L'école doctorale SDSV

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MàJ 14/03/17

 

Les posters doivent être rédigés en anglais et imprimés au format A0 vertical.

 

 

 

1. Idranil ADHYA
Studying the role of Ty1 retroelement integration site selection on Pol III transcription in yeastLTR

Dir.: Joël Acker, Service de Biologie Intégrative et Génétique Moléculaire, Paris-Sud

Retrotransposons are widespread transposable elements in eukaryotes. They share analogies with retroviruses in their structure and mode of replication. However, their life cycle is exclusively intracellular since they do not encode an envelope glycoprotein. They contain two open reading frames analogous to the GAG and POL retroviral genes, flanked by two long terminal repeats (LTRs). Like retroviruses, they replicate by reverse transcription of their RNA into cDNA, which is integrated into the host genome by their own integrase (IN). The choice of the integration site is critical both for the host survival and the retroelement replication since its integration can alter the structure of cellular genomes. Integration does not occur randomly throughout the host cell genome. Ty1-LTR will be our working model.A direct interaction between Ty1 IN and AC40, one subunit of Pol III, determines Ty1 integration site choice upstream of Pol III-transcribed  genes. The major ami of the project is to decipher the novel cofactors of the Ty1 IN involved in its specific targeting.

2. Ipek ALTINOGLU
Organization of Bacterial Cell Pole in Vibrio cholerae

Christien Merrifield, Mickael Poidevin, Yoshiharu Yamaichi

Dir.: Frédéric Boccard, I2BC, Gif

Cell poles in rod-shaped  bacteria constitute key subcellular domains that are often critical for several cellular processes including motility, chemotaxis, protein secretion, antibiotic resistance, and chromosome segregation. Our group previously identified a polar anchor protein HubP in Vibrio cholerae, a Gram negative highly motile with single polar flagellum and causative agent of cholera disease. HubP has been shown to organize the cell pole by tethering multiple proteins such as  ParA1, ParC, and  FlhG that  are involved in chromosome segregation, chemotaxis and flagella biosynthesis, respectively.
Further  understanding  of  precise  organization  of  the  cell  pole,  we  aimed  to  identify comprehensive interaction partners of HubP, characterize biochemical and cellular functions of identified polar proteins by comparative proteomics approach, and draw conceivable picture of the cell pole and imply the cellular functions by super-resolution  photoactivated localization microscopy (PALM).
Isobaric tags for relative and absolute quantitation (iTRAQ) was carried out to identify proteins that are differentially expressed in the minicells (in which cell poles are enriched) prepared from HubP+ and HubP- V. cholerae. In addition to previously known interaction partners (including many downstream chemotaxis machinery proteins), several proteins were shown to be enriched in HubP+ minicells. After experimental validation, four proteins were confirmed to localize/enrich at cell pole(s) in HubP- dependent manner. Current investigations for characterization of their biological function will be discussed.
To understand the precise subcellular localization and organization of the cell pole, we constructed strains with PALM-compatible fluorescent tags. We realized that determination of cell outline is critical for our analysis, and current protocol using out-of-focus brightfield image and MicrobeTracker software are not sufficient enough. We use inner membrane labeling technique (PamCherry-fused Membrane targeting sequence from Bacillus subtilis) and in-house program to overcome these issues. Dual-color 3D-PALM confirmed that vast majority of HubP molecules were indeed localized at the tip of the cell pole.
Taken together, we  are aiming to understand how HubP and its  interaction partner proteins organize the cell pole and coordinate the cellular functions.

3. Anissa BELFETMI
Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

Dir.: Olivier Mauffret, Laboratoire de Biologie et Pharmacologie Appliquée, ENS Paris-Saclay

The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reversetranscription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem–loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them. These latter properties are important in the context of genome packaging, during which NC is part of the Gag precursor. We used NMR to investigate destabilization of the full-length TAR (trans activatingresponse element) RNA by NC, which is involved in the first strand transfer step of reverse transcription. NC was used at a low protein:nucleotide (nt) ratio of 1:59 in these experiments. NMR data for the imino protons of TAR identified most of the base pairs destabilized by NC. These base pairs were adjacent to the loops in the upper part of the TAR hairpin rather than randomly distributed. Gel retardation assays showed that conversion from the initial TAR–cTAR complex to the fully annealed form occurred much more slowly at the 1:59 ratio than at the higher ratios classically used. Nevertheless, NC significantly accelerated the formation of the initial complex at a ratio of 1:59.

4. Diletta CIARDO
A numerical model for the spatio-temporal program of DNA replication in Xenopus early embryos

Dir.: Kathrin Marheineke, I2BC, Gif

In all cells, chromosomes are replicated with high fidelity to allow the transmission of the genetic material to progeny cells. The initiation of DNA replication in multicellular organisms begins at thousands genomic positions known as replication origins which are activated at different times during the replicative phase (S phase) of the cell cycle in a strictly regulated manner. The ATR-Chk1 dependent checkpoint pathway, so-called intra-S phase checkpoint, globally inhibits origin activation when replication forks are arrested due to DNA damage. It has been suggested that the kinase Plk1 locally suppresses this pathway by inhibiting Chk1.In western blot analysis I found that Plk1 is recruited to chromatin when replication forks are arrested in a vertebrate replication in vitro system. Inhibition of Plk1 by small chemical inhibitors increases the activity of Chk1 showing the role of Plk1 in the intra S phase checkpoint. Then, I focused on the study of the spatial-temporal program of DNA replication in this system and I tested different models by comparing numerical simulations and DNA combing data of Chk1 inhibition, previously obtained by the laboratory. The best accordance with experimental data was obtained with a model that combines three notions: 1) a random initiation by an increasing limiting factor, 2) a strong global inhibition of origins firing by Chk1 protein and 3) an enhanced initiation probability near active replication forks together with a repression of the Chk1 action. The latter is consistent with the fact that replication origins are grouped in different temporal clusters.

5. Nadine EL BANNA
Investigating redox-based anticancer mechanisms of anticancer drugs

Dir.: Meng-Er Huang, Stress génotoxiques et Cancer, Paris-Sud

Reactive oxygen species (ROS) homeostasis and dynamic intracellular reductive/oxidative (redox) balance play a key role in maintaining genome stability and regulating cell survival/death. One of the main features of cancer cells, when compared to the normal ones, is a persistent pro-oxidative state that can lead to intrinsic oxidative stress. The administration of an anticancer ROS-generating reagent should increase ROS levels to a certain threshold leading to cell death. Our team is interested in redox-based anticancer properties of different drugs used in chemotherapy such as platinum drugs widely used in chemotherapy for the treatment of many tumors including lung, colorectal and ovarian cancers and ascorbic acid a natural molecule repurposed for significant anti-cancer activity.In this project, we are performing studies to better understand the implication of redox-based mechanisms in the anticancer activity of these drugs. This study could open new avenues for discovering possible combinations of redox-modulating molecules to synergistically enhance anticancer effect and bypass the drug resistance.

6. Zhong HAN
Biochemical and structural analyses provide new insights into the mechanisms of non-coding transcription termination by the helicase Sen1

Dir.: Domenico Libri, IJM, Paris

Pervasive transcription is widespread and needs to be controlled in order to avoid the interference with canonical gene expression. In Saccharomyces cerevisiae, the highly conserved helicase Sen1 plays a key role in restricting pervasive transcription by eliciting early termination of non-coding transcription. However, many aspects of the mechanisms of termination remain unclear. In this study we characterize both biochemically and structurally the helicase activity of Sen1 and its role in termination. We have analysed several truncated versions of Sen1 and we have found that the helicase domain (HD) is sufficient for dissociation of the elongation complex in vitro. Both full-length Sen1 and its HD can translocate along single-stranded nucleic acids in the 5’ to 3’ direction. However, we have found that Sen1 is a relatively low-processivity enzyme, implying that in vivo Sen1 should be recruited in close proximity to the RNA polymerase II for efficient termination. In addition, we have observed that Sen1 is significantly more processive on DNA than on RNA. Our in vitro analyses indicate that Sen1 requires exclusively the interaction with the RNA and not with the DNA to elicit termination. Yet, substituting the nascent RNA by ssDNA improves termination in vitro, further supporting the notion that the processivity of translocation is a limiting parameter in the termination reaction. We have solved the structure of the HD and we have identified several regions that are critical for translocation. Importantly, mutations that specifically impair translocation without affecting RNA binding or ATP hydrolysis provoked a dramatic decrease in termination efficiency. Taken together, our results support a model in which Sen1 translocation along the RNA is required for termination and provide a mechanistic rationale for the kinetic competition between termination and elongation.

7. Mounia KORTEBI
A vacuolar persistence stage in the Listeria monocytogenes life-cycle in epithelial cells

Dir.: Hélène Bierne, MICALIS

Over the past 30 years, the study of L. monocytogenes has produced major advances in microbial pathogenesis and eukaryotic cell biology. In particular, L. monocytogenes has become a paradigm for intracellular pathogens entering and replicating in the cytoplasm of mammalian cells. Here, we report that besides this well-known replicative and motile stage, bacteria can shift to another intracellular lifestyle during long-term infection of epithelial cells, such as human hepatocytes (including primary hepatocytes) and trophoblast cells, by entering a quiescent stage in vacuoles. In addition, this phenotype can be reversible, as infected cells and vacuolar bacteria remain alive after cell subculturing and bacteria can escape from these vacuoles to re-enter into a replicative and motile stage. We also identify two bacterial factors playing key roles in the switch between active and latent stages: ActA, the actin-polymerizing and locomotion factor, and PtsI, the major enzyme of the phosphotransferase systems (PTS) involved in sugar transport in Firmicutes. This novel facet of the L. monocytogenes intracellular life may changes our view of Listeria infections and their outcomes, because intracellular silent infection in epithelial tissues might promote an asymptomatic carriage, delayed infections and side-effects in humans. In addition, latent listeriosis might not be easily detected if intracellular quiescent Listeria acquire a metabolic state that renders them non-culturable under standard plating-based methods, leading to underestimation or non-detection of bacteria in clinical samples. Therefore, there is a need to develop easy-to-use diagnostic methods and animal models to detect and study latent Listeria infection in epithelial tissue samples.

8. Ganna KYCHIGINA
Connection between nuclear envelope dysfunction and DNA damage response at telomeres

Dir.: Laure Crabé, I2BC

Pas de résumé disponible

 

9. Francesco LAVECCHIA
Exploring bacteriophage peptide deformylase (PDF) to decode dynamic interplay among cotranslational proteostasis networks in bacteria

Dir.: Carmela Giglione, I2BC, Gif

Peptide deformylase (PDF) and methionine aminopeptidase (MetAP) correspond to the enzymes sequentially involved in an essential and ubiquitous process called N-terminal Methionine Excision (NME). NME occurs in all compartments where protein synthesis takes place i.e., the cytosol, chloroplasts and mitochondria. NME is the first modification occurring on the majority of proteins and corresponds in bacteria and in the organelles of eukaryotes to the early removal of the N-formyl group from nascent chains by PDF, immediately followed by the cleavage (in bacteria and in eukaryotes) of the first methionine by MetAP. NME is essential for the normal growth and function of all living organisms and affects a huge number of proteins (99% and 30-60% of the whole proteome for deformylation and methionine excision, respectively).
Recently it has been revealed that E. coli PDF interacts with the ribosome at the exit tunnel and its C-terminal a3-helix acting as the master regulator of PDF-ribosome binding. While seeking to understand how other PDF enzymes that do not belong to the same class as the E. coli PDF (i.e., PDFs displaying different C-termini from the E. coli one), fulfill their interaction with ribosome, a metagenomic study of viruses within oceanic microbial samples has allowed the group to identify the existence of unpredicted PDF genes into many phage genomes. Global sequence comparisons with other known PDFs implied that these viral PDFs could correspond to active enzymes and belong to subtype IB PDF, the same class of E. coli PDF. Unexpectedly, all the identified viral PDFs displayed variable shorter C-termini with none of them exhibiting the C-terminal a3-helix, conserved in many subtype IB PDFs.
Why viruses should carry a PDF acquired from the host genome remains unclear at the moment. The evidence that viral proteins also undergo NME led us to propose that encoded phage PDF could be important for viral fitness. Aside from this intriguing hypothesis, which goes against the established view that control of host gene expression by phages occurs at the transcriptional level, not much is known about this particular phage PDF and how it is able to control viral and host protein expression.

10. Émilie LEBRAUD
Mediator's and Cohesins's role in the repair of oxidative DNA damage

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

Our laboratory focuses on the base excision repair (BER) mechanism that is responsible for the removal of damaged bases in DNA. Oxidative DNA damage is generated spontaneously by the endogenous metabolism of the cells or induced exogenously by chemical agents or ionizing radiation. The BER pathway has been characterized by biochemical and genetic approaches. We found that BER proteins are assembled after treatments generating oxidized bases into cellular DNA. These complexes include BER enzymes and the scaffold protein that coordinates the process, XRCC1. We already confirmed the model by a microscopy approach, in which, for the case of the 8-oxoguanine (8-oxoG) mutagenic lesion, assembly of the BER complex depends on the recruitment of OGG1 on chromatin, OGG1 being the DNA glycosylase that recognizes and excise the lesion. Surprisingly, the recruitment of this initiator protein does not require the recognition of the damage by the protein. This implies that there are signals other than the recognition of the modified base by OGG1 that allow recruitment of the enzyme to the chromatin and thus initiation of the repair of the 8-oxoG by the BER. We have performed a high-throughput siRNA screen in human cells to identify proteins required for the recruitment of OGG1 to chromatin. Among the candidates issued from the screen two groups of proteins were selected for further study. They include members of the Mediator and cohesin complexes. In order to shed light on the link between this two essential nuclear complexes and BER, we study the molecular mechanisms through which cohesin and Mediator direct the recruitment of OGG1, and subsequently the rest of the BER machinery, to chromatin. Through a variety of technical approaches, going from live cell imaging and biochemistry to global genomic techniques, we want to identify the subunits of Mediator and cohesin implicated, and sequential mode of action required for an efficient recruitment of the DNA glycosylase and repair of 8-oxoG. We also want to explore their putative role in the coordination between transcription and repair in response to a genotoxic stress. The identification of molecular mechanisms and new factors involved in the repair of oxidized bases could be used in the design of new therapies.

11. Xing LUO
VSR217, a sRNA potentially involved in the regulation of carbohydrate metabolism in Vibrio tasmaniensis

Xing Luo1, Yiqin Deng2, Philippe Bouloc1 and Annick Jacq1
1: Institute de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud, Université Paris-Saclay, Orsay
2: Key Laboratory of Tropical Marine Bio-resources and Ecology (LMB), South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China

Dir.: Annick Jacq, I2BC, Orsay

The Gram-negative bacteria, Vibrio tasmaniensis (formerly named V. splendidus) has been associated with oyster summer mortality outbreaks over the past 15 years, causing major economic losses in aquaculture. V. tasmaniensis has been shown to be a facultative intracellular pathogen, which can survive and multiply within hemocytes, the oyster defense cells.
Bacterial regulatory small RNAs, usually 50-300 nt long, are important elements which regulate a variety of processes, including carbohydrate and intermediate metabolism, virulence or iron homeostasis. They act generally by base-pairing with specific mRNA targets.  A previous transcriptomic study of V. tasmaniensis LGP32 allowed us to identify hundreds of sRNAs in this species. Among them, Vsr217, was found to be very conserved within vibrios and highly upregulated within the oyster hemolymph.
We determined the 5' and 3'ends of Vsr217 by mRACE and showed that Vsr217 is highly induced by maltose. Interestingly, Vsr217, which is located upstream of malK, a gene involved in maltose import, results from the processing of the long 5' UTR of the malK mRNA. A vsr217 deletion mutant has been constructed and is being currently phenotypically characterized. In addition, a transcriptomic approach allowed us to identify Vsr217 specific potential targets.
Oysters are especially rich in glycogen, the precursor of maltose. Our results point toward an important role for Vsr217 in adapting bacterial cells to this nutrient when infecting oysters, illustrating the importance for virulence of metabolic adaptation to the new environment encountered within the host. 

12. Laura MEYER
The cellular response of Deinococcus radiodurans to genotoxic stresses

Dir.: Pascal Servant, I2BC, Orsay

Pas de résumé disponible

 

13. Florian PLAZA ONATE
Abundance-based reconstitution of microbial pan-genomes from shotgun metagenomic data. Application to the human gut ecosystem

Dir.: S. Dusko Ehrlich (INRA MetaGenoPolis) & Frédéric Magoules (Ecole Centrale Paris)

Shotgun metagenomics has revolutionized microbiology by allowing culture-free characterization of microbial communities at strain level. Capturing gene content variability in a strain of a given species is crucial in many contexts as it can impact significantly its phenotype by providing selective functional advantages such as complex carbohydrates, antibiotic resistance or virulence.

However, tools for analyzing metagenomic data are limited by the extent of available reference genomes. For example, 50% of the species present in the gut microbiota of western individuals lack reference genomes.

Metagenomic assembly where overlapping reads are merged into longer sequences called contigs, is a powerful reference-free technique for overcoming these limitations. To retrieve a set of genes representative of the studied ecosystem, metagenomic assembly is performed on several samples. However, obtained genes catalogs are not structured as the link between genes and the microorganisms they belong to is often unknown.

We present MSPminer, a new method that structure genes catalogs and reconstitute Metagenomic Species Pan-genomes by binning co-abundant genes across metagenomic samples. It distinguishes species core genes from accessory and shared genes even when rarely observed. The method relies on a new robust score that is only limited by the number of reads mapped on a gene and its prevalence across samples.

MSPminer was applied to the integrated catalog of reference genes of the human gut microbiome made up of 9.9 millions genes. Preliminary results will be exposed in the poster.

14. Renaud POURPRE
Control of chromatin-repressive complexes by the pathogenic bacterium Listeria monocytogenes

Dir.: Hélène Bierne, MICALIS

This invasive pathogen produces an arsenal of virulence factors, which promote its intracellular lifestyle by hijacking diverse cellular pathways and controlling host cell defenses. Among these virulence factors, the secreted protein LntA has been shown to target BAHD1, a BAH-containing protein acting as a epigenetic co-repressor within an HDAC-HMT multi-subunit complex (1,2). Secreted bacterial factors that target the nucleus belong to the family of nucleomodulins (3). These microbial molecules can manipulate transcription, chromatin modification, RNA splicing, as well as DNA replication, repair or stability. Nucleomodulins have now been described in various pathogenic bacteria, and investigating their functions is an important novel topic to understand the bacterial control of eukaryotic organisms.My PhD project aims at characterizing some chromatin-repressive complexes controled by Listeria to promote an efficient infection. I will first described my contribution to the in-depth characterization of the BAHD1 complex and the identification of a new subunit: MIER1 (4). I will then present my first results on InlN, a potential listerial nucleomodulin. I will present evidence that InlN targets a chromatin regulator, RERE, which like BAHD1 is a BAH-containing protein defining a HDAC-HMT repressive complex.

References:

1. Bierne et al. PNAS 2009 Aug 18;106(33):13826-31.
2. Lebreton et al. Science 2011 Mar 11;331(6022):1319-21.
3. Bierne and Cossart, Cellular Microbiology 2012 May;14(5):622-33.
4. Lakisic et al. PLoS genetics 2016 Mar 3;12(3):e1005898

15. Guillaume REBOUL
Microbial eukaryotes in the Movile Cave chemosynthetic ecosystem

Reboul G.1, Hillebrand-Voiculescu A.M. 2, Bertolino, P. 1, Moreira D. 1 López-García P. 1
1 Ecologie Systématique Evolution, Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud, Orsay, France
2 Group for underwater and speleological exploration, Emil Racovita Institute of Speleology, Bucharest, Romania

Dir.: Purification Lopez-Garcia, ESE, Orsay

Located near the coast of the Black Sea on the Dobrogea Plateau in Mangalia, Romania, the Movile Cave is a unique underground cave ecosystem. This karstic cave is thought to have been sealed off from the outside world for several million years. Discovered in 1986, several studies have been carried out to study troglodytic metazoa but also the diversity of bacterial and archaeal communities responsible for carbon fixation in this chemosynthetic ecosystem. However, the presence of microbial eukaryotes (generally, protists) has never been investigated. Compared to animals, plants or fungi, the diversity of protists remains poorly known. Molecular studies based on 18S rRNA gene markers in natural communities have unveiled unexpected protist diversity in many different environments in the past decade. Gaining information about poorly sampled clades and about new protist clades with early diverging positions may be particularly useful to improve phylogenetic reconstructions and improve the resolution of the eukaryotic tree of life.  Here, we present the first results about the diversity and activity of microbial eukaryotes in the unique ecosystem of Movile Cave by using a dual approach. We used metabarcoding approaches based on 18S rRNA gene amplicon sequencing using high-throughput techniques (Illumina) to characterize protist diversity and we have also generated metatranscriptomic data in order to i) identify active lineages and ii) carry out phylogenomic analysis of these microbial eukaryotes using concatenated marker genes.

 

 

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