CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A viscossuplementaÃÃo à uma tÃcnica terapÃutica utilizada no tratamento de osteoartrite, a qual busca substituir o lÃquido sinovial (lubrificante das articulaÃÃes) por soluÃÃes viscoelÃsticas de derivados do Ãcido hialurÃnico com propriedades reolÃgicas melhores que a deste lÃquido. Este tratamento à caro e comercialmente sà se utilizada o Ãcido hialurÃnico. O estudo de outros polissacarÃdeos, tais como a goma guar e a quitina, para aplicaÃÃo em viscossuplementaÃÃo, aumenta a possibilidade de substituir o Ãcido hialurÃnico por outros de fontes mais abundantes e viÃveis. A goma guar (GGNP) foi purificada por vÃrios mÃtodos com a finalidade de reduzir contaminantes, como polissacarÃdeos (que nÃo galactomanana) e principalmente proteÃnas. O mÃtodo de maior eficiÃncia na reduÃÃo destes contaminantes foi o mÃtodo fÃsico+Fehling, resultando em uma guar (GGFISFEH) com 96% de galactomanana e 0% de proteÃnas. A goma comercial (GGNP) e a purificada (GGFISFEH) foram reticuladas com glutaraldeÃdo para originar hidrogÃis fluidos de alta viscosidade. O aumento da viscosidade do polissacarÃdeo com o tempo de reaÃÃo de reticulaÃÃo à gradativo e pode ser interrompido no momento em que se obtÃm um hidrogel fluido, por diÃlise contra Ãgua. Os hidrogÃis obtidos tem viscosidade quarenta vezes (GelGGNP) e oitocentas vezes (GelGGFISFEH) a viscosidade das soluÃÃes iniciais das gomas. A goma guar purificada (GGFISFEH) foi modificada por reaÃÃo de oxidaÃÃo com reagente TEMPO e por reaÃÃo de sulfataÃÃo, com a finalidade de introduzir grupos carboxilato e sulfato, assemelhando a estrutura desta ao Ãcido hialurÃnico e ao condroitim sulfato. As reaÃÃes foram eficientes na modificaÃÃo, resultando em grau de substituiÃÃo (GS) de 0,36 para a goma oxidada (GGOXID) e 0,60 para a sulfatada (GGSULF). AnÃlises de RMN de 13C e 1H indicam que a sulfataÃÃo ocorreu no C6 da galactose e a oxidaÃÃo no C6 da manose. A quitina foi carboximetilada para originar um polieletrÃlito solÃvel em Ãgua e com grupo acetil glicosamina semelhante ao do Ãcido hialurÃnico. Esta reaÃÃo foi testada em vÃrias condiÃÃes reacionais, resultando em GS diferentes. O melhor rendimento foi de 92% com um GS de 0,53. Os testes farmacolÃgicos foram realizados em ratos Wistar. Avaliou-se o efeito per si na cavidade articular de GGNP e GGFISFEH, ambas em soluÃÃo e gel. A GGNP causou incapacitaÃÃo e influxo celular dosedependente, efeito nÃo demonstrado por GGFISFEH. A incapacitaÃÃo articular foi maior com a administraÃÃo do GelGGNP. Avaliou-se tambÃm, o efeito terapÃutico da GGFISFEH em soluÃÃo e gel, e Hilano G-F20 em osteoartrite induzida pelo modelo de transecÃÃo do ligamento cruzado anterior. A inibiÃÃo da incapacitaÃÃo articular obtida pelo gel ou pela soluÃÃo de guar foram absolutamente semelhantes ao resultado obtido com o Hilano G-F 20. A goma guar apresenta potencial como agente viscossuplementador. / The viscosupplementation is a therapeutic technique used in the osteoarthritis treatment, in which the sinovial fluid (lubricant of the ligaments) is replaced by viscoelastic solutions derived from hyaluronic acid with rheological properties better than the starting material. This treatment is expensive and commercially, only hialuronic acid is used. The research of other polysaccharides such as the guar gum and chitin, for the application on viscosupplementation, increases the possibility of substituting the hyaluronic acid with other from more abundant sources and more viable. The guar gum (GGNP) was purified through various methods with the aim to reduce contaminants, such as polysaccharides (but not galactomannan) and most importantly, proteins. The method with best efficiency in the reduction of these contaminants was the physic+Fehling method, resulting in a guar (GGFISFEH) with 96% of galactomannan and 0% of proteins. The commercial gum (GGNP) and the purified (GGFISFEH) were crosslinked with glutaraldehyde to originate fluids hydrogel of high viscosity. The increase in viscosity of the polysaccharides with the increase of the crosslinking reaction time is gradual and can be interrupted at the moment a hydrogel fluid is obtained through dialysis against water. The hydrogels obtained have viscosity forty times (GelGGNP) and eight hundred times (GelGGFISFEH) than the initial viscosities of the gums. The purified guar gum (GGFISFEH) was modified through oxidative reaction with a TEMPO reagent and through a sulfatation reaction, with the aim of introducing carboxylated and sulphate groups, making their structure similar to the one from the hyaluronic acid and the chondroitin sulfate. The reactions were efficient in the modification, resulting in degree of substitution (DS) of 0.36 for the oxidated gum (GGOXID) and 0.60 for the sulfated gum(GGSULF). 13C and 1H NMR analyses indicated that the sulfatation occurred in C6 of galactose and the oxidation on C6 of mannose The chitin was carboxylated to form a water soluble polyeletrolite and with a group of acetyl glucosamine similar to the hyaluronic acid. This reaction was tested in various conditions, resulting in different DS. The best yield was of 92% with a DS of 0.53. The pharmacological tests were conducted on rats Wistar. The effect, per si, in the articulate cavity of GGNP and GFISFEH, both in solution and gel was also studied. The GGNP caused incapacitation and dosage-dependant cell influx, an effect not demonstrated by GGFISFEH. The articular incapacitation was greater with the administration of GelGGNP. It was also evaluated the therapeutic effect of GGFISFEH in solution and gel, and Hilano G-F20 in osteoarthritis induced by the transection of the anterior cruciate ligament. The inhibition of the articular incapacitation obtained through gel or through the guar solution were entirely similar to the results obtained with the Hilano G-F20. The guar gum presents potential as a viscosuplemented agent.

No description available.

Sugar complexes with anions and cations are reviewed. Evidence is presented for the formation of a complex of stoichiometry 1:1 in aqueous solution between methyl-α-Dglucopyranoside and magnesium perchlorate. The complex may be detected by the method of continuous variations. Measurements of changes in the proton chemical shifts of the glucoside in dauterium oxide, indicate that the complex is probably formed between the vicinal hydroxyl groups at C-2 and C-3 of the pyranoside ring and the hydrated cation. At elevation temperatures there is evidence for the presence of a complex of stoichiometry 2:1. Experiments conducted on cellulose indicate the possible formation of cellulose - magnesium-ion complexes . These complexes provide an explanation for the "protective action" of mEgnesium compounds on the cellulose portion of pulp during alkali-oxygen bleaching.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

No description available.

No description available.

No description available.

Les films multicouches de polyélectrolytes sont des auto-assemblages de polymères chargés formant des films dont l'épaisseur peut être variée de quelques nm à quelques µm. Un nombre croissant de travaux concerne la compréhension de leur mécanisme d'auto-assemblage et leur utilité pour modifier les propriétés physico-chimiques, topographiques ou mécaniques de surface de (bio)matériaux. Dans cette thèse, nous avons étudié les propriétés de films à base de poly(L-lysine) et de polysaccharides connus pour leur rôle physiologique, notamment le hyaluronane, la chondroïtine sulfate et l'héparine. Les compositions internes de ces films mono-constituants ou à base de mélanges de polyélectrolytes ont été sondées. L'influence de la chimie des polyélectrolytes sur la formation des films, en particulier l'importance des groupements sulfates, a été mise en évidence. Leur potentiel comme vecteur de délivrance d'un facteur de croissance, la BMP-2, a été évalué. De fortes quantités de BMP-2 ont pu être chargées dans les films à base de hyaluronane. Nous avons pu contrôler les quantités insérées en faisant varier la composition chimique des films, leur épaisseur ou la concentration en BMP-2 de la solution de chargement. Puis nous avons mis en évidence une différenciation contrôlée de façon dose-dépendante de cellules C2C12 pluripotentes sur les films bioactifs : différenciation myogénique (en absence de facteur) ou ostéoblastique. De plus, nous montrons qu'un contact des cellules avec le film bioactif est nécessaire pour induire leur différenciation. La protéine est donc présentée par « la phase solide », ce qui constitue un mode de présentation du facteur proche des conditions physiologiques. Des résultats préliminaires obtenus en recouvrant des biomatériaux orthopédiques par les films bioactifs laissent penser que ces films offrent des perspectives intéressantes dans le domaine de la régénération osseuse in vivo. / Polyelectrolyte multilayer films are self-assembled architectures forming nm to µm thick films. During the last decade, they have emerged as an efficient way of modifying materials surface properties such as chemistry, physico-chemical properties, topography as well as mechanical properties. Thanks to the technology's versatility and ease of use, polyelectrolyte multilayer films are now recognized as a new tool for modifying biomaterial surfaces and mediating cell behaviours and implant bio-integration. In this thesis, we studied the properties of poly(L-lysine) and polysaccharide-based multilayer films and focused on their physical-chemical properties as well as on their internal composition. In particular, we studies the influence of their chemistry (presence of carboxylic or sulfate groups) on film formation and characteristics. Three polysaccharides with increasing sulfate group content were chosen for this purpose: hyaluronan, chondroitin sulfate and heparin. The capacity of these films to act as a drug delivery vehicle for BMP-2 (a growth factor able to induce osteo-differentiation) was then assessed. High BMP-2 amounts were successfully loaded and retained in the films in a controlled manner. The loaded amounts could be modulated by varying the film's chemistry, film thickness or BMP-2 concentration in the loading solution. We showed that it is possible to control the extent of C2C12 cell differentiation in osteoblasts when cultured on the bioactive films. Importantly, when no BMP-2 is loaded in the films, the cells differentiated in to myotubes, their most common differentiation pathway. Cells needed a direct contact with the bioactive films to respond to BMP-2, suggesting that BMP-2 is mainly presented to the cells from the solid phase. Preliminary in vivo tests on film-coated orthopaedic biomaterials are encouraging. They showed that these films are interesting candidates for surface modification of orthopaedic biomaterials and may foster bone regeneration.

CARACTERISATION DE VACCINS A BASE DE GLUCIDESVariables influençant l'immunogénicité et propriétés physico-chimiques des vaccins glycoconjuguésDe nombreux aspects peuvent influer sur l'immunogénicité des vaccins conjugués et les principales variables étudiées jusqu'ici sont la taille du fragment saccharide et la nature des glycosides: taux de protéine dans le conjugué purifié, la stratégie de conjugaison, nature de l’espaceur et la protéine porteuse.La taille de la partie saccharidique et le ratio de cette partie / protéine a été étudiée dans différents travaux de Seppälä et Mäkelä,.Dans l'une des premières études sur l'effet de la taille sur l'immunogénicité de la protéine conjuguées à des dextrans, il a été montré que des dextrans de faible poids moléculaire conjugué à l’albumine sérique de poluet induit des réponses anti-dextran fortes chez la souris. L’augmentation de la taille du dextran, a abouti à une réduction de l’immunogénicité. Peeters et al. a montré qu’un tétramère synthétique de Hib unité capsulaire polysaccharide, conjuguée à un support protéique, induit chez des souris adultes, des niveaux d'anticorps primates non humains comparables à un conjugué Hib commercial. Ces niveaux sont plus élevées que ceux induits par un trimère répété, ce qui indique que pour le Hib un minimum de huit sucres est nécessaire pour une bonne réponse immunologique. Laferrière et al. a trouvé peu d'influence de la longueur de la chaîne glucidique sur l'immunogénicité des vaccins antipneumococciques conjugués chez la souris. Pozsgay et al. a étudié chez la souris, l'immunogénicité de l’oligosaccharides du LPS de Shigella dysenteriae de type 1 conjugué à l'albumine sérique humaine (HSA). Les auteurs ont constaté que les octa-, dodéca-, et des fragments de hexadécasaccharides induit des niveaux élevés d'anticorps IgG après trois injections. Ces niveaux sont supérieurs à ceux obtenus avec un conjugué tétrasaccharidique. L'influence du ratio glucides / protéine est différente pour les trois conjugués. Le conjugué octasaccharide-HSA avec la plus forte densité provoque une bonne réponse immunitaire, tandis que dans le cas des conjugués dodéca- et hexadécasaccharides, la densité médiane est optimal. Ces études suggèrent que la longueur de la chaîne d'oligosaccharides et le chargement de l’haptène peuvent être liés entre eux pour déterminer l'immunogénicité des vaccins glycoconjugués.L'espaceur est une molécule linéaire courte qui est généralement liée à la chaîne polysaccharidique et la protéine ou de fragments. Il ya des évidences dans la littérature qui suggèrent que les espaceurs rigides, contraints comme le maléimide cyclohexyle, provoquent une importante quantité d'anticorps indésirables, avec le risque de conduire à une réponse immunitaire éloignée de l'épitope ciblé sur la haptène. L'utilisation d'un alkyle souple type maleimido a été rapporté comme un moyen de surmonter l'immunogénicité observée précédemment. Un certain nombre de transporteurs protéiques ont été utilisés jusqu'ici dans l'évaluation préclinique et clinique de vaccins conjugués. Des protéines telles que les anatoxines diphtériques et tétaniques, qui dérivent des toxines respectives, après la décontamination chimique avec le formaldéhyde, ont été initialement choisies comme transporteur en raison de inocuité (tétanos et la vaccination contre la diphtérie). CRM 197, un mutant non toxique de la toxine 61 de la diphtérie a été largement utilisé comme support pour Hib. Un complexe protéique de la membrane externe de méningocoque du sérogroupe B a été utilisé par Merck comme support pour leur vaccin conjugué Hib. GSK dans leur vaccin antipneumococcique, conjugué multivalent, introduit la protéine D Hib liée à la plupart des polysaccharides inclus dans le vaccin. L'équipe de John Robbins fait un large usage de la forme recombinante non toxique de l’exo-toxine de Pseudomonas aeruginosa comme support contre Staphylococcus aureus de type 5 et 8 ainsi que pour Salmonella. / CHARACTERIZATION OF CARBOHYDRATE BASED VACCINES Variables influencing the immunogenicity and physicochemical properties of glycoconjugate vaccinesMany aspects can influence the immunogenicity of conjugate vaccines and the main variables investigated so far are the size of the saccharide moiety, the saccharide:protein ratio in the purified conjugate, the conjugation strategy, the nature of the spacer and the protein carrier. The size of the saccharide moiety and saccharide/protein ratio were investigated in different works such as Seppälä and Mäkelä in one of the first studies on the effect of size and chemistry on the immunogenicity of dextrans-protein conjugates found that dextrans of low molecular weight conjugated to chicken serum albumin, induced strong anti-dextran responses in mice, while increasing the dextrans' size resulted in reduced immunogenicity.47 Peeters et al. showed that a synthetic tetramer of Hib capsular polysaccharide repeating unit, conjugated to a protein carrier, induced in adult mice and non-human primates antibody levels comparable to a commercial Hib conjugate and higher than those induced by a trimer, indicating that for Hib a minimum of eight sugars is needed for a proper immunological response.48 Laferriere et al. found little influence of the carbohydrate chain length on the immunogenicity of pneumococcal conjugate vaccines in mice.49 Pozsgay et al. studied the immunogenicity in mice of synthetic Shigella dysenteriae type 1 LPS oligosaccharides conjugated to human serum albumin (HSA). The authors found that octa-, dodeca-, and hexadecasaccharide fragments induced high levels of lipopolysaccharide binding IgG antibodies in mice after three injections and were superior to a tetrasaccharide conjugate. The influence of the carbohydrate/protein ratio was different for the three conjugates. The octasaccharide-HSA conjugate with the highest density evoked a good immune response, while in the case of dodeca- and hexadecasaccharide conjugates, the median density was optimal.50 These studies suggest that oligosaccharide chain length and hapten loading might be interconnected in determining the immunogenicity of glycoconjugate vaccines. The spacer is a short linear molecule that is generally linked to the polysaccharide chain or to the protein or to both moieties, depending on the chemistry, used to facilitate the coupling between the protein and sugar. There are evidences in the literature which suggest that rigid, constrained spacers like cyclohexyl maleimide, elicit a significant amount of undesirable antibodies, with the risk of driving the immune response away from the targeted epitope on the hapten.51 52 The use of a flexible alkyl type maleimido spacer has been reported as a way to overcome the previous observed immunogenicity of cyclic maleimide linkers.53 A number of protein carriers have been used so far in preclinical and clinical evaluation of conjugate vaccines. 54 55 56 57 58 59 60Proteins such as diphtheria and tetanus toxoids, which derive from the respective toxins after chemical detoxification with formaldehyde, were initially selected as carrier because of the safety track record accumulated with tetanus and diphtheria vaccination. CRM197, a non-toxic mutant of diphtheria toxin61 which instead does not need chemical detoxification, has been extensively used as carrier for licensed Hib, pneumococcal, meningococcal conjugate vaccines and for other vaccines being developed. An outer membrane protein complex of serogroup B meningococcus has been used by Merck as carrier for their Hib conjugate vaccine.62 GSK in their multivalent pneumococcal conjugate vaccine introduced the use of the Hib-related protein D as carrier for most of the polysaccharides included into the vaccine.63 64 The team of John Robbins made extensive use of the recombinant non toxic form of Pseudomonas aeruginosa exo-toxin as carrier for Staphylococcus aureus type 5 and 8 as well as for Salmonella

Des solutions d'acides carboxyliques contenant principalement de l'acide formique sont obtenues par oxydation photo-Fenton des polysaccharides. Une irradiation dans le visible de 60 W sous des conditions douces suffit à fournir des résultats reproductibles. Les produits d'oxydation issus d'amidon de pomme de terre et d'amidon de blé ont montré des capacités de complexation comparables aux solutions commerciales d'acide gluconique et glucuronique.Un système d'oxydation innovant, comprenant des sels d'halogénures et du DMSO, convertit quantitativement le HMF en DFF. Cette méthode permet aussi la transformation one-pot du fructose en DFF avec de bons rendements. Selon l'étude mécanistique, le bromo-HMF serait un intermédiaire réactionnel. / Solutions of carboxylic acids, containing mainly formic acid, are produced under photo-Fenton conditions. Visible irradiation with a 60 W spot is sufficient to provide reproducible results under mild conditions. The oxidation products of potato starch and wheat starch have shown Ca sequestering properties similar to those of gluconic and glucuronic acids.Using halide salts and DMSO, an innovating method has been elaborated for the selective oxidation of HMF to DFF with quantitative yields. The one-pot transformation of fructose to DFF occurs with fair yields. Based on the mechanistic study, Br-HMF would be the reaction intermediate.