Rozprawy doktorskie na temat „POLYSACCHARIDES PLANT”

Arabinogalactan proteins (AGPs) are proteoglycans heavily substituted by arabinogalactan polysaccharides. These are composed of arabinose and galactose, and minor sugars such as glucuronic acid (GlcA), fucose and xylose. The arabinogalactan polysaccharides do not decorate classical AGPs exclusively, but they can also be found decorating a wide range of proteins. Arabinogalactan proteins have been implicated in many processes of plant development. Recently, AGPs were proposed to bind and store calcium at the plasma membrane. They are extracellular, and are localised mainly at the plasma membrane via a GPI-anchor. They can also be soluble in the apoplast. Their low abundance, chemical similarity and high functional redundancy have hindered their study. My strategy to overcome these difficulties was to study knock-out Arabidopsis thaliana plants of glycosyltransferases that transfer sugars specifically onto AG-polysaccharides. Glucuronic acid makes up about 10% of the arabinogalactan polysaccharide structure in Arabidopsis thaliana cell culture AGPs. Previously, the glucuronic acid transferase A TGLCA T14A, a member of the CAZy Glycosyl Transferase 14 family, was shown to transfer GlcA specifically onto AGPs, and knock-out Arabidopsis plants showed a 30% reduction in [Me]GlcA substitution in AGP-enriched preparations. However, no clear growth phenotype was observed. The characterisation of knock-out plants of other GT14 family members and combinations thereof is described here. Based on previous studies (Lamport and Várnai, 2013), I assayed in vitro the calcium binding capacity of AGP extracts from WT and knock-out plants. The results showed that AGP extracts from knock-out plants can hold less calcium than WT plants in vitro. A wide range of plant growth phenotypes were identified. Growth phenotypes can be explained by changes in the cytoskeleton and deficiencies in calcium signaling. Our evidence suggests links between structural deficiencies of extracellular proteoglycans to extracellular calcium and cytoskeleton. This research has the potential to create a new model system for the study of molecular mechanisms dependent on calcium that drive cell expansion, division and differentiation in plants.

The plant cell wall determines cell shape and is essential for plant growth during development. Pectin is an important component of cell walls and, like many wall polysaccharides, is synthesized in the Golgi apparatus and secreted by vesicles. In Arabidopsis thaliana seed coats, pectin-rich mucilage is secreted in a polarized fashion during a specific stage of development. How the Golgi apparatus in seed coat cells accommodates the large increase in pectin-rich mucilage provides a unique window into the cellular machinery that supports cell wall polysaccharide biosynthesis and secretion. Examination of seed coat cells, using cryo-fixation and transmission electron microscopy and electron tomography, showed that Golgi stacks undergo dramatic changes in structure during mucilage production. Initiation of mucilage biosynthesis also correlated with increased numbers of Golgi stacks per cell. To understand if these cellular changes were dependent on pectin biosynthesis, the cell structure of a reduced mucilage mutant, mum4, was studied by similar methods and revealed that, while the morphology of Golgi stacks was dependant on mucilage, the increased stack number was not. To determine what proportion of the scattered Golgi stacks were producing mucilage, immunogold labeling with the novel mucilage-specific antibody CCRC-M36 was used to detect the pectin cargo. The large percentage of labeled Golgi stacks found suggests that many stacks produce pectin synchronously, rather than a subset of specialist Golgi. To test if a pectin modifying enzyme, MUM2, is co-secreted with pectin, a tagged MUM2 was engineered and introduced into mum2 mutants, where it rescued the mutant phenotype. However, the tag was not detectable using antibodies in immunofluorescence. Although mucilage was secreted to the top of the cell, antibody label demonstrated that pectin-producing stacks were randomly distributed throughout the cytoplasm, indicating that the destination of cargo has little effect on location of the Golgi stack producing it. The mechanism of targeting of vesicles with the domain of the plasma membrane exclusively at the mucilage pocket is unknown, although the correlation of a population of densely staining vesicles and abundant cortical microtubules in the cell cortex at the site of secretion was documented.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第21808号
農博第2321号
新制||農||1066(附属図書館)
学位論文||H31||N5180(農学部図書室)
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 充美, 教授 渡邊 隆司, 教授 栗原 達夫
学位規則第4条第1項該当

It has long been proposed that a high intake of dietary fibre promotes good intestinal health. Work performed previously by our research group suggests that soluble dietary fibre might have a particularly beneficial impact on intestinal health via its ability to inhibit potentially harmful interactions between bacterial pathogens and the gut epithelium. The aims of this thesis were to evaluate soluble plantain NSP for its ability to disrupt the epithelial interactions of diarrhoeal pathogens C. difficile and Enterotoxigenic Escherichia coli (ETEC), as well as other bacterial components implicated in diarrhoeal disease. Work was also performed to characterise the specific inhibitory fraction of plantain NSP, and in addition, to establish the molecular mechanism underlying its inhibitory activity. A range of soluble dietary fibres were shown to significantly inhibit the in vitro epithelial adhesion of C. difficile and ETEC, but out of all the fibres tested, soluble plantain NSP exhibited the highest efficacy. Plantain NSP also significantly inhibited the epithelial adhesion of eleven C. difficile clinical isolates, irrespective of their toxin expression or ribotype status. Furthermore, plantain NSP blocked the epithelial interactions of five purified C. difficile spore preparations. In addition to its anti-adhesive effects, soluble plantain NSP significantly down-regulated the pro-inflammatory, cytotoxicity and apoptotic response induced by C. difficile and its toxins. Similar effects were also found with respect to mucosally-associated E. coli isolated from ulcerative colitis (UC) patients, as well as bacterial components such as flagellin and LPS. Results demonstrated that the inhibitory activity of plantain NSP was mediated by its acidic polysaccharide fraction, which is mainly composed of pectic material. In addition, it was shown that soluble plantain disrupted bacterial-epithelial interactions via an interaction with the intestinal epithelium. Whilst plantain NSP induced increased cellular chloride secretion, this mechanism was not responsible for inhibitory activity. It was also hypothesised that plantain NSP might mimic intestinal MUC2 glycans by interacting with cell-surface galectin-3, with consequent nuclear localisation of β-catenin and down-regulation of inflammatory cytokines. Whilst plantain NSP was shown to induce activation of β-catenin, the knockdown of surface galectin-3 expression had no effect on inhibitory activity. Thus, the specific mechanism underlying the inhibitory activity of plantain NSP requires further investigation. This work supports the hypothesis that soluble plantain fibre can inhibit harmful interactions between bacteria and the human intestinal epithelium. Indeed, these studies provide convincing evidence to suggest that soluble plantain fibre, acting as a ‘contrabiotic’, could be developed as a potential prophylaxis or treatment against C. difficile and ETEC, which represent the main cause of antibiotic-associated diarrhoea and traveller’s diarrhoea, respectively. In addition, dietary supplementation with soluble plantain NSP may also confer a therapeutic benefit in inflammatory bowel disease (IBD).

La paroi végétale assure des fonctions biologiques majeures définissant la singularité des plantes ; elle est également à l'origine de multiples applications en tant que ressource agro-alimentaire, source de biomatériaux ou encore pour la production de biocarburants. Malgré cette importance fondamentale et pratique de la paroi végétale, la connaissance de sa biosynthèse apparaît à ce jour toujours très limitée. En effet, la faible abondance des glycosyltransférases (GTs) responsables de sa biosynthèse, l'absence de substrat spécifique et les difficultés à obtenir certains nucléotides-sucres nécessaires aux tests enzymatiques, a souvent rendu difficile les approches de biochimie classiques. Cependant, le séquençage de génomes (Arabidopsis thaliana, Oryza sativa, Poplar populus), la création de banques de mutants d'insertion et la classification des activités glycosyltransférases dans la base de données CAZy (www.cazy.org) sont autant d'outils récents ayant permis des avancées significatives vers la compréhension de la biosynthèse de la paroi des végétaux. Le CERMAV a participé à ce type d'avancée en 2009, en publiant une liste de 24 gènes candidats, nommés " NGT " pour " Nouvelles GlycosylTransférases ", présentant des signatures caractéristiques des glycosyltransférases. Afin de démontrer l'implication des gènes NGT dans les processus d'édification de la paroi végétale, nous avons développé une approche de génomique fonctionnelle, analysant en parallèle des lignées mutantes d'Arabidopsis altérées pour les gènes NGT et testant l'activité GT de ces protéines exprimées en systèmes hétérologues. Durant mes travaux de thèse j'ai pu caractériser 15 lignées mutantes à l'état homozygote pour 7 des 24 gènes NGT. Ces lignées homozygotes ont été criblées afin de rechercher un phénotype d'altération du développement ou de la composition en sucres de leur paroi qui soit corrélé à l'altération des gènes NGT. Ce travail de criblage a conduit à s'intéresser plus particulièrement aux mutants ngt1-1 et ngt1-2 altérés pour le gène NGT1 (At5g28910). La caractérisation des lignées mutantes ngt1-1 et ngt1-2 a permis de quantifier un phénotype de croissance foliaire réduit de 38%, par comparaison au développement des feuilles de la plante sauvage. Par ailleurs, la caractérisation biochimique de la paroi des mutants a révélé des réductions significatives et quantitatives de l'arabinose, du galactose et du rhamnose dans la paroi des mutants, ainsi que des modifications qualitatives marquées principalement des arabinanes. L'altération des arabinanes a d'ailleurs pu être confirmée par microscopie après immuno-marquage de sections d'hypocotyle de mutants à l'aide des anticorps monoclonaux LM6 et LM13 dirigés contre des épitopes α-1,5-arabinanes. Il a pu être montré également que la complémentation des mutants par une construction 35S::NGT1 permet de restaurer un phénotype sauvage à ces mutants. Par ailleurs, de façon à tester l'activité glycosyltransférase de la protéine NGT1, nous avons réalisé son expression en système hétérologue. A ce jour, malgré des résultats préliminaires encourageants, il n'a pas été possible de déterminer des conditions de tests permettant d'observer une activité glycosyltransférase suffisante et reproductible pour la protéine NGT1, que ce soit une activité fucosyltransférase (correspondant à la signature de la séquence du gène) ou bien une activité arabinosyltransférase (correspondant au phénotype biochimique des mutants ngt1).

L’analyse d’échantillons du Patrimoine Culturel est primordiale pour la compréhension des techniques, la conservation des œuvres et leur restauration. Ces échantillons sont rares et précieux et l’analyse doit être effectuée sur une faible quantité de matière, ce qui nécessite le développement et l’optimisation de méthodes analytiques appropriées. L’objectif de ce travail de thèse a donc été de développer de nouvelles méthodes analytiques dans le but d’étudier les glycoprotéines et les polysaccharides de gommes végétales des échantillons du Patrimoine Culturel. Les macromolécules ont été séparées par chromatographie d'exclusion stérique et électrophorèse sur gel de polyacrylamide modifié, révélant la présence de poids moléculaires très élevés pouvant atteindre jusqu’à 1 à 2 millions de Dalton. Une stratégie analytique innovante basée sur la spectrométrie de masse a permis d’obtenir des empreintes caractéristiques de chaques gommes. La stratégie analytique développée a été appliquée avec succès sur un échantillon d'aquarelle daté de 1870 du Metropolitan Museum of Art (New York, USA)
The analysis of Cultural Heritage samples is critical for the understanding of the arstists' technique, the conservation and restoration of artworks. These objects under investigation are rare and precious and the amount of sample available for the analysis is usually very low, thus implying the development and the optimization of adapted analytical methodologies. The objective of this PhD was to develop new analytical methods to study glycoproteins and polysaccharides from plant gums in Cultural Heritage samples.These macromolecules have been separated by size exclusion chromatography and modified polyacrylamide gels, which allowed to reveal the presence of proteins with molecular weights up to 1-2 million Dalton. A novel analytical strategy based on mass spectrometry allowed to obtain the caracteristic fingerprint of each plant gum. This developed method was successfully applied on a watercolor sample dated from 1870 supplied by the Metropolitan Museum of Art (New York, USA)

Plant gums are naturally occurring compounds coming from several plant species and their application as binding media and adhesives in the field of cultural heritage traces back to the third millennium B.C. On the contrary of other organic materials, information on gums are fairly limited since their complex nature which consists of a mixture of polysaccharide and polypeptide chains. The analytical study of these materials is important to shed light on artists’ techniques, on the methods of manufacture of ancient artefacts, and to inform appropriate strategies for their preservation. In the cultural heritage field, characterization of plant gums is usually based on the monosaccharide composition as determined by gas chromatography mass spectrometry (GCMS) after complete acid hydrolysis of the gum. However, inorganic and/or organic compounds simultaneously present in the sample were demonstrated to alter the gum chromatographic profile thus preventing the correct identification. The present research aims to evaluate a proper analytical approach for plant gums identification in samples from works of art based on their chromatographic, electrophoretic and mass spectrometric profile. Analytical parameters have been optimized for the analysis of arabic, tragacanth, karaya, guar, ghatti, locust bean and fruit tree gums raw samples. In the first part of the thesis, size exclusion chromatography (SEC) was used to highlight the proteinaceous component of plant gums thus revealing the presence of glycoproteins with a molecular weight of 1-2 million Da. Complementary information were obtained by higher resolution techniques such as polyacrylamide gel electrophoresis (PAGE) that revealed to be a promising method for discriminating plant gums according to their protein electrophoretic profile. In the second part of the research, a novel method involving partial enzymatic digestion of the plant gums polysaccharide component, followed by analysis of the released oligosaccharides by mass spectrometry was developed. Due to the different polysaccharide structure of the gums, the obtained MALDI mass spectra represented the unique profile or “saccharide mass fingerprint” of each gum, thus allowing their discrimination. The efficacy of the strategy was also successfully tested on an aged gum arabic sample and on fresh and old watercolors.

The extension of frozen-thawed epidermal strips prepared from the first centimetre below the hypocotyl hook of six day old dark grown Phaseolus vulgaris seedlings while immersed in various buffers and under various tensions was characterized. This was done in an attempt to determine if the acid wall loosening phenomenon, which according to the Acid-growth theory (Taiz, 1984) is thought to mimic part of the auxin mechanism of action, is mediated by unspecified wall loosening enzymes. Epidermal strips were found to be significantly loosened by media pH 6.0 to pH 2.6 (0.05M citric acid-O.lOM disodium phosphate) relative to pH 7.5. A minimum stress between 1.6 and 7.6 grams was required for the acid-extension of strips 4.5±0.5 mm wide. Regardless of tension, extension by tissues in an acid medium was largely transient For example, tissues tensioned by a 16.0 gram load reached a maximum extension rate of 6.18 ±1.37% of initial length per hour (L°/hr) between 4 and 6 minutes after immersion in pH 4.8. The rate was 1.29±0.17% L°/hr between 55 and 60 minutes and 1.05±0.14% L°/hr between 220 and 240 minutes. Total acid-extension over four hours was 4.24±0.57% L°. The extension response was found to be stable; newly harvested tissues whether frozen or not performed similarly to strips aged up to 15 days at -12°C before being extended. The performance of strips immersed in unbuffered solutions indicated that tissues were self-buffering at an acid pH probably because of the fixed carboxyls within the wall. The capacity for acid-extension by epidermal strips was lost in mature tissues harvested 4-5 cm below the hypocotyl hook. Temperature coefficients from extension rates were determined at several pHs. The results were highly variable. The acid-extension of strips boiled 15 minutes in ethanol or extracted in 3M NaCl for 4 hours at 4°C or 6M LiCl for 8 hours was determined in several pHs. The impact of the treatments was largely a suppression of the initial burst of acceleration. Extension rates following the initial surge were relatively unaffected. Glycosidase activities in untreated, ethanol-boiled, or salt extracted strips were determined. β-glucosidase was found to be most active in untreated strips with lesser levels of β-galactosidase and β-xylosidase and a trace of α-galactosidase being detected. Ethanol-boiling and LiCl-extraction removed or deactivated all four activities from the strips and NaCl-extraction lowered all four activities 70-80%. NaCl proved to have solubilized most of the missing β-glucosidase and β-galactosidase when the extraction solution was assayed following desalting and concentration. LiCl solubilized most of β-xylosidase. It was concluded that glycosidases and any other similarly soluble enzyme cannot be responsible for long term acid wall loosening in bean epidermis. If an enzyme is involved, it must be extremely stable and tightly bound to the wall. The acid-extension performance of frozen-thawed longitudinally halved hypocotyl sections in comparison to epidermal strips, as well as other evidence was considered support for another hypothesized mechanism of acid wall loosening, the displacement of calcium bridges.
Science, Faculty of
Botany, Department of
Graduate

Les fucosyltransférases sont les enzymes responsables du transfert d'un groupement fucose à partir du GDP-fucose sur des accepteurs variés (oligosaccharides, protéines,...). Chez l'homme, les rôles de ces glycosyltransférases sont importants dans un grand nombre de processus biologiques et pathologiques. De nombreuses fucosyltransférases existent chez les végétaux. Notamment FuT1, qui transfert un fucose en 1,2 sur un résidu galactose du xyloglucane : l'une des hémicelluloses majeures de la paroi des dicotylédones. Ce polysaccharide ramifié est très étudié en raison de ses applications actuelles et potentielles dans différents secteurs de l'industrie : textile, alimentation, pharmaceutique, etc. Les objectifs de ce doctorat ont été d'obtenir des informations biochimiques et structurales sur la fucosyltransférase AtFuT1 de la plante modèle Arabidopsis thaliana. Pour cela, une forme recombinante soluble de l'enzyme a été produite avec le système baculovirus /cellules d'insectes. De manière à obtenir suffisamment de protéines pour les études structurales, une méthode de culture en suspension des cellules a été mise en place au laboratoire. Un protocole de purification en deux étapes, impliquant une chromatographie par affinité puis par exclusion de taille, a permis d'obtenir à partir d'un litre de culture cellulaire entre un à deux mg de protéine pure et homogène. Ce résultat a permis de mieux comprendre le comportement de l'enzyme vis-à-vis de ses substrats (GDP-fucose et xyloglucane), d'obtenir des cristaux de la protéine et de résoudre la structure 3D d'AtFuT1 en complexe avec le GDP et un oligosaccharide dérivé du xyloglucane, à une résolution de 2,2 Å. La forme AtFut1 produite se comporte en solution et dans le cristal sous forme d'un dimère non covalent. La protéine adopte un repliement qui est un variant du type GT-B classique. Parallèlement à ces travaux, des tests d'activité glycosyltransférase ont été mis au point permettant le criblage de nombreuses conditions de réactions. L'ensemble des méthodes et techniques développées constitue une base utile, devant faciliter la caractérisation d'autres glycosyltransférases
Fucosyltransferases are enzymes that transfer a fucose residue from GDP-fucose on varied acceptors (oligosaccharides, proteins). In Human, these glycosyltransferases are involved in many biological and pathological processes. Numerous fucosyltransferase exist in the plant kingdom. Among them, FuT1 transfers a fucose linked in 1,2 onto a galactose of xyloglucan: a major hemicellulose of dicots cell wall. This branched polysaccharide is intensively studied because of its current and potential industrial applications in textile, food, pharmaceuticals, etc. The main objective of this PhD program was to obtain biochemical and structural information on the fucosyltransferase AtFuT1 from the model plant Arabidopsis thaliana. A recombinant form of this protein has therefore been produced, using the baculovirus/insect cell system. In order to get sufficient amount of protein for structural studies, a suspension cell culture method has been set-up in the lab. A two-step purification protocol, involving affinity and size exclusion chromatography was established. The active, and highly pure recovered protein was used to determine the biochemical properties of the protein towards its substrates (GDP-fucose and xyloglucan), to get protein crystals and hence to solve its 3D structure in complex with GDP and a xyloglucan derived oligosaccharide (2.2 Å resolution). AtFuT1 behaves in solution and in crystallo as a non-covalent dimer. The protein adopts a variant of the classical GTB fold. In addition, novel glycosyltransferase assay have been designed allowing the screening of numerous reaction conditions. Methods and techniques that were developed during this study should be a useful base for the characterization of other glycosyltransferase

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Using the osteoarthritis (OA) model induced by anterior cruciate ligament transection (ACLT) in rats, we have demonstred that guar gum (GG), a galactomannan extracted from Cyamopsis tetragonolobus seeds, displays analgesic activity similar to Hylan G-F 20, a hyaluronate derivate used as local therapy in human OA. In the same model, we objective to develop a parametric biochemical method for joint lesion evaluation, and to investigate the mechanism for analgesia and the chondroprotective effect of GG. Wistar rats subjected to ACLT (OA group) were sacrificed at different endpoints. Joint pain was daily measured using the test for articular incapacitation in rats, until 70 days. Joint lavage was used for NO determination. Articular cartilage was evaluated by the chondroitin-sulfate (CS) content and determination of its molar weight. Histophatologic analysis using the OARSI score system was also performed. Sham-operated groups were used for comparison. In the studies of joint pain, OA groups received indomethacin (2mg/kg/d s.c.), meloxicam (6mg/kg/d i.p.), tadalafil (0.5mg/kg/d p.o.) between days 4 and 7.Morphine (200Âg i.art.) was given at day 4 only, 30 min before the pain evaluation. Naloxone (500Âg i. art.) was given 15 min prior morphine. Sodium alendronate (30 or 240Âg i. art.) was given prophilactically, starting 3 days prior ACLT, and repeated at each 3 days, until day 6. Original or chemically modified GG (100μg/50μl i. art.) was given as a single dose at day 4. Galactose or mannose was co-administred (500μg/50μl i. art.) with original GG. In the study of chondroprotection, GG (100μg/50μl i. art.) was given once weekly, starting at day 14, during 8 weeks. Non-treated animals (NT) received vehicle (saline). OA animals presented maximal joint pain and NO release in the first week. Both CS content and molar weight were higher at day 70 (p<0.05). At this endpoint, important histopathologic changes were found in the OA group. Indomethacin, meloxican, tadalafil and morphine significantly reduced joint pain (p<0.05). Naloxone reverted the effect of morphine. Alendronate prevented the joint pain development. Modified GG structures were unable to promote analgesia. The analgesic effect of unmodified GG could be reverted by galactose (p<0.01), but not by mannose. GG treatment prevented the CS changes, and it reduced the histopatologic lesion (p<0.05). CS changes seem to reflect the tecidual damage, and may be used as an index for the joint lesion. The analgesic efficacy of GG is due to galactose residues. In addition, the chondroprotective effect by GG was demonstrated. This is an important contribution to propose GG as an antiarthrosic drug
No modelo de osteoartrite (OA) induzida por transecÃÃo do ligamento cruzado anterior em ratos (TLCA), havÃamos demonstrado que a goma guar (GG), uma galactomanana extraÃda das sementes de Cyamopsis tetragonolobus goma guar, apresenta atividade analgÃsica em magnitude semelhante à exibida pelo Hilano G-F 20, um derivado do Ãcido hialurÃnico utilizado em terapia intra-articular da OA humana. Utilizando o referido modelo, objetivamos desenvolver um mÃtido bioquÃmico paramÃtrico para avaliaÃÃo da lesÃo articular no referido modelo, e investigar o mecanismo para a analgesia e a eficÃcia condroprotetora da GG. Ratos Wistar submetidos à TLCA (grupo OA) foram sacrificados em diferentes perÃodos. A dor articular foi avaliada diariamente pelo teste de incapacitaÃÃo para ratos, por atà 70 dias. O lavado articular foi usado para determinaÃÃo da liberaÃÃo de NO. A cartilagem foi avaliada pela determinaÃÃo do teor de condrotin-sulfato (CS) na matriz, alÃm da avaliaÃÃo da massa molar do mesmo. A lesÃo articular foi avaliada tambÃm por anÃlise histopatolÃgica, segundo os escores OARSI. Grupos falso-operados (sham) foram utilizados para comparaÃÃo. Para estudos sobre a dor articular, animais do grupo OA receberam terapeuticamente indometacina (2mg/kg/d s.c.), meloxicam (6mg/kg/d i.p.), ou tadalafila (0,5mg/kg/d p.o.), do quarto ao sÃtimo dias. Morfina (200Âg i. art.) foi administrada apenas no quarto dia, 30 min antes da avaliaÃÃo da dor. Naloxona (500Âg i. art.) foi administrada 15 min antes de morfina. Alendronato sÃdico (30 ou 240Âg/kg s.c.) foi administrado profilaticamente trÃs dias antes da induÃÃo, e repetido a cada trÃs dias, atà o sexto dia. GG original ou quimicamente modificada foi administrada em dose Ãnica no quarto dia (100μg/50μl i. art.). Galactose ou manose (500μg/50μl i. art.) foram co-administrados à GG original. Para avaliaÃÃo sobre a lesÃo da cartilagem, GG (100μg/50μl i. art) foi administrada como dose Ãnica semanal, do 14 ao 63 dias. Animais nÃo tratados (NT) receberam veÃculo (salina) nas respectivas vias. O grupo OA apresentou dor articular mÃxima durante a primeira semana, perÃodo no qual houve a maior liberaÃÃo de NO. 70 dias apÃs TLCA, o teor e a massa molar do CS da matriz da cartilagem mostraram-se ambos aumentados (p<0,05). Importantes alteraÃÃes histopatolÃgicas foram encontradas no grupo OA nesse perÃodo. Indometacina, meloxicam, tadalafila e morfina reduziram significantemente a dor (p<0,05), sendo o efeito desta Ãltima revertido por naloxona. Alendronato sÃdico preveniu a ocorrÃncia da dor. As estruturas modificadas de GG nÃo exibiram eficÃcia analgÃsica. Galactose, mas nÃo manose, reverteu significativamente o efeito analgÃsico da GG nÃo-modificada (p<0,01). GG preveniu as alteraÃÃes do CS da cartilagem, e reduziu significativamente a lesÃo histopatolÃgica (p<0,05). As alteraÃÃes sofridas pelo CS parecem refletir o dano tecidual, validando tal metodologia para avaliaÃÃo da lesÃo estrutural. A eficÃcia analgÃsica da GG decorre de um efeito farmacolÃgico dependente de galactose. Mais ainda, demonstramos a eficÃcia condroprotetora in vivo para a GG, indispendÃvel para sua validaÃÃo como droga anti-artrÃsica.

L’objectif de cette thèse était de comprendre comment les caractéristiques structurales des pommes peuvent être liées aux facteurs structurels des purées après cuisson et fragmentation tissulaire. Les caractéristiques structurales du fruit ont été modulées par les cultivars, les pratiques culturales et la maturation, et les conditions du procédé (thermique : 50–95 °C et mécanique : 100–3000 tr/min) ont été modulées grâce à un cuiseur-broyeur. La structure de la purée (volume occupé par les particules, taille des particules, viscosité du sérum) et la texture (viscosité, seuil d’écoulement, G’ et G’’) ont ensuite été analysées et comparées entre les matières premières et les conditions du procédé. Les pectines ont été extraites et leur composition chimique ainsi que leur structure ont été corrélées à la structure de la purée. La taille des particules s´est montré être le déterminant majeur de la texture des purées en absence de dilution ou de concentration. Le degré d’adhésion cellulaire (défini par la structure et la composition des pectines) a eu un impact plus important sur la taille des particules que la taille des cellules individuelles (définie par les cultivars ou les pratiques culturales). D’autres facteurs structuraux, tels que la viscosité du sérum ou la quantité de pulpe, n’ont contribué à la texture des purées qu’à taille de particules constante. La fragmentation tissulaire, déterminant la taille des particules pendant le procédé, a été principalement affectée par l’intensité du cisaillement. Le stockage post-récolte des pommes et des températures élevées (95 °C) ont induit une dégradation et une solubilisation des pectines, en particulier par l'hydrolyse des chaînes latérales des rhamnogalacturonanes I. Cela a réduit l’adhésion cellulaire et la fragmentation tissulaire a ainsi été favorisée. Ces résultats ont permis d´approfondir la compréhension de la fragmentation tissulaire et des changements de texture au cours du procédé ce qui permettra de fournir des directives à l’industrie pour mieux gérer la diversité et l’hétérogénéité des fruits pendant le procédé de transformation des fruits en purée
The objective of this thesis was to understand how structural characteristics in raw apples can be linked to structural factors in purees after cooking and tissue fragmentation. Structural characteristics of the fruit were modulated by cultivars, agricultural practices and maturation, and process conditions (thermal: 50–95 °C and mechanical: 100–3000 rpm) were modulated in a cooker-cutter during processing. Puree’s structure (volume occupied by particles, particle size, serum viscosity) and texture (viscosity, yield stress, G’ and G’’) were then analysed and compared between raw materials and process conditions. Pectins were extracted and their chemical composition and structure were correlated to puree’s structure. Particle size appeared to be the most important determinant of puree’s texture when there is no dilution or concentration of the fruit tissue. The extent of cell adhesion (defined by pectin structure and composition) determined particle size more than individual cell size (defined by varietal effects or agricultural practices). Other structural factors only contributed to puree’s texture once particle size was constant. Tissue fragmentation, determining particle size during processing, was principally affected by shear intensity. Post-harvest maturity of the raw apples and high temperatures (95 °C) induced pectin degradation, especially rhamnogalacturonan I side chain hydrolysis, and solubilisation. This led to reduced cell adhesion and tissue fragmentation was additionally favoured. The results deepened the understanding of tissue fragmentation and textural changes during processing and provided guidelines for industry to manage diversity and heterogeneity of raw fruits during processing

Abstract: Drug dosage forms contain many components in addition to the active pharmaceutical ingredient(s) to assist in the manufacturing process as well as to optimise drug delivery. Due to advances in drug delivery technology, excipients are currently included in novel dosage forms to fulfil specific functions and in some cases they directly or indirectly influence the extent and/or rate of drug release and absorption. Since plant polysaccharides comply with many requirements expected of pharmaceutical excipients such as non-toxicity, stability, availability and renewability they are extensively investigated for use in the development of solid oral dosage forms. Furthermore, polysaccharides with varying physicochemical properties can be extracted from plants at relatively low cost and can be chemically modified to suit specific needs. As an example, many polysaccharide-rich plant materials are successfully used as matrix formers in modified release dosage forms. Some natural polysaccharides have even shown environmental-responsive gelation characteristics with the potential to control drug release according to specific therapeutic needs. This review discusses some of the most important plant-derived polymeric compounds that are used or investigated as excipients in drug delivery systems.

O etanol celulósico a partir da palhada de cana pode elevar a produção do bioetanol, porém esta é normalmente decomposta no campo. A degradação da parede celular no campo não foi elucidada e compreender este processo auxiliará na produção de etanol celulósico. O objetivo deste trabalho foi caracterizar a degradação da palhada de cana de açúcar no campo durante um ano. Foi analisada a composição da parede celular por fracionamento e composição dos monossacarídeos. Na parede celular, observou-se redução de 26% no teor de celulose enquanto houve aumento de 13% na fração de hemiceluloses mais solúveis. Mudanças na composição dos monossacarídeos das frações mostraram que o arabinoxilano (AX) foi o primeiro polímero a ser solubilizado (após 3 meses) seguido dos b-glucanos e celulose (após 6 meses). Isto sugere que o AX é a hemicelulose mais exposta e sua solubilização permitiu a degradação da celulose após 6 meses. A partir dos dados obtidos, sugeriu-se a utilização de xilanases seguidas de glucanases numa possível ordem de enzimas para produção de etanol celulósico.
The sugarcane straw cellulosic ethanol can increase bioethanol production, but the straw is usually degraded in the field. However, the process that leads the cell wall disassembly under field conditions is unknown and understanding how this happens can improve cellulosic ethanol production. In the present work we aimed at studying how sugarcane straw is degraded in the field during a year. Cell wall composition was determined by fractioning and determination of monosaccharide composition. Results showed a decrease (ca.26%) in cellulose content and an increase of 13% in high solubility hemicelluloses fraction. Changes in monosaccharide composition showed that the first polymer to be solubilised is the arabinoxylan (AX) (after 3 months) followed by b-glucans and cellulose (after 6 months). This suggests that AX is the most exposed hemicelullose and its solubilisation allowed cellulose degradation after 6 months. Our data suggest the use of xylanases followed by glucanases as an enzyme order to be used in cellulosic ethanol production from sugarcane straw.