L'école doctorale SDSV

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MàJ 23/02/17

 

Rappel aux orateurs : Les exposés dureront 10 mn et seront suivis de 5 mn de question. En raison de l'afflux de doctorants étrangers, la langue officielle de la journée est l'anglais pour les communications scientifiques : exposés, diaporamas et posters.

 

9h00 : Saïd MAMMAR, Vice-président de l'Université d'Évry-Val d'Essonne
9h15 : Pierre CAPY, Directeur de l'ED

9h30-10h30 : SESSION 1 - modératrices : Zoé LAMA & Ilham SEFFOUH

9h30 : Céline AUBRY
Towards combinatorial biosynthesis of pyrrolamides in Streptomyces

Dir.: Sylvie Lautru, I2BC, Orsay

Due to the emergence of bacterial resistance to antibiotics, there is an urgent need to develop new molecules. Combinatorial biosynthesis is one of the tools available to develop new bioactive metabolites. It consists in combining biosynthetic genes from the pool of genes known to be involved in the biosynthesis of various natural products to generate natural product analogs. Although a number of studies have established feasibility of combinatorial biosynthesis [1], the yield of the natural product analogs greatly varies among these studies. This demonstrates the insufficiency of the current knowledge regarding the factors (domains / protein interactions, substrate specificity of domains / enzymes) determining the outcome of combinatorial biosynthesis approaches [2]. In this study, we aim at filling this gap using the pyrrolamide family as a model. Like many antibiotic compounds, the pyrrolamides are synthetized by non-ribosomal peptide synthetases (NRPS). Usually, these enzymes are mega-complexes formed of modules and domains, but the NRPS of the pyrrolamide family are non-canonical NRPS made of stand-alone modules and domains, which makes them easier to engineer genetically. Furthermore, there are examples of natural combinatorial biosynthesis among the pyrrolamide family [3]. In this work, we will present the different groups of genes of pyrrolamides that have been or are being characterized and the tools constructed for combinatorial biosynthesis.

References:

9h45 : Imène B. BOUHLEL
Role of human Sfi1 and Centrins in Centriole biogenesis

Dir.: Anne Paoletti, Compartimentation et dynamique cellulaires, Paris-Sud

The centrosome is the main microtubule organizing center. It nucleates and organizes interphase microtubule and contributes to the assembly of the bipolar mitotic spindle. To do so, the centrosome, present in one copy at the beginning of the cell cycle, duplicates to produce a second copy. The duplication process is tightly controlled and regulated since centrosome over-duplication can lead to multipolar mitotic spindles and promote genome instability and tumorigenesis.

The duplication of the yeast centrosome, the SPB (Spindle pole body), begins with the duplication of the half bridge. This appendage is composed of Sfi1/Cdc31 complex organized in a parallel array attached to the core SPB. SPB duplication relies on the assembly of a second array of Sfi1/Cdc31, anti-parallel to the first one, creating thereby an assembly site for the new SPB. Therefore Sfi1 is essential for SPB duplication and our previous work defined the timing of half-bridge duplication and some of the regulatory mechanisms that favor bridge splitting to release duplicated centrosomes and allow spindle assembly at mitotic onset.

Sfi1 and Cdc31/Centrins are conserved in human cells where the centrosome is composed of two centrioles surrounded by the pericentriolar material.

Centrins are concentrated in the distal end of centrioles. Sfi1 has also been localized to centrioles, but its function remained unknown. Thus, we started investigating Sfi1 function in human cells. We found that Sfi1 depletion leads to an asymmetry in Centrin recruitment to the centrioles. It also leads to a cell cycle arrest in G1 in RPE1 cells, an event previously observed in presence of defects in centriole biogenesis. Moreover, Sfi1 depletion leads to cilium assembly defects. To conclude, these results altogether point towards a role of human Sfi1 in centriole biogenesis.

10h00 : Cyril CARVALHO
Inhibition of oncogene-induced senescence by glucocorticoïds and implication of the EGR1 transcription factor

Dir.: Carl Mann, Service de Biologie Intégrative et Génétique Moléculaire, CEA

mportant tumor-suppressor mechanism. We use an inducible B-RAF-V600E to trigger Oncogene Induced Senescence (OIS) in human immortalized fibroblast. B-RAF-V600E is a mutation commonly found in various types of cancer (80% of malignant melanoma). We noticed that when we induce senescence through the expression of our oncogene and at the same time treat the cells with glucocorticoïds, the cells were able to continue to proliferate for a certain amount of time. We have been able to show that the glucocorticoïds effects on the proliferation were due to the regulation of p15-cdkn2b and p21 cdkn1a. By using transcriptomic analysis and a screening of SiRNA, we identified EGR1 as an important regulator of senescence induction.

10h15 : Drice CHALLAL
Unexpected role for yeast transcription factors in termination

Dir.: Domenico Libri, IJM, Paris

Transcription termination is a key step required for 3’ end processing of RNA and is also important to precisely delimit transcriptional units and prevent the occurrence of overlapping transcription events. Overlapping transcription has been shown to represent a danger for the cell because transcription through the promoter of a neighboring gene leads to its downregulation. In yeast, mRNA coding genes are terminated by the Cleavage and Polyadenylation Factor complex (CPF), which is associated with the production of stable RNAs that are exported in the cytoplasm to be translated.

We recently described a new transcription termination mechanism by which a transcription factor binding to the DNA can roadblock the polymerases. We have generated high resolution transcription maps of the yeast genome upon depletion of roadblock factors and studied the impact of this termination pathway on the transcriptome. We show that roadblock termination functions to restrict transcription of polymerases that naturally fail to terminate at CPF termination sites, thus protecting promoters against transcriptional interference.

10h30 - 11h00 : pause

11h00-12h00 : SESSION 2 - modérateurs : Benjamin GRANDON & Tatiana MAROILLEY

11h00 : Dorian COLLOT
Feedback loops between traits under selection and the environment in batch experimental evolution: a mathematical analysis

Dir.: Olivier Martin & Judith Legrand, GQE-Le Moulon, Gif

In complex environments, such as seasonal environments, adaptation relies on several traits interacting with the environment. Using mathematical modeling of batch experimental evolution, we explored the link between selection, traits and season length.In yeast glucose batch cultures, population dynamics can be separated in three seasons: a fermentation season, during which glucose is consumed and ethanol produced; a respiration season, where ethanol is consumed and a last one where resources are exhausted. Using an ordinary differential equations model calibrated on data of a yeast experimental evolution in successive batch cultures (Spor et al., Evolution, 2014), we showed that the seasonality drives the selection.

We found that: (a) a long phase increases selection on the growth rate during this phase, (b) traits that drive response to environmental changes are also under selection (c) different types of feedback between environmental changes and selection pressures occur. Indeed, selection affects the composition of the population, which in turn leads to changes in resources (glucose and ethanol) and poison (ethanol) dynamics and consequently modifies season length and thus selective pressures on traits. We will present these different feedbacks and discuss their impact on life-history traits evolution.

11h15 : Olivier FOGEL
Epigenetic changes in Spondyloarthritis: The EPISPA project

Dir.: Jorg Tost, Centre National d eGénotypage, UEVE

Spondyloarthritis (SpA) is a complex disease involving genetic, epigenetic and environmental contributions to disease risk. Genetic variations explain only 25 % of SpA heritability suggesting an important role for epigenetic factors such as DNA methylation or microRNAs. Computational analyses showed a large overlap of active gene regulatory regions with genetic variation associated with SpA for CD4 T cells and CD14 monocytes. No comprehensive analysis of genome-wide DNA methylation or dysregulated miR expression in specific cell populations have so far been reported in SpA.

Objective: This study aimed to perform a genome-wide DNA methylation analysis and to assess the miRNA expression patterns in the two relevant cell populations CD4and monocytes from SpA patients compared with controls.

Methods: 24 SpA patients and 16 age and sex-matched controls were monocentrically recruited in the department of rheumatology (Cochin hospital). Genome-wide DNA methylation patterns were analyzed in cell-sorted (MACS) monocytes and CD4 T-lymphocyte populations from SpA patients and controls using the Illumina Infinium Human Methylation 450K BeadChip allowing the quantitative assessment of more than 480,000 CpG positions. Expression of 360 miRNAs was assessed by qPCR using the Exiqon MicroRNA Ready-to-Use Human Panel I.

Results: 122 CpGs in 82 promoter regions in CD4+ cells and 158 CpGs in 86 promoter regions in monocytes were found to be differentially methylated including several genes involved in disease-relevant signaling cascades such as Wnt-signaling or bone metabolism, genes implicated in related disease such as psoriasis and genes in which genetic polymorphisms have previously been associated with susceptibility to SpA. A single gene was found differentially methylated in both monocytes and T-lymphocytes underlining the importance to perform these epigenetic analyses in purified cell populations. Six and 25 miRNAs were found to be differentially expressed in CD4 and monocytes respectively including miRNAs previously involved in ulcerative colitis and autoimmune diseases or in the degradation of TNFα.

Conclusions: This study is the first to analyze epigenetic changes in purified disease-relevant blood cell populations in SpA bringing into evidence a number of interesting miRNAs and promoters whose deregulation might contribute to the pathogenesis of the disease.

Authors: Olivier Fogel, Andreas Bugge Tinggaard, Shu-Fang Wang-Renault, Florence Busato, Maxime Dougados, Corinne Miceli-Richard, Jörg Tost

11h30 : Malika FOY
PRL-3/PTP4A3 phosphatase regulates integrin β1 in adhesion structures during migration of human ocular melanoma cells

Dir.: Simon Saule, Signalisation normale et pathologique : de l'embryon aux thérapies innovantes des cancers , Institut Curie, Paris-Sud</p>

In a previous transcriptomic analysis of 63 ocular melanomas of the uvea, we found that expression of the PRL-3/PTP4A3 gene, encoding a phosphatase that is anchored to the plasma membrane, was associated with the risk of metastasis, and a poor prognosis. We also showed that PRL-3 overexpression in OCM-1 ocular melanoma cells significantly increased cell migration in vitro and invasiveness in vivo, suggesting a direct role for PRL-3 in the metastatic spreading of uveal melanoma.Here, we aimed to identify PRL-3 substrates at the plasma membrane involved in adhesion to the extracellular matrix. We focused on integrin b1, which is the most highly expressed integrin in our cohort of uveal melanomas.We show that preventing PRL-3 anchorage to the plasma membrane i) abolishes PRL-3-induced migration in OCM-1 cells, ii) specifically enhances the spreading of OCM-1 cells overexpressing PRL-3, and iii) favors the maturation of large focal adhesions (FAs) containing integrin b1 on collagen I. Knockdown experiments confirmed integrin b1 involvement in PRL3-induced migration. We identified interactions between PRL-3 and integrin b1, integrin b1, a partner for integrin b1 to bind collagens, as well as with FAK P-Y397, an auto-activated form of Focal Adhesion Kinase found in FAs. We also show that integrin b1 may be dephosphorylated by PRL-3 in its intracytoplasmic S/T region, an important motif for integrin-mediated cell adhesion. Finally, we observed that PRL-3 regulated the clustering of integrin b1 in FAs on collagen I but not on fibronectin.This work identifies PRL-3 as a new regulator of cell adhesion structures to the extracellular matrix, and further supports PRL-3 as a key actor of metastasis in uveal melanoma, of which molecular mechanisms are still poorly understood.

11h45 : Transito GARCIA GARCIA
Functional study of phosphorylation of the replication controller YabA in Bacillus subtilis

Dir.: Sandrine Poncet, MICALIS

To ensure cell viability, chromosomal replication must be limited to once and only once per cell cycle. Multiple regulatory pathways therefore govern initiator proteins and replication origins. In Bacillus subtilis, a model of Gram positive bacteria including many human pathogens, YabA plays a major role in down regulating initiation replication through interaction with the initiator protein DnaA and the clamp polymerase DnaN. YabA is also a structural hub protein which interact with many protein partners, indicating it might be multifunctional. We showed that its unique overall tri-dimentional structure composed of N-terminal four helix-bundle tetramer connected to four monomeric C-terminal domains by a highly flexible linker makes YabA able to physically contact more than one protein at a time. We showed that YabA is phosphorylated by the Hanks-kinase YabT at the residue Threonine 71 localized within the flexible inter-domain region; the phosphorylation of YabA is not involved in initiation control, but rather modulates differenciation processes such as sporulation, biofilm formation and colony morphology. Our data suggest that YabA may have a role in gene regulation through its interaction with DnaA, and that YabT-dependent phosphorylation of YabA regulates this interaction.

12h00 - 12h30 : présentation de l'association I2BC
12h30 - 14h00 : déjeuner et posters

14h00-15h15 : SESSION 3 - modérateurs : Anissia AIT SAADA & Francesco LAVECCHIA

14h00 : Diyavarshini GOPAUL
Study of the molecular mechanisms linking transcription and DNA repair in Saccharomyces cerevisiae

Dir.: Julie Soutourina, Service de Biologie Intégrative et Génétique Moléculaire 

Nucleotide excision repair (NER) is a well conserved pathway that removes DNA lesions arising namely upon UV irradiation. There are two pathways of NER: global genome repair (GGR) and transcription coupled repair (TCR). GGR removes DNA lesions in the genome overall and TCR removes DNA lesions interfering with RNA polymerase II through actively transcribed genes.Defects in the NER pathway may lead to severe human pathologies. For instance in humans, mutations in the XPG gene give rise to xeroderma pigmentosum (XP) sometimes associated with Cockayne syndrome (CS). Recently, we have identified a functional link between Rad2/XPG, a 3’ endonuclease, and Mediator. The latter is a well characterized transcriptional co-regulator complex conserved in eukaryotes that recruits RNA polymerase II to promoters in response to transcriptional activators.The main goal of my project is to elucidate the molecular mechanisms behind this new role of Mediator in linking transcription and DNA repair in the budding yeast Saccharomyces cerevisiae. In particular, we aim at determining if other NER factors are functionally linked to Mediator. We have identified a physical interaction between Mediator and Rad1/XPF, Rad10/ERCC1 and with Rad26/CSB by co-immunoprecipitation without UV stress. These experiments are currently being conducted after UV irradiation. To further study the functional link between Mediator and the NER machinery, we investigated the distribution of different NER proteins on a genomic scale by ChIP-sequencing.In conclusion, we aim to better comprehend the functional interplay between transcription and DNA repair, two fundamental processes, dysfunctions of which lead to serious human diseases.

14h15 : Laura LAENCINA
The ESX-4 locus: a type VII secretion pathway involved in Mycobacterium abscessus intracellular survival

Dir.: Jean-louis Hermann, Infection et Inflammation Chronique, UVSQ

The Mycobacterium genus brings together species ranging from harmless saprophytic organisms to major human pathogens. M. abscessus, a rapid growing mycobacterium (RGM) was recently considered as an opportunistic human pathogen, responsible for a wide spectrum of infections. Genome analysis of M. abscessus showed that this mycobacterium is endowed with several virulence genes, not found in saprophytic and non-pathogenic RGM. We aimed to find if M. abscessus has acquired genomic advantages to survive in antigen presenting cells such as macrophages, or in environmental protozoa such as free-living amoeba.A transposon mutant library of a clinical strain of M. abscessus subsp. massiliense was screened based on the ability of the mycobacterium to persist or not in amoeba and macrophages. 136 out of 6000 individually screened mutants exhibited a reduced ability to survive in macrophages and/or amoeba. Among them, 16 were mutated in genes belonging to M. abscessus esx4 gene locus, belonging to the type VII secretion system. The locus ESX4 is composed of 8. One gene, eccB, was chosen to create a deletion mutant by replacing eccB by a zeocin resistance cassette of the M. abscessus Type strain. These mutants allowed confirming the attenuated phenotype in amoeba and in macrophages, and this defect was complemented by introducing a wild-type copy of eccB.The defective phenotype observed indicates that ESX4 locus participates in growth and survival of M. abscessus inside macrophages and amoeba. In addition, we were able to show in a well described zebrafish model of infection, a significate decreased of granuloma formation after infection of ZF embryos by eccB mutants, further demonstrating the impact of eccB mutation on M. abscessus behaviour in vivo. As we recently showed the potential release of M. abscessus into the cytosol, investigations towards a similar role for ESX4 in M. abscessus, as the one described for ESX1 in M. tuberculosis are presently ongoing.

14h30 : Valentine LAGAGE
Regional control of Pseudomonas aeruginosa ParABS system

Dir.: Isabelle Vallet-Gély, I2BC, Orsay

Different studies had revealed a complex organization of the bacterial chromosome inside the cell which is maintained during its replication and segregation. In many bacteria, a partition system named ParABS mediates chromosome segregation via two proteins ParA and ParB, and specific DNA sequences named parS sites which are often found near oriC. We are studying the functioning of this system in thegamma-proteobacteria Pseudomonas aeruginosa. This opportunistic human pathogen presents a unique circular chromosome of 6.3 Mb oriented from the old pole of the cell to the division plane/new pole along the oriC-dif axis, and replicated near midcell. In this bacteria, four parS sites located near oriC are strongly bound by ParB. Using molecular biology, genetics and microscopy, we established that one of the four sites is sufficient for the correct segregation of the chromosome. We demonstrated that its position on the chromosomal map is important for the parS site ability to sustain chromosome segregation, and delimited a “competence zone” of the parS site which goes from -200 kb to + 400 kb from oriC. To identify genetic elements defining this “competence zone”, we used strains in which a parS is inserted outside of it and generated programmed chromosome rearrangements. More precisely, we used the λ derived site-specific recombination system to invert chromosomal fragments, in order to bring specific sequences from the competence zone closer to the non-functional parS site, and analyze their impact on chromosome segregation. Our results indicate that one determinant of the competent zone may be oriC. We also observed that the asymmetry relative to oriC of the “competence zone” is due to the presence of a ribosomal operon at -220 kb. Finally we showed that displacing a parS site leads to a global repositioning of the chromosome inside the cell and to a delay in the segregation (until the parS site is replicated) showing that the parS is the site of force exertion.

14h45 : Perrine LAVALOU
Synteny as a determinant of lincRNA function during vertebrate melanoma formation and evolution

Dir.: Alena Shkumatava, Génétique et biologie du Développement, Institut Curie

LincRNAs are molecularly similar to messengers RNAs but lacking protein coding potential. Although the function of most lincRNAs is unknown, a subset has been implicated in human cancers. Recently, more than 550 lincRNAs were identified in the zebrafish genome, a vertebrate model used in developmental and cancer biology. Comparative genomics reveals that 5% of zebrafish lincRNAs share sequence conservation with mammalian transcripts, whereas >50% of them are maintained in syntenic positions despite 450 million years of evolutionary distance. To understand the selective pressure to maintain lincRNA genomic position, I have used Crispr-CAS9 genome editing to generate zebrafish deletion mutants for four syntenically conserved lincRNAs. As the level of some of our candidate lincRNAs is deregulated in patients suffering from melanoma, I am currently assessing their functions and roles in this deadly human cancer. Combining both zebrafish melanoma model, obtained through the insertion of the human NRAS oncogene, and syntenic lincRNA mutants, I am evaluating our candidate genes impact on melanocytic lesion and nodules formation and frequency, as well as their tumour metastatic potential using histological and molecular biology techniques. Thanks to this in vivo model, we will be able to decipher, for the first time, the role of synteny and the function of lincRNAs in melanoma.

15h00 : Debora LIA
Role of αOGG1 in the maintenance of mitochondrial function

Dir.: Anna Campalans, Stabilité génétique, cellules souches et radiations  

8-oxoguanine has been shown to accumulate in the mitochondrial genome (mtDNA) during our lifespan and this accumulation is even more pronounced during cancerogenic processes. 8-oxoG is removed by the base excision repair (BER) pathway that starts with the recognition of this damage by a specific DNA glycosylase, OGG1. My project aims first at determining if and which form of OGG1 is present in mitochondria, using both biochemical and microscopy approaches. I will then, characterize the mechanisms involved in the addressing of this enzyme in mitochondria and determine the consequences of the absence of OGG1 in the organelle physiology. Mitochondria are essential for cellular survival and apoptosis equilibrium. Alterations of these organelles are emerging as new molecular markers for cancer detection and monitoring. Moreover, in various types of human cancer a high frequency of mutations has been identified in both coding and non-coding regions of the mtDNA. Therefore, a better understanding of how mitochondrial genome is preserved is fundamental, because a misbalance of this delicate equilibrium between survival and apoptosis could lead to the development of a cancer prone environment.

15h15 - 15h45 : pause

15h45-16h45 : SESSION 4 - modérateurs : Tatiana MAROILLEY & Charbel SOUAID

15h45 : Wenfeng LIU
Staphylococcus aureus regulatory RNAs driving fitness upon antibiotic

Dir.: Philippe Bouloc, I2BC, Orsay

Staphylococcus aureus is an opportunistic pathogen, one of the major bacteria responsible for nosocomial infection, whose treatment is often complicated by the emergence of multidrug resistant strains. Our project aims at identifying and characterizing S. aureus small regulatory RNAs (sRNAs) impacting virulence and antibiotic resistance. Bacteria response to infections and treatments are indeed complex events comprising many input effectors. The regulation network driving bacterial response is multi layers. Among them, sRNAs contribute to the "fine-tuning" of gene expression. Consequently, sRNA-dependent phenotypes are in general, difficult to detect. Minor sRNA-mediated phenotypes conferring modest advantages, however, may emerge as dominant traits after a few generations under selective pressure such as growth within the host or in presence of antibiotics. sRNA mutations affecting fitness could be determined by measuring growth rates in batch cultures but tiny differences are difficult to determine with this method. However, sRNA-mediated phenotypes are expected to emerge during competitive fitness experiments i.e. by growing several strains together (each strain bearing a single sRNA deletion) and evaluating their proportions in the population at different times. Finding phenotypes associated with sRNA deletions usually require testing of many conditions for each mutant, with no assurance of success. However, introducing specific DNA sequences in each engineered sRNA mutant allow monitoring many mutants simultaneously. We adapted our protocol and the use of these DNA barcoded sequences to use deep sequencing technology. With primers specific for each experiment we detect quantitatively the mutant ratios within the mixed cultures. 12 growth conditions have been tested so far with 39 tagged S. aureus sRNA mutants. We will submit around 80 sRNA mutant library to competition fitness experiments with sublethal antibiotic concentrations and in two mice infection models (bacteremia and biofilm-mediated implant infection models). These experiments will lead to the identification of S. aureus sRNAs influencing its antibiotic resistance and virulence. The targets of these sRNAs will be identified and the molecular mechanism of their action determined by techniques that we recently developed to decipher the sRNA-associated gene networks.

16h00 : Xavier RAFFOUX
Diversity and genetic determinism of meiotic recombination rate in S. cerevisiae

Dir.: Matthieu Falque, GQE-Le Moulon, Gif

Meiotic recombination is a major driver of genome dynamics and thus evolution in sexually reproducing organisms. The number of crossing-over events and their position along the chromosomes are tightly regulated, but the mechanisms involved are still not well understood. Getting more insights into the regulation of recombination rate and crossover distribution would be beneficial for many fields of fundamental and applied genetics, in particular to improve the efficiency of plant breeding. To study the natural genetic diversity of recombination rate, we developed a high throughput method to measure crossover rates in S. cerevisiae. We constructed 8 tester strains carrying three different fluorescent markers at chosen loci, and we used flow cytometry to measure the segregation of the three markers in the progenies of hybrids between the testers and yeast strains to phenotype for recombination. Using this method, we measured the recombination rate of yeast strains representing a large part of the intraspecific diversity, and investigated some aspect of its genetic control.

16h15 : Jean ROMAN
Modification de nanopores solides pour la détection et l'analyse de nanoparticules

Dir.: Laurent Bacri, LAMBE, UEVE

Les techniques par impulsion résistive (Resistive Pulse Sensing) sont très prometteuses tant elles devraient permettre une analyse fine et rapide de nanoparticules en concentration très faible. Ces techniques se basent sur la mesure d'un courant ionique passant dans un unique nanopore percé dans une membrane fine (4-30nm) et isolante. Une particule passant dans ce pore le bouche momentanément (1µs-1s) changeant ainsi sa conductance de manière caractéristique. Seules quelques centaines de particules passant effectivement dans le pore suffisent à sonder plusieurs de leurs caractéristiques (charge, forme), d'où la très haute sensibilité attendue de la technique. L'utilisation de nanopores solides est la voie d'application de cette technique que nous étudions. Très robustes et de taille ajustable, les nanopores solides présentent bien des avantages. Néanmoins, leur durabilité est limitée. Aussi je vais vous présenter nos avancées récentes pour répondre à cette problématique.

16h30 : Julie VENDOMELE
Anti-Transgene Cellular Immune Reponses can be Induced by Subretinal Gene Transfer with rAAV in a Dose-Dependent Manner

Dir.: Sylvain Fisson, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares, UEVE

Reponses can be Induced by Subretinal Gene Transfer with rAAV in a Dose-Dependent MannerPurpose. From animal experiments to the first human clinical trials in 2007, recombinant adeno-associated virus (rAAV)-mediated ocular gene therapy has shown successful results which have been attributed in part to the immune-privileged situation of the eye. Recently, some ocular gene therapy clinical trials have reported that visual acuity returned to baseline 6 to 12 months after therapy. The involvement of anti-transgene immune responses may lead to loss of therapeutic efficacy. This prompted us to evaluate in a murine model if rAAV gene transfer leads to an anti-transgene immunization. In this study, we characterized the CD4 and CD8 T cell responses specifically directed toward the transgene product in a murine model following rAAV2/8-mediated subretinal gene transfer.Methods. A rAAV2/8 encoding for the GFP-HY fusion protein under the ubiquitous PGK promoter was used. The transgene expresses the HY male antigen which contains MHC class I and MHC class II-restricted T cell epitopes (UTY and DBY, respectively), that are immuno-dominant in female mice. Two µL of endotoxin-free, PBS-formulated rAAV2/8 PGK GFP-HY were injected subretinally in C57Bl/6 female mice. At day 7, mice were challenged subcutaneously with the UTY and DBY peptides adjuvanted in CFA, and the immune response was analyzed at day 14 by IFN ELISpot, cytokine titration and proliferation assays.Results. Our results revealed that: (i) The subretinal injection of 10E8 to 2.10E9 vg/mouse of rAAV2/8 PGK GFP-HY did not induce a significant HY-specific peripheral immune modulation in contrast to the ACAID obtained after subretinal injection of HY peptides (50µg each); (ii) Higher doses of rAAV2/8 PGK GFP-HY (5.10E10 vg/mouse) triggered increased Th1 and Tc1 cellular immune responses against the transgene product in peripheral lymphoid organs.Conclusion. We show that rAAV2/8 vector-mediated subretinal gene transfer is not necessarily ignored at the immunological level. High doses of vector can effectively trigger anti-transgene T-cell responses with the potential for elimination of transgene-expressing cells. Clearly, anti-transgene-specific immune monitoring should be refined at least in preclinical models, to improve the biosafety and the long-term efficacy of rAAV-mediated ocular gene transfer. Moreover, immunosuppressive strategies concomitant to the AAV injection are currently being tested.

16h45 : remise des prix aux deux meilleurs exposés et deux meilleurs posters

17h00-18h00 : conférence de Maureen O'Malley "From microbiota to human brains: questions about causality, control and coevolution"

Microbiota research is a recent sequencing-based development that allows the compositional and functional analysis of microbial communities in specific niches. Some of the most intensive research in this area has been carried out on the gut microbiota of animals. Many associations have been made between microbiota composition and various health or disease states. A rapidly expanding area of investigation is concerned with the connections between animal gut microbiota, the enteric nervous system, and the brain and behaviour. Links have been made between microbiota composition and disorders such as autism, anxiety and depression. Many strong interpretations have been made of these findings, including claims that microbiota control animal behaviour. I will examine these claims in the context of how this research has been carried out, and the various problems in microbiota research methodology.

 

Présentation de la Journée

Les posters des doctorants

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