Heparan sulfate (HS), a linear negatively charged polysaccharide located at the cell surface and in the extracellular matrix, interacts with, and thereby regulates the functions of numerous proteins. HS-protein interactions depend on the fine structure of HS, especially its sulfation pattern. This thesis aimed to understand how differently sulfated domains in HS are generated. Specifically, the substrate specificities of HS hexuronic acid 2-O-sulfotransferase (2OST) and HS glucosaminyl 6-O-sulfotransferases (6OSTs) were investigated. Three different 6OSTs (6OST1-3) have been cloned and characterized. To study the mechanisms controlling 6-O-sulfation we incubated the recombinant purified 6-OST isoforms with different 6-O-desulfated poly- and oligosaccharide substrates and the active sulfate donor 3'-phosphoadenosine 5'-phospho[35S]sulfate (35S-labeled PAPS). All three enzymes catalyzed 6-O-sulfation of both N-acetylated (GlcNAc) as well as N-sulfated (GlcNS) glucosamines next to a nonreducing iduronic acid (IdoA) or glucuronic acid (GlcA). Similar specificities were demonstrated, although some differences in substrate preferences were noted. To understand how pre-existing 2-O-sulfates affects 6-O-sulfation, 6OST2 and 6OST3 were incubated with pair-wise mixed octasaccharide substrates with different contents of 2-O-sulfates. The specificities for substrates with two or three 2-O-sulfates were higher compared to octasaccharides with no or one 2-O-sulfate indicating that 2-O-sulfate groups substantially promote the subsequent 6-O-sulfation. Overexpression of the 6OSTs in a mammalian cell line resulted in increased 6-O-sulfation of -GlcA-GlcNS- and -GlcA-GlcNAc- sequences. The results were not isoform specific, but affected by the overexpression level. The 2OST catalyzes 2-O-sulfation of both IdoA and GlcA residues, with high preference for IdoA units. To study how 2-O-sulfation of GlcA and IdoA is regulated, we incubated the enzyme with different substrates and 35S-labeled PAPS. Our findings revealed that the 2OST almost exclusively sulfated IdoA also with a ratio of GlcA to IdoA of 99:1, suggesting that 2-O-sulfation of GlcA occurs before IdoA is formed.

Thesis (doctoral)--Christian-Albrechts-Universität zu Kiel, 1997.

Submitted by MARCIO TULIO MATHEUS SANTOS null (marciotulio@globo.com) on 2018-04-03T12:14:14Z No. of bitstreams: 1 DISSERTAÇÃO - MARCIO TULIO - 26-03-2018 CORRIGIDA VF.pdf: 2812609 bytes, checksum: 292ece5efee66c9b2d09ec3d1956148c (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-04-03T12:55:34Z (GMT) No. of bitstreams: 1 santos_mtm_me_bot.pdf: 2812609 bytes, checksum: 292ece5efee66c9b2d09ec3d1956148c (MD5) / Made available in DSpace on 2018-04-03T12:55:34Z (GMT). No. of bitstreams: 1 santos_mtm_me_bot.pdf: 2812609 bytes, checksum: 292ece5efee66c9b2d09ec3d1956148c (MD5) Previous issue date: 2018-02-23 / Os biopolímeros podem combinar a degradabilidade “in vivo” com a boa biocompatibilidade e, desta forma, podem ser usados como agentes encapsulantes com o objetivo de libertarem compostos bioativos. Dentro do grupo dos biopolímeros, os polissacarídeos apresentam vantagens importantes, como alta estabilidade, segurança, hidrofilicidade e biodegradabilidade. Os polissacarídeos têm sido objetos de estudos, apresentando resultados promissores. Este trabalho tem como objetivo, analisar o comportamento de uma matriz biopolimérica a base de amido, quando submetida à radiação ionizante visando seu uso em moldes planares para radioterapia de lesões cutâneas. Foram confeccionadas amostras de matrizes biopoliméricas de amido, constituído de água, amido, glicerina, duas variedades de açúcares na forma de pó, totalmente solúveis em água e produzida através da conversão do amido de milho. Durante o preparo das amostras variou-se as formulações para se obter melhor resistência da película plástica formada. As formações dos filmes de amido, foram realizadas através do método Casting com baixa viscosidade. As amostras foram submetidas à radiação em um acelerador linear clínico (linac) com feixes de raios-X de 10 MV e feixe de elétrons de 4 MeV, 6 MeV, 9 MeV e 12 MeV, e em uma fonte telecobaltoterapia com 1,25 MeV de energia. A dose total de radiação aplicada variou de 20 Gy a 140 Gy, um grupo controle foi mantido sem exposição à radiação. Após o processo de irradiação, foi realizada uma dosimetria das amostras, usando câmaras de ionização e detector Geiger Muller, não houve leitura de radiação residual nas amostras. Em seguida, as amostras foram submetidas a análise espectrofotométrica para avaliação de sua estabilidade e verificar se a radiação induzida alterou os grupos orgânicos e a estrutura primária do biomaterial. A análise de FTIR, mostrou que a radiação aplicada sobre o molde biopolimérico não alterou sua estrutura ou grupos químicos, sendo preservadas as propriedades plastificantes do biomaterial. / Biopolymers can combine "in vivo" degradability with good biocompatibility and, in this way, can be used as encapsulating agents in order to release bioactive compounds. Within the group of biopolymers, polysaccharides have important advantages, such as high stability, safety, hydrophilicity and biodegradability. Polysaccharides has been object of studied, presenting promising results. This work aims to study the properties of biopolymers for use in pharmaceutical applications in the treatment of pathologies and to analyze the behavior of the biopolymer matrix based on starch when submitted to ionizing radiation for its use in planar models for radiotherapy of cutaneous lesions. An extensive bibliographic research was carried out aiming to understand the characteristics, properties and applications of the biopolymers in the health area, having as starch the base of the polymer matrices. Samples of biopolymeric were made matrices, consisting of water, starch, glycerin, two sugar varieties in the form of powder, totally soluble in water and produced through the conversion of corn starch were made. During the preparation of the samples the formulations were varied to obtain better resistance of the plastic film formed. The formations of the starch films were carried out using the casting method with low viscosity. The samples were subjected to radiation in a clinical linear accelerator (linac) with x-ray beams of 10 MV and electron beam of 4 MeV, 6 MeV, 9 MeV and 12 MeV, and in a telecobaltotherapy source with 1.25 MeV of energy. The total applied radiation dose varied from 20 Gy at 140 Gy, a control group was maintained without radiation exposure. After the irradiation process, a dosimetry of the samples was carried out, using ionization chambers and Geiger Muller detector, there was no residual radiation reading in the samples. The samples were then subjected to spectrophotometric analysis to evaluate their stability and to verify if the induced radiation altered the organic groups and the primary structure of the biomaterial. The FTIR analysis showed that the radiation applied on the biopolymer mold did not alter its structure or chemical groups, and the plasticizing properties of the biomaterial were preserved.

Orientador: Marco Antônio Rodrigues Fernandes / Resumo: Os biopolímeros podem combinar a degradabilidade “in vivo” com a boa biocompatibilidade e, desta forma, podem ser usados como agentes encapsulantes com o objetivo de libertarem compostos bioativos. Dentro do grupo dos biopolímeros, os polissacarídeos apresentam vantagens importantes, como alta estabilidade, segurança, hidrofilicidade e biodegradabilidade. Os polissacarídeos têm sido objetos de estudos, apresentando resultados promissores. Este trabalho tem como objetivo, analisar o comportamento de uma matriz biopolimérica a base de amido, quando submetida à radiação ionizante visando seu uso em moldes planares para radioterapia de lesões cutâneas. Foram confeccionadas amostras de matrizes biopoliméricas de amido, constituído de água, amido, glicerina, duas variedades de açúcares na forma de pó, totalmente solúveis em água e produzida através da conversão do amido de milho. Durante o preparo das amostras variou-se as formulações para se obter melhor resistência da película plástica formada. As formações dos filmes de amido, foram realizadas através do método Casting com baixa viscosidade. As amostras foram submetidas à radiação em um acelerador linear clínico (linac) com feixes de raios-X de 10 MV e feixe de elétrons de 4 MeV, 6 MeV, 9 MeV e 12 MeV, e em uma fonte telecobaltoterapia com 1,25 MeV de energia. A dose total de radiação aplicada variou de 20 Gy a 140 Gy, um grupo controle foi mantido sem exposição à radiação. Após o processo de irradiação, foi realizada uma dosime... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Biopolymers can combine "in vivo" degradability with good biocompatibility and, in this way, can be used as encapsulating agents in order to release bioactive compounds. Within the group of biopolymers, polysaccharides have important advantages, such as high stability, safety, hydrophilicity and biodegradability. Polysaccharides has been object of studied, presenting promising results. This work aims to study the properties of biopolymers for use in pharmaceutical applications in the treatment of pathologies and to analyze the behavior of the biopolymer matrix based on starch when submitted to ionizing radiation for its use in planar models for radiotherapy of cutaneous lesions. An extensive bibliographic research was carried out aiming to understand the characteristics, properties and applications of the biopolymers in the health area, having as starch the base of the polymer matrices. Samples of biopolymeric were made matrices, consisting of water, starch, glycerin, two sugar varieties in the form of powder, totally soluble in water and produced through the conversion of corn starch were made. During the preparation of the samples the formulations were varied to obtain better resistance of the plastic film formed. The formations of the starch films were carried out using the casting method with low viscosity. The samples were subjected to radiation in a clinical linear accelerator (linac) with x-ray beams of 10 MV and electron beam of 4 MeV, 6 MeV, 9 MeV and 12 MeV, and in ... (Complete abstract click electronic access below) / Mestre

LAGE, Luis Paulo Araújo. Avaliação dos imunoparâmetros do camarão branco Litopenaeus vannamei submetido a banhos de imersão com polissacarídeos sulfatados de Spirulina platensis. 2011. 66 f . : Dissertação (mestrado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Engenharia de Pesca, Fortaleza-CE, 2011 / Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-19T13:27:36Z No. of bitstreams: 1 2011_dis_lpalage.pdf: 566157 bytes, checksum: 76d1f5323c56ff43c8b9dccac48a34d9 (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-19T13:28:33Z (GMT) No. of bitstreams: 1 2011_dis_lpalage.pdf: 566157 bytes, checksum: 76d1f5323c56ff43c8b9dccac48a34d9 (MD5) / Made available in DSpace on 2016-07-19T13:28:33Z (GMT). No. of bitstreams: 1 2011_dis_lpalage.pdf: 566157 bytes, checksum: 76d1f5323c56ff43c8b9dccac48a34d9 (MD5) Previous issue date: 2011 / Aquaculture is the food-producing activity that presented the fastest grow the worldwide and the cultivation of marine shrimps had the highest average growth rate over the past 50 years. The search for growing up the production, obtained through the increase on the density, carrying the animals on condition stress and the appearance of diseases. The use of immunostimulants on aquaculture, like the sulfated polysaccharides (SP), resulted in an enhancement of the animal’s resistance against pathogenic microorganism and stress situations. The aim of this study was to evaluate the effect of the SP of Spirulina platensis in post-larvae (PL's) and juvenile of the white shrimp Litopenaeus vannameithrou gh immersion baths. The administration of the SP to L. vannamei’s PL's was performedat concentrations of 0 (control), 50, 100 and 200 mg L-1, two hours before the evaluation of their resistance to the salt stress test. The juveniles of L. vannamei were exposed for three hours to the SP of S. platensisat concentrations of 0 (control), 60, 120 and 240 mg L-1 followed by a hemolymph collection for analysis of the following immune parameters: haemocytes total (THC) and differential (DHC) counts, serum protei n concentration, activity of phenoloxidase (PO), activity of α2-macroglobulin (α2-M) and serum hemagglutinating activity. After the salt stress test it was observed that the PL’s that were exposed to the SP at a concentration of 100 mg L -1 showed a tendency of a higher survival rate but there was not any significant difference between the treatments. Regarding the immune parameters, THC was reduced when the animals were exposed to the SP, while the exposure of shrimp to higher concentrations of PS resulted in normal values of the THC. However, no significant differen cebetween treatments and control. But, the significant difference(p≤0.05) was observed between the treatments that were exposed to PS. The number of granular haemocytes (GH) showed adownward trend in the concentration of 60 mg L -1 and, with increasing concentration of SP, the values of HG also tended to increase, but without significant differences compared to that of the control. However, significant differences were observed between the number of HG at concentrations of 60 and 240 mg L - 1. The PO activity was significantly reducedat a concentration of 60 mg L - 1 which may be related to the low value of THC as well as the high activity of the protease inhibitor α2 -M. The PO activity was again similar to that obtained in control with increasing concentration of SP to 120 mg L -1 and was further reduced in the highest concentration (240mg L-1). The activity of the protease inhibitor α2-M was significantly higher when the shrimps were exposed at concentrations of 60 e 240 mg L -1, while this activity was similar to that of control at a concentration of 120 mg L -1. Thus, there was no need for regulation of the proPO system, when the shrimp were exposed to an intermediate concentration of SP of S. platensis. The administration of SP of S. platensis to shrimps L. vannamei was able to modulate the immune response of these animals in different forms depending on the concentration used. So analyzing the results it is possibl e to conclude that the intermediate concentration of 120 mg L -1 was the most efficient, since it maintained the levels of circulating hemocytes and the basal activities of PO and α 2 -M. / A aquicultura é uma das atividades produtoras de alimento que mais cresce a nível mundial e o cultivo de camarões marinhos apresentou a maior taxa de crescimento média nos últimos 50 anos. A busca por produção, muitas vezes, requer um incremento na densidade utilizada, levando os indivíduos cultivados a condições de estresse e ao surgimento de doenças oportunistas. O uso de imunoestimulantes na aquicultura, entre eles os polissacarídeos sulfatados (PS), tem resultado em um aumento na resistência dos animais a patógenos e a situações de estresse. O objetivo deste trabalho foi avaliar o efeito dos PS de Spirulina platensis em pós-larvas (PL’s) e juvenis do camarão branco Litopenaeus vannamei, por meio de banhos de imersão. A administração do PS para as PL’s de L. vannamei foi realizada nas concentrações de 0 (controle); 50; 100 e 200 mg L-1, duas horas antes da avaliação da resistência das mesmas ao teste do estresse salino. Os juvenis de L. vannamei foram expostos, durante três horas, aos PS de S. platensis nas concentrações de 0 (controle); 60; 120 e 240 mg L-1 seguida da coleta da hemolinfa para a análise dos seguintes imunoparâmetros: contagem total (CTH) e diferencial (CDH) de hemócitos, concentração protéica no soro, atividade da fenoloxidase (PO), atividade da α2-macroglobulina (α2-M) e atividade hemaglutinante do soro. Após o teste do estresse salino, foi observado que as PL’s expostas aos PS na concentração de 100 mg L-1 apresentaram uma tendência de maior sobrevivência, mas não houve diferença significativa entre os tratamentos. Com relação aos imunoparâmetros, a CTH foi reduzida quando os animais foram expostos aos PS e nas concentrações mais altas a CTH voltou aos valores normais. Porem, não houve diferença significativa entre os tratamentos e o controle. No entanto, a diferença significativa foi observada entre os tratamentos que foram expostos ao PS. O número de hemócitos granulares (HG) apresentou uma tendência de queda na concentração de 60 mg L-1 e, com o incremento da concentração de PS, os valores de HG também tenderam a aumentar, porém não houve diferenças significativas em relação ao controle. No entanto, foi observada diferença significativa entre as contagens de HG nas concentrações de 60 e 240 mg L-1. A atividade da PO foi significativamente reduzida na concentração de 60 mg L-1 o que pode estar relacionado ao baixo valor de CTH como também à elevada atividade do inibidor de protease α2-M. A atividade da PO voltou a ser semelhante à obtida no controle com o aumento da concentração de PS para 120 mg L-1 e foi novamente reduzida na maior concentração (240 mg L-1). A atividade do inibidor de protease α2-M foi significativamente elevada quando os animais foram expostos aos PS nas concentrações de 60 e 240 mg L-1, enquanto essa atividade se manteve semelhante ao controle na concentração de 120 mg L-1. Dessa forma, não houve necessidade de regulação do sistema proPO, quando os camarões foram expostos à concentração intermediária dos PS de S. platensis. A administração dos PS de S. platensis aos camarões L. vannamei foi capaz de modular a resposta imunológica desses animais de maneira diferente, dependendo da concentração utilizada. Com a realização deste experimento pode-se concluir que a concentração intermediária de 120 mg L-1 foi a mais eficiente, já que manteve os níveis de hemócitos circulantes e as atividades basais da PO e α2-M.

Orientador: Iracilda Zeppone Carlos / Banca: Angela Maria Victoriano de Campos Soares / Banca: Eduardo Bagagli / Resumo: A esporotricose é uma micose de distribuição universal cujo agente é o fungo termodimórfico Sporothrix schenckii. A forma mais comum da doença é a linfocutânea, que compromete pele, tecido subcutâneo e gânglios linfáticos regionais. Os principais constituintes da parede celular de S. schenckii são compostos peptídeo-polissacarídicos contendo ramnose, manose e galactose. Estes compostos se arranjam na parede celular de modo a formar duas subcamadas distintas na forma leveduriforme do fungo, uma das quais, a mais interna e fixa delas, é denominada como peptídeo-polissacarídeo da parede celular (PPC) e foi utilizada como antígeno neste estudo, juntamente com a levedura termo-inativada (SSTI). Os antígenos liberados pelo fungo participam diretamente do processo de escape do sistema imune e também servem como alvos para a eliminação do mesmo por anticorpos ou ligação a receptores presentes em células da imunidade inata, como os macrófagos. Nossos resultados mostraram que o PPC induz citocinas de padrão inflamatório de forma mais pronunciada que o SSTI, que por sua vez induziu maior liberação de IL-10. Sugerimos também que no modelo experimental utilizado a IL-10 não atua provocando supressão da resposta proliferativa dos esplenócitos, e que a IL-4 atua apenas na fase de resolução da infecção, não sendo induzida como forma de escape imunológico pelo fungo. Também mostramos que os antígenos testados são capazes de induzir a liberação de IL-17 em culturas de esplenócitos, num perfil semelhante àquela das citocinas Th1 / Abstract: Sporotrichosis is a micotic infection of universal distribution, which is caused by dimorphic fungus Sporothrix schenckii. It usually happens as a lymphocutaneous disease, compromising skin, subcutaneous tissues and regional lymphatic nodules. Major constituents of S. schenckii cell wall are peptido-polysaccharide complexes containing rhamnose, mannose and galactose. These complexes are organized in two distinct layers in the fungus yeast cell wall, of which the inner one is called cell wall peptido-polysaccharide (PPC), being used as antigen in this study, along with the heat-killed yeast (SSTI). Antigens released by the fungus directly participate in evasion of the immune system, also serving as targets for fungus elimination by binding of antibodies or innate immune cells like macrophages. Our results showed that PPC induces inflammatory cytokines in a more pronounced way when compared to SSTI, which induced higher levels of IL-10. We also suggest that, in the animal model used, IL-10 doesn't act as a suppressor of splenocyte proliferative response, and that IL-4 play a role at the resolution phase of the infection, being not induced as a means of immunologic escape by the fungus. Also, we showed that the assayed antigens are capable of inducing IL-17 secretion in splenocyte cultures, in a fashion close to that of Th1 cytokines / Mestre

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / O cÃncer tem se configurado como um grande problema de saÃde pÃblica mundial. Como alternativa terapÃutica, muitos produtos naturais tÃm sido avaliados para seleÃÃo de compostos ativos com menos efeitos indesejÃveis e capazes de reduzir tumores malignos de modo mais eficiente. Morinda citrifolia, conhecida popularmente como noni, à uma espÃcie nativa do sudeste asiÃtico e da AustrÃlia que tem sido utilizada popularmente para diversos fins terapÃuticos e nutricionais. O polissacarÃdeo extraÃdo da polpa do fruto de Morinda citrifolia Linn (Noni-ppt 1) foi purificado por reprecipitaÃÃo em Ãlcool etÃlico, originando as subfraÃÃes: Noni-ppt 3 e Noni-ppt 5. O objetivo do presente trabalho foi investigar a atividade antitumoral in vitro e in vivo do Noni-ppt, bem como realizar sua caracterizaÃÃo fÃsico-quÃmica utilizando tÃcnicas de cromatografia lÃquida de alta eficiÃncia (CLAE), espectroscopia de infravermelho (FT-IR), cromatografia de permeaÃÃo em gel (GPC), anÃlise termogravimÃtrica (TGA), anÃlise elementar de nitrogÃnio e ressonÃncia magnÃtica nuclear de 13C (RMN). A atividade citotÃxica in vitro das trÃs fraÃÃes de Noni-ppt (50 μg/mL) foi avaliada sobre trÃs linhagens de cÃlulas tumorais (HCT-116; SF-295 e OVCAR-8) por meio do mÃtodo colorimÃtrico brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolio (MTT). O efeito antitumoral in vivo do Noni-ppt 3 foi analisado em camundongos com tumor Sarcoma 180 nas doses de 25 e 50 mg/Kg/dia por via oral e intraperitoneal. As anÃlises fÃsico-quÃmicas demonstraram que o Noni-ppt à composto por 12,1-13,7% de proteÃnas, 44,2-58,8% de Ãcidos urÃnicos e 65,4-71,3% de carboidratos totais. A anÃlise da composiÃÃo monossacarÃdica demonstrou que o Noni-ppt à um heteropolissacarÃdeo constituÃdo, predominantemente, por ramnose, arabinose, galactose e Ãcido galacturÃnico. Os resultados da avaliaÃÃo da atividade antitumoral in vitro demonstraram que as amostras de Noni-ppt 1, 3 e 5 nÃo apresentam nenhuma atividade citotÃxica direta sobre as cÃlulas tumorais na concentraÃÃo testada. AlÃm disso, nÃo foi observada qualquer inibiÃÃo do tumor nos camundongos inoculados com as cÃlulas tumorais. / Currently, cancer is a global public health problem. As an alternative therapy, many natural products have been evaluated for selection of active and with less undesirable effects capable of reducing malignant tumors more efficiently compounds. Morinda citrifolia, commonly known as noni, is a species native to Southeast Asia and Australia that has been popularly used for many therapeutic and nutritional purposes. The polysaccharide extracted from the pulp of Morinda citrifolia Linn fruits (Noni-ppt 1) was purified by reprecipitation in ethanol, yielding the subfractions: Noni-ppt 3 and Noni-ppt 5. The objective of this study was to investigate the antitumor activity in vitro and in vivo of Noni-ppt and carry out their physico-chemical characterization by high performance liquid chromatography (HPLC), gas chromatography (GC), fourier transform infrared spectrometry (FTIR), gel permeation chromatography (GPC), thermal gravimetric analysis (TGA), elemental analysis and 13C nuclear magnetic resonance (NMR). The in vitro cytotoxic activity of the three fractions of Noni-ppt (50 μg/mL) was evaluated on three tumor cell lines (HCT-116, SF-295 and OVCAR -8) by colorimetric method bromide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The in vivo antitumor effect of Noni-ppt 3 was analyzed in mice with tumor Sarcoma 180 at doses of 25 and 50 mg/kg/day orally and intraperitoneally. The physico-chemical analyzes showed that Noni-ppt consists of protein (12.1-13.7%), uronic acids (44.2-58.8%) and of total carbohydrates (65.4-71.3%). The monosaccharide composition analysis showed that Noni-ppt is a heteropolysaccharide composed predominantly of rhamnose, arabinose, galactose and galacturonic acid. The Noni-ppt samples showed no direct cytotoxic activity against tumor cells in vitro. Moreover, the treatment with Noni-ppt 3 did not show any inhibition of tumor growth in mice inoculated with Sarcoma 180 cells.

Abstract The purpose of this study was to develop sensitive and specific laboratory diagnostic methods for the demonstration of pneumococcal surface antigens or pneumococcus-specific antibodies in clinical samples. The work took account of epidemiological aspects of both pneumococcal disease and nasopharyngeal carriage of pneumococcus. We first compared the sensitivity of pneumococcal culture and antigen detection methods in nasopharyngeal samples in a developing country setting and then investigated the possibility of improving the sensitivity of the antigen detection by introducing an enrichment step in the procedure. — Further investigations were designed to determine the validity of pneumolysin-specific immune complex bound antibody assay as a tool for diagnosing pneumococcal ALRI in a developing country setting. Finally, we developed an enzyme immunoassay for the detection of pneumococcal capsular polysaccharide antigens, using type-specific antibodies produced in-house in rabbits through immunisation with an in-house-produced pneumococcal whole cell vaccine. The method was tested in nasopharyngeal and middle ear fluid samples. The first results indicated that antigen detection might be more sensitive than culture in demonstrating pneumococci in URT, particularly in children with prior antimicrobial therapy. Antigen detection is a feasible method for studies on pneumococci in developing countries. For type-specific demonstration of S. pneumoniae, detection of pneumococcal antigen after an enrichment step proved a sensitive method that can be applied for epidemiologic study purposes, e.g., in vaccine trials, in areas without ready access to a good microbiology laboratory. Determination of IC-bound pneumolysin IgG antibodies appears to be a useful method for species-specific diagnosis of pneumococcal infections. The results indicating pneumococcal aetiology in ALRI patients in this study compare well with the best results obtained by the use of lung aspirates. Increasing the number of serial samples improves the sensitivity of the assay, but even two samples provide more positive findings than other methods currently in routine use. Criteria of positivity need to be confirmed in subsequent larger studies with both healthy controls and patients with confirmed pneumococcal disease. It is also important to control the findings in patients with pneumonia of non-pneumococcal origin. The novel enzyme immunoassay was shown to work well with enrichment culture samples, with an almost 100% sensitivity compared with the culture. Middle ear fluid samples were too diluted for the enzyme immunoassay method used, and only 74% sensitivity compared with culture was achieved. Provided that adequate samples can be obtained, the method will be a useful complement to the current laboratory methods used to diagnose pneumococcal disease. With the existence of a broad spectrum of microbiological and immunological methods, it is imperative to seek international consensus for standard methods to demonstrate pneumococcus. Otherwise it is very difficult to compare results from different clinical studies. A WHO Working Group recently proposed a standard method for detecting upper respiratory carriage of pneumococcus, but a lot of work remains to be done in other areas of research on pneumococcal infections.

Ce travail de thèse a pour objectif de formuler un emballage comestible à base d’un mélange de chitosan et de gélatine (bœuf ou poisson), de mieux comprendre l’influence d’irradiations par faisceaux d’électrons et de l’incorporation d’antioxydants naturels sur les propriétés physico-chimiques et fonctionnelles des films. Une étude de l’effet de l’irradiation sur la cinétique de libération des antioxydants dans un milieu liquide simple a été étudiée pour validation. Une étude préliminaire a montré d’abord que la densification de la solution filmogène puis du gel pendant le séchage ne dépend ni de l’épaisseur, ni de la concentration, ni du temps et laisse supposer une diffusion Fickienne de l’eau dans la matrice. La perméabilité à la vapeur d’eau augmente linéairement avec l’épaisseur de films et d’autant plus lorsque l’étendue du différentiel d’humidités relatives est élevé. Ce phénomène est attribué à un mécanisme de gonflement et de plastification du réseau de gélatine-chitosan par l’eau. L’effet du taux de mélange de deux biopolymères a montré une amélioration des propriétés mécaniques et barrières à l’eau et à l’oxygène des films de chitosan. Ces gains de performances sont dus à la bonne miscibilité des deux macromolécules et aux interactions moléculaires établies suite à la formation d’un complexe polyélectrolytique, confirmé par analyse FTIR. L’irradiation des films après séchage accroit la polarité de surface et l’hydrophilie des films et ainsi induit une augmentation des propriétés barrières à la vapeur d’eau et à l’oxygène, mais aussi des mécaniques et thermiques des films. Toutefois, l’irradiation ne modifie pas la cristallinité des films. L’incorporation des antioxydants (acide férulique, coumarine, quercétine et tyrosol) avec ou sans irradiation agit différemment, selon leur nature, sur l’organisation du réseau macromoléculaire et donc sur les propriétés des films. Ainsi, l’acide férulique et le tyrosol réduise la perméabilité à la vapeur d’eau mesurée à un gradient de 0-30% d’humidité relative, alors qu’ils l’augmentent jusqu’à 90 fois avec un gradient 30-84% et un traitement d’irradiation. La perméabilité à l’oxygène diminue d’une façon significative après ajout de quercétine ou de tyrosol et après irradiation. L’acide férulique et la coumarine augmente la force à la rupture des films alors que ce sont la quercétine et l’acide férulique qui permettent d’accroitre la résistance thermique des films. Ces résultats mettent ainsi en évidence la complexité des interactions mises en jeu entre les antioxydants et les chaînes de chitosan et/ou de gélatine, leur dépendance au niveau d’hydratation du système et à l’impact du traitement de réticulation par irradiation. Il faut noter que l’irradiation a permis de protéger les molécules actives contre l’oxydation durant une longue période de stockage des films. La libération en milieu aqueux de l’acide férulique est la plus ralentie avec une rétention dans les films la plus élevée à l’équilibre (27%). Les coefficients de diffusion des antioxydants, déterminés à partir des cinétiques de libération ont pu être modulés (50%) grâce à l’irradiation. / This thesis aims to develop an edible packaging made of a mixture of chitosan and gelatin (beef or fish), to better understand the influence of the electron beam irradiation and of the incorporation of natural antioxidants on the physico-chemical and functional properties of the films. A study of the effect of irradiation on antioxidants release kinetics in a simple liquid medium was studied for validation. A preliminary study first showed that the densification of the film-forming solution and the gel during drying does not depend on the thickness or concentration or time and suggests a Fickian diffusion of water in the matrix. The permeability to water vapor increases linearly with film thickness and especially when the extent of the relative humidity differential is high. This phenomenon is attributed to a swelling mechanism and plasticization of the gelatin-chitosan network by the water. The effect of the chitosan-gelatin ratio showed an improvement of the mechanical properties and barrier to water and oxygen of the films compared to chitosan films. These performance gains are due to the good miscibility of the two macromolecules and to the molecular interactions established after the formation of a polyelectrolyte complex, as confirmed by FTIR analysis. Irradiation of films after drying increases the polarity of the surface and the film hydrophilicity, and thus induces an increase in barrier properties to water vapor and oxygen, and also of the mechanical and thermal films. However, irradiation does not change the crystallinity of the films. The incorporation of antioxidants (ferulic acid, coumarin, quercetin and tyrosol) with or without irradiation acts differently on the organization of the macromolecular network and thus on the film properties. Thus, ferulic acid and tyrosol reduce the permeability of water vapor measured in a gradient of 0-30% relative humidity, while it increases up to 90 times with a gradient 30-84 % and an irradiation treatment. The oxygen permeability decreases significantly after addition of quercetin or tyrosol and after irradiation. Ferulic acid and coumarin increases the tensile strength of the films while they are quercetin and ferulic acid which allow to increase the thermal resistance of the films. These results thus demonstrate the complexity of the interactions involved between antioxidants and the chains of chitosan and/or gelatin, their dependence on the moisture level of the system and on the impact of cross-linking treatment by irradiation. It is noticed that the irradiation has protected the active molecules against oxidation for a long period of storage of the films. Release in aqueous medium of ferulic acid is the more delayed with the highest retention in the film at equilibrium (27%). The antioxidants diffusion coefficients, determined from the release kinetics, could be modulated (about 50%) by the irradiation treatments.

The expression of polysaccharide capsules is common in bacteria and associated with virulence in some pathogenic strains. Strains of the Gram-negative bacterium Escherichia coli express a structurally diverse range of capsular polysaccharides. E. coli strains expressing group 2 capsules are associated with a number of extra-intestinal infections, including sepsis, urinary tract infections, and neonatal meningitis. Group 2 capsular polysaccharides are synthesised on the cytoplasmic face of the inner membrane. Evidence from previous work suggests that export of polysaccharides across the Gram-negative membranes involves four transport proteins which interact to form a continuous membrane-spanning translocation complex (the KpsMTED translocon). Polysaccharide translocation across the inner membrane requires the ABC transporter KpsMT, in which KpsM is the integral inner membrane component and KpsT is the ATPase. Transport across the periplasmic space and outer membrane involves the integral inner membrane protein KpsE and the outer membrane protein KpsD, respectively. This thesis addressed some of the key areas in the study of group 2 polysaccharide transport by employing the K5 capsule as a model system. Using biochemical and molecular genetics approaches, the study focused on establishing functional and structural characteristics of the translocon members and analysing protein-protein interactions within the complex. This study demonstrated that KpsE can self-associate as dimers, tetramers and possibly higher order oligomers in the absence of other capsule gene products and the K5 substrate. A mutagenesis study of KpsE revealed that the periplasmic, membrane-associated C-terminus is essential for correct protein function. Work presented here confirmed previous data, which suggested a direct interaction between KpsE and KpsM, by alternative methods, and demonstrated that the C-terminal domain of KpsE is required for this interaction. Further experiments suggested that KpsE and KpsM can both form higher order oligomers when interacting as a complex. The C-terminus of KpsE is not required for an interaction between KpsE and KpsD, and the two proteins are thus more likely to interact via their respective periplasmic domains. Generation of a theoretical model of the secondary structure and topology of KpsD predicted that KpsD is made primarily of β-sheets with some interspersed α-helices, including a larger coiled coil region. The theoretical topology model proposed an N-terminal transmembrane domain made of eight membrane-spanning regions, and a large periplasmic domain. Substituted-cysteine accessibility method and myc-epitope insertion analysis were both assessed for their suitability for topology analysis of KpsD. Myc-epitope insertion was identified as the recommended approach for future topology study. Myc-epitope tagging of the periplasmic C-terminus of KpsD revealed that a native C-terminus is essential for correct KpsD function.In conclusion, this thesis contributes to the model of group 2 polysaccharide export in E. coli, and, more generally, provides clues about the transport of high-molecular weight molecules across Gram-negative membranes. It is hoped that a thorough understanding of polysaccharide transport might reveal therapeutic targets to block capsule export in pathogenic E. coli in the future.