N011 Culture et délivrance au niveau du tissu cardiaque de cardiomyocytes issus de cellules souches embryonnaires humaines au moyen de matrices tridimensionelles poreuses à base de polysaccharides

Un intérêt particulier a été porté ces dernières années à la thérapie cellulaire réparatrice cardiaque. Les cellules souches embryonnaires humaines (hES) sont une source prouvée de cardiomyocytes et les premières données in vivo suggèrent leurs capacités fonctionnelles à type d’effet pacemaker ou réparatrices d’infarctus du myocarde. Nous avons étudié un mode de délivrance des cellules hES dans le tissu cardiaque basé sur une matrice 3D servant de support à la fois pour la culture des cellules et pour leur implantation au contact du myocarde.Des matrices poreuses de polysaccharides (pullulane et dextrane) ont été préparées par réticulation chimique permettant de réaliser des films avec des pores de 100 à 200 microns. Les matrices ont été recouvertes de différentes protéines; les cellules hES indifférenciées ont été cultivées sur fibroblastes murins, en milieu supplémenté avec du sérum knock-out et du FGF2. Dans une première partie in vitro, nous avons mis en évidence par q-RT-PCR, observation microscopique et imagerie confocale, la différenciation en cardiomyocytes de cellules hES directement cultivées dans les matrices en présence d’un milieu inducteur de différentiation; les matrices permettaient aussi la culture, l’expansion et la survie à long terme de parties battantes obtenues à partir de corps embryoïdes issus d’hES et isolées manuellement. Nous avons ensuite étudié le devenir des cellules hES dans un modèle de lésions cardiaques par dépôt de films poreux cellularisés sur les cœurs infarcis de souris NOD SCID. L’identification est confirmée pour les cardiomyocytes issus d’ES d’une lignée de cellules hES H9 GFP+ ainsi que d’une lignée de cellules hES dans laquelle l’expression de la GFP est sous contrôle d’un promoteur spécifique du tissu cardiaque, Nkx2.5. Nous avons ainsi mis en évidence la migration des cellules ES à différents stades de différenciation à partir des matrices 3D vers les souris NOD SCID ainsi que leur différenciation en cardiomyocytes. Les données de PCR quantitative sur la base du transgène GFP mettent en évidence une meilleure survie des ES délivrées par l’intermédiaire des matrices 3D en comparaison avec une administration directe. Une étude fonctionnelle comparative est en cours

Social acceptability of a marine protected area: The case of Reunion Island

This paper examines variations in social acceptability of a Marine Protected Area (MPA) prior to implementation. The influence of a number of factors, including socio-economic characteristics, perception of coral resources state of health and attitudes towards non-compliance with regulations are analysed. During May 2006, 640 questionnaires were distributed to school children around Reunion Island, Western Indian Ocean, for completion by their parents, following an informal educational activity made in school. From a 73% (n = 469) response rate, results showed that 78% of participants were in favour of the MPA. Analysis further identified that those supportive of the MPA were generally from higher socio-professional categories, had a negative perception of the coral reef ecosystem's health and were not originally from Reunion. In contrast, locals (born in Reunion) from lower socio-professional categories or with no employment activity and having a positive perception of the health status of coral reefs offered no opinion on the MPA. Attitudes towards enforcement and compliance highlighted that SCUBA divers, fishers and jet skiers attributed a higher value to the protection of the coral reef environment through enforcement of MPA regulations than to their own use of the coral reef resource. When asked about the use of penalties to deter non-compliance, swimmers were awarded the lowest fines, followed by SCUBA divers, fishers then jet skiers being awarded the highest fines. Thus, the more severe the act of non-compliance by a resource user group was perceived to be, the more these users themselves disapproved of non-compliant behaviour and supported use of high penalties. The survey design through focusing on school children's parents, demonstrated a simple and cost-effective method for data collection while providing environmental education, which could be employed in similar case studies elsewhere

Steric masking of a dilysine endoplasmic reticulum retention motif during assembly of the human high affinity receptor for immunoglobulin E.

International audienceSignals that can cause retention in the ER have been found in the cytoplasmic domain of individual subunits of multimeric receptors destined to the cell surface. To study how ER retention motifs are masked during assembly of oligomeric receptors, we analyzed the assembly and intracellular transport of the human high-affinity receptor for immunoglobulin E expressed in COS cells. The cytoplasmic domain of the alpha chain contains a dilysine ER retention signal, which becomes nonfunctional after assembly with the gamma chain, allowing transport out of the ER of the fully assembled receptor. Juxtaposition of the cytoplasmic domains of the alpha and gamma subunits during assembly is responsible for this loss of ER retention. Substitution of the gamma chain cytoplasmic domain with cytoplasmic domains of irrelevant proteins resulted in efficient transport out of the ER of the alpha chain, demonstrating that nonspecific steric hindrance by the cytoplasmic domain of the gamma chain accounts for the masking of the ER retention signal present in the cytoplasmic domain of the alpha chain. Such a mechanism allows the ER retention machinery to discriminate between assembled and nonassembled receptors, and thus participates in quality control at the level of the ER.Signals that can cause retention in the ER have been found in the cytoplasmic domain of individual subunits of multimeric receptors destined to the cell surface. To study how ER retention motifs are masked during assembly of oligomeric receptors, we analyzed the assembly and intracellular transport of the human high-affinity receptor for immunoglobulin E expressed in COS cells. The cytoplasmic domain of the alpha chain contains a dilysine ER retention signal, which becomes nonfunctional after assembly with the gamma chain, allowing transport out of the ER of the fully assembled receptor. Juxtaposition of the cytoplasmic domains of the alpha and gamma subunits during assembly is responsible for this loss of ER retention. Substitution of the gamma chain cytoplasmic domain with cytoplasmic domains of irrelevant proteins resulted in efficient transport out of the ER of the alpha chain, demonstrating that nonspecific steric hindrance by the cytoplasmic domain of the gamma chain accounts for the masking of the ER retention signal present in the cytoplasmic domain of the alpha chain. Such a mechanism allows the ER retention machinery to discriminate between assembled and nonassembled receptors, and thus participates in quality control at the level of the ER

PHIL Accelerator at LAL - Diagnostic status

http://accelconf.web.cern.ch/AccelConf/BIW2010/papers/tupsm100.pdfInternational audienceThe "Photo-Injector at LAL" (PHIL : http://phil.lal.in2p3.fr/) is a new electron beam accelerator at LAL. This accelerator is dedicated to test and characterise electron photo-guns and high-frequency structures for future accelerator projects (like the next generation lepton colliders, CLIC, ILC). This machine has been designed to produce low energy (E<10 MeV), small emittance (epsilon < 10 pi.mm.mrad), high current (charge 2 nC/bunch) electrons bunch at low repetition frequency (frep<10Hz) [1]. The first beam has been obtained on the 4th of November 2009. This paper will describe the current status and the futures developments of the diagnostics devices on this machine

Low Energy Beam Measurements Using PHIL Accelerator at LAL, Comparison with PARMELA Simulations

http://accelconf.web.cern.ch/AccelConf/PAC2011/papers/wep210.pdfInternational audiencePHIL ("PHo­to-In­jec­tor at LAL") is a new elec­tron beam ac­cel­er­a­tor at LAL. This ac­cel­er­a­tor is ded­i­cat­ed to test and char­ac­ter­ize elec­tron RF-guns and to de­liv­er elec­tron beam to users. This ma­chine has been de­signed to pro­duce and char­ac­terise low en­er­gy (E<10 MeV), small emit­tance (e<10 p.​mm.​mrad), high bril­liance elec­trons bunch at low rep­e­ti­tion fre­quen­cy (n<10Hz). The first beam has been ob­tained on the 4th of Novem­ber 2009. The cur­rent RF-gun test­ed on PHIL is the Al­phaX gun, a 2.5 cell S-band cav­i­ty de­signed by LAL for the plas­ma ac­cel­er­a­tor stud­ies per­formed at the Strath­clyde uni­ver­si­ty. This paper will pre­sent the first Al­phaX RF-gun char­ac­ter­i­za­tions per­formed at LAL on PHIL ac­cel­er­a­tor, and will show com­par­isons be­tween mea­sure­ments and PARMELA sim­u­la­tions

Combined transcriptomic-(1)H NMR metabonomic study reveals yhat monoethylhexyl phthalate stimulates adipogenesis and glyceroneogenesis in human adipocytes

Adipose tissue is a major storage site for lipophilic environmental contaminants. The environmental metabolic disruptor hypothesis postulates that some pollutants can promote obesity or metabolic disorders by activating nuclear receptors involved in the control of energetic homeostasis. In this context, monoethylhexyl phthalate (MEHP) is of particular concern since it was shown to activate the peroxisome proliferator-activated receptor γ (PPARγ) in 3T3-L1 murine preadipocytes. In the present work, we used an untargeted, combined transcriptomic-(1)H NMR-based metabonomic approach to describe the overall effect of MEHP on primary cultures of human subcutaneous adipocytes differentiated in vitro. MEHP stimulated rapidly and selectively the expression of genes involved in glyceroneogenesis, enhanced the expression of the cytosolic phosphoenolpyruvate carboxykinase, and reduced fatty acid release. These results demonstrate that MEHP increased glyceroneogenesis and fatty acid reesterification in human adipocytes. A longer treatment with MEHP induced the expression of genes involved in triglycerides uptake, synthesis, and storage; decreased intracellular lactate, glutamine, and other amino acids; increased aspartate and NAD, and resulted in a global increase in triglycerides. Altogether, these results indicate that MEHP promoted the differentiation of human preadipocytes to adipocytes. These mechanisms might contribute to the suspected obesogenic effect of MEHP

Is the 6 kDa tobacco etch viral protein a bona fide ERES marker?

The claim that the 6 kDa viral protein (VP) of Tobacco Etch Virus is a marker for ER exit sites (ERES) has been investigated. When transiently expressed as a CFP tagged fusion construct in tobacco mesophyll protoplasts, this integral membrane protein co-localizes with both the COPII coat protein YFP-SEC24 and the Golgi marker Man1-RFP. However, when over-expressed the VP locates to larger spherical structures which co-localize with neither ER nor Golgi markers. Nevertheless, deletion of the COPII interactive N-terminal D(X)E motif causes it to be broadly distributed throughout the ER, supporting the notion that this protein could be an ERES marker. Curiously, whereas brefeldin A (BFA) caused a typical Golgi-stack response (redistribution into the ER) of the VP in leaf epidermal cells, in protoplasts it resulted in the formation of structures identical to those formed by over-expression. However, anomalous results were obtained with protoplasts: when co-expressed with the non-cycling cis-Golgi marker Man1-RFP, a BFA-induced redistribution of the VP-CFP signal into the ER was observed, but, in the presence of the cycling Golgi marker ERD2-YFP, this did not occur. High resolution images of side-on views of Golgi stacks in epidermal cells showed that the 6 kDa VP-CFP signal overlapped considerably more with YFP-SEC24 than with Man1-RFP, indicating that the VP is proportionately more associated with ERES. However, based on a consideration of the structure of its cytoplasmic tail, the scenario that the VP collects at ERES and is transported to the cis-Golgi before being recycled back to the ER, is supported

A genome-wide approach reveals novel imprinted genes expressed in the human placenta

Genomic imprinting characterizes genes with a monoallelic expression, which is dependent on the parental origin of each allele. Approximately 150 imprinted genes are known to date, in humans and mice but, though computational searches have tried to extract intrinsic characteristics of these genes to identify new ones, the existing list is probably far from being comprehensive. We used a high-throughput strategy by diverting the classical use of genotyping microarrays to compare the genotypes of mRNA/cDNA vs. genomic DNA to identify new genes presenting monoallelic expression, starting from human placental material. After filtering of data, we obtained a list of 1,082 putative candidate monoallelic SNPs located in more than one hundred candidate genes. Among these, we found known imprinted genes, such as IPW, GRB10, INPP5F and ZNF597, which contribute to validate the approach. We also explored some likely candidates of our list and identified seven new imprinted genes, including ZFAT, ZFAT-AS1, GLIS3, NTM, MAGI2, ZC3H12Cand LIN28B, four of which encode zinc finger transcription factors. They are, however, not imprinted in the mouse placenta, except for Magi2. We analyzed in more details the ZFAT gene, which is paternally expressed in the placenta (as ZFAT-AS1, a non-coding antisense RNA) but biallelic in other tissues. The ZFAT protein is expressed in endothelial cells, as well as in syncytiotrophoblasts. The expression of this gene is, moreover, downregulated in placentas from complicated pregnancies. With this work we increase by about 10% the number of known imprinted genes in humans