No description available.

No description available.

Xanthan is a well-known extracellular polysaccharide, produced by a Gram negative bacterium Xanthomonas campestris (X. campestris) under aerobic conditions. Solutions of xanthan exhibit high viscosities and non-Newtonian behaviour even at low concentrations. This biopolymer has a wide range of valuable commercial and industrial applications, for example; it can be used as a food thickening agent and a stabilizer in some other industries. Traditionally the production of xanthan has predominantly been performed in stirred tank fermenter (STR). This study sought to compare the cultivation of the bacterium, X. campestris for the production of the viscous biopolymer xanthan gum in two different reactor systems, a novel oscillatory baffled reactor (OBR) and the conventional industry workhorse, the stirred tank reactor (STR). Overall biopolymer production occurred at similar rates in the well stirred and aerated STRs, albeit at the cost of higher energy inputs for mixing and aeration. Despite much previous literature promoting the use of the OBR for transporting and reacting very viscous systems, this was the first actual study attempting to investigate the use of the OBR for a highly viscous non-Newtonian fermentation process. The experimental results show that xanthan production was similar in the OBR than in the STR, the OBR is however readily suitable for the cultivation of xanthan. The probable reasons for the inability of the OBR to match the production rates of the STR may well lie in the complex nature of this fermentation process. Unlike a previous study on pullulan production (Gaidhani 2004) where the OBR outperformed the STR, X. campestris initially needs high oxygen transfer rates and the OBR, although it provides good bulk mixing and low energy consumption, seemed unable to equal the STR in this respect, especially in a very viscous system. The result shows that xanthan production in the OBR was similar to the equivalent process in the STR. In order to attempt to improve the OBR a number of technical modifications were made including a novel sparger design to improve gas dispersal. These were not successful in improving xanthan production. Similarly, attempts to achieve improvements via wider amplitude ranges led to damage to the equipment. The conclusion was that significant improvements to the physical robustness of the OBR were necessary before it could be successfully used to process highly viscous bio-fluids.

No description available.

Alginates are polysaccharides extracted from the cell walls of brown seaweed or from certain bacteria such as Azotobacter vinelandii. Alginates have been shown to reduce the activity of pepsin and initial data suggest that it may affect the activity of pancreatic lipase. Pancreatic lipase plays an important role in the breakdown of triacylglycerol, if the activity of pancreatic lipase can be reduced then the breakdown of triacylglycerol would be reduce which in turn would lower the amount absorbed by the body. A pharmaceutical treatment for obesity called orlistat inhibits lipase and accounts for 66% of all prescriptions for treatments for obesity in 2008 in the UK. However the side effects of orlistat (uncontrolled diarrhoea and steatorrhea) can reduce the compliance with the treatment. If orlistat is taken with a high fibre product it has been shown that this can greatly reduce or eliminate the side effects. Since alginate is a dietary fibre it is believed that this could be an effect treatment for obesity. Colourimetric and turbidimetric assays were used to determine the effect of a wide range of alginates and other dietary fibres on pancreatic lipase. Alginates have the ability to inhibit pancreatic lipase, with a maximum inhibition of 72.2% (±9.9). Pectin could also inhibit lipase by a maximum of 71.8% (±22.3), however, specific enzymactically modified alginates could activate the enzyme by up to 22.0% (±10.1). Carrageenan and Hydroxypropyl methylcellulose could also activate the enzyme by a maximum of 37.3% (±24.5). The structure of the alginate is key to the inhibition or activation of the enzyme. Consecutive guluronate residues are important for inhibition whereas alternating mannuronate and guluronate blocks are detrimental to inhibition. The alginate that activated the enzyme was almost entirely consisting of poly-alternating uronate residues. The level of esterification was important for pectins inhibition of lipase with higher levels causing little inhibition. Alginate causes sustained inhibition at low concentrations of biopolymer compared to pectin. Therefore alginate could play a potential role in the management of obesity. ii

In this study, the structure-function relationships of the polysaccharides extracted from Ganoderma applanatum were investigated. This involved the extraction of polysaccharides, testing their immunomodulatory properties, structurally characterising the immunomodulatory polysaccharides and correlating the structures determined with their immunomodulatory activity. Three bio chemically different polysaccharide preparations obtained from both the fruiting body and cultured mycelium of G.applanatum using a sequential extraction method employing three solvents (water, ammonium oxalate and sodium hydroxide), under conditions of varying temperatures and pH accompanied by constant stirring. This 'mild to harsh' method of extraction facilitated the extraction of polysaccharides from the outermost to the innermost layers of the fungal cell wall. As markers of immunomodulation, the levels of stimulation/inhibition of reactive oxygen species, nitric oxide and tumour necrosis factor-alpha production by macrophages and neutrophils, were measured in the presence of the polysaccharide extracts. The polysaccharides extracted through different solvents had different immunomodulatory activities, which varied with their concentrations. In general, all mycelial polysaccharides had stimulatory effects on macrophages and neutrophils while the fruiting body polysaccharides had inhibitory effects. Composition and linkage analysis of the mycelial polysaccharides confirmed that they were composed of at least three species of hetero-polysaccharides (P-1,3-glucans, cc-1,4-glucans and C(- 1.6-mannans) with different degrees of branching. molecular weight:. anionic charge. monosaccharide composition and linkages. A significant finding from this study was that. with a variation in the conditions of extraction and the solvent used. the complexity of the polysaccharides changed. While polysaccharides with relatively low molecular weight:. fewer branches and low negative Charge were extracted under mild and neutral conditions. polysaccharides with higher molecular weight:. more branches and more negative charge were extracted under the harsh. alkaline conditions. Taking into account the higher reproducibility of the mycelial polysaccharides and their significant immunomodulatory activity compared with those from the fruiting bodies. the mycelial polysaccharides were further purified on the basis of charge and molecular weight The immunomodulatory effects of the purified low molecular weight neutral and charged sub-fractions of the mycelial extracts suggested that the negative charge of polysaccharides - due to the presence of uronic acids and low molecular weight - played a vital role in their activity. This study confirmed that the three-solvent sequential extraction method facilitates polysaccharide extraction from both the cultured mycelium and the fruiting bodies of G.applanatum. The composition and linkages of these extracts, which govern their solubility, conformation. charge. molecular weight:. degree of branching and degree of polymerisation, play a vital role in their immunomodulatory activity. G.applanatum polysaccharides modulate immune responses by triggering immune cell (macrophages/neutrophils) activation and cytokine induction by binding to carbohydrate specific receptors on macrophages and neutrophils. Based on all the analysis and inferences made during this study it was conclUded that (a) all mycelial and fruiting body polysaccharides are 'immunomodulators' and (b) the nature of the relationships between the polysaccharide structures and their immunomodulatory activity is specific to each fungal species and therefore, cannot be generalised, Mycelial polysaccharides from medicinal fungi such as G.applanatumcan therefore be used as health supplements, immune boosters or ac!juvants in radio- and/or chemo-therapy to improve the quality of life.

Ces 20 dernières années ont été consacrées à la dissection du réseau enzymatique complexe aboutissant à l’édification de l’amidon alors que bien moins de données ont été obtenues en ce qui concerne la dégradation ou les phénomènes de régulation de cette voie métabolique. Le métabolisme de ce polysaccharide nécessite plus de 40 gènes aussi bien chez la microalgue la plus primitive que chez les plantes les plus évoluées. Ce résultat est surprenant si on considère que l’amidon n’est composé que d’un seul sucre, le glucose, et contient uniquement que deux types de liaisons chimiques. En sus de son intérêt pour les industries alimentaire et des matériaux, un intérêt grandissant pour la compréhension des phénomènes de mobilisation de l’amidon chez les microalgues est apparu du fait de l’importance de ces organismes pour la production de bioénergies. Tandis que la majorité des données disponibles à ce jour proviennent d’approches de génétique inverse chez la plante modèle Arabidopsis thaliana, nous proposons d’étudier le catabolisme de l’amidon via une stratégie de génétique formelle dans un système plus convenu pour une telle approche que les plantes modèles : l'algue verte unicellulaire Chlamydomonas reinhardtii. La construction et le crible d’une banque de mutants d’insertion nous ont permis d’isoler plus de 40 souches déficientes pour la mobilisation et représente une ressource essentielle pour une compréhension complète des mécanismes de dégradation de l’amidon. Nos caractérisations biochimiques, enzymologiques et moléculaires sur ces mutants nous ont permis d’identifier un nombre appréciable de fonctions impliquées plus ou moins directement dans le processus. / During the last two decades of research, efforts have been focused on the understanding of the complex pathway of starch biosynthesis with comparatively less emphasis on the genes and regulatory networks responsible for mobilization in planta of this important source of storage polysaccharide. However the starch metabolism network includes over 40 genes highly conserved from green algae to land plants. This comes as a surprise when one considers that this storage polysaccharide is made solely of glucose residues with only two types of chemical linkages. In addition to its central importance in food and material science, a particular interest in the understanding of starch mobilization in green algae rather than crops has been generated from recent developments in the production of bioenergies. While successful reverse genetic approaches have been mostly applied to Arabidopsis, we propose to complete and reassess our vision of starch catabolism by applying a high throughput forward genetic strategy in the system which among all model plants is most suited for such an approach: the unicellular green alga Chlamydomonas reinhardtii. We thus constructed and screened an insertional mutant bank and identified more than 40 mutant strains altered in their ability or in the kinetics of starch mobilization. The combination of biochemical, enzymological and molecular approaches allowed us to identify several of the mutations responsive of the catabolic phenotype.

Les héparanes sulfates (HS) sont produits sous la forme d’un précurseur non sulfaté, qui subit ensuite une phase de maturation catalysée par plusieurs sulfotransférases. Une fois synthétisés, ils peuvent subir une dernière modification par les Sulfs, des 6-O-endosulfatases sécrétées. Ces modifications n’ont pas lieu uniformément sur le polymère : il en résulte une large diversité structurale, ce qui va influencer la fixation et les fonctions de nombreux ligands protéiques. Des études récentes ont mis en exergue l’implication des HS dans la régulation de la réponse inflammatoire. Toutefois, les mécanismes qui contrôlent l’expression de leurs enzymes de modification ont été peu étudiés. Dans ce contexte, mon travail de thèse a porté sur l’influence des conditions inflammatoires sur la machinerie de biosynthèse des HS. La première partie a été consacrée à l’étude des variations d’expression des 3-O-sulfotransférases (3-OSTs) dans les monocytes. Nous avons montré que la 3-OST3B est augmentée de façon précoce en réponse à des agonistes des TLRs et au TNF-α, puis son expression est stabilisée par des mécanismes post-transcriptionnels. Dans la deuxième partie de ma thèse, j’ai étudié la régulation de la 6-O-sulfatation des HS. Alors que l’expression des 6-O-sulfotransférases ne varie pas en réponse à des stimuli inflammatoires, Sulf-1 est fortement induite dans des fibroblastes stimulés par le TNF-α. La forte expression s’accompagne d’une diminution des HS 6-O-sulfatés et est corrélée à la désensibilisation des cellules au FGF-1. Ces résultats suggèrent que la 3-OST3 et Sulf-1, en modifiant la structure des HS, participent à la régulation de la réponse inflammatoire. / Heparan sulfates (HS) are produced from a non-sulfated precursor, which is then subjected to the actions of several sulfotransferases. Thereafter, HS can undergo a last modification catalyzed by secreted 6-O-endosulfatases named Sulfs. These modifications do not take place uniformly in the same chain, resulting in a complex organization that influences the functions of many binding proteins. Recent works have highlighted the involvement of HS in the regulation of the inflammatory response. However, little is known about the mechanisms that regulate the expression of HS-modifying enzymes. Thus, my thesis focused on the regulation of the HS biosynthetic machinery in response to inflammatory stimuli. In the first part, I studied the variations of expression of 3-O-sulfotransferases (3-OSTs) in monocytes. This study showed that 3-OST3B was rapidly up-regulated in response to TLR agonists and TNF-α. Thereafter, the high level of expression was stabilized by post-transcriptional mechanisms. The second part of my thesis was dedicated to the regulation of HS 6-O-sulfation. Although the expression of 6-O-sulfotransferases was not modified in response to inflammatory stimuli, the expression of Sulf-1 was strongly induced in fibroblasts exposed to TNF-α. The high expression of Sulf-1 was accompanied by a decrease in the rate of 6-O-sulfated HS and by the desensitization of fibroblasts to FGF-1. Altogether, these results suggest that 3-OST3B and Sulf-1 could participate in the regulation of the inflammatory response by modifying HS structure.

Les Archaeplastides sont apparus il y a environ 1,6 milliard d'années lorsqu'une cellule eucaryote a établi une symbiose avec deux autres organismes: une cyanobactérie et une chlamydiales. Durant l'endosymbiose, la cyanobactérie a évolué vers le plaste, tandis que les chlamydiales ont laissé pour empreintes une cinquantaine de gènes chez les Archaeplastides. En parallèle les Archaeplastides ont substitué leur biosynthèse de glycogène, par celle de l'amidon. Si le rôle de la cyanobactérie est évident dans l'émergence des Archaeplastides, qu'en est-il des bactéries parasitaires? Un effecteur sécrété par les chlamydiales correspond à l'activité de la glycogène synthase (GlgA). Des analyses phylogénétiques nous signalent que les isoformes d'amidon synthases III/IV ont probablement évolué à partir d'une GlgA provenant d'une chlamydiale. Nous avons formulé l'hypothèse que l'activité GlgA des chlamydiales possède une propriété biochimique unique expliquant pourquoi elle a été sélectionnée chez les Archaeplastides. Dans ce manuscrit, nous avons premièrement rapporté la caractérisation de la mutation d'une souche de cyanobactérie, ne synthétisant plus que du glycogène. Ceci est corrélé avec une défectuosité dans GlgA2. Ces résultats suggèrent que GlgA2 est indispensable à la synthèse d'amidon chez Cyanobacterium sp. CLg1. Nous nous sommes également occupés de caractériser les activités synthases des chlamydiales. L'activité GlgA de certaines chlamydiales est capable d'utiliser les deux nucléotides sucres : ADP-glucose et UDP-glucose. Ces résultats démontrent que l'activité GlgA de certaines chlamydiales a évolué de manière à mieux détourner le stockage des carbohydrates de l'hôte. / The Archaeplastida appear approximately 1.6 billion years ago when a heterotrophic eukaryote cell established a symbiotic relationship with two partners: a cyanobacterium and a chlamydiales. During the endosymbiosis process, the cyanobacterium has evolved to plastid, while chlamydiales have left in Archaeplastida a fingerprint of 50 genes. In parallel Archaeplastida have substituted the glycogen biosynthesis by starch. If the role of ancestral cyanobacteria is pretty obvious in the emergence of Archaeplastida, what about those parasitic bacteria? One effectors secreted by the chlamydiales corresponds to glycogen synthase activity (GlgA). Phylogenetic analyses point out that starch synthase III/IV isoforms have probably evolved from GlgA of chlamydiales. We hypothesize that GlgA activity of chlamydiales displays unique biochemical properties that could explain why this activity has been selected in the Archaeplastida. In this manuscript, we, first, report the characterization of mutant strain of cyanobacterium strain, which produce only glycogen. This phenotype is correlated with a defect in the GlgA2 isoform, which belongs to the same family of SSIII/IV of plants. Altogether, results suggest that GlgA2 is indispensable to starch in the Cyanobacterium sp. CLg1. We also carry out the biochemical characterization of synthase activities of the chlamydiales. We show that GlgA activities of some chlamydiales are able to use both nucleotide-sugars: ADP-glucose and UDP-glucose. These results stress out that GlgA activities of some chlamydial species have evolved to be more efficient to hijack the carbohydrate storage of infected cell.

La synthèse d’amidon fait intervenir de multiples enzymes dont des enzymes de débranchement qui clivent certaines liaisons glucosidiques et donnent au polysaccharide en formation une structure dense et insoluble. Chez Arabidopsis, cette activité est dépendante de la présence des protéines ISA1 et ISA2 qui s’assemblent en complexe hétéromultimérique. Chez le maïs, ISA1 peut également fonctionner sous forme d’homomultimères et la synthèse d’amidon est maintenue dans l’albumen même en absence d’ISA2. La première partie de mon travail de thèse a consisté à entreprendre une analyse comparative des activités isoamylases de maïs et d’ A. thaliana. Ces différents travaux nous ont permis de conclure que la capacité d’ISA1 de maïs à fonctionner en absence d’ISA2 constitue une caractéristique évolutive probablement acquise lors de la divergence entre monocotylédones et dicotylédones. Dans un second temps je me suis intéressée à l’initiation de la synthèse de nouveaux grains. Des travaux antérieurs avaient mis en évidence le rôle essentiel de l’amidon synthase 4 (SS4) dans ce processus. L’analyse in silico de la séquence et différentes observations phénotypiques nous ont conduit à émettre l’hypothèse de l’existence d’un dialogue moléculaire entre division du plaste et métabolisme de l’amidon. Nous avons initié différents travaux visant à identifier les fonctions des parties Cter et Nter de la protéine dans le processus d’initiation. La recherche de partenaires potentiels de l’enzyme et l’étude de certains candidats nous ont permis d’ouvrir de nouvelles pistes de recherche quant à une régulation possible de l’activité de SS4. / Starch synthesis needs a debranching enzyme activity that will cleave some of the glucosidic bounds. This activity allows the formation of a dense and insoluble cluster-like structure. In Arabidopsis, this activity is strictly dependent on the presence of both ISA1 and ISA2 proteins that assemble into active hetero-oligomers. However, in maize, ISA1 is able form active homo-oligomers and starch synthesis is maintained in the endosperm even when ISA2 is missing. In a first part of my thesis, I undertook a comparative analysis of maize and Arabidopsis isoamylases. This work allowed us to conclude that the ability of maize ISA1 to function without ISA2 represent a characteristic acquired during evolution probably at the time of divergence between monocots and dicots.In a second part of my thesis work, I focused my research on the initiation of new starch granules. Previous work already demonstrated the essential role of starch synthase 4 (SS4) in this process. In silico protein sequence analysis and phenotypic observations led us to hypothesize the existence of a molecular dialogue, involving SS4, linking plastid division to starch metabolism. We initiated in vitro and in vivo analysis with the objective to identify the role of SS4 C-terminal or N-terminal moiety. Finally, the search for potential interacting partners of the enzyme and study of some candidates allowed us identify a potential new regulation process of SS4.