Sialylation in planta (Potentiel endogène et reconstitution par transgénèse)

Les plantes sont des systèmes d'expression de glycoprotéines thérapeutiques recombinantes performants, ne présentant pas d'agents pathogènes transmissibles à l'homme. Au contraire des mammifères, les N-glycannes végétaux ne possèdent pas d'acides sialiques terminaux, comme le Neu5Ac. Ces résidus contrôlent la demi-vie de nombreuses glycoprotéines circulantes. L'absence de Neu5Ac peut entraîner la perte d'activités de ces protéines. Après avoir vérifié l'absence d'acides sialiques dans la cellule végétale, nous avons tenté de détourner l'activité de la Kdo-8-P synthase végétale pour synthétiser du Neu5Ac. Nos résultats montrent que la voie de biosynthèse du Kdo ne peut pas être redirigée vers la synthèse de Neu5Ac. Enfin, deux enzymes bactériennes, la Neu5Ac lyase et la Neu5Ac synthase NeuB2 ont été exprimées chez les cellules BY2 de tabac et la luzerne. Nos tests d'activité ont démontré que ces enzymes sont fonctionnelles chez les plantes et permettent la synthèse du Neu5Ac in vitro.Plants are low cost and contamination safe factories for the production of recombinant pharmaceutical proteins. However, plant glycoproteins differ from their mammalian counterparts. For instance, mammalian N-glycans harbour terminal sialic acids, such as Neu5Ac, that control their half-life in the blood stream. Absence of sialylation in plants is of major concern as non sialylated pharmaceuticals may not be biologically active in humans. In this context, we have investigated the synthesis of Neu5Ac in the plant cytosol by the re-routing of the endogenous Kdo-8-P synthase. We have demonstrated that the plant Kdo-8-P synthase is not able to use the Neu5Ac precursor as substrate and thus, that the re-routing of the Kdo pathway to synthesise Neu5Ac is not possible. Microbial Neu5Ac-synthesising enzymes were then expressed in two model plants, BY2 tobacco cells and alfafa. This resulted in the production of functional cytosolic enzymes in plants able to synthesise Neu5Ac in vitro.ROUEN-BU Sciences (764512102) / SudocSudocFranceF

Complément et neuroprotection

Pendant longtemps, l'activation du complément exprimé dans le système nerveux central a été considérée comme délétère pour les cellules environnantes puisque permettant une amplification de l'inflammation et surtout la lyse cellulaire par le complexe d'attaque membranaire. Mais le concept d'un rôle du complément dans la neuroprotection a émergé suite à des données plus récentes. Le travail de cette thèse a consisté à explorer les mécanismes que le complément met en place également par son activation pour protéger les cellules avoisinantes, les neurones essentiellement. Cela peut passer par une augmentation d'expression de CD55 au niveau neuronal ou par la libération de NGF par les astrocytes grâce à un effet synergique des anaphylatoxines et IL-1b. En revanche, la phagocytose des cellules en apoptose, processus important pour éviter le déclenchement d'un réaction inflammatoire n'est pas régulée par le C3a.For a long time, the classical dogma has considered complement activation in brain pathology deleterious for surrounding cells since it promotes inflammation processes and triggers membran attack complexe formation resulting in cell lysis. A growing body of evidence implicates complement in neuoroprotection processes. My PhD work consisted in exploring the different mecanisms set up by complement activation to protect cells, neurons essentially. The up-regulation of neuronal CD55 or NGF secretion by astrocytes following a simultaneous anaphylatoxins and IL-1b stimulation may contibute to neuronal protection . On the other hand, phagocytosis of apoptotic cells, which is an important process to prevent inflammatory reaction is not regulated by C3a.ROUEN-BU Sciences (764512102) / SudocSudocFranceF

Erythropoietine et neuroprotection

Erythropoietin (Epo) has long been recognised for its role in the control of erythropoiesis and therefore in the treatment of anemia including anemia of prematurity. The erythropoietin receptor (Epo-R) though is expressed in many other organs including the CNS. This review focuses on the role of erythropoietin during the development of the CNS and its potential role as a neuroprotective agent. Epo-R is expressed in many different cellules of the CNS during development including neural progenitor cells, neurons, astrocytes and oligodendrocytes. In the event of hypoxia CNS cells respond with increase of erythropoietin release with subsequent stimulation of neurogenesis through Epo-R on neural progenitor cells. In an Epo-R knock-out model therefore cerebral development is severely impaired. In models of hypoxia-ischemia exogenous Epo has been shown to reduce lesion size and improve structural and functional recovery. Human studies are emerging using Epo as a neuroprotective agent both for the term infant with hypoxia-ischemia as well as for the extremely preterm infant

Lactoferrin during lactation protects the immature hypoxic-ischemic rat brain

Lactoferrin (Lf) is an iron-binding glycoprotein secreted in maternal milk presenting anti-inflammatory and antioxidant properties. It shows efficient absorption into the brain from nutritional source. Brain injury frequently resulting from cerebral hypoxia-ischemia (HI) has a high incidence in premature infants with ensuing neurodevelopmental disabilities. We investigated the neuroprotective effect of maternal nutritional supplementation with Lf during lactation in a rat model of preterm HI brain injury using magnetic resonance imaging (MRI), brain gene, and protein expression

Interleukin-1beta and anaphylatoxins exert a synergistic effect on NGF expression by astrocytes.

C3a and C5a anaphylatoxins are proinflammatory polypeptides released during complement activation. They exert their biological activities through interaction with two G protein-coupled receptors named C3aR and C5aR, respectively. In the brain, these receptors are expressed on glial cells, and some recent data have suggested that anaphylatoxins could mediate neuroprotection. In this study, we used RT-PCR and ribonuclease protection assays (RPA) to investigate the role of anaphylatoxins on neurotrophin expression by the human glioblastoma cell line T98G and by rat astrocytes. Our data show that for both cell types, anaphylatoxins upregulate expression of NGF mRNA. This response depended on a G protein-coupled pathway since pre-treatment of cells with pertussis toxin (PTX) completely blocked NGF mRNA increases. This effect was anaphylatoxin-specific since pre-incubation with anti-C3a or anti-C5aR antibodies abolished the effects of C3a and C5a, respectively. The regulation of NGF mRNA by anaphylatoxins was not accompanied by translation into protein expression, but there was a significant synergic effect of anaphylatoxins/IL-1b costimulation. Our demonstration of involvement of anaphylatoxins in the NGF release process by astrocytes suggests that C3a and C5a could modulate neuronal survival in the CNS

DTI analysis of microstructure.

<p>Direction encoded color maps of typical P25 Control, EPO and NaCl pup rat brain at four different image-planes corresponding to the four levels of DTI indices measurements: Splenium, Body, Body and Genu of the corpus callosum. The different ROIs analyzed are overlaid on the Control map: external capsule (EC), internal capsule (IC), corpus callosum (CC) and cortex (Cx).</p