L'école doctorale SDSV

Le doctorat


Direction de thèse

Liens utiles


MàJ 24/02/16


Rappel aux orateurs : les exposés des doctorants peuvent être en français ; par contre, les diapos doivent être en anglais. L’exposé devra durer 10 minutes et 5 minutes seront consacrées aux questions.


9h00 - 9h15 : Pierre Capy
Accueil des participants

9h15-10h30 : SESSION 1, modératrice : Ilham SEFFOUH

9h15 : Alessandro Berto
Nup133 deficiencies affect nuclear envelope architecture and embryonic stem cell survival upon differentiation

In eukaryotes, nucleocytoplasmic transport occurs through the nuclear pore complexes (NPCs), large assemblies composed of ~30 distinct proteins (Nups). The Y-complex, a major structural NPC component, encompasses 9 distinct Nups, including Nup133. Studies on the mermaid (merm) mouse mutant previously revealed that lack of Nup133 does not affect mouse embryonic stem cell (ESCs) self-renewal but impairs their neural differentiation a.
To further investigate the differentiation properties of Nup133-/- ESCs, we used neural but also mesoendodermal differentiation protocols. Our study revealed that starting at day 2-4 and continuing on subsequent days of differentiation, cell number was strongly decreased in Nup133-/- relative to WT ESCs cultures. However, the few Nup133-/- ESCs that escaped cell death properly repressed the pluripotency markers and acquired a neural or mesoendodermal fate based on morphology and markers expression. These assays thus highlighted poor cell survival upon differentiation as the first overt in vitro phenotype of Nup133-/- ESCs.
To determine which domain of Nup133 is critical for ES cell differentiation, we next developed a “rescue strategy” using truncated alleles of Nup133 stably expressed in Nup133-/- ESCs. This study indicated that the N-terminal domain (NTD) of Nup133 is necessary for proper ESC differentiation.
In view of previous studies revealing the contribution of Nup133-NTD to interphasic NPC assembly and distribution in vertebrates and yeasts, we combined various approaches to visualize pore assembly and dynamics in Nup133 mutants ESCs. This analysis revealed numerous and extended nuclear envelope invaginations containing NPCs in Nup133-/-ESCs.
Nup133-NTD is also known to interact with CENP-F, contributing to centrosomes tethering at the nuclear envelope in prophase HeLa cells b. To investigate whether this interaction contributes to the merm differentiation defect, I generated mutations in Nup133-NTD specifically inhibiting its interaction with CENP-F.
Studies of various Nup133 mutants cell lines are in progress to determine the extent to which the altered nuclear envelope architecture and/or interaction with CENP-F contribute to the differentiation failure of ES cells lacking Nup133-NTD. a Lupu et al.  Dev. Cell, 2008 - b Bolhy et al.  JCB, 2011
Valérie Doye, IJM

9h30 : Marion Chevallier
Degradation of a persistent pesticide: chlordecone

Chlordecone (Kepone®) is a synthetic organochlorine pesticide (C10Cl10O) which has been used more than two decades ago in the French West Indies to control the banana black weevil. Persistence of chlordecone in soils and water still causes global public health and socio-economic problems. Chlordecone biodegradation has been investigated under anaerobic conditions using microbial enrichment cultures. GC-MS and LC-MS monitoring demonstrated the accumulation of chlorinated compounds coming from chlordecone degradation, in a number of cultures [1]. More interestingly, two bacterial consortia led to complete chlordecone disappearance and simultaneous accumulation of three chlorinated metabolites families: non-polar C9 compounds, polar C10 compounds and hydrochlordecones. Their production, purification and structural elucidation are carried out in order to better understand the degradation mechanism. This work also shed light on previous intriguing observations reported respectively forty and twenty years ago: a chemical approach using vitamin B12 as catalyst [2] and a biological approach using Methanosarcinathermophila bacteria [3] for chlordecone degradation that generate incompletely elucidated chlorinated metabolites highly similar to our characterized metabolites.The elucidation of chlordecone degradation pathwaycould be extremely useful to consider depollution of contaminated soils.
[1] Chaussonnerie S. *1, Saaidi P. L. *2, Ugarte E.*1, Barbance A.1, Fossey A.1, Barbe V.1, Gyapay G.1, Brüls T.1, Chevallier M.1, Couturat L.1, Marie L.1, Fouteau S.1,Pateau E.1,Cohen G. N.3, Fonknechten N.1, Weissenbach J.1, Le Paslier D.2,4, Microbialdegradation of a recalcitrantpesticide: chlordecone, submission in progress.
[2] Schrauzer G. N., and Katz R. N., Reductive Dechlorination of Mirex and Kepone with Vitamine B12s, Bioinorganic Chemistry 1978, 9, 123-143.
[3] Jablonski P. E., Pheasant D. J., Ferry J. G., Conversion of Kepone by Methanosarcinathermophila, FEMS Microbiology letters 1996, 139, 169-173.
Denis Le Paslier et Pierre-Loïc Saaidi, Génomique métabolique, Génoscope

9h45 : Clément Coudereau
H2AJ : a novel histone variant implicated in the DNA-damaged induced senescence of human cells

In mammalian cells, cellular senescence has been defined as a stress response. It ischaracterized by a stable cell cycle arrest, morphological transformation, a secretion of pro-inflammatoryfactorstermed the SASP (senescence associatedsecretoryphenotype) and the alteration of the chromatin structure.
This phenotype can be induced by various stimuli. Originally, telomere loss or dysfunction was shown to trigger the onset of senescence. However, the senescence state canal so result from inadequate culture conditions, oncogene induction or genotoxic stresses.
Work in the lab focuses on mechanisms governing the onset and maintenance of senescence and on the search for new markers of senescence. Recent results identified chromatin modifications and epigenetic regulations during cellular senescence, such as post-translational modifications of histones and changes in the histone variants composition of nucleosomes.
Mass spectrometry revealed the accumulation of a specific histone variant in DNA-damage induced senescence. This variant, H2A.J, makes up to 1% of the H2A histone content during proliferation, but reaches 20% of H2A species during deep senescence.
The goal of mythesis workis to determine the function of this histone variant, the mechanism leading to its accumulation and its mode of action.
Microarray and RNA-sequencing analyses have shown that H2AJ-depleted fibroblasts have an altered transcriptome. In particular, such cells show a greatly delayed derepression in senescence of several SASP genes coding for some key cytokines and chemokines. This result indicates that accumulation of H2A.J in senescence is important for efficient expression of the SASP phenotype.
Finally, the accumulation of senescent cells in aged tissues has often been inferred using surrogate markers such as DNA damage or increased SA-B-Galactosidase activity. Our data suggest that H2AJ accumulation maybe a novel in-vivo biomarker of aging for certain cell types.
Carl Mann, SBIGeM

10h00 : Sara Filipa Dias Henriques
Identification of targeted therapies in the rescue of mutated proteins through the CRISPR/Cas9 technology

Protein quality control (QC) is a crucial process in cells: it keeps proteins in check, recognizing and destroying potential pathogenic misfolded proteins that would eventually have deleterious consequences. In the case of missense mutations, this pathway can destroy proteins that – although imperfect – still retain their biological activity. Rescue of such proteins can lead to improvement of the disease.
It has been demonstrated that the degradation of misfolded transmembrane  glycoproteins - the sarcoglycans – is associated with the pathogenicity of a group of muscular diseases, the sarcoglycanopathies. These proteins are involved in the protein complex that allows the muscle fibers to resist to contraction, it is therefore important to study the degradation mechanisms in order to develop a strategy to ameliorate the disease progression.
For a better understanding of the QC of these proteins, we are using a recent and powerful gene editing tool: the CRISPR/Cas9 system. This system comprises a Cas9 endonuclease and a small-guide RNA (sgRNA) molecule that guides the Cas9 into a targeted site in the genome, causing the disruption and knock-out (KO) of the targeted gene via a double-strand break.
Two strategies are used: a targeted KO of lead-candidates of the QC and, in a more extensive approach, the use of a genome-wide KO library targeting hundreds of human genes. The two approaches, combined with a selection screening, should reveal key players of the degradation pathway relevant to the sarcoglycanopathies.
Using this strategy, we have started to elucidate some partners of the sarcoglycan protein degradation pathway which will be further studied.
Isabelle Richard, UMR_S 951 - Approches génétiques intégrées et nouvelles thérapies pour les maladies rares

10h15 : Malika Foy
PRL-3/PTP4A3 phosphatase promotes uveal melanoma cell migration through the regulation of integrin β1 in focal adhesion

Using transcriptomic analysis, we found that PRL-3 overexpression is highly correlated with metastatic tumors and predicts poor prognosis in patients with uveal melanoma. PRL-3 is a dual specific phosphatase with a carboxy-terminal farnesylation motif, allowing anchorage to the plasma membrane. PRL-3 overexpression in ocular melanoma cell line-1 (OCM1) significantly increases cell migration in vitro and invasiveness in vivo, suggesting a direct role of PRL-3 in metastasis (Laurent et al, Cancer Res 2011). Despite its role in the metastatic process of various cancers, its mechanism of action and its intracellular substrates remain largely unknown.  It has been shown that the anchorage of PRL-3 to the plasma membrane plays an important role in the metastatic process, suggesting that the phosphatase regulates transmembrane substrates. Among potential targets involved in extracellular matrix adhesion, we focused on integrin β1 which we found overexpressed in the transcriptomic analysis of our uveal melanomas cohort.
We showed that FTI-277, a farnesyltransferase inhibitor that prevents PRL-3 anchorage to the plasma membrane, abolishes PRL-3-induced OCM1 cell migration on collagen I in vitro, specifically enhances the spreading of OCM1 cells overexpressing PRL-3, and allows the formation of large focal adhesions involving integrin β1. Knockdown of integrin β1 in OCM1-PRL3 cells partially restores the spreading and migration. Furthermore, in focal adhesions, we observed that PRL-3 specifically regulates the aggregation of integrin β1 but does not affect integrin β3. Our results suggest the involvement of integrinb1 in PRL-3-mediated cell migration. To go further, we are currently investigating the phosphorylation status of specific integrin β1 residues, known to be involved in cell adhesion.The present study highlights the importance of PRL-3 membrane localization in uveal melanoma cell migration, by regulating adhesion to the extracellular matrix through integrin β1.
Simon Saule, Curie Orsay

10h30 - 11h00 : pause

11h00-12h15 : SESSION 2, modératrice : Anissia AIT SAADA

11h00 : Rafael Galupa
The structural and regulatory puzzle of the X-inactivation centre

The regulation of X-chromosome inactivation (XCI), a mammalian developmental epigenetic phenomenon, is highly complex and dynamic, being coordinated by the X-inactivation centre locus, Xic. It involves trans-acting elements, such as pluripotency factors, as well as numerous cis-acting elements, such as noncoding genes, enhancer-like regions and transcriptional interference. Transgene experiments have failed to define a complete autonomous Xic, implying that the full extent of the regulatory landscape of XCI still misses crucial elements. Using 5C, a multitude of new long-range interactions with the promoters of Xist, the key long noncoding RNA (lncRNA) of XCI, and of Tsix, its negative antisense regulator, have been uncovered (Nora et al, 2012). In particular, we are now focusing on the role of the newly identified lncRNALinx, which lies adjacent to Tsix and interacts with its enhancer Xite. Mutant ESCs and mice harbouring specific Linx deletions have been generated to explore the role of this locus during initiation of XCI in the early embryo, when Tsix is thought to play a critical role in repressing Xist and in the choice of the X-chromosome to be inactivated.
Edith Heard, Curie Paris

11h15 : Libera Latino
Selective pressure imposed on Pseudomonas aeruginosa by bacteriophages: the importance of pseudolysogeny

Pseudomonas aeruginosa is an opportunistic pathogen possessing a large genome with high level of plasticity, allowing the bacteria to adapt to hostile environments. Bacteriophages have been shown to drive the emergence of P. aeruginosa variants playing a significant role in bacterial survival, activity, and evolution. Although phage therapy has proven to be promising in several animal case studies and in some human clinical trials, it has always been considered with skepticism by the medical community because of the emergence of the phage-resistant variants.
The purpose of this study was to investigate the mechanisms and frequency of resistance in response to infection by cocktails of virulent phages belonging to different genera. Phages belonging to four genera were combined to perform infections. Phage Ab05, a podovirus (ФKMV-like), phage Ab09, a podovirus (N4-like), phage Ab27, a myovirus (PB1-like) and phage Ab17, a myovirus (KPP10-like), isolated from waste water in Abidjan (Ivory Coast), were used to perform single or multiple infection of P. aeruginosa strain PAO1 allowing the selection of several phage resistant variants whose genome was, then, fully sequenced through Illumina technology.
Several phenotypic alterations were observed in the phage-resistant variants. Alterations in bacterial motility and reduction in biofilm formation due to mutations in type IV pilus biosynthesis genes, characterized variants selected by Ab05 alone or as a component of a cocktail. Mutations in genes involved in the LPS production and in the alginate regulation were also identified. Cross resistance was systematically observed for phages using LPS as a receptor. Pseudolysogeny was a frequent outcome of infections and in some case was stable enough to allow the emergence of new mutations. If phage predation selects for variants with alterations in genes involved in biogenesis or regulation of virulence determinants, the resulting phage-resistant variants could potentially exhibit altered levels of virulence, in a beneficial or detrimental way. The use of cocktail does not lower significantly the frequency of phage-resistance and in addition we show that pseudolysogeny is a major actor in the selection of mutations.
We thank Direction Générale de l’Armement (DGA) and ANR “Resisphage” for financial support to this project.
Gilles Vergnaud, I2BC

11h35 : Tatiana Maroilley
Analysis of the genetic architecture of blood gene expression in pigs: the first step in the research of biomarkers of immune capacity

Antibiotics were until very recently regularly used as growth factors in livestock, provinding protection against diseases in farms also. However, current and future EU regulations tend to limit or even eliminate antibiotic use in farms systems. In this context, combining animal production and health traits while reducing the use of antibiotics becomes a main objective in livestock breeding. Blood is a source of biological information linked to health and physiological state of each individual. Furthermore, it is an interesting surrogate tissue for animal phenotyping because its sampling is non-invasive and reproducible. We have already shown that several innate and adaptive immune traits are heritable and that the blood transcriptome is informative to quantify some of them in pigs.
In this study, our aim is to identify genetic markers associated with gene expression variations in blood, based on expression Genome-Wide Association Studies (eGWAS). We have used 243 Large White pigs at 60 days of age, each of them being genotyped with the Illumina iSelect 60K Chip. The blood transcriptome analysis was performed by using a custom gene expression 8X60K microarray (Agilent Technologies).
After association analysis between SNPs and gene expression levels, 4,191 significant associations were found, including 62% cis-associations (that is, with the associated SNP in a 2 Mb window around the microarray probe).
We are currently performing Allele Specific Expression analysis, using blood RNA-Seq data of 38 other pigs to validate our cis-results. These analyses are the first step toward the identification of blood biomarkers of immune capacity and candidate causal mutations involved in their variation. The next step consists in integrating immune phenotypic data to potentially find genetic markers linked both to immune traits and gene expression variation.
Claire Rogel-Gaillard, GABI INRA

11h45 : Caroline Midonet
The vibriophage TLC activates the Xer machinery for its integration

The cholera toxin is one of the main factors of pathogenicity of Vibriocholerae. The genes encoding the toxin are carried by the genome of an integrative filamentous phage: the cholera toxin phage CTX. The genomic context of the CTX phage suggests that its lysogeny requires the prior integration of an another phage targeting the same integration site: TLC (toxin linked cryptic element). Thus, the environmental integration site (difA) would be inefficient for CTX integration whereas TLC could exploit it. The recombinant site dif1 after TLC integration would be then the one used by CTX. (Hassan F. et al, 2010)
However, the mechanism discriminating between TLC and CTX integration is unclear. Indeed, both phages (CTX and TLC) use the same Xer machinery, two tyrosine recombinases encoded by the host, to integrate into the chromosome (Das B. et al, 2012). Secondly, as the TLC attachment site is highly degenerated such as only one of the two recombinase binding sites could be detected, TLC should have more difficulties to integrate. Thirdly, because of its sequence, TLC attachment site could allow a double strand exchange during recombination suggesting that TLC could be excised with CTX from the chromosome.
We demonstrated that TLC integration/ excision could explain the evolution of the cholera toxin in the vibrio pathogenic strains (Midonet C. et al, 2014). Then, we found that TLC genome encodes a protein able to activate the Xer machinery between the TLC attachment site and the host site whereas the normal cellular activator was only able to activate the recombination between two host sites. We reproduced in vitro the reaction of recombination and we demonstrated that this protein interacts specifically with the Xer recombinases of Vibriocholerae.
Fr.-Xavier Barre, I2BC

12h00 : Appel à candidatures pour l’élection des doctorants au Conseil Scientifique de l’Ecole Doctorale
12h30 - 14h00 : déjeuner et posters
14h00 : Point sur les procédures ADUM

14h30-15h30 : SESSION 3, modératrice : Zoé LAMA

14h30 : Nam Nguyen Quang
Ingénierie d'aptamères par séquençage à haut-débit

Les aptamères sont des structures d’acides nucléiques capable de se fixer spécifiquement à une cible d’intérêt (ex : molécules, protéines, cellules). Souvent comparés aux anticorps, ils ont l’avantage d’être peu immunogènes et sont plus faciles à produire. Leur champ d’application est varié : chromatographie d’affinité, régulation de l’expression des gènes, thérapie, imagerie etc. L'identification d'aptamères repose sur une technique d'évolution moléculaire appelée SELEX qui consiste à enrichir une banque en séquences ayant de l’affinité pour une cible, ceci à travers plusieurs cycles de sélection. Le passage du séquençage Sanger au séquençage HD représente une avancée technologique majeure qui change la manière d’identifier des aptamères. La possibilité de séquencer les banques de chaque cycle, sans passer par des clones bactériens, permet de suivre l’enrichissement de millions de séquences au cours du SELEX au lieu d’identifier une centaine de séquences de la dernière banque évoluée. Cependant, cette technique de séquençage est encore peu utilisée dans la communauté « aptamères » et ces retombées en termes d’efficacité d’identification d’aptamères restent à découvrir.
Dans le cadre de mon projet de thèse, j'ai utilisé le séquençage à haut débit afin d'améliorer l'affinité d'un aptamère préalablement identifié au laboratoire. Pour ce faire, nous avons réalisé un SELEX à partir d'une banque composée de millions de mutants de cet aptamère (banque dégénérée). Suite au séquençage de cette banque aux différents tours de SELEX, il a été possible d’identifier l'évolution des mutations sur toutes les positions de la séquence d'aptamère. Ce travail nous a permis de prédire des mutations susceptibles d’améliorer l’affinité de l’aptamère. A l’inverse, nous avons également des positions qui ne tolèrent pas de mutation. L’importance de ces mutations a été validée par des mesures d'affinité. Suite à ce travail, nous avons également délimité une région conservée de la séquence qui nous a permis d'identifier le motif structural minimum de notre aptamère. En conclusion, ce travail montre que le séquençage HD donne accès à plus d’informations sur l’évolution de la banque durant le SELEX et de ce fait améliore sensiblement la prédiction et l’optimisation d’aptamères.
Frédéric Ducongé, Molecular Imaging Research Center, Ingénierie d'aptamères par séquençage à haut-débit

14h45 : Florian Plaza Onate
Étude par métagénomique quantitative de la variabilité du contenu en gènes dans le microbiote intestinal humain

L’intestin, organe longtemps négligé, abrite un écosystème complexe : la microbiote intestinal.
Le microbiote intestinal joue un rôle primordial sur la santé humaine en complémentant le métabolisme de l’hôte et stimulant son système immunitaire.
Des publications montrent que le microbiote intestinal est impliqué dans des maladies chroniques telles que les maladies inflammatoires chronique de l’intestin, l’obésité, le diabète etc. 
La compréhension du lien entre ces maladies et le microbiote intestinal nécessite de caractériser ce dernier.  La méthode classique consistant  à isoler, cultiver et séquencer des souches provenant d’échantillons de selles s’est révélée fastidieuse et limitée par la difficulté à cultiver des espèces anaérobies. L’avènement de la métagénomique a permis l’étude directe du matériel génétique contenu dans un échantillon de selles sans passer par l’étape de culture en laboratoire.
En 2015, la compilation de données de séquençage provenant des métagénomes de 1267 échantillons de selles a abouti à la création d’un catalogue de 10 millions de gènes microbiens. Parmi ceux-ci, seulement 300 000 sont détectés chez au moins 50% individus. Ce résultat illustre une forte variabilité du contenu en gènes entre individus.
Cette variabilité peut s’expliquer par diversité des espèces  colonisant l’intestin mais aussi par la diversité des souches de ces différentes espèces.  Les nombreux gènes accessoires présents chez seulement certaines souches  d’une espèce peuvent changer son phénotype en lui conférant par exemple une résistance aux antibiotiques.
Dans cet exposé, on présentera une méthode quantitative permettant de grouper au sein d’un ensemble appelé espèce métagénomique (MGS) les gènes provenant de la même entité biologique. En particulier, la méthode est capable de détecter et distinguer les gènes indispensables à la MGS  des gènes accessoires rares. Finalement, on présentera quelques résultats d’analyses s’appuyant sur cette méthode.
Dushko Ehrlich et Frédéric Magoulès, MICALIS

15h00 : Charles-Adrien Richard
Cartographie et caractérisation du domaine d’interaction de la phosphoprotéine P avec la protéine M2-1 du Virus respiratoire syncytial humain

Le virus respiratoire syncytial (VRS) est le principal agent responsable de maladies respiratoires graves (bronchiolites, pneumonies) chez les jeunes bovins et chez l’homme, principalement chez les nouveau-nés, personnes âgées ou immunodéprimées. En l’absence de vaccin efficace contre ce virus, le développement rationnel de traitements antiviraux constitue un enjeu de taille.
Appartenant à la famille des Paramyxoviridae, le VRS est un virus enveloppé dont le génome est constitué d’un ARN simple brin de polarité négative, codant pour 11 protéines. Le génome est répliqué et transcrit par le complexe ARN polymérase viral. Ce complexe est composé de la nucléoprotéine N, de la polymérase L, de la phosphoprotéine P et du facteur de transcription M2-1 qui assure la « processivité » de la polymérase au cours de la transcription virale. Ne présentant pas d’équivalent dans la cellule, lecomplexe polymérase constitue une cible privilégiée pour le développement d’inhibiteurs spécifiques. Une meilleure compréhension des mécanismes d’interaction entre les différentes protéines ducomplexe polymérase est donc indispensable afin d’identifier des cibles potentielles.
Mason SW et al. (J Virol. 2003 77(19)) ont mis en évidence que la région [100-120] de la phosphoprotéine P était nécessaire pour interagir avec la protéine M2-1 du VRS. Ornos résultats obtenus par RMN indiquaient que cette région était plutôt localisée entre les résidus 90 et 110 de la protéine P. L’étude de ce domaine a été poursuivie en utilisant des outils de biochimie et de biologie moléculaire. Ainsi, par GST-pulldown et en utilisant des protéines recombinantes tronquées produites chez E. Coli, nous avons déterminé que la région [93-110] de la protéine P constitue un domaine minimal pour  interagir avec M2-1.
Ce domaine a été ensuite validé in cellula par transfection de vecteurs d’expression dans des cellules de mammifère. Des mutations ciblées ont été introduites dans cette région de P (Alanine scanning) et l’activité ARN polymérase virale a été dosée grâce à un test fonctionnel appelé minigénome. Ainsi nous avons identifié 3 nouveaux résidus hydrophobes critiques dans l’interaction avec la protéine M2-1. De plus, par immunofluorescence et microscopie, nous avons observé que ces variants de P ne recrutaient plus la protéine M2-1 dans les corps d’inclusion.
Enfin des expériences de co-immunoprécipitation ont confirmé l’importance des résidus nouvellement identifiés de la protéine P dans l’interaction avec M2-1. Mais cette approche a surtout mis en évidence une corrélation entre l’état de phosphorylation de M2-1 et l’interaction avec P. De façon surprenante, un résidu de P situé en amont à la région [93-110] s’est avéré critique pour la déphosphorylation de M2-1 par P sans impacter sur l’interaction M2-1–P.
Jean-François Eléouët, VIM

15h15 : Ilham Seffouh
Proteomic analysis of human endosulfatases HSulfs, key enzymes in the modulation of the sulfation pattern of heparan sulfate

The human endosulfatases HSulf-1 and HSulf-2 are two extracellular 6-O-endosulfatase enzymes recently discovered. They remove 6-O sulfate group from glucosamine residue in heparan sulfate (HS), a glycosaminoglycan located on cell surface. It is assumed that HS sulfation pattern may host an informational content governing the recognition and the interaction of HS with various ligands. By varying the HS sulfation pattern, HSulf-1 and HSulf-2 have a strong impact on HS-ligand interactions, and consequently they are involved in many physiological and pathological processes.
To date, the structural organization of HSulfs remains elusive. Some pieces of information revealed that both HSulf isoforms have a common molecular organization consisting of two chains presumably linked by disulfide bonds, and their maturation into active form requires a cleavage by furin. The objectives of this work are to achieve the fine structural characterization of HSulfs and elucidate their properties of interaction using top-down and bottom-up mass spectrometry (MS) approaches. Particular attention will be paid to the determination of post-translational modifications (disulfide bond, glycosylation) using differential chemical labeling and electron transfer dissociation (ETD) MS. Moreover, the enzymes will be also studied by hydroxyl radical footprinting combined with MS analysis.
All the data obtained will help to determine the structural organization of HSulfs, and to establish the molecular basis of HSulfs / HS interaction.
Régis Daniel et Florence Gonnet, LAMBE

15h30 - 16h00 : pause

16h00-16h45 : SESSION 4, modérateur : Benjamin GRANDON

16h00 : Zehua Song
Yeast-based mutational analysis of the bc1 complex Qo and Qi site

The bc1 complex of the respiratory chain is central to mitochondrial bioenergetics and the target of antiprotozoals. We characterized a modified yeast bc1 complex that more closely resemble the Plasmodium falciparum enzyme. The mutant version was generated by replacing ten yeast cytochrome bQo site residues by their P. falciparum equivalents. The Plasmodium-like changes caused a major dysfunction of the catalytic mechanism of the bc1 complex resulting in superoxide overproduction and respiratory growth defect. The defect was corrected by substitution of the conserved residue Y279 by a phenylalanine, or by mutations in or in the vicinity of the hinge domain of the iron-sulphur protein. It thus appears that these modifications prevented side-reactions. Interestingly, P. falciparum - and all the apicomplexan - contains an unusual hinge region. We replaced the yeast hinge region by the Plasmodium version and combined it with the Plasmodium-like version of the Qo site. This combination restored the respiratory growth. It could be suggested that, in the apicomplexan, the hinge region and the cytochrome bQo site have co-evolved to maintain catalytic efficiency of the bc1 complex Qo site.
Brigitte Meunier, I2BC

16h15 : Shoutang Wang
The transcription factor Lyl-1 regulates the production of yolk sac macrophages and is involved in the development of microglia

Hematopoietic cells are generated during embryonic development and sequentially colonized the fetal liver, the spleen, and finally the bone marrow. The first hematopoietic site is the YS, the hematopoietic progenitors are generated by the YS at E7.5, and they differentiate in situ. A second and independent hematopoietic site occurs in the P-Sp/AGM at E10.
Tal-1/SCL is a bHLH transcription factor and comfirmed to be required for the generation of extra- and intra-embryonic precursors. Lyl-1 is also a bHLH transcription factor and share a 84% homology with Tal-1/SCL in the bHLH domain. In our team, we use lyl-1 Lac Z knock-in mouse to research the functions for lyl-1 in hematopoiesis. Lyl-1/LacZ encodes a lyl-1 fusion protein devoid of the HLH and C-terminal domains and fused to β-Gal. It has been reported that Lyl-1/lacZ mice are viable, but display: a reduced number of HSC and immature precursors in the foetal liver and adult bone marrow; a impaired reconstitution capacity in competitive assay and an impaired Lymphoid cell specification and maintenance. However, it is still unclear for the function of lyl-1 in developmental hematopoiesis.
Isabelle Godin, Hématopoïèse normale et pathologique

16h45 : Aurélien Saghaï
Phylogenetic characterisation of microbial communities associated to modern microbialites

Microbial mats are structures made of phylogenetically and metabolically diverse microbial communities. Particular, lithified, mats called microbialites and including the well-known stromatolites, are abundant in the fossil record. Their distribution span over all the history of life on Earth, the oldest fossil identified so far being also the oldest indisputable trace of life (3.5 Ga old). Contemporary microbialites can thus be used as models to study the early evolution of life and to identify the type of potential biosignatures that might be preserved in the fossil record.
Microbialite distribution is today restricted to a few locations, including marine settings such as the extensively studied Shark Bay (Australia) and Highbourne Cay (Bahamas). Continental water bodies (e.g. hypersaline ponds, crater lakes) have, on the contrary, received less attention. Yet, some elements tend to indicate that Precambrian microbialite probably formed in lacrustine environmnts. Compared with marine systems, where accretion is important, microbialites from freshwater environments seem better models to study the in situ precipitation of minerals due to microbial activity, since this is the major process leading to their formation. Two major types of  energy metabolism are thought to promote carbonate precipitation, photosynthesis and sulfato-reduction, with a dominant role attributed to Cyanobacteria.
We recently (Saghaï et al. 2015) investigated the alkaline (pH~9) crater lake Alchichica, located at 2,300 m a.s.l on the Mexican Central Plateau. By coupling molecular biology techniques and mineralogical analyses we  aimed at (i) characterising the diversity of microorganisms from the three domains of life, (ii) gain insight into the relative abundance of taxa potentially involved in microbialite formation and (iii) correlate the observed microbial diversity with the mineral composition of the microbialites.
Purification Lopez-Garcia, Écologie, Systématique, Évolution

16h45 : remise des prix aux deux meilleurs exposés et deux meilleurs posters
17h00-18h00 : conférence de Patrick WINCKER
Génomique holistique du plancton marin : l'expédition Tara Océans


Présentation de la Journée

Les posters des doctorants

Contact : secretariat_sdsv@universite-paris-saclay.fr