L'école doctorale SDSV

Le doctorat


Direction de thèse

Liens utiles

MàJ 23/02/16


Les posters doivent être rédigés en anglais. Les posters seront au format A0.


1. Aude BRETON
All LPS are not endotoxins: structure to activities relationship on different examples

Endotoxins are the main components of the outer membrane of Gram-negative bacteria. Each bacteria possesses a specific lipopolysaccharides (LPS) structure which is characteristic and unique like a finger print for human can be. Chemically, endotoxins are LPS, but all LPS are not endotoxins. They are composed of two moieties: the polysaccharidic (PS) and the lipid A. The PS are made of the so-called O-antigens linked to core oligosaccharides. They carry the antigenic properties of the molecule and can be used in vaccines as well as in bacterial detection tests. The lipid A, is responsible for most of the biological activities of the molecule. These activities can be beneficial at low doses, or toxic, even leading to septic shock and death when present at higher quantities. Earlier structure to activity relationship studies have shown that the smallest structural detail can make all the difference in terms of biological activities. Vitreoscilla filiformis is a Gram-negative bacterium isolated from spa waters and described for its beneficial effects on the skin. Since its endotoxin was a potential biologically active contributor to the observed skin properties, we characterized the detailed structure of its lipid A moiety. We compare here the structure to activity relationships of these LPS to others differing by the chain-length of their fatty acids, their linkage position on the GlcN, and their impact on the IL-6 and TNF-α inflammatory responses and cytotoxicity.
I2BC, Martine Caroff

2. Olivier BUGAUD
Translational non-sense suppression, a therapeutic strategy against cancer

Nonsense mutations, also known as premature termination codons (PTCs) are responsible for 10% of all genetic diseases including cystic fibrosis (CF), Duchenne muscular dystrophy (DMD) and several cancers. Most PTCs found in cancer cells in activate tumor suppressor genes leading to tumorigenesis. Translational nonsense suppression strategy aims to re-express a functional full-length protein to restore normal cellular functions. However, the low number of currently available molecule constitutes the main barrier to the development of this approach. Our objectives aim at identifying new read through inducer drugs and understanding their mechanisms of action. We have focused on two different strategies: First, we tested new synthetic aminoglycoside derivatives specifically developed for PTC suppression. The chemical structure of these molecules has been modified to improve their efficiency. Our work revealed a new molecule that efficiently suppresses PTC and allows the re-expression of a functional protein that promotes apoptosis of cancer cells. Our second strategy was to screen large chemical libraries using a new reporter system, based on stable cell line expressing a secreted luciferase. This gene, interrupted by a nonsense mutation, is expressed only if read through is stimulated. Among 17 000 molecules screened, we identified 48 candidates increasing luciferase activity. We used several secondary assays to discriminate true stop codon inducers from false positive hits. This leads to the identification of 3 compounds that specifically increase read through up to 20 times. These results pave the way for the development of new treatments for diseases linked with non-sense mutations. We will now identify the mechanism of action of these new drugs. A more detailed characterization of the mechanisms targeted by these molecules should help us to understand how the ribosome works and mechanisms involved in the fidelity of the translation.
I2BC, Olivier Namy

Modulation of Oncogene Induced Senescence

Senescence is a cellular response to stress, and corresponds to a stable cell cycle arrest associated with characteristic changes in gene expression or morphological modification. Several types of stress are capable of inducing senescence, including loss/deprotection of telomeric sequences, oxidative stress, DNA damage and oncogene expression (Oncogene Induced Senescence). All of these stresses may occur during human oncogenesis and the senescence response is an important tumor-suppressor mechanism that blocks the proliferation of potentially cancerous cells.
While screening the Prestwick Chemical Library for modulators of BRAF-V600E OIS, we observed that all glucocorticoids in the library were able to allow prolonged proliferation after senescence induction. We therefore set out to characterize the effects and mechanisms of glucocorticoids on senescence induction and are interested in evaluating consequences on a model of tumorigenesis.
SBIGeM, Carl Mann

4. Transito GARCIA
Functional characterization of YabA phosphorylation by the ser/thr kinase YabA

Chromosome replication is tightly regulated to ensure that initiation at the chromosomal origin takes place only once per cell cycle. In bacteria, the widely conserved initiator protein DnaA acts at the chromosomal replication origin by opening the DNA duplex to allow the loading of the replication machinery. Bacteria evolved different mechanisms for initiation control that mostly regulate the activity and the availability of the initiator protein DnaA in the cell. In Bacillus subtilis, a model of Gram-positive bacteria, the regulation of initiation is not well understood and appears to be very different from that of E. coli. DnaA is control negatively through interaction with several proteins during the bacteria growth cycle and development. During vegetative growth, the main regulatory protein YabA acts to down regulate replication initiation by forming a heterocomplex with DnaA and with the replication processing protein DnaN.
Upon environmental/nutritional changes bacterial cells must integrate DNA replication with growth. Reversible protein phosphorylation is a widespread strategy to transduce extracellular signals. This is achieved by a variety of bacterial protein kinases able to phosphorylate proteins on different residues. We found that YabA is phosphorylated by the Ser/Thr Hanks kinase YabT. YabT does not possess external sensing domain like the typical Hanks kinases, but is activated by DNA. However, YabT is strongly up-regulated during spore development and glucose exhaustion.
So far, we have established in vitro that YabA is phosphorylated by YabT at the residue T71. We also detected that YabA exhibits an atypical ATP/GTPase activity that seems to be modulated by phosphorylation on T71. Nevertheless our recent in vivo results confirm that phosphorylation of the YabA T71 residue by YabT is probably not involved in the replication initiation control and possibly has a role in sporulation.
MICALIS, Sandrine Poncet

5. Quentin KHEBIZI
Regulation of anti-transgene cellular immune responses following systemic gene transfer by Anterior Chamber Associated Immune Deviation (ACAID)

Anterior Chamber-Associated  Immune Deviation (ACAIO), is an antigen specific peripheral immunomodulatory mechanism described when an antigen is introduced into the anterior segment of a mammalian eye. It is not known if the ACAID mechanism could impact on cellular immune responses induced by gene therapy.
Our study was conducted in a murine model to evaluate if ACAID induction would trigger immune tolerance to the product of a transgene injected by systemic route (i. v.) and if this could impact on the destruction of the transduced cells. The model consists of ACAID induction by injecting intracameraly soluble peptides (DBY and UTY) of the HY male antigen into wild type female C57BI/6 mice (H-2b background), followed by an intravenous injection of a recombinant AAV218 vector (rAAV218-GFP-HY) containing a transgene coding for a fusion GFP-HY molecule.
Our results show that an ocular injection of HY peptides 7 days before i.v. infusion of vector is able to reduce the number of IFNγ producing spleen cells up to 80%. ln addition, cytokine secretion assays showed an impairment of the Th1 profile in ACAIO condition with a reduction in the secretion of IFNγ and TNFα (8 and 2 times lower, respectively). Moreover, a model of in vivo cytotoxic assay suggests that almost all HY-expressing cells can survive in ACA/0-induced mice in contrast to the control mice.
ln conclusion, ACAIO seems to be a powerful inhibitory mechanism in the context of systemic gene transfer, limiting cellular immune responses against the transgene and promoting transgene expressing cells survival.
UMR S 951 - Approches génétiques intégrées et nouvelles thérapies pour les maladies rares, Sylvain Fisson

Interaction between telomeres and the nuclear envelope inhuman cells : Dynamics and molecular mechanism

Telomeres, the natural ends of linear chromosomes, protect our genetic material from degradation and our chromosomes from fusion. They consist of tandem TTAGGG repeats and are regulated by a protein complex that controls telomere length and telomere integrity, called shelterin. The interphase nucleus is highly compartmentalized, and during the last decades it has become clear that chromosomes occupy specific discrete territories. This spatial organization of the genome has emerged as an essential aspect of gene regulation and genome stability. In budding east, telomeres are organized in clusters enriched at the nuclear periphery. This localization is thought to be critical for telomere silencing, likely by creating a nuclear sub‐compartment enriched for silencing factors. Binding of budding yeast telomeres to the nuclear envelope also protects telomeric repeats from recombination, adding a new dimension to telomere protection. Telomere dynamics also play an essential function during meiosis in yeast and mammals. Their clustering to the nuclear envelope has been shown to be crucial for proper meiotic pairing and the recombination of homologous chromosomes. In human cells, telomeres are tethered to the nuclear envelope specifically during post-mitotic nuclear assembly.
The goal of this project is to investigate how human telomeres are tethered to the nuclear envelope in a cell cycle dependent manner. This project will determine whether proteins from the Shelterin complex at telomeres are able to interact with proteins from the nuclear envelope to initiate and maintain telomere tethering during nuclear assembly. It will also address the significance of such interactions, for telomere localization but also for genome organization and regulation. These experiments will be performed on human cells: primary cells, cancer cells, and cells from premature aging syndromes.
I2BC, Sébastien Bloyer

Type VII Secretion Pathway Involved in Mycobacterium abscessus Intracellular Survival

The Mycobacterium genus brings together species ranging from harmless saprophytic organisms to major human pathogens. The well-known pathogenic species such as Mycobacterium tuberculosis, Mycobacterium marinum and Mycobacterium ulcerans belong to the subgroup of slowly growing mycobacteria (SGM). By contrast, rapidly growing mycobacteria (RGM) form a group of over 150 species essentially composed of non-pathogen saprophytic organisms. However, Mycobacterium abscessus, an RGM, was recently considered as an opportunistic human pathogen, responsible for a wide spectrum of infections, from pulmonary to skin and soft tissues infections. M. abscessus is considered, with Mycobacterium avium, as the major mycobacterial pathogen in patients with cystic fibrosis.
Mycobacterium abscessus has been shown to behave as an intracellular pathogen, able to survive to the bactericidal response of macrophages and fibroblasts in lungs and skin, survival usually not observed for RGM. Sequence analysis of M. abscessus genome showed that this bacterium is endowed with the metabolic pathways typically found in environmental micro-organisms with contact to soil, plants, and aquatic environments, where free-living amoeba are frequently present. Very little is known about the genetic requirements that M. abscessus has developed or acquired to survive in an intracellular environment. We aimed to find if M. abscessus possess genomics advantages to survive in antigen presenting cells such as macrophages or in environmental protozoa such as amoeba.
A transposon mutant library of a clinical strain of M. abscessus subspecies massiliense was screened by its ability to persist in amoeba and macrophages. Several mutants were identified to have a reduced ability to survive in those intracellular environments. Interestingly, twelve on 6,000 mutants screened appeared to be muted in genes homologs to the type VII secretion pathway of M. tuberculosis also present in M. abscessus. These twelve mutants were all in the ESX-4 locus. This evolutionary system of secretion could manipulate the host cell biology to make macrophages and amoeba attractive hosts for M. abscessus. We showed here for the first time, genes allowing M. abscessus to survive inside phagocytic cells present in the environment and in the human host.
Infection et inflammation chronique, Jean-Louis Hermann et Fabienne Girard-Misquich

8. Ionna Polydorou
Molecular targets of BMP signaling in skeletal muscle stem cells

Bone morphogenetic proteins (BMPs) are secreted signaling molecules that belong to the transforming growth factor β (TGFβ) superfamily. BMPs are known for their critical role in skeletogenesis as well as various processes during embryonic development and adult tissue homeostasis. Recent evidence suggested that BMP signaling is active and mediates important functional consequences in adult skeletal muscle stem cells, also known as satellite cells (Friedrichset al., 2011; Ono et al., 2011; Stantzouet al., unpublished). Additionally, the BMP-Smad1/5/8 signaling axis has been highlighted as a major positive regulator of adult skeletal muscle mass (Sartoriet al., 2013; Winbankset al., 2013). Although the mechanistic insights of this role are slowly revealed, the direct targets of BMP signaling remain largely unknown. Elucidating the molecular targets of BMP signaling in adult myogenic cells will allow a better understanding of how the signaling regulates these cells, and how it thereby influences skeletal muscle physiology and pathology.
Therefore, our main aim is to define the major signaling networks that are mediated by BMPs in adult satellite cells. To meet this aim our objectives are:

To meet these objectives, we have developed a multi-scale approach that will allow the role of BMPs to be elucidated from the gene to the organism. Thus far, we have performed RNA Sequencing (RNA-seq) analysis on BMP-treated and untreated mouse cultured satellite cells to identify differentially expressed genes. The enriched genes were confirmed by qPCR and subjected to gene ontology analysis to assign their functional significance. Our initial findings have revealed JunB and Hes1, the main effector gene of Notch signaling, to be under the control of BMP4. Given the importance of the Notch signaling pathway in satellite cell behaviour (Conboy and Rando, 2002) our future work will aim to interrogate the how BMP and Notch pathways interact to control satellite cell fate and ultimately influence skeletal muscle homeostasis.
Handicap Neuromusculaire : Physiopathologie, Biothérapie et Pharmacologie appliquées,

9. Noémie Ranisavljevic
How to tidy up your X chromosome(s) as a pre implantation embryo?

Chromosome conformation capture techniques recently highlighted a new structural level of chromosomal organization, at the megabase level and called Topologically Associating Domains (TADs). This partitioning of the genome is well conserved among cell differentiation and species, however it is not known when this organization is set during development of multicellular organisms. In mouse, preimplantation development is a specific timeframe for a global genome reorganization along with the embryonic genome activation. Our goal is to cover these particular development stages to understand if the TADs could be inherited from gametes or if they are set de novo, along with transcriptional activation. For this purpose, we focus on investigating the X chromosome organization in mouse pre implantation embryos, using a combination of 3D DNA FISH and super-resolution microscopy, as well as chromosome conformation capture (HiC) approach at a single cell level. A locus encompassing two TADs on the X chromosome was found to display a similar structural organization in 8-cell stage embryos as previously described in mESCs (9 embryos examined, 41 signals examined and p = 1,9.10-9, two samples t-test). However, at an anterior developmental stage (2-cell), a very different organization is observed (59 embryos examined, 214 signals examined and p = 1,8.10-3, two samples t-test). This suggests that TAD partioning may occur sometime between the 2- and 8-cell stages. This implies that genome reorganization may occur following zygotic genome activation rather than being inherited from the gametes, although this requires further investigation. We are now examining different regions of the genome, on the X chromosome and autosomes. We also started a collaboration with Peter Fraser lab so as to perform single cell HiC analysis to obtain a more genome-wide information of this organization in pre implantation embryos.
Curie Paris, Edith Heard et Catherine Patrat

10. Jérôme Rouvière
Nuclear pore-associated deSUMOylation determines the composition and the cytoplasmic fate of mRNPs

mRNAs packaging into messenger ribonucleoparticles (mRNPs) involves the recruitment of a battery of proteins controlling mRNA stability, localization or translation, and, as such, represents a critical aspect of gene expression processes. However, despite major advances in the description of mRNPs composition, the molecular mechanisms that underlie mRNP remodelling, i.e. the association/dissociation of mRNA-associated proteins, remain to be fully understood. In this context, we have asked which remodelling steps could occur up on mRNP docking at nuclear pore complexes (NPCs), prior to export out of the nucleus.
In view of the localization of the conserved SUMO (small ubiquitin-like modifier)-deconjugating enzyme Ulp1 at the nuclear side of NPCs, we asked whether modification of mRNA-associated proteins by SUMO could impact mRNP composition at this stage. For this purpose, we have designed a proteomic screen to compare mRNP composition between wt and ulp1 mutant yeast cells. This approach identified a limited number of proteins which require Ulp1 activity for their association with mRNPs. Among them, the THO complex, an early mRNP biogenesis factor, was found to be recruited onto mRNPs in a SUMO-dependent manner, thereby regulating the stability of a subset of mRNAs (1). In addition, we found that Ulp1 controls the association of a conserved hnRNP with mRNAs. We further established that this hnRNP is itself SUMOylated on its C-terminus and that NPC-associated Ulp1 has to remove this modification to favor mRNA binding. Ulp1-dependent deposition of the hnRNP onto certain mRNAs allows in turn proper mRNA translational repression and asymmetrical localization in the cytoplasm. Our work thus reveals that SUMO-dependent, nuclear pore-associated remodeling events can influence the cytoplasmic fate of mRNPs.
IJM, Benoît Palacande

11. Matteo Tosolini
The different states of mouse in vitro pluripotency adopt diverse epigenetic programs to tightly control heterochromatin regions

Embryonic stem cells (ESC) and epiblast stem cells (EpiSC) are in vitro representatives of the different states of pluripotency in mouse, the naïve, early epiblast at blastocyst stage and the primed, late epiblast of a post-implantation embryo, respectively. They are well characterized at the molecular level but few is known about their chromatin features particularly concerning heterochromatin compartments and their regulations. In Eukaryote, constitutive heterochromatin is a gene-poor compartment constituted of repeat DNA sequences, generally silenced and located at telomeres, centromeres and pericentromeric regions. Tight control of these regions is extremely important for chromosomal stability. Beside the telomeric sequences, there are two types of DNA sequences in mouse: major satellite and minor satellite repeats at the pericentromeric and centromeric regions, respectively. In mouse somatic cells, these regions from different chromosomes organize in clusters. The first insight about different epigenetic regulations of these sequences according to the pluripotent state comes from our observation thatH3K9me3 forms foci in EpiSC, like differentiated cells, while ESCs cultured in 2i medium present mostly H3K27me3 clusters. Transcription of major and minor satellite repeats is strongly repressed in EpiSCs whereas ESCs transcribe at high level these repeats, even if 2i condition leads to a considerable down- regulation particularly of minor satellites. To further investigate the interplay of these two histone modifications on the transcription of major and minor satellites we studied ESCs and EpiSCs lacking either H3K9me3 or H3K27me3, or both. Interestingly our results reveal that the absence of both marks in EpiSCs impairs the normal silencing of satellite repeats while the same condition in ESCs is transcriptionally repressive. Ongoing work suggests that differential involvement of DNA methylation could play a role in such contrasting phenomena. We show that ESCs and EpiSCs differ in the epigenetic regulation of pericentromeric and centromeric regions. Altogether, our study addresses the question of the functional link between pluripotency status and heterochromatin organization.
BDR INRA, Alice Jouneau

12. Jerzy Witwinowski
Characterization of a diketopiperazine biosynthetic gene cluster of Streptomyces venezuelae

Diketopiperazines constitute a large class of secondary metabolites characterized by the presence of a piperazine-2,5-dione core. They are produced either by non-ribosomal peptide synthetase- or by cyclodipeptide synthase (CDPS)-dependent pathways [1]. A few complete biosynthetic pathways involving a CDPS and tailoring enzymes have been characterized [2]. One, discovered in Mycobacterium tuberculosis, involves a CDPS producing cyclo-(l-tyrosyl-l-tyrosyl) (cYY) and CYP121, an unusual cytochrome P450 catalysing the formation of a carbon-carbon bond between the two tyrosyl side chains to yield mycocyclosin [3,4,5]. The gene encoding CYP121 is essential in M. tuberculosis, making CYP121 an interesting drug target [6]. A cluster homologous to that of M. tuberculosis is present in Streptomyces venezuelae: the genes Sven_1864 and Sven_1865 encode products sharing respectively 59% and 60% identity with the CDPS and CYP121 from M. tuberculosis. We have undertaken the study of this cluster. Our findings demonstrate that the CDPS Sven_1864 catalyses cYY formation and preliminary results indicate that Sven_1865 is able to transform cYY into mycocyclosin. Contrarily to M. tuberculosis, the cytochrome gene Sven_1865 is not essential in S. venezuelae. The biological role of this cluster in S. venezuelae remains elusive.
I2BC, Jean-luc Pernodet

[1] Belin et al. 2012 Nat. Prod. Rep., 29, 961-979
[2] Giessen et Marahiel 2014 Int. J. Mol. Sci. 15, 14610-14631
[3] Gondry et al. 2009 Nature Chem. Biol. 5, 414-420
[4] Belin et al. 2009 PNAS 106, 7426-7431
[5] Vetting et al. 2010 Nature Chem. Biol 6, 797-799
[6] McLean et al. 2008 J. Biol. Chem. 283, 33406-33416


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