Sterilization of Exopolysaccharides Produced by Deep-Sea Bacteria: Impact on Their Stability and Degradation

Polysaccharides are highly heat-sensitive macromolecules, so high temperature treatments are greatly destructive and cause considerable damage, such as a great decrease in both viscosity and molecular weight of the polymer. The technical feasibility of the production of exopolysaccharides by deep-sea bacteria Vibrio diabolicus and Alteromonas infernus was previously demonstrated using a bioproduct manufacturing process. The objective of this study was to determine which sterilization method, other than heat sterilization, was the most appropriate for these marine exopolysaccharides and was in accordance with bioprocess engineering requirements. Chemical sterilization using low-temperature ethylene oxide and a mixture of ionized gases (plasmas) was compared to the sterilization methods using gamma and beta radiations. The changes to both the physical and chemical properties of the sterilized exopolysaccharides were analyzed. The use of ethylene oxide can be recommended for the sterilization of polysaccharides as a weak effect on both rheological and structural properties was observed. This low-temperature gas sterilizing process is very efficient, giving a good Sterility Assurance Level (SAL), and is also well suited to large-scale compound manufacturing in the pharmaceutical industry

Régulation des transporteurs vésiculaires du glutamate chez les rongeurs

Les transporteurs vésiculaires du glutamate, VGLUT1, -2 et -3, sont des protéines présentes à la membrane des vésicules synaptiques des neurones glutamatergiques. Ils sont responsables de l'accumulation du neuromédiateur dans les vésicules. VGLUT1 et VGLUT2 sont les sous-types majoritaires dans le cerveau et couvrent la totalité des neurones glutamatergiques avec des profils d'expression complémentaires. VGLUT3 est exprimé de façon plus discrète dans des populations de neurones contenant d'autres neuromédiateurs. Au cours de cette thèse, j'ai participé à la description détaillée de la localisation de VGLUT3 dans le cerveau adulte de rat, ainsi qu'à l'étude de l'ontogenèse des trois sous-types. J'ai également réalisé un crible double-hybride avec une partie cytoplasmique de VGLUT1 à la recherche de différences fonctionnelles entre les trois sous-types. J'ai ainsi caractérisé l'interaction entre VGLUT1 et l'endophiline, une protéine impliquée dans l'endocytose des vésicules synaptiques. Cette interaction suggère un lien fonctionnel entre l'accumulation de neuromédiateur et le recyclage des vésicules synaptiques dans la terminaison nerveuse.The vesicular glutamate transporters, VGLUT1, -2 and -3, are proteins present at the membrane of synaptic vesicles in glutamatergic neurons. They are responsible for the accumulation of neurotransmitter into the vesicles. VGLUT1 and VGLUT2 are the majoritary subtypes in the brain and their expression covers all known glutamatergic neurons with complementary expression patterns. VGLUT3 is expressed in a more discrete fashion in neuron populations which were not previously thought to be glutamatergic. During this thesis, I contributed to the detailled description of the localisation of VGLUT3 in the adult rat brain, as well as the ontogenic study of all three subtypes. I also carried out a yeast two-hybrid screen in order to search for putative functionnal differences between the subtypes. Thus, I characterised an interaction between VGLUT1 and endophilin, a protein involved in the endocytosis of synaptic vesicles. This interaction suggests a functional link between the loading in neurotransmitter and the recycling of synaptic vesicles in the nerve terminal.PARIS12-CRETEIL BU Multidisc. (940282102) / SudocSudocFranceF

Purification of the exopolysaccharide produced by Alteromonas infernus: identification of endotoxins and effective process to remove them

Alteromonas infernus bacterium isolated from deep-sea hydrothermal vents can produce by fermentation a high molecular weight exopolysaccharide (EPS) called GY785. This EPS described as a new source of glycosaminoglycan-like molecule presents a great potential for pharmaceutical and biotechnological applications. However, this unusual EPS is secreted by a Gram-negative bacterium and can be therefore contaminated by endotoxins, in particular the lipopolysaccharides (LPS). Biochemical and chemical analyses of the LPS extracted from A. infernus membranes have shown the lack of the typical LPS architecture since 3-deoxy-d-manno-oct-2-ulopyranosonic acid (Kdo), glucosamine (GlcN), and phosphorylated monosaccharides were not present. Unlike for other Gram-negative bacteria, the results revealed that the outer membrane of A. infernus bacterium is most likely composed of peculiar glycolipids. Furthermore, the presence of these glycolipids was also detected in the EPS batches produced by fermentation. Different purification and chemical detoxification methods were evaluated to efficiently purify the EPS. Only the method based on a differential solubility of EPS and glycolipids in deoxycholate detergent showed the highest decrease in the endotoxin content. In contrast to the other tested methods, this new protocol can provide an effective method for obtaining endotoxin-free EPS without any important modification of its molecular weight, monosaccharide composition, and sulfate content