissue formation after 42 days of tissue maturation. Following IL treatment, the mechanical, biochemical, and media analyses revealed that the sex of the donor cells did not influence the engineered cartilage’s response to IL insult. By understanding how sexual dimorphism impacts cartilage growth and susceptibility to proinflammatory cytokine insult, we may better direct cell-based strategies for cartilage repair that are personalized to account for patient sex.

Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells and early osteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECMwasmost prominent in early osteoblasts. [ABSTRACT FROM AUTHOR]

Este estudo avaliou a efetividade de uma nova membrana bioabsorvível com propriedades mecânicas e químicas mais adequadas para o uso em tendões. A avaliação foi realizada em coelhos submetidos a reconstrução da porção gastrocnêmia do tendão calcâneo. Foi feita uma avaliação prévia das propriedades mecânicas da membrana com uso de dinamômetro digital que demonstrou capacidade de deformação elástica mínima de 100%. Todos os coelhos foram submetidos a tenotomia e reparo da porção gastrocnêmia do tendão direito, após isso foram randomicamente separados em grupos envelopado com membrana e controle. A extração foi realizada nos seguintes períodos: sete, 14 e 28 dias. A avaliação foi realizada através da macroscopia, histologia, mensuração objetiva do colágeno à luz polarizada pelo image-J®, mensuração de glicosaminoglicanos sulfatados e expressão gênica de proteoglicanos. Na avaliação macroscópica o grupo com membrana apresentou menos aderência e melhor direcionamento das fibras e tecido mais homogêneo em 14 e 28 dias, p=0,02 e 0,03 respectivamente. Na histologia a Classificação de Watkins modificada apresentou as seguintes médias: 14,67±0,42 membrana e 12,67±0,56 sem membrana, p=0,03 em 14 dias, e 19,88±0,83 membrana e 17,25±0,62 sem membrana, p=0,02 em 28 dias. Na mensuração do colágeno as médias dos valores de cinza(mvc) o colágeno tipo III foram de 17,97±1,83 membrana e 12,63±1,07 sem membrana p=0,03 em 14 dias. Para o colágeno tipo I as médias foram de 2,41±0,33mvc membrana e 1,31±0,18mvc sem membrana p=0,01 em 14 dias e 7,30±0,63mvc membrana e 2,92±0,32mvc sem membrana p < 0,0001 em 28 dias. A média dos GAGs foi avaliada em três porções do tendão, proximal, central e distal, em ug/mg de tecido seco. Em sete dias apresentou diferença significativa apenas na porção distal 0,80±0,04 com e 0,38±0,04 sem membrana para condroitin-sulfato em 14 dias não apresentou diferenças entre os grupos. O dermatan-sulfato apresentou diferença estatisticamente significante em 7 dias apenas na porção central 0,42±0,09 com membrana e 1,29±0,67 sem membrana p=0,02. Em 14 dias não foram observadas diferenças entre os grupos. Houve grande variabilidade na expressão gênica no teste das amostras com beta-Actina e GAPDH levando a resultados inconclusivos ou não variação entre os grupos que pode sugerir não variabilidade na expressão gênica dos GAGs no período de 28 dias. Os dados fornecidos por esse trabalho mostram que a envelopagem com a membrana bioabsorvível promoveu aceleração dos processos cicatriciais da porção gastrocnêmia do tendão calcâneo de coelhos / This study assessed the effectiveness of a new absorbable membrane, that presents mechanical and chemical features more suitable to tendons, in rabbit tendons. Before the animal model assessments a mechanical study of the membrane was carried out demonstrating that the minimal capability for elastic deformation of the membrane was more than 100%. All rabbits underwent to tenotomy and reconstruction of the right gastrocnius tendons, thereafter they were randomly divided in tendon wrapped by the membrane and control groups. Extraction was performed in the following periods of time: seven, 14 and 28 days. Assessments used macroscopy, histology, objective collagen assessment by using polarized light and Image-J® program in mean of gray values(mgv), sulphated glycosaminoglycans, genetic expression of proteoglycans. In the macroscopic 14 and 21-day assessments the membrane group presented less adherences p=0.02 and p=0.03 respectively. The modified Watkins classification: 14,67±0,42 membrane and 12,67±0,56 without membrane p=0,03 for 14 days; 19,88±0,83 membrane and 17,25±0,62 without membrane p=0,02 for 28 days. The type III collagen were 17,97±1,83 membrane and 12,63±1,07 without membrane p=0,029 for 14 days. For type I collagen were 2,41±0,33 membrane and 1,31±0,18 without membrane p=0,01 for 14 days and 7,30±0,63 memebrane and 2,92±0,32 without membrane p < 0,0001 for 28 days. The glycosaminoglycans were measured in 3 tendon portions, distal, central and proximal, by using ug/mg of dry tissue. In seven days just the distal part presented statistical differences 0,80±0,04 membrane and 0,38±0,04 without membrane, for 14 days no differences were found for Chondroitin-Sulphate. For Dermatan-Sulphate the central part of the tendon 0,42±0,09 and 1,29±0,67 p=0,02, for 14 days no differences were found. There was high variability for beta-actin and GAPDH for the samples in 28 days with inconclusive results that may mean no variability in gene expression of GAGs at this time period. Results as mentioned above demonstrated that the wrapped tendons by the new membrane presented acceleration in the healing processes for gastrocnemius tendons of New Zealand Rabbits

No infarto, o remodelamento da região ventricular remota tem sido pouco examinado. Previamente, observamos que a redução da pressão de perfusão coronariana (PPC) associa-se ao aparecimento de fibrose subendocárdica. O objetivo do presente estudo foi investigar o papel da PPC no remodelamento e na função ventricular esquerda após o infarto. Medidas hemodinâmicas foram determinadas em ratos Wistar imediatamente após o infarto (IM) ou cirurgia fictícia (SH) e ao final dos seguimentos de 1, 3, 7, e 28 dias. Cortes teciduais do coração foram submetidos a coloração com HE, Sirius red, e a imunohistoquímica para a-actina. Duas regiões distintas do ventrículo esquerdo remotas ao infarto foram examinadas: subendocárdio (SE) e interstício (INT). A necrose de miócitos, a infiltração leucocitária e de miofibroblastos, e a fração de volume do colágeno foram determinadas. Comparados com SH, os grupos IM apresentaram menor PPC e disfunção sistólica e diastólica do ventrículo esquerdo. A necrose foi evidente em SE no dia 1. A inflamação e a fibroplasia ocorreram predominantemente em SE até o dia 7. A fibrose foi restrita a SE e evidente desde o dia 3. Em INT, a inflamação ocorreu predominantemente nos dias 1 e 3, mas em menor grau comparada a SE. A queda da PPC associou-se à dilatação progressiva do ventrículo esquerdo. Em SE, a PPC relacionou-se inversamente com a necrose (r =- 0,65, p = 0,003, no dia 1), com a inflamação (r = -0,76, p < 0,001, no dia 1), com a fibroplasia (r = -0,47, p = 0,04, no dia 7) e com a fibrose (r = -0,83, p < 0,001, no dia 28). A necrose no dia 1, a inflamação nos dias 3 e 7 e a fibroplasia no dia 7 relacionaram-se inversamente com a função do ventrículo esquerdo. A PPC é um fator determinante da integridade de SE e interfere com o remodelamento e a função do ventrículo esquerdo. / Left ventricular (LV) subendocardial remodeling has been poorly investigated after myocardial infarction. Previously, we have demonstrated that low coronary driving pressure (CDP) early in the course of infarction was associated with the subsequent development of subendocardial fibrosis. The present study aimed at the investigation of the role of CDP in LV remodeling and function following infarction. Hemodynamics were determined in Wistar rats immediately after infarct surgery (MI group) or sham (SH group) and at days 1, 3, 7, and 28. Heart tissue sections were stained with HE, Sirius red and immunostained for a- actin. Two distinct LV regions remote to MI were examined: subendocardium (SE) and interstitium (INT). Myocyte necrosis, leukocyte and myofibroblast infiltration, and collagen volume fraction were determined. Compared with SH, MI groups showed lower CDP and LV systolic and diastolic dysfunction. Necrosis was evident in SE at day 1. Inflammation and fibroplasia occurred predominantly in SE as far as day 7. Fibrosis was restricted to SE and was evident beginning from day 3. Inflammation occurred predominantly at days 1 and 3 in INT, but at a lower extent than in SE. CDP fall was associated with progressive LV dilatation. In SE, CDP correlated inversely with necrosis (r = -0.65, p=0.003, at day 1), inflammation (r = -0.76, p < 0.001, at day 1), fibroplasia (r = -0.47, p = 0.04, at day 7) and fibrosis (r = -0.83, p < 0.001, at day 28). Necrosis at day 1, inflammation at days 3 and 7, and fibroplasia at day 7 correlated inversely with LV function. CDP is a key factor to SE integrity following infarction and interferes with LV remodeling and function.

Les glycosyltransférases (GTs) sont une famille importante d’enzymes responsable de la biosynthèse des chaînes de glycosaminoglycane (GAG) des protéoglycanes, composants clés de la matrice extracellulaire et de la membrane plasmique cellulaire impliqués dans la communication, l'adhésion, la migration et la prolifération cellulaires. Les GTs jouent donc un rôle central dans de nombreux processus physiologiques et physiopathologiques tels que les cancers ou encore les maladies dégénératives et génétiques. Parmi ces GTs, la ß1,4-galactosyltransférase 7 (ß4GalT7) est une cible thérapeutique potentielle puisqu'elle : i) catalyse une étape précoce et majeure de la biosynthèse des chaînes de GAG, ii) est impliquée dans une forme rare de maladie génétique des tissus conjonctifs, le syndrome d’Ehlers-Danlos, iii) prend en charge des xylosides exogènes modulant son activité in vitro et in vivo. Ce travail de thèse s'organise sur une étude structure/fonction de cette enzyme afin de cerner les résidus d'acides aminés clés dans l'interaction de l'enzyme avec un substrat modèle, le 4-methylombelliferyl-ß-D-xylose (4-MOX). Les résidus Y194, Y196 et Y199 ont ainsi été identifiés comme clés dans l'architecture du site de fixation du substrat accepteur et dans l'interaction de l'enzyme avec le xyloside. Au contraire, les résidus H195, R226 et le résidu R270, muté dans la forme progéroïde du syndrome d'Ehlers-Danlos, apparaissent comme des résidus « modulant » l'activité de l'enzyme, notamment du fait d'interactions moléculaires impliquant leur squelette peptidique pour H195 et R226 et une boucle flexible pour R270. Ces travaux ont permis de guider la synthèse d’analogues xylosidiques visant à inhiber l'activité de la ß4GalT7 humaine. Parmi les propositions, un dérivé fluoré du 4-MOX apparaît comme un inhibiteur efficace de la ß4GalT7 in vitro et in cellulo. Faiblement cytotoxique, ce dérivé réduit la prolifération des cellules de lignées cancéreuses SW1353 et MB MDA 231. Ces résultats ouvrent la perspective de nouvelles stratégies thérapeutiques utilisant les xylosides comme agents potentiels dans le traitement de cancers ou encore des maladies génétiques des tissus conjonctifs / Glycosyltransferases (GTs) are an important family of enzymes involved in the biosynthesis of glycosaminoglycan (GAG) chains of proteoglycans which are key components of cell plasma membranes and of the extracellular matrix, and are thus implicated in cell communication, adhesion, migration and proliferation. GTs are thus key players in numerous pathophysiological processes such as cancers, degenerative and genetic diseases. Among these GTs, ß1,4-galactosyltransférase 7 (ß4GalT7) is a potential therapeutic target since : i) it catalyzes a rate-limiting step in the early phase of the GAG chains biosynthesis, ii) it is implicated in a rare genetic connective tissue disorder (Ehlers-Danlos Syndrome), iii) its in vitro and in vivo activity can be modulated by exogenous xyloside molecules. This PhD work is focused on a structure/function study of the enzyme aiming to identify key amino acid residues that interact with 4-methylumbelliferyl-ß-D-xylose (4-MUX), taken as reference substrate. Y194, Y196 and Y199 have been identified as key residues for the architecture of the acceptor substrate binding site and establish interactions with 4-MUX. By contrast, H195, R226 and R270, a residue mutated in the progeroid form of Ehlers-Danlos Syndrome, should rather be considered as “modulating” residues towards the ß4GalT7 activity, H195 and R226 interacting with 4-MUX with their polypeptide backbone, and R270 via a flexible loop. This work guided the design of xylosidic compounds that would potentially inhibit the ß4GalT7 activity. Thus, a fluorinated derivative of 4-MUX appeared as an efficient in vitro and in cellulo inhibitor of the enzyme. Poorly cytotoxic, this compound also reduced the proliferation rate of cancer cells SW1353 and MB MDA 231. Altogether, these results offer new therapeutic strategies using xylosides as potential therapeutic agents in the treatment of cancer or rare genetic disorders