Pasteur Erasmus 2018 2019career.ut.ee/failid/2018/detsember/17.12/Pasteur Erasmus...Institut Pasteur...

Projects 2018-2019

Institut Pasteur 2018-2019

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RESEARCH CENTRE

Legal name: Institut Pasteur

Address: 25-28 rue du Dr. Roux, 75724 Cedex 15, PARIS

Country: France

Website: https://www.pasteur.fr/education

Contact person: Elisabetta Bianchi, David Itier, Sylvie Malot

Contact person e-mail: [email protected]

Brief description of your Institution The Institut Pasteur is a private non-profit foundation that contributes to the prevention and treatment of diseases through research, education, and public health activities. Its campus in Paris hosts almost 2600 individuals. Research: priority is given to fight infectious diseases, such as viral, bacterial, and parasitic diseases, as well as certain types of cancer, genetic, neurodegenerative, and allergic diseases. Education: every year 550 young scientists from all over the world follow high-level courses in various fields related to research in microbiology, immunology, cellular biology, epidemiology, genetics, and disease control. Over 850 trainees from 60 different countries come to perfect their skills or conduct their Master or Doctoral trainings in the Institute's laboratories. Description of the work program(s) See projects on following pages N° of placements available for work programs a), b), c) etc: The laboratories at Institut Pasteur, Paris, France have proposed 34 projects for Erasmus internships (see following pages). In addition, two projects have been presented from the International Pasteur Network, and are mainly reserved for PhD level students to be discussed by the candidates with their University). Students may also contact other laboratories at Pasteur to apply for an internship, even if the laboratories have not presented a project.


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FACILITIES (not compulsory for the host centre) at Institut Pasteur, Paris

• Accommodation (some centres offer it) X YES 1 NO a limited number of rooms for rent are reserved for Pasteur at the student residence

Cité Universitaire http://www.ciup.fr/ • Support in finding accommodation (many centres offer it) X YES 1 NO • Canteen (most centres offer it) X YES 1 NO • Additional salary X YES 1 NO Institut Pasteur Paris offers an additional salary of approximately 550 euros/month,

which is paid by the host lab (3.75 euros/hour, 7 hours/working day). NOTE: internship conditions at the International Pasteur Network may vary and have to be

discussed directly with the host lab.


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Title of the work program 1 Deep learning for medical diagnosis of brain tumors Description of the work program Brain tumors are among the most devastating forms of cancer, and extracting diagnostic information from the tumor accurately and rapidly is key to inform therapeutic decision making. Currently, this process requires labor-intensive inspection of very large histology images by trained pathologists and suffers from relatively high error rates and large differences in accuracy between local and reference pathologists. Deep learning methods use artificial neural networks to learn relationships between complex numerical data sets and underlie the current renaissance in artificial intelligence (AI)1. In recent years, deep learning methods have achieved remarkable success in image classification, including for medical applications such as dermatology, radiology or ophthalmology2,3. In collaboration with a team of neuropathologists led by F. Chrétien (Institut Pasteur and Hôpital Sainte Anne, Paris), we seek to explore the potential of deep learning methods to automate and improve the classification of tumors based on histological images. Using training data obtained by our collaborators, a previous project has led to the development of a deep learning algorithm that classifies incoming histological images into one of three types of tumors (ependymoma, glioma grade III, glioma grade IV). According to our quantitative evaluations, the algorithm currently achieves a classification accuracy of ~80%, slightly lower than the accuracy of a local pathologist (85%). The main goal of this project is to improve the classification accuracy to the level of a reference pathologist (95%). To this end, we propose to explore multiple avenues, including the following: (i) The current neural network is pretrained on general imaging data (from Imagenet4). We will test pretraining on more similar histological data from the Cancer Genome Atlas project, which should yield better learning performance. (ii) The current classification scheme operates on 300 small image patches extracted from each histology slide and uses a majority voting procedure for final classification. This is certainly suboptimal, we therefore propose to test changes to the voting procedure, for example by automatically learning how to weigh results from different patches for better overall classification and by incorporating patch-wise classification uncertainty. (iii) We will use multiresolution decompositions of the image to test the predictive power of large-scale cellular patterns and leverage access to an Nvidia DGX GPU station to increase the size of input images. (iv) We will take into account human diagnostic uncertainty (as determined based on an expert consensus) to adjust the weights of distinct training data In addition to improving classification accuracy, another goal is to determine the image patterns that are most informative for accurate cancer diagnosis, using techniques to probe the sensitivity of individual neurons in the trained neural network5.


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We expect that this project will result in an improved AI-based tool for the diagnosis, and ultimately treatment, of brain cancer. References: 1. LeCun, Y., Bengio, Y. & Hinton, G. Deep learning. Nature 521, 436–444 (2015). 2. Esteva, A. et al. Dermatologist-level classification of skin cancer with deep neural networks. Nature 542, 115–118 (2017). 3. Kermany, D. S. et al. Identifying Medical Diagnoses and Treatable Diseases by Image-Based Deep Learning. Cell 172, 1122–1131.e9 (2018). 4. Krizhevsky, A., Sutskever, I. & Hinton, G. E. ImageNet Classification with Deep Convolutional Neural Networks. in Advances in Neural Information Processing Systems 1097–1105 (2012). 5. Zeiler, M. & Fergus R. Visualizing and Understanding Convolutional Networks. Lect. Notes Comput. Sci. 8689, 818–833 (2014).

Tutor/supervisor

First name, Last name Christophe, Zimmer Phone E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://www.researchgate.net/profile/Christophe_Zimmer

Lab websites: https://sites.google.com/site/imagingandmodeling/ https://research.pasteur.fr/en/team/imaging-and-modeling/ Selected publications or patents of the Research Group offering the work program

A. Aristov, B. Lelandais, E. Rensen, C. Zimmer. ZOLA allows flexible 3D localization microscopy over an adjustable axial range. Nature Communications, 8:2409 (2018). doi: 10.1038/s41467-018-04709-4 W. Ouyang, A. Aristov, M. Lelek, X. Hao, C. Zimmer. Deep learning massively accelerates super-resolution localization microscopy. Nature Biotechnology, 36 (5), 460–468, 2018. doi:10.1038/nbt.4106. S. Herbert, A. Brion, J.-M. Arbona, M. Lelek, A. Veillet, B. Lelandais, J. Parmar, F. Fernandez, E. Alamyrac, Y. Khalil, E. Birgy, E. Fabre, and C. Zimmer. Chromatin stiffening underlies enhanced locus mobility after DNA damage in budding yeast EMBO Journal. Sep 1; 36(17):2595-2608 (2017). doi: 10.15252/embj.201695842 Arbona J-M, S. Herbert, E. Fabre, C. Zimmer. Inferring the physical properties of yeast chromatin through Bayesian analysis of whole nucleus simulations. Genome Biology. 18:81, doi: 10.1186/s13059-017-1199-x (2017).


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Scientific or technical background required for work program We expect a background in computer science, physics, mathematics, or a related field, and

good programming skills. Prior training or experience in machine learning and ability to

communicate with biologists and clinicians are a big plus.


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Title of the work program 2 Double-strandbreakrepairoftrinucleotiderepeats:frommechanismstogenetherapy Description of the work program Trinucleotide repeat expansions are involved in a number of neurodegenerativedisorders, includingHuntington disease,myotonic dystrophy type 1 (DM1or Steinertdisease) and several ataxias. Expansion mechanisms include DNA slippage duringreplication, homologous recombination and DNA repair. It was recently shown thatbreak-inducedreplication–aspecifictypeofhomologousrecombination–wasamajordriveroftrinucleotiderepeatexpansions(reviewedin[1]).Giventhatthepathologyisalwaysassociatedtotheexpansionofonesingletrinucleotiderepeattract,shorteningthisexpandedrepeattonon-pathologicallengthshouldsuppressclinicalsymptoms,andcouldthereforebetheoreticallyusedasagenetherapyapproach[2].WerecentlyshowedthataTALENdesignedtorecognizeandcutaCTGtripletrepeatfromaDM1patient,integratedintoayeastchromosome,wasveryefficientatshorteningtherepeat(>99%cellsshowedcontraction)andhighlyspecifictoo,sincenoothermutationwasdetectedinyeastcellsinwhichtheTALENwasinduced[3].Onthecontrary,similarexperimentswereperformedwithaCRISPR-Cas9nucleaseandshowedareducedefficacyand a lack of specificity leading to large chromosomal rearrangements (our ownunpublishedresults).Hence,itappearsthatTALENsandCRISPR-CasnucleasesbehavedifferentlyonthesameDNAsubstrate,but thereasonfor thisdifference isnot totallyunderstoodyet.Wehavealso recentlyshown thatadouble-strandbreakmade inyeastwithaTALENwithinaCTGrepeatneededtheproductof threegenes,RAD50,RAD52andSAE2 toberepaired,suggestingthatthebreakwasrepairedbysingle-strandannealingbetweenbothrepeat ends. In addition, resection of the breakwas impeded by the repeat tract andabsolutelyrequiredSAE2tobecorrectlyprocessed[4].Theprojectreliesontwocomplementaryapproaches:1)ShorteningCTGrepeattractsinmammaliancellsusinghighlyspecificendonucleases.TheTALENusedinyeastwasclonedintolentivirusesthatmaybeusedtotransducethenuclease intohumanpatientcells.CellsobtainedfromapatientaffectedbyDM1wereimmortalizedandwillbe transfectedwith theTALEN inorder toassess its efficacyatshorteningthepathologicalrepeattract.Thenucleasespecificitywillbedeterminedusinggenomewidehigh-throughputIlluminasequencing.ExperimentswithatransgenicmicemodelofDM1willalsobeperformedincollaborationwiththeteamofGenevièveGourdon(InstitutImagine).2)Nuclease-inducedrecombinationoftrinucleotiderepeattractsinyeastWedesignedanexperimentalrecombinationreporterinyeast,inordertotesttheefficacyand specificity of different Cas9-like nucleases. Ten different repeat tracts involved inhuman disorders were introduced into this reporter assay. The student will have to


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express every nuclease in each of the ten repeat tract constructs and determinerecombinationefficaciesandspecificitiesusingyeastgeneticsandmolecularmethods.References[1] V.Mosbach,L.Poggi,G.-F.Richard,Trinucleotiderepeatinstabilityduringdouble-strandbreakrepair:frommechanismstogenetherapy,Curr.Genet.(2018).doi:10.1007/s00294-018-0865-1.[2] G.-F. Richard, Shortening trinucleotide repeats using highly specific endonucleases: apossibleapproachtogenetherapy ?,TrendsGenet.31(2015)177–186.[3] G.-F. Richard, D. Viterbo, V. Khanna,V.Mosbach, L. Castelain, B. Dujon, Highly specificcontractionsofasingleCAG/CTGtrinucleotiderepeatbyTALEN inyeast,PLoSONE.9(2014)e95611.[4] V.Mosbach, L. Poggi,D.Viterbo,M.Charpentier,G.-F.Richard,TALEN-induceddouble-strandbreakrepairofCTGtrinucleotiderepeats,CellRep.22(2018)2146–2159.

Tutor/supervisor

First name, Last name

Guy-Franck RICHARD

Phone +33-1-45-68-84-36 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://www.researchgate.net/profile/Guy_Franck_Richard?ev=hdr_xprf&_sg=o4FK9eMccqGKl6kF1PGWBuh6CunxuYY79UK1bNvKNxwlCT-Xr-jn4XY7YFh1C-sQOAXOHxclP-QI5YKImkKqsRa7

Selected publications or patents of the Research Group offering the work program

PUBLICATIONS

1-G.-F.Richard,D.Viterbo,V.Khanna,V.Mosbach,L.CastelainandB.Dujon(2014)HighlyspecificcontractionsofasingleCAG/CTGtrinucleotiderepeatbyTALENinyeast.PLoSONE9:e95611

2-G.-F.Richard(2015)Shorteningtrinucleotiderepeatsusinghighlyspecificendonucleases:apossibleapproachtogenetherapy?TrendsinGenetics31:177-186

3-S.Descorps-Declère,C.Saguez,A.Cournac,M.Marbouty,T.Rolland,L.Ma,C.Bouchier,I.Moszer,B.Dujon,R.KoszulandG.-F.Richard(2015)Genome-widereplicationlandscapeofCandidaglabrata.BMCBiology13:69

4-D.Viterbo,G.Michoud,V.Mosbach,B.DujonandG.-F.Richard(2016)Replicationstallingandheteroduplex formation within CAG/CTG trinucleotide repeats by mismatch repair. DNARepair42:94-106

5- J.Nguyen,D.Viterbo,R.Anand,L.Verra,L.Sloan,G.-F.RichardandC.Freudenreich(2017)DifferentialrequirementofSrs2helicaseandRad51displacementactivitiesinreplicationofhairpin-formingCAG/CTGrepeats.NucleicAcidsResearch45:4519-4531

6- V. Mosbach, L. Poggi, D. Viterbo, M. Charpentier and G.-F. Richard (2018) TALEN-induceddouble-strandbreakrepairofCTGtrinucleotiderepeats.CellReports22:2146-2159


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7-D.Viterbo,A.Marchal,V.Mosbach,L.Poggi,W.Vaysse-ZinkhöferandG.-F.Richard(2018)Afast,sensitiveandcost-effectivemethod fornucleicaciddetectionusingnon-radioactiveprobes.BiologyMethods&Protocols3:1-6

8-V.Mosbach,L.PoggiandG.-F.Richard(2018)Trinucleotiderepeatinstabilityduringdouble-strandbreakrepair:frommechanismstogenetherapy.CurrentGeneticshttps://doi.org/10.1007/s00294-018-0865-1

PATENT

TALEffectormeans useful for partialor full deletion ofDNA tandem repeats. (PatentNumber:WO2015/078935A1).

Scientific or technical background required for work program Thestudentneedssomebackgroundineukaryoticgeneticsandgenomics.He/shewillbe

workingwithyeastandhumancells.Knowledgeofdouble-strandbreakrepairwillbe

appreciated but not mandatory. Fluent english speaking (or french) is required. Lab

meetingswillbegiveninenglishorinfrench,atstudent'schoice.


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Title of the work program 3 Targeting G proteins by cancer-promoting immunomodulatory virulence factors Description of the work program The Cytotoxic Necrotizing Factor 1 (CNF1) from pathogenic Escherichia coli catalyzes a post-translational modification (PTM) of Rho family members through site-specific deamidation of a glutamine residue into a glutamic acid which places Rho in a constitutively active state. Missense mutations of residues C886, N835 and S864 (Buetow et al, 2001) and the role of loop L8 and P968 of CNF1 in discerning Rac1 from RhoA have been unveiled (Buetow et al, 2003; Hoffman et al, 2007), as well as the importance of a specific peptide region in RhoA (Lerm et al, 1999). We propose to explore by structural bioinformatics approaches the molecular basis of the modification by CNF1 of the subfamily of Rho GTPases that belong to the super-family of p21-Ras GTPases. This integrated study will open new avenues for a comprehensive view of several hot topics in the field. The first aspect concerns the potential connection of pathogens to cancer development. The second aspect is the role played by the disordered or partially disordered protein regions in the cell signalisation processes (Csizmok et al, 2016). There is a remarkable convergence of bacterial virulence toward host Rho GTPases to promote their activation or inactivation. Activation of oncogenic pathways downstream Rho or Ras GTPases are at risk in cancer progression. Recent in silico analysis has unveiled an unexpected wide distribution of CNF1-like factors among the armamentum of Enterobacteriacae, thereby raising the question of the specificity of targeting of Rho GTPases by these virulence factors (Ho et al., 2018). The deciphering of the structure of the catalytic region of CNF1 (Buetow et al, 2001), reveals a sandwich beta structure surrounded by few alpha helices. The C866 and H881 forming the catalytic site, are surprisingly located at the bottom of a very narrow groove, poorly accessible for G-proteins. The molecular modeling study of CNF1 catalytic domain has revealed that a conformational transition of CNF1 makes the catalytic site more accessible, thereby allowing us to propose models of interactions between CNF1 and the switch-II target region of Rho proteins. The M2 project intends to use molecular modeling approaches to explore the interactions between CNF1 and small G proteins to shade light on the specificity of recognition of Rho proteins by this toxin. Starting from the model proposed for CNF1/RhoA interaction, molecular dynamics (MD) and TAMD (Maragliano and Vanden-Eijnden, 2006; Cortes-Ciriano et al, 2015) trajectories will be recorded to analyze the interaction between CNF1 and other small G proteins. Molecular modeling approaches (Lamothe and Malliavin, 2018 ; Alam and Schueler-Furman, 2017) will be used to explore the conformational space of the interaction partners along with the possible interactions they can establish with each other. This project is driven by an already established collaboration with the Unit of Bacterial Toxins at Institut Pasteur. References: • Alam, Schueler-Furman (2017) Modeling Peptide-Protein Structure and Binding


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• Using Monte Carlo Sampling Approaches: Rosetta FlexPepDock and FlexPepBind. Methods Mol Biol 1561:139

• Buetow, Flatau, Chiul, Boquet, Gosh (2001) Structure of the Rho-activating domain of Escherichia coli cytotoxic necrotizing factor 1. Nat Struct Biol 8:584

• Buetow, Ghosh (2003) Structural Elements Required for Deamidation of RhoA by Cytotoxic Necrotizing Factor 1. Biochem 42:12784

• Cortes-Ciriano, Bouvier, Nilges, Maragliano, Malliavin (2015) Temperature Accelerated Molecular Dynamics with Soft-Ratcheting Criterion Orients Enhanced Sampling by Low-Resolution Information. JCTC 11:3446

• Csizmok, Follis, Kriwacki, Forman-Kay (2016) Dynamic Protein Interaction Networks and New Structural Paradigms in Signaling. Chem Rev 116:6424

• Doye, Mettouchi, Bossis, Clément, Buisson-Touati, Flatau, Gagnoux, Piechaczyk, Boquet, Lemichez (2002) CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. Cell 111:553

• Ho, Mettouchi, Wilson, Lemichez (2018) CNF1-like deamidase domains: common • Lego bricks among cancer-promoting immunomodulatory bacterial virulence factors.

Pathog Dis 76(5) doi: 10.1093/femspd/fty045. • Lerm, Schmidt, Goehring, Schirmer, Aktories (1999) Identification of the Region of Rho

Involved in Substrate Recognition by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1). JBC 274:28999

• Maragliano, Vanden-Eijnden (2006) A temperature accelerated method for sampling free energy and determining reaction pathways in rare events simulations. CPL 426:168

• Prevost, Delarue-Cochin, Marteaux, Colas, van Renterghem, Blondel, Malliavin, Corringer, Joseph (2013) Identification of cinnamic acid derivatives as novel antagonists of the prokaryotic proton-gated ion channel GLIC. J Med Chem 56:4619

Tutor/supervisor

First name, Last name Thérèse Malliavin Phone +33 1 45 68 88 54 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://www.researchgate.net/profile/Therese_Malliavin


• Cortes-Ciriano, Bouvier, Nilges, Maragliano, Malliavin (2015) Temperature Accelerated Molecular Dynamics with Soft-Ratcheting Criterion Orients Enhanced Sampling by Low-Resolution Information. JCTC 11:3446

• Doye, Mettouchi, Bossis, Clément, Buisson-Touati, Flatau, Gagnoux, Piechaczyk, Boquet, Lemichez (2002) CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. Cell 111:553

• Duclert-Savatier, Bouvier, Nilges, Malliavin (2016) Building Graphs To Describe Dynamics, Kinetics, and Energetics in the d-ALa:d-Lac Ligase VanA. J Chem Inf Model 56(9):1762-75.


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• Ho, Mettouchi, Wilson, Lemichez (2018) CNF1-like deamidase domains: common Lego bricks among cancer-promoting immunomodulatory bacterial virulence factors. Pathog Dis 76(5) doi: 10.1093/femspd/fty045.

• Lamothe, Malliavin (2018) re-TAMD: exploring interactions between H3 peptide and YEATS domain using enhanced sampling. BMC Struct Biol 18(1):4.

• Malliavin (2017) Molecular Modeling of the Catalytic Domain of CyaA Deepened the Knowledge of Its Functional Dynamics. Toxins (Basel) 9(7).

• Martinez, Duclert-Savatier, Betton, Alzari, Nilges, Malliavin (2016) Modification in hydrophobic packing of HAMP domain induces a destabilization of the auto-phosphorylation site in the histidine kinase CpxA. Biopolymers 105(10):670-82.

Scientific or technical background required for work program The candidate could be a student with a solid background in structural bioinformatics

(molecular modeling, physical chemistry) and willing to invest himself/herself in

learning mutagenesis and biochemistry techniques relevant for handling the proposed

biological system.

No heavy computer programming is considered in the project, the informatics tools will

be usual Linux tools, the student is expected to prepare and run small scripts.

Technical support will be provided by the two involved teams, in structural

bioinformatics and in biochemistry and molecular biology.

The student is expected to be able to work at the interface of several scientific fields

and to interact with scientists from various backgrounds. Intellectual flexibility and

curiosity are essential in that frame.


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Title of the work program 4 Single-Molecule Visualization and Analysis in Virtual Reality Description of the work program The DIVA project aids scientific users in the interpretation of their imaging data using virtual reality (VR). This multidisciplinary project is localized between the research teams of Maxime Dahan (Institut Curie) and Jean-Baptiste Masson (Institut Pasteur). More information is available https://goo.gl/myWD57. We are proposing a 3 to 6 month internship for a motivated student to contribute to a VR software project intended for scientific researchers. Specifically, this role will involve developing tools for visualization and analyzing single-molecule imaging data (i.e. point clouds) in virtual environments. Novel strategies for interacting, representing and exporting data will be developed. The successful candidate will have a background in object-oriented programming and an interest in data visualization and computer graphics.

Figure 1) Visualizing data in VR}. A) Schematic of different usage configurations for VR. The user can either be in a standing (room-scale) (left) or sitting (right) posture. The user wears a specialized stereoscopic headset that permits total immersion in a computer-generated environment. External infrared sensors and internal sensors within the headset communicate to allow precise rotational and translational motion tracking. The user visualizes data, here a set of cells in 3D, and can interact with the representation using one or two VR controllers. B)


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Example of visual representations of scientific data using VR. On the left, a maximum intensity projection of an image stack of neurons imaged by spinning disk confocal microscopy [brault_2017]. On the right a capture of the same dataset in VR. A video of the user exploring this dataset is available at http://goo.gl/dnNueu. There is a significant increase in volumetric cues and information in the VR mode; 3D geometry is accessible, boundaries can be enhanced and complex geometrical relation between neurons is clearly observed. C) Single-molecule point cloud originating from multifocus microscopy represented in VR [hajj_2014b]. In blue-white, the wall of a Saccharomyces cerevisiae yeast, in red-yellow a filament of alpha tubulin passing between the dividing mother and daughter cells. D) A volumetric representation of the simulated positions of sixteen Saccharomyces cerevisiae yeast chromosomes (distinguished by colour) based on the statistical properties in a as Hi-C map [schalbetter_2017].

Primary themes for this internship will include:

o Software Design o Virtual Reality o Data Visualization

Duration

Up to 12 months

Tutor/supervisor

First name, Last name Jean-Baptiste Masson Phone + 33 (0)1 44 38 92 67 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://research.pasteur.fr/en/team/decision-and-bayesian-computation/


• El Beheiry et al, Virtual Reality: Beyond Visualisation JMB (2018, in Review) • M. El-Beheiry et al, InferenceMAP, Nature Methods 12, 594–595 (2015) • M. El-Beheiry et al,VISP, Nature Methods, vol 10, pages 689–690 (2013) • Software DIVA, Pasteur DI-2018-33, depot APP (ongoing) (2018), lean mapper for DIVA

software DI-2018-33 (patent filing ongoing) • Software: IMMERSE, depot APP IDDN.FR.001.530017.000.S.C.2017.000.21000 (2017) • Software: Escale, open source, https://goo.gl/aZSPZF (2017) • Software: InferenceMAP, https://goo.gl/HiwoxC, Depot APP

IDDN.FR.001.350042.000.S.P.2014.000.20700 (2015)


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Scientific or technical background required for work program Required Skills

Object-oriented programming experience (e.g. C++, C#, Java)

“Nice-to-Have” Skills

Game design

Computer graphics

Interface design


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Title of the work program 5 Image Segmentation Using Virtual Reality Description of the work program

The DIVA (Data Integration and Visualization in Virtual and Augmented Environments) project aims to create environments for data treatment that leverages virtual reality (VR), human-data interaction and automated algorithms. It is a joint initiative between the Institut Pasteur and the Institut Curie. The proposed post-doctoral fellowship involves developing machine-aided segmentation approaches to volumetric imaging data. Key to our segmentation approach is the relation between user intervention and data treatment. It is tailored to handle data in limited quantities, possibly very noisy, and to allow rapid and robust learning of the required procedures to extract relevant information from data.

Development of the proposed segmentation tool will be based on an in-house software platform that is designed to load any type of image stack (e.g. microscopy image or MRI/CT scan) into VR environments. The platform generates entire scenes allowing real-time interaction with data (i.e. representation modifications, annotations and projections of data). Currently the platform is available in alpha version at both DIVA host institutes.

More information on applications of the DIVA project is available https://goo.gl/myWD57.

We are proposing an internship for a motivated candidate. They will be expected to contribute to our efforts of designing segmentation procedures for noisy data coming from microscopy and biomedical images. The successful candidate will employ Bayesian pixel feature learning (within the VR environment) and amortized active membrane segmentation to user-data interactions. Good experience in programming is recommanded and previous experience in pixel classification and segmentation would be beneficial (bit not mandatory). The approach will be tested on large-scale multimodal images of neurons (e.g. via light-sheet and two-photons microscopy techniques) and, if times permits, on CT scans of breast and hepatic cancers. The successful candidate will be working directly with both designers and coders of the DIVA platform: Mohamed El Beheiry and Sébastien Doutreligne. He or she will work directly with various members of the team of Jean-Marie Lledo to validate performance on experimental data.


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Figure 1: Example of use of DIVA for data treatment. B) Demonstration tracing of a one month old adult-born neuron in the complete reconstructed hippocampus using an early prototype of the DIVA platform. The tissue was imaged using serial end-block imaging (SEBI) with a two-photon microscope (dimensions 2.5 × 2.3 × 3.1 mm).

The successful candidate will join a highly multidisciplinary lab of physicists and computer scientists who use computational approaches to study random walks of biomolecules in living cells and study the relationship between neural architecture and behavior in drosophila larva. Localized in the world-renowned Institut Pasteur, the successful candidate will have privileged access to multiple GPU clusters and custom platforms in the lab.

Duration


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Up to 12 months

Tutor/supervisor




• El Beheiry et al, Virtual Reality: Beyond Visualisation JMB (2018, in Review) • M. El-Beheiry et al, InferenceMAP, Nature Methods 12, 594–595 (2015) • M. El-Beheiry et al,VISP, Nature Methods, Vol 10, pages 689–690 (2013)

• Software DIVA, Pasteur DI-2018-33, depot APP (ongoing) (2018), lean mapper for DIVA software DI-2018-33 (patent filing ongoing)

• Software: IMMERSE, depot APP IDDN.FR.001.530017.000.S.C.2017.000.21000 (2017) • Software: Escale, open source, https://goo.gl/aZSPZF (2017) • Software: InferenceMAP, https://goo.gl/HiwoxC, Depot APP

IDDN.FR.001.350042.000.S.P.2014.000.20700 (2015)

Scientific or technical background required for work program

Required Skills

◦ C, C++ or C# ◦ Scripting (e.g. Python, Bash) ◦ Statistical Physics ◦ Inference and Numerical Optimization

Recommended Skills

◦ Image Processing ◦ Machine Learning Frameworks (e.g. PyTorch, TensorFlow) ◦ Computer Graphics (e.g. OpenGL, GLSL) ◦ General-Purpose GPU Programming (e.g. CUDA, OpenCL)


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Title of the work program 6 Bayesian approaches to Biomolecules Random Walks with mixed properties Description of the work program The TramWay project aims at designing an entirely automated procedure to study the random walks (RWs) of biomolecules in living cells. At the nanoscale, the dynamics of individual biomolecules is inherently random, governed by thermal noise and stochastic molecular interactions. By giving access to the full distribution of molecular properties, rather than simply their average value, the great advantage of single molecule measurements is thus their ability to identify static and dynamic heterogeneities, and rare behaviours. Local heterogeneities, numerous specific and non-specific interactions, and unknown number of molecular partners in the cellular environment lead to extremely unusual properties of biological RWs, making the inference of their nature a challenging statistical and computational problem. More information on the application of TRamWAy to synapses is available at the following address: https://research.pasteur.fr/en/project/mapping-receptor-dynamics-in-synapses/ We are proposing 2 internships for graduate students and 1 internship for an undergraduate student Internship 1 We are proposing an internship of up to 6 months for a motivated applicant. She/he is expected to contribute to our mixed temporal/spatial analysis of RW. She/ he will combine Bayesian time series analysis and point process modelling to infer temporal and spatial processes from single molecule recordings in 2D/3D at the full cell scale. She/he will also develop a variational inference scheme to simultaneously infer multiple dynamical maps from single datasets. We are looking for either a physicist with a strong statistical physics background or a computer scientist with experience in complex, large-scale variational inferences. Internship 2 We are proposing an internship of up to 6 months for a motivated applicant. She/he is expected to contribute to our effort to characterize unusual RWs that cannot be described by canonical model, e.g. such as a fractional Brownian motion or a hidden Markov model. She/he will develop Bayesian non-parametric approaches to characterize and classify the temporal and spatial characteristics of these unusual RW. We are looking for either a physicist with a strong statistical physics background or a computer scientist with experience in complex, large-scale variational inferences. Internship 3 We are offering an internship for a motivated undergraduate applicant. She/he is expected to contribute to our effort to analyze biomolecules RW at the big data scale. She/he will develop pipelines to entirely automate single molecule analysis. The pipelines will leverage TRamWAy and will also perform post analyses in order to automatically generate reports providing


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interpretation of the results. We are looking for a physicist with a background in statistical physics or a computer scientist with experience in inferences. The successful candidates will have privileged access to multiple GPU clusters in the Pasteur institute as well as a CPU cluster. Furthermore, she/he will join a full team working on the project composed of Christian L. Vestergaard, who developed a non-tracking algorithm using belief propagation and unseen graph summation to infer biomolecule dynamics at high density, Francois Laurent, who designed the TRamWAy software platform and is currently developing stochastic gradient approaches to infer time varying 2D/3D maps of biomolecule dynamics at the full cell scale, and Alexander Serov, who after designing Bayesian evidence tests for Ito-Stratonovitch dilemma is developing amortized approaches to detect out-of-equilibrium dynamics from recorded RWs. Tutor/supervisor




• Floderer et al, Single molecule localisation microscopy reveals how HIV-1 Gag proteins sense membrane virus assembly sites in living host CD4 T cells, Sci. Rep. (2018, in press)

• Remorino A et al, Gradients of Rac1 Nanoclusters Support Spatial Patterns of Rac1 Signaling, Cell Rep 2017 Nov;21(7):1922-1935

• Knight S. C et al, Dynamics CRISPR-CAS9 genome interrogation in living cells. Science vol 350, Issue 6262, p823-826 (2015).

• M. El-Beheiry et al, InferenceMAP, Nature Methods, 12, 594–595 (2015) • M. El-Beheiry et al,VISP, Nature Methods, 10, pages 689–690 (2013) • Software DIVA, Pasteur DI-2018-33, depot APP (ongoing) (2018), lean mapper for DIVA

software DI-2018-33 (patent filing ongoing) • Software: IMMERSE, depot APP IDDN.FR.001.530017.000.S.C.2017.000.21000 (2017) • Software: Escale, open source, https://goo.gl/aZSPZF (2017) • Software: InferenceMAP, https://goo.gl/HiwoxC, Depot APP

IDDN.FR.001.350042.000.S.P.2014.000.20700 (2015)

Scientific or technical background required for work program For a physicist: statistical physics and random walk theory

For a computer scientist: Bayesian Inference, structured and variational inferences

Scripting language experience: Bash, Pytho


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Title of the work program 7 “Consequences of H. pylori infection on mitochondrial DNA repair: impact on gastric carcinogenesis” Description of the work program H. pylori is a gastric pathogen responsible for the most common infection in the world population. This bacterium is the major risk factor for gastric cancer development. Up to now it is the only one bacterium classified as a group 1 carcinogenic agent. The induction of genetic instabilities during H. pylori infection plays an essential role in the promotion of the gastric carcinogenesis process. During its interaction with gastric epithelial cells, H. pylori targets mitochondria through its pro-apoptotic VacA cytotoxin, leading to increase mitochondrial membrane permeability and release of cytochrome c. Mitochondria are a strategic target for pathogens, to draw host cellular functions to their benefit. Alterations of these organelles are also associated to human diseases including cancer. Our group previously demonstrated a mutator effect of H. pylori infection to the gastric epithelial cells, at both nuclear and mitochondrial genome (1-3). This mutagenic effect of H. pylori infection is correlated with the impairment of DNA repair systems including mismatch repair and base excision repair. These two DNA repair systems as well as the recombinational repair have also been identified in mitochondria. Our recent study demonstrated that H. pylori induced a dramatic increase of two essential components of the mitochondrial DNA replication/transcription machinery, the mitochondrial DNA polymerase POLG and the mitochondrial transcription factor TFAM (5). Based on these data, the objectives of this project are to investigate the role of components of mitochondrial DNA replication/transcription machinery on the mitochondrial DNA repair activity in response to H. pylori infection. The consequences of H. pylori on the mitochondrial DNA damage and mutation will be also investigated. This project will include microbiology, cellular and molecular biology and imaging approaches. In addition, some aspects will be investigated in the mouse model.

Tutor/supervisor

First name, Last name Eliette, TOUATI Phone 00 33 140613785 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):


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Selected publications or patents of the Research Group offering the work program Selected publications: 1) Touati E, Michel V, Thiberge JM, Wuscher N, Huerre M and Labigne A (2003) Chronic

Helicobacter pylori infection induce gastric mutations in mice. Gastroenterology, 124, 1408-1419.

2) Machado AM, Figueiredo C, Touati E, Máximo V, Sousa S, Michel V, Carneiro F, Nielsen FC, Seruca R, and Rasmussen LJ (2009) Helicobacter pylori infection influences genetic stability of nuclear and mitochondrial DNA. Clinical Cancer Research, 15 : 2995-3002.

3) E.Touati (2010) When bacteria are mutagenic and carcinogenic: lessons from H. pylori. Mutation Research. 703: 66-70

4) Fernandes J, Michel V, Carmolinga-Ponce M, Gomez A, Maldonada C, De Reuse H, Torres J, Touati E. (2014) Circulating mitochondrial DNA level as a potential non-invasive biomarker to the early detection of gastric cancer. Cancer Epidemiology, Biomarkers and Prevention, 23 : 2430-2438.

5) Chatre L, Fernandes J, Michel V, Fiette L, Ave P, Arena G, Jain U, Haas R, Wang TC, Ricchetti M, Touati E (2017) Helicobacter pylori targets mitochondrial import and components of mitochondrial DNA replication machinery through an alternative VacA-dependent and a VacA-independent mechanisms. Scientific Reports, 7: 15901

• Patent: PCT/EP2014/071239: "Method for in vitro investigating mitochondrial DNA levels in a biological sample, kits and uses thereof" E. Touati, J. Fernandes, V. Michel and J. Torres (2013)

Scientific or technical background required for work program We are looking for a very motivated student with a confirmed knowledge in infectious

disease, host-pathogen interaction, cellular and molecular biology. A technical expertise in

cell culture and imaging will be appreciated.


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Title of the work program 8 T cell immunity to dengue virus infection in transgenic mice expressing human HLA molecules Description of the work program Based on the recent identification of the non-structural proteins NS3, NS4B and NS5 of dengue virus (DENV) as the main targets for T cell responses in humans, we have designed a minimal DENV antigen, called PolyDV-NS, enriched in conserved and highly antigenic epitopes (patent WO2015/197565, initially filed on June 23, 2014). This PolyDV-NS vaccine can induce a strong CD8 T-cell response and a protection against DENV1 infection in transgenic mice that express different human HLA class I molecules (Roth et al., manuscript in preparation). To optimize immunogenicity as much as possible against the 4 DENV serotypes, we will measure the magnitude of T-cell responses (mediated by CD4 and/or CD8 T cells) by quantifying ex vivo IFN-g responses against peptides from DENV1 and from their serotype variants covering the whole PolyDV-NS sequence by enzyme linked immunosorbent spot assay (ELISPOT assay). The contribution of DENV-specific CD8+ T cells elicited by the PolyDV-NS vaccination will also be determined by analyzing the phenotype and frequency of peptide-specific CD8+ T cells that are CD44hi CD62Llo (i.e. effector memory cells) and express IFN-g alone, both IFN-g and TNF-a, or Granzyme B. Altogether, these analyses should allow us to better predict and enhance the efficiency of the DV-NS vaccine against the different DENV serotypes. Tutor/supervisor

First name, Last name Claude Roth Phone 33 1 45 68 89 62 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):


Publications

- Roth C, Delgado FG, Simon-Lorière E, Sakuntabhai A, Immune Responses to Dengue and Zika Viruses-Guidance for T Cell Vaccine Development, Int J Environ Res Public Health 2018 02;15(2).

- Delgado FG, Torres KI, Castellanos JE, Romero-Sánchez C, Simon-Lorière E, Sakuntabhai A, Roth C, Improved Immune Responses Against Zika Virus After Sequential Dengue and Zika Virus Infection in Humans, Viruses 2018 09;10(9).

- Manet C, Roth C, Tawfik A, Cantaert T, Sakuntabhai A, Montagutelli X, Host genetic control of mosquito-borne Flavivirus infections, Mamm. Genome 2018 Aug;29(7-8):384-407.

Patents - A dengue virus chimeric polyepitope composed of fragments of non-structural proteins and its use in an immunogenic composition against dengue virus infection (E14305984.8)


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Scientific or technical background required for work program - The student should have a good knowledge in Immunology, more specifically the fundamental mechanisms involved in the cellular and humoral immune responses.

- A good laboratory practice in cell culture in sterile conditions.

- A technical experience in staining with antibodies for fluorescence analysis would be appreciated.


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Title of the work program 9 Characterization of the entry of Streptococcus gallolyticus in colonic cells Description of the work program

Streptococcus gallolyticus (also known as S. bovis type I) is one of the few opportunistic pathogens, which has been unambiguously linked to colonic malignant diseases. Colorectal cancer (CRC) is the third most common cause of cancer mortality in the world, with more than 600 000 deaths every year. CRC is typically a genetic disease that develops over many years via a sequence of genetic changes known as the adenoma-carcinoma sequence. Tumors are more frequent in the distal large intestine, the habitat of a vast and complex community of microorganisms referred as “gut microbiota” whose contribution in the development of CRC is increasing.

In this project, we aim at characterizing the entry process of Streptococcus gallolyticus (Sgg) into human colonic cells HT-29 (our unpublished data). Interestingly, no invasion has been seen in related human colonic Caco-2 cells. The student will test the WT strain UCN34 and various isogenic mutants available in the laboratory to identify bacterial surface components important for this process. Pharmacological inhibitors of classical internalization pathways will also be assayed to decipher the invasion process using classical gentamicin survival assay and immunofluorescence microscopy. The student may also contribute to the development of fluorescent tools for tracking Sgg during in vivo studies.

Tutor/supervisor

First name, Last name Shaynoor Dramsi Phone +33140613280 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://www.researchgate.net/profile/Shaynoor_Dramsi


1- Pasquereau-Kotula E, Martins M, Aymeric L, Dramsi S*. Significance of Streptococcus

gallolyticus subsp. gallolyticus association with colorectal cancer. Front Microbiol. 2018 Apr 3;9:614.

2- Aymeric L, Donnadieu F, Mulet C, du Merle L, Nigro G, Saffarian A, Bérard M, Poyart C, Robine S, Regnault B, Trieu-Cuot P, Sansonetti PJ, Dramsi S*. Colorectal cancer specific conditions promote Streptococcus gallolyticus gut colonization. Proc Natl Acad Sci USA 2018 Jan 9;115(2):E283-E291.

3- Martins M, Porrini C, du Merle L, Danne C, Robbe-Masselot C, Trieu-Cuot P, Dramsi S*. 4- The Pil3 pilus of Streptococcus gallolyticus binds to intestinal mucins and to fibrinogen.

Gut Microbes 2016 Nov;7(6):526-532.


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5- Martins M, Aymeric L, du Merle L, Danne C, Robbe-Masselot C, Trieu-Cuot P, Sansonetti P, Dramsi S*. Streptococcus gallolyticus Pil3 is required for adhesion to colonic mucus and for colonization of mouse distal colon. J. Infect. Dis. 2015 Nov 15; 212(10):1646-55.

Scientific or technical background required for work program The applicant should be familiar with basics in molecular cloning techniques and work under sterile conditions. Skills in microbiology techniques or microscopy will certainly be a plus.


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Title of the work program 10 Deciphering the role of m6A methylation in Plasmodium falciparum mRNA homeostasis Description of the work program Keywords: epitranscriptomics; translation control; non-coding RNA; RNA-protein interactions; CRISPR/Cas9 genome editing Malaria is the deadliest disease in human history. In 2015, nearly half a million died and more than 200 million people were infected with Plasmodium falciparum - the causative agent of the most severe form of malaria. Malaria pathogenesis results from the asexual reproduction inside erythrocytes where the parasite differentiates and replicates up to 32 daughter cells within 48 hours. The developmental stages are characterized by specific profiles of gene expression that are highly coordinated by an as-yet poorly-defined regulatory mechanism. Interestingly, transcription factors seem to play a small role in transcriptional regulation, and gene activation/repression is believed to be mainly achieved by reversible histone modifications and spatial nuclear organization. However, an emerging body of evidence demonstrates that post-transcriptional regulation (i.e. at the RNA level) through specific protein-RNA interactions significantly contributes to coordinated gene expression in the human host and might also play a role in other life cycle stages (i.e. in the mosquito vector). While these processes seem to be key to parasite virulence, many questions remain concerning the extent of protein-RNA interactions, the manner in which proteins recognize specific RNA transcripts, and the consequences of these interactions.

Most intriguingly, recent findings in model organisms point towards a high level of post-transcriptional regulation through chemical modifications on mRNA and non-coding RNA transcripts. We set out to characterize the so-called ‘epitranscriptome’ in P. falciparum with mass spectrometry and identified dozens of RNA modifications throughout the parasite life cycle. Of those, we found methylation of adenosine at N6 (m6A) to be the most abundant and highly dynamic RNA modification. Currently, we are attempting to characterize the m6A methylation machinery, which includes knock down of the putative m6A methyltransferase and genome-wide identification of individual m6A sites.

Our current efforts and the main focus of the proposed project will be on characterizing the m6A methylation-dependent processes and phenotypes in P. falciparum. Specific questions include:

1) Which proteins are involved in ‘writing’ m6A methylation on RNA and which proteins specifically recognize this modification? 2) How do m6A modifications affect mRNA translation and stability? 3) Does m6A occur on types of RNA transcripts other than mRNA?

The Master student will learn and apply several targeted and genome-wide

approaches to answer these questions. Protein immunoprecipitations will be used to identify new members of the m6A methylation complex and RNA pull-downs will help to identify


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specific m6A ‘reading’ proteins. Genome-wide approaches such as ribosome profiling will be used to measure translation efficiencies across the parasite life cycle in methylation-deficient versus wild-type parasites. In follow-up experiments, the student will have the opportunity to characterize individual proteins involved in RNA methylation by creating inducible knock out or knock down cell lines with CRISPR/Cas9. Importantly, the student will have the flexibility to investigate unforeseen and interesting developments in the proposed project.

The proposed project takes the emerging hot topic of post-transcriptional regulation

and applies it to a pathogen that has a huge impact on global public health. The student will receive training in a wide variety of techniques in parasitology, cell and molecular biology, and genetics, ranging from parasite cell culture to generation and bioinformatic analysis of NGS data. To facilitate scientific interactions, BIHP has weekly lab meetings and participates in an annual lab and departmental retreat. Finally, each student in BIHP is encouraged to attend international workshops and conferences where she/he will have the opportunity to present her/his data and interact with experts from all over the world.

Tutor/supervisor

First name, Last name Artur Scherf Sebastian Baumgarten

Phone +33 145688616 (A. Scherf) +33 145683516 (S. Baumgarten)

E-mail [email protected] [email protected]

Profile on http://www.researchgate.net/ (if applicable):

not applicable


• Droll D, Wei G, Guo G, Fan Y, Baumgarten S, Zhou Y, Xiao, Y, Scherf A, Zhang Q.

Disruption of the RNA exosome reveals the hidden face of the malaria parasite transcriptome. RNA Biology, 2018, 1-9

• Bryant, J.M., Regnault, C., Scheidig-Benatar, C., Baumgarten, S., Guizetti, J., and Scherf, A. (2017) CRISPR/Cas9 genome editing reveals that the intron is not essential for var2csa gene activation or silencing in Plasmodium falciparum, mBio.

• Guizetti, J., Barcons-Simon, A., and Scherf, A. (2016) Trans-acting GC-rich non-coding RNA at var expression site modulates gene counting in malaria parasite, Nucleic Acids Res.

• Zhang, Q., Siegel, T.N., Martins, R.M., Wang, F., Cao, J., Gao, Q., Cheng, X., Jiang, L., Hon, C.C., Scheidig-Benatar, C., Sakamoto, H., Turner, L., Jensen, A.T., Claes, A., Guizetti, J., Malmquist, N.A., and Scherf, A. (2014) Exonuclease-mediated degradation of nascent RNA silences genes linked to severe malaria, Nature.

• Ghorbal, M., Gorman, M., Macpherson, C. R., Martins, R. M., Scherf, A., and Lopez-Rubio, J. J. (2014) Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system, Nat Biotechnol.


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Scientific or technical background required for work program Basic experience in molecular and cell biology. Proficiency in English is a requirement


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Title of the work program 11 Regulation of immune-related genes and interleukin 10 expression by type I interferon in human CD4+ T cells Description of the work program We study the immunomodulatory effect of type I interferon family (IFNa/b) on the T cell

adaptive immune response of healthy donors and patients with multiple sclerosis (MS).

The latter is a chronic, neuro-inflammatory and autoimmune disease, which leads to

progressive neurodegeneration and physical disability. Type I interferon, IFNb, is

commonly prescribed to patients with the relapsing-remitting form of MS but around

30% of patients do not respond to the therapy. Therefore, one major goal is to uncover

cellular and/or molecular signatures that could help to predict patient responsiveness

to the treatment.

Using whole blood cultures and the Nanostring approach, we have obtained a series

of new data on the modulatory effect of IFNb on the expression of immune related

genes. A first objective will be to validate by qPCR some of these data obtained in

whole blood, using purified CD4+ T cells. The second objective concerns the molecular

mechanisms by which IFNa/b promotes expression of the potent anti-inflammatory

cytokine IL-10 and development of type 1 regulatory-like cells (Tr1-like cells). Tr1 cells

secreting IL-10 have been shown to play an important role in immune homeostasis by

limiting the immune response and contributing to tolerance to self-antigens. We have

recently identified transcription factors that promote the TCR/IFN crosstalk towards IL-

10 expression. More recently, we have investigated by RNA-Seq the impact of IFNa

on the TCR-induced landscape of non-coding microRNAs in CD4+ T cells of healthy

donors. We found that a number of differentially expressed microRNAs was predicted

or reported to regulate IL-10 expression in distinct immune cell types. Expression and

impact of these miRNAs on IL-10 expression will be gained using primary CD4+ T cells

of healthy donors and T cell lines. Insights from this study may be translated to multiple

sclerosis patients, taking advantage of the project that we are developing.


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(https://research.pasteur.fr/en/program_project/milieu-interieur-labex/).

Tutor/supervisor

First name, Last name Frédérique MICHEL Phone 33 1 45 68 86 38 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

Selected publications or patents of the Research Group offering the work program - U. Govender, B. Corre, Y. Bourdache, S. Pellegrini and F. Michel. 2017.Type I interferon-enhanced IL-10 expression in human CD4 T cells is regulated by STAT3, STAT2, and BATF transcription factors. J. Leukoc. Biol. Doi :10.1189/jlb.2A0416-187RR - Zhang X., Bogunovic D., Payelle-Brogard B., Francois-Newton V., Speer S, Yuan C, Volpi S, Li Z, Sanal O, Mansouri D, Tezcan I, Rice GI, Chen C, Mansouri N, Mahdaviani S, Itan Y, Boisson B, Okada S, Zeng L, Wang X, Jiang H, Liu W, Han T, Liu D, Ma T, Wang B, Liu M, Liu J, Wang QK, Yalnizoglu D, Radoshevich L, Uzé G, Gros P, Rozenberg F, Zhang S-Y, Jouanguy E, Bustamante J, García-Sastre A, Abel L, Lebon P, Notarangelo L, Boisson-Dupuis S, Crow YJ, Casanova J-L and Pellegrini S. 2015. Human intracellular ISG15 prevents IFN-a/b over-amplification and auto-inflammation. Nature, 517: 89-93 - B. Corre, J. Perrier, M. El Khouri, S. Cerboni, S. Pellegrini and F. Michel. 2013. Type I interferon potentiates T-cell receptor mediated induction of IL-10-producing CD4⁺ T cells. Eur. J. Immunol., 43(10):2730-40. - Z. Li, M. Gakovic, J. Ragimbeau, M-L Eloranta, L Rönnblom, F Michel and S Pellegrini. 2013. Two rare disease-associated Tyk2 variants are catalytically impaired but signaling competent. J. Immunol., 190(5):2335-44. - Francois-Newton V., Livingstone M., Payelle-Brogard B., Uzé G., and Pellegrini S. 2012. USP18 establishes the transcriptional and anti-proliferative interferon α/β differential. Biochem. J. 446, 509-516. - Francois-Newton V., de Freitas Almeida G., Payelle-Brogard B., Monneron D., Pichard-Garcia, L. Piehler, J., Pellegrini S., and Uzé G. 2011. USP18-based negative feed-back control is induced by Type I and Type III Interferons and specifically inactivates interferons a response. PLoS ONE 6(7):e22200.

Scientific or technical background required for work program Experience in transcriptomic studies, regulation of gene expression, microRNA and/or

T cell adaptive immunity would be an advantage.


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Title of the work program 12 Structural studies of the archaeal replisome Description of the work program

DNA polymerases (DNAPs) are molecular motors directing the synthesis of DNA from nucleotides. On the basis of their amino acid sequence and structural analysis, DNAPs have been classified into seven families, A, B, C, D, X, Y and reverse transcriptases. In addition to their fundamental biological functions, DNAPs are versatile tools used in important molecular biology core technologies. Recently, a novel family (D-family) of archaeal thermostable DNAP, named PolD, was discovered and shown to have significant commercial value in PCR technology. PolD is composed of a large catalytic subunit (DP2) and a smaller subunit with proofreading exonuclease activity (DP1). We have determined the first crystal structures of both individual DP1 and DP2 catalytic subunits of PolD from P. abyssi, and showed that it is structurally unrelated to any other DNAP family.

However, the DP1 and DP2 crystal structures were obtained separately and do not provide a comprehensive description of the molecular mechanisms of DNA polymerization and proofreading by PolD. Indeed, the DNAP active site is located at the interface between the DP1 and DP2 subunits, and the interaction between both subunits is essential for the full activity of PolD. Unlike other DNAPs used in PCR whose crystal structure have been solved in complex with various DNA substrates, the molecular basis of DNA and nucleotide-recognition by PolD remains poorly characterized. Elucidating the specificities of the PolD active site at the molecular level is required in order to fully exploit the biotechnological potential of this unusual thermostable DNAP.

Using a multi-disciplinary approach combining primarily electron microscopy, X-ray crystallography, we aim to determine the DNA-bound structures of the native full DP1-DP2 PolD complex in both elongation and proof-reading modes, as well as in complex with the proliferating cell nuclear antigen (PCNA), a key actor of the replication fork, which has been shown to considerably improve the processivity of PolD.

The Unit for Structural Dynamics of Macromolecules at the Pasteur Institute is a well published and highly dynamic research environment that applies a multidisciplinary research strategy including structural biology, biochemistry, and biophysics to address fundamental questions about the molecular mechanisms of DNA replication. The laboratory is located at the Pasteur Institute in Paris that has an outstanding range of facilities and instrumentation, including a state-of-the-art Titan Kryos equipped with a K3 camera, a F20 (200kV) microscope equipped with a Falcon 2 camera, and two T12 (120 kV) microscopes. Additional resources for specimen preparation and image processing are in place. No prior experience in X-ray crystallography and Cryo-electron microscopy is required, the host lab will provide full training to the successful candidate.


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Tutor/supervisor

First name, Last name Ludovic Sauguet Phone +33140613565 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://www.researchgate.net/profile/Ludovic_Sauguet

Selected publications or patents of the Research Group offering the work program Shared active site architecture between archaeal DNA polymerase D and multisubunit RNA polymerases revealed by X-ray crystallography. Sauguet L, Raia P, Henneke G, Delarue M Nature communications 2016 (7) 12227


The candidate should have a scientific back ground in biochemistry, molecular biology,

or biophysic. No prior experience in X-ray crystallography and Cryo-electron microscopy is

required, the host lab will provide full training to the successful candidate.


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Title of the work program 13 CRISPR/dCas9 elucidates the var gene interactome in Plasmodium falciparum Description of the work program Mutually exclusive transcription within the ~60 member var gene family is important for antigenic variation and pathogenesis of the malaria parasite Plasmodium falciparum. It has been shown that transcriptional control of var genes is primarily epigenetic, with the single active var gene associated with euchromatin while all silent var genes are embedded in heterochromatin. However, only a few chromatin-associated factors have been implicated in this process. To investigate how a single var gene is targeted for transcription while all others are kept in a transcriptionally silent state, we attempted to identify new proteins and/or RNAs that bind to putative transcription control regions of var genes. We developed a multi-locus, CRISPR-mediated chromatin immunoprecipitation technology to isolate var gene loci in their natural chromatin environment in vivo. Using a tagged, catalytically inactive Cas9 (“dead” or dCas9), we targeted ~30% of var genes with a single guide RNA. After crosslinking, immunoprecipitation of dCas9 allowed for co-purification of all proteins, DNA, and RNA closely associated with the targeted genomic DNA region. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed that dCas9 binding was highly robust and specific, with an average 500-fold enrichment of target loci. Quantitative proteomics analysis of active dCas9-targeted var genes identified both general chromatin-associated proteins and specific var gene-interacting factors. This study provides a new tool for the unbiased and in vivo characterization of locus-specific cis- and trans-regulatory elements in one of the major virulence gene families of the malaria parasite. The Erasmus student would help to functionally characterize the proteins and factors identified in the proteomics analysis with regard to var gene transcription. The student could also help develop other dCas9 technologies in the lab. The skills learned during this project include, but are not limited to, plasmid cloning, parasite culture and transfection, RNA/DNA sequencing, and chromatin immunoprecipitation.

Tutor/supervisor

First name, Last name Jessica Bryant, head of unit: Artur Scherf Phone +33 1 45 68 86 22 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://research.pasteur.fr/en/team/biology-of-host-parasite-interactions/


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Selected publications or patents of the Research Group offering the work program • Bryant JM, Regnault C, Scheidig-Benatar C, Baumgarten S, Guizetti J, Scherf A. 2017. CRISPR/Cas9

Genome Editing Reveals That the Intron Is Not Essential for. MBio 8(4) • Martins RM, Macpherson CR, Claes A, Scheidig-Benatar C, Sakamoto H, Yam XY, Preiser P, Goel S,

Wahlgren M, Sismeiro O, Coppée JY, Scherf A. 2017. An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum. Sci Rep 7(1):14042.

• Guizetti J, Barcons-Simon A, Scherf A. 2016. Trans-acting GC-rich non-coding RNA at var expression site modulates gene counting in malaria parasite. Nucleic Acids Res 44(20):9710-9718.

• Ghorbal, M., Gorman, M., Macpherson, C. R., Martins, R. M., Scherf, A., and Lopez-Rubio, J. J. (2014) Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system, Nat Biotechnol.

• Guizetti, J., and Scherf, A. (2013) Silence, activate, poise and switch! Mechanisms of antigenic variation in Plasmodium falciparum, Cell Microbiol 15, 718-726.

• Zhang, Q., Huang, Y., Zhang, Y., Fang, X., Claes, A., Duchateau, M., Namane, A., Lopez-Rubio, J. J., Pan, W., and Scherf, A. (2011) A critical role of perinuclear filamentous actin in spatial repositioning and mutually exclusive expression of virulence genes in malaria parasites, Cell Host Microbe 10, 451-463.

• Lopez-Rubio, J. J., Mancio-Silva, L., and Scherf, A. (2009) Genome-wide analysis of heterochromatin associates clonally variant gene regulation with perinuclear repressive centers in malaria parasites, Cell Host Microbe 5, 179-190.

Scientific or technical background required for work program Basic experience in molecular and cell biology and willingness to perform parasite culture in

human blood is required. Proficiency in English is a requirement.


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Title of the work program 14 Targeting methyltransferases in human diseases Description of the work program Currently there is a large interest in the role of epigenetic changes in human diseases and great expectations from drugs bridling these changes. The main epigenetic processes are DNA methylation, histone modifications, nucleosome positioning and noncoding RNA-associated silencing. Proteins involved in “writing”, “erasing” and “reading” of the chemical chromatin modifications conveying the epigenetic regulation have been identified and studied. Inhibitors have been designed and are in clinical trials for cancer. However, the chemical scaffolds of the actual drugs are limited. We develop chemical probes to target the epigenetic methyltransferases and use them to better understand the mechanisms that are aberrant in cancer. We are synthetizing a focused chemical library to identify new scaffolds able to inhibit methyltransferases and designing biological assays apt to screen such library. In our team, chemists and biologists work together to reach these aims. The student will either participate to the design and synthesis of the chemical library and optimisation of the hits or to assay development and characterisation of the biological activity of the compounds in cancer cells, or both depending on her/his background and interests. Tutor/supervisor

First name, Last name Paola B. Arimondo Phone +33186467869 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

NA

Selected publications or patents of the Research Group offering the work program -Gros C, Fleury L, Nahoum V, Faux C, Valente S, Labella D, Cantagrel F, Rilova E, Bouhlel

MA, David-Cordonnier M, Dufau I, Ausseil F, Mai A, Mourey L, Lacroix L, Arimondo PB. New Insights on the Mechanism of Quinoline-based DNA Methyltransferase Inhibitors J Biol Chem 2015 290(10):6293-302.

-Desjobert C, El Maï M, Gérard-Hirne T, Guianvarc'h D, Carrier A, Pottier C, Arimondo PB, Riond J. Combined analysis of DNA methylation and cell cycle in cancer cells Epigenetics 2015 2;10(1):82-91.

-Vispé S, Deroide A, Davoine E, Desjobert C, Lestienne F, Fournier L, Novosad N, Bréand S, Besse J, Busato F, Tost J, De Vries L, Cussac D, Riond J, Arimondo PB. Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2’-deoxycytidine in human leukemic KG1 cells. 2015 OncoTarget 6(17):15265-82.


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-Halby L, N Marechal, D Pechalrieu, V Cura,D-M Franchini, C Faux, F Alby, N Troffer-Charlier, S Kudithipudi, A Jeltsch, W Aouadi, E Decroly, J-C Guillemot, P Page, C Ferroud, L Bonnefond, D Guianvarc’h, J Cavarelli and PB Arimondo Hijacking DNA methyltransferase transition state analogues to produce chemical scaffolds for PRMT inhibitors Phil. Trans RS B 2018, 373(1748) pii: 20170072.

-Halby L, Menon Y, Rilova E, Pechalrieu D, Masson V, Faux C, Bouhlel MA, David-Cordonnier MH, Novosad N, Aussagues Y, Samson A, Lacroix L, Ausseil F, Fleury L, Guianvarc'h D, Ferroud C, Arimondo PB. Rational Design of Bisubstrate-Type Analogues as Inhibitors of DNA Methyltransferases in Cancer Cells. J Med Chem. 2017 60(11):4665-4679.

Scientific or technical background required for work program Skills either in organic synthesis and analytical characterisation of chemical compounds or in

molecular and cellular biology.


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Title of the work program 15 Morpho-dynamics analysis of Entamoeba histolytica, a human pathogenic amoeba, in a 3D environment mimicking the extracellular matrix and showing chemotactic migration towards TNF. Description of the work program Entamoeba histolytica, a pathogenic highly motile cell, is the etiological agent of amoebiasis. The amoeba resides in the human intestinal lumen as a commensal and invades in 10% of the cases the intestinal mucosa. E. histolytica infection initiates by parasite adherence to the mucus, which is depleted and then the parasite binds to the epithelium provoking cell death and production of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-12, INF-γ and TNF) that in turn act on immune cells, promoting their activation and recruitment1. How E. histolytica converts human tissue components and mechanical clues are still an open question to understand its pathogenic process. We have shown using human colon explants that Entamoeba takes advantage of a dense collagen scaffold at the sub-epithelial level to migrate until the crypts of Lüberkhun and then penetrates the mucosa via the loose collagen meshwork causing a remodeling and the destruction of the extracellular matrix (ECM)2, 3. E. histolytica exhibits amoeboid movement during random migration. Interestingly, we have found that TNF is a chemoattractant for the parasite and modifies the parasite mode of migration (on a two-dimensional surface) that becomes mesenchymal like4, 5. This phenomenon reminds us the switch between mesenchymal and amoeboid movement described for cancer cells in vivo. One of our aims is to analyze whether/how the parasite penetration and migration within the mucosa is directed/affected by human elements. Knowledge on the migration behavior of E. histolytica during its tissue invasive process should bring new insights into the mechanobiology of migrating cells and to fight the disease. To this goal, we proposed in this project to characterize E. histolytica morpho-dynamics during chemotactic migration towards TNF within 3D collagen I fibers matrices that mimic human colon ECM. Cell biology, real time imaging and image quantification tools will be used to analyze the impact of a TNF gradient on the cell morpho-dynamics. - The 3D morphology of migrating cells and intra-cellular biophysical quantities (pressure, force, flux velocity) will be determined in real time using dedicated in house-made image quantification softwares 6, 7. Fluorescent-labeled amoeba will be acquired with a spinning disk confocal microscopy system. - The dynamics of key cytoskeleton proteins will be analyzed in live with amoeba expressing a Halo-Tag-Actin (available in the lab) and/or with fixed sample by immunofluorescence and confocal microscopy. - The role of candidate proteins (found in our previous works) in the “switch” will be tested by analyzing the behavior of E. histolytica in the presence of specific inhibitors or affected for one of these proteins. This study should help to correlate morpho-dynamic features with biological intracellular information.


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Tutor/supervisor

First name, Last name Elisabeth Labruyère Phone 01 45 58 88 87 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

Yes

Selected publications or patents of the Research Group offering the work program 1-Labruyère E, Thibeaux R, Olivo-Marin JC, Guillén N. Crosstalk between Entamoeba histolytica and the human intestinal tract during amoebiasis. (2017). Review. Parasitology.

2-Thibeaux R, Avé P, Bernier M, Morcelet M, Frileux P, Guillén N, Labruyère E. The parasite Entamoeba histolytica exploits the activities of human matrix metalloproteinases to invade colonic tissue. (2014). Nat Commun.

3-Thibeaux R, Dufour A, Roux P, Bernier M, Baglin AC, Frileux P, Olivo-Marin JC, Guillén N, Labruyère E. Newly visualized fibrillar collagen scaffolds dictate Entamoeba histolytica invasion route in the human colon. (2012). Cell Microbiol.

4-A. Silvestre, A. Plaze, P. Berthon, R. Thibeaux, N. Guillen and E. Labruyère. In Entamoeba histolytica, a BspA family protein is required for chemotaxis toward tumor necrosis factor. (2015). Microbiol Cell.

5-S. Blazquez, G. Guigon, C. Weber, S. Syan, O. Sismero, J. Y. Coppée, E. Labruyère and N. Guillén. Chemotaxis of Entamoeba histolytica towards the pro-inflammatory cytokine TNF is based on PI3K signalling, cytoskeleton reorganization and the Galactose/Nacetylgalactosamine lectin activity. (2008). Cell Microbiol.

6- Dufour A, Thibeaux R, Labruyère E, Guillen N, Olivo JC. 3-D active meshes: fast discrete deformable models for cell tracking in 3-D time-lapse microscopy. (2011). IEEE Trans Image Process.

7- A. Boquet-Pujadas, T. Lecomte, M. Manich, R. Thibeaux, E. Labruyère, N. Guillén, J-C Olivo-Marin, and A. C. Dufour. BioFlow: a non-invasive, image-based method to measure speed, pressure and forces inside living cells. (2017). Nature Scientific report


- Microbiology or Cell biology or Biophysics.


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Title of the work program 16 P. vivax infections in humanized mice: a model for studying P. vivax ligands involved in host cell invasion Description of the work program Plasmodium vivax is a major cause of malaria outside Africa with possibly up to 130 million clinical cases annually. While vivax malaria has been considered as a benign infection compared to P. falciparum, it is responsible for long-term chronic infections that have significant consequences for the health and economy of endemic areas. Erythrocyte invasion is a complex process involving multiple interactions between Plasmodium merozoites and host erythrocytes. Unlike P. falciparum, which can use multiple erythrocyte receptors for invasion and has merozoite proteins with overlapping and redundant receptor-binding functions, invasion of human erythrocytes by P. vivax merozoites primarily relies on the interaction between the P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy antigen receptor for chemokines (DARC). Consequently, PvDBP is considered a promising candidate for a P. vivax malaria vaccine. However, recently, a growing body of studies have reported PCR-positive vivax malaria cases in Duffy-negative individuals around the world, specifically across Africa and South America. These novel observations highlight the emerging issue of P. vivax infection in Duffy-negative populations, which questions the essential role of the PvDBP-DARC interaction and raises the possibility of alternative invasion mechanism(s) with implications for the design of blood-stage vaccines for P. vivax malaria. To identify P. vivax ligands involved in host cell invasion and understand how P. vivax has gained the capacity to infect reticulocytes from Duffy-negative individuals, we aim at developing a strategy based on P. vivax infection in a unique model of humanized mice (along with ex vivo invasion assays). Previously engineered alymphoid RAG-/- γc-/- alymphoid mice transgenic for HLA-A2 and HLA-DR1 and for human SIRPa were further modified in our unit to increase the development of human erythrocytes upon human CD34+ hematopoietic stem cell engraftment collected from Duffy-negative and Duffy-positive cord blood. The objective of this project will be to obtain preliminary data on P. vivax infections in this chimeric mouse (median parasitemia, lengh of the infection) and develop ex vivo protocols (ex vivo P. vivax culture) to identify P. vivax ligands involved in host cell invasion.


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Tutor/supervisor

First name, Last name Didier Menard and Sylvie Garcia (Biology of Host-Parasite Interactions Unit)

Phone +33 1 44 38 91 35 E-mail [email protected] - [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://research.pasteur.fr/fr/member/didier-menard/ https://research.pasteur.fr/fr/member/sylvie.garcia/

Selected publications or patents of the Research Group offering the work program 1. Menard D, et al. (2010) Plasmodium vivax clinical malaria is commonly observed in Duffy-

negative Malagasy people. Proc Natl Acad Sci U S A 107(13):5967-5971. 2. Chan ER, et al. (2012) Whole genome sequencing of field isolates provides robust

characterization of genetic diversity in Plasmodium vivax. PLoS Negl Trop Dis 6(9):e1811. 3. Hester J, et al. (2013) De novo assembly of a field isolate genome reveals novel Plasmodium

vivax erythrocyte invasion genes. PLoS Negl Trop Dis 7(12):e2569. 4. Menard D, et al. (2013) Whole genome sequencing of field isolates reveals a common

duplication of the Duffy binding protein gene in Malagasy Plasmodium vivax strains. PLoS Negl Trop Dis 7(11):e2489.

5. Serra-Hassoun M, et al. (2014) Human hematopoietic reconstitution and HLA-restricted responses in

6. nonpermissive alymphoid mice. J Immunol 193(3):1504-1511. 7. Garcia S & Freitas AA (2012) Humanized mice: current states and perspectives. Immunol Lett

146(1-2):1-7. 8. Patent 2018/9894888/ 20180160662. TRANSGENIC IMMUNODEFICIENT MOUSE EXPRESSING

HUMAN SIRPalpha, S. Garcia et M. Serra.

Scientific or technical background required for work program Have experience or willing to work on on mouse model; skills in in vitro culture; ability to

work in a team and on own initiative; excellent leadership and communication skills; an

analytical approach to problem solving.


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Title of the work program 17 Role of human factors in the transmission of dengue virus to mosquitoes Description of the work program Dengue virus (DENV) infection is the most common mosquito-borne viral infection, infecting approximately 390 million people per year worldwide with one quarter developing dengue disease. There are two major obstacles in efficiently controlling dengue epidemics: 1) knowledge on dengue disease pathogenesis and 2) knowledge on dengue transmission from man to mosquito. Most studies focus on the first obstacle, which lead to the identification of several factors involved in dengue pathogenesis: namely antibody-dependent enhancement, DENV viral antigens and human genetic factors. However, little is known on factors modulating dengue viral transmission. Anti DENV immunoglobulins are known to reduce transmission and the cytokine storm accompanying symptomatic dengue also likely impacts upon transmission. However, such immune effector impacts cannot explain all the observed variation, especially in asymptomatic or sub-clinical infections that occur majoritarily following infection with DENV. Our work on human genetics has demonstrated their predominant role in determining the infection outcome and different pathways contribute to the spectrum of possible outcomes from asymptomatic to fever to hemorrhage to shock. These pathways include the xenobiotic and lipid metabolism pathways as well as specific genes involved in the innate immune response. The role of lipids is of particular interest as DENV non-structural protein 1 (NS1), a barrel shaped high-density lipoprotein, is implicated in dengue-associated vascular leakage and transmission success. Moreover, low level of serum high-density lipoprotein at admission showed high predictability for the development of severe dengue in clinical settings. Thus, lipoprotein complexes are good candidates for both dengue pathogenesis and transmission. Benefiting from well-characterized human samples collected from previous dengue case cohort studies, we have access to blood samples for studying viral transmission to mosquitoes in the laboratory. This project will involve hypothesis-free screening of proteins (proteomics) and metabolites (metabolomics including lipidomics) in human samples and artificial feeding to mosquito vectors in the laboratory to identify human factors associated with viral transmission success. Tutor/supervisor

First name, Last name Richard, PAUL Phone 0140613626 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://www.researchgate.net/profile/Richard_Paul4


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Selected publications or patents of the Research Group offering the work program Duong V, et al. (2015) Asymptomatic humans transmit dengue virus to mosquitoes. Proc Natl Acad Sci

U S A. 112(47):14688-93. doi: 10.1073/pnas.1508114112.

Oliveira M, et al. (2018) Joint ancestry and association test indicate two distinct pathogenic pathways involved in classical dengue fever and dengue shock syndrome. PLoS Negl Trop Dis. 12(2):e0006202.

Sakuntabhai A, et al. (2005) A variant in the CD209 promoter is associated with severity of dengue disease. Nat Genet. 2005 May;37(5):507-13.

Sierra B, et al. (2017) OSBPL10, RXRA and lipid metabolism confer African-ancestry protection against dengue haemorrhagic fever in admixed Cubans. PLoS Pathog. 27;13(2):e1006220.

Simon-Loriere E, et al. (2015) High anti-dengue viral activity of OAS gene family is associated with increased severity of dengue disease. Journal of Infectious Diseases. pii: jiv321.

Simon-Lorière E, et al. (2017) Increased adaptive immune responses and proper feedback regulation protect against clinical dengue. Sci Transl Med.; 9(405). pii: eaal5088.

Scientific or technical background required for work program No particular requirements, although experience of laboratory work and the associated

hygiene and safety requirements would be desirable.


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Title of the work program 18 In utero transplantation of human neural stem cells as a relevant model of Alzheimer’s disease Description of the work program In our laboratory, we are using an integrated approach to study central neuronal function and dysfunction. One major objective is to improve our understanding on the pathogenesis of Alzheimer’s disease (AD). AD is the most common neurodegenerative disorder which has been characterized by a progressive loss of cognitive abilities and memory as well as the loss of specific neuronal populations such as the pyramidal neurons of cortex and hippocampus. These two brain structures are particularly vulnerable in the early stages of AD. An accumulation of the β-amyloid (Aβ) peptide to form amyloid plaques occurs within the brain. However, the direct correlation between the levels of amyloid plaques and AD progression is still unknown. By contrast, the loss of cholinergic neurons has been shown to contribute to memory and attention deficits. Cholinergic signaling proceeds through the activation of cholinergic receptors which include the nicotinic acetylcholine receptors (nAChR). Brain nAChR play crucial roles as they modulate the release of a variety of neurotransmitters including GABA, glutamate and dopamine. The molecular mechanisms of AD have not yet been fully characterized. None of the published studies so far allows to precisely determinate molecular sites that could represent therapeutic drug targets. Our initial studies clearly demonstrate the existence of a direct binding interaction between Aβ and nAChR that may take place in the normal physiology of the cells and in AD as well. These mechanisms are very promising and should be further analyzed more extensively. In the laboratory, we have developed relevant in vitro models using neurons derived from induced pluripotent stem cells (iPSC) by considering the genetic background of patients as well as that of control individuals. Our current project is to expand such approach using iPSC transplantation. The student will participate and take over initial experiments using in utero transplantation of neural stem cells in mouse brain. He or she will compare the resulting data with those obtained in our laboratory using a slightly different protocol which consists of the use of transplantation in neonates. The student will therefore become familiar with the use of human stem cells by comparing different approaches and following these cells innate formation, development and connections overtime in in vivo models. These data in a context of a true team work should contribute to a deeper understanding of AD molecular mechanisms as well as addressing the key challenges of such technology. The data should also allow to elaborate future directions for the use of iPSC-neurons in AD research.


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Tutor/supervisor

First name, Last name Isabelle Cloëz-Tayarani (“Integrative Neurobiology of Cholinergic Systems Unit”, Uwe Maskos)

Phone +33 1 45 68 88 04 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable): N/A See following links:

https://research.pasteur.fr/fr/member/isabelle-cloez-tayarani/ and https://research.pasteur.fr/fr/team/integrative-neurobiology-of-cholinergic-systems/


A Vitrac & I Cloëz-Tayarani (2018) Induced pluripotent stem cells as a tool to study brain circuits in autism-related disorders. Stem Cell Research and Therapy 9, 226 https://doi.org/10.1186/s13287-018-0966-2.

F Koukouli, M Rooy, D Tziotis, KA Sailor, HC O’Neill, J Levenga, M Witte, M Nilges, JP Changeux, CA Hoeffer, JA Stitzel, BS Gutkin, DA DiGregorio & U Maskos (2017) Nicotine reverses hypofrontality in animal models of addiction and schizophrenia. Nature Medicine 23, 347-354.

S Lombardo, J Catteau, M Besson & U Maskos (2016) A role for ß2* nicotinic receptors in a model of local amyloid pathology induced in dentate gyrus. Neurobiology of Aging 46, 221-234.

C Morel*, L Fattore*, S Pons, A Hay, F Marti, B Lambolez, M De Biasi, M Lathrop, W Fratta, U Maskos* & P Faure* (2014) Nicotine consumption is regulated by a human polymorphism in dopamine neurons. Mol Psychiatry 19, 930-936.

S Tolu, R Eddine, F Marti, V David, M Graupner, S Pons, M Baudonnat, M Husson, M Besson, C Reperant, J Zemdegs, C Pagès, YAH Hay, B Lambolez, J Caboche, B Gutkin, AM Gardier, J-P Changeux, P Faure & U Maskos (2013) Co-activation of VTA DA and GABA neurons mediates nicotine reinforcement. Mol Psychiatry 18, 382-393.

Scientific or technical background required for work program The student should have a strong background in biological science in general and the central nervous system, in particular. The technical background should include skills in laboratory techniques, including cell culture and molecular biology. Students should also be motivated in experimentations with mice.


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Title of the work program 19 Unravelling the role of the histone methyltransferase SET9 in the malaria parasite Plasmodium falciparum Description of the work program Malaria is a parasitic tropical disease responsible for more than 400.000 deaths each year. Five different species of Plasmodium can infect humans, the deadliest being P. falciparum. The increasing proportion of P. falciparum parasites resistant to artemisinin, the most potent antimalarial, is of major concern in Southeast Asia. Finding new drugs with activity against parasites including multi-drug resistant strains is urgently needed. Epigenetic mechanisms, such as histone methylation, are involved in the cell cycle progression, antigenic variation and adaptation of the parasite in its two different hosts bearing completely different environments (human and mosquito). We are thus interested in targeting these mechanisms. In collaboration with the laboratory of Matt Fuchter (Imperial College London), we shown that an inhibitor of the human histone methyltransferase G9a (BIX-01294) is highly potent against P. falciparum asexual stages. There are 10 putative histone methyltransferases annotated in Plasmodium genome, 5 of them being essential for parasite survival. As the protein target of BIX-01294 is unknown in Plasmodium, we have used a chemical genomic approach to successfully induce resistant strains to BIX-01294 and by comparing the genomic sequences of resistant and sensitive parasites we hope to identify the putative target. So far, we have identified a gene duplication of the histone methyl transferase SET9. The aim of this internship would be to confirm the role of SET9 in BIX-01294 resistance and better understand the role of this protein using CRISPR-Cas 9 and other genome editing tools. During this internship the Erasmus student will use different techniques:

- Parasite culture and transfection - Immunofluorescence, Western-Blot and Chip-seq using SET9-tagged parasites - Analysis of parasite growth in overexpressing or inducible-KO lines through flow

cytometry

Tutor/supervisor

First name, Last name Flore NARDELLA (unit director Artur Scherf) Phone 01 40 61 35 07 E-mail [email protected] Profile on http://www.researchgate.net/ https://www.researchgate.net/profile/Flore_Nardella


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Selected publications or patents of the Research Group offering the work program Doerig, C., Rayner, J.C., Scherf, A., and Tobin, A.B. (2015). Post-translational protein modifications in malaria parasites. Nature Reviews Microbiology 13, 160–172. Malmquist, N.A., Sundriyal, S., Caron, J., Chen, P., Witkowski, B., Menard, D., Suwanarusk, R., Renia, L., Nosten, F., Jiménez-Díaz, M.B., et al. (2015). Histone methyltransferase inhibitors are orally bioavailable, fast-acting molecules with activity against different species causing malaria in humans. Antimicrob. Agents Chemother. 59, 950–959. Malmquist, N.A., Moss, T.A., Mecheri, S., Scherf, A., and Fuchter, M.J. (2012). Small-molecule histone methyltransferase inhibitors display rapid antimalarial activity against all blood stage forms in Plasmodium falciparum. Proceedings of the National Academy of Sciences 109, 16708–16713. Chen, P.B., Ding, S., Zangh?, G., Soulard, V., DiMaggio, P.A., Fuchter, M.J., Mecheri, S., Mazier, D., Scherf, A., and Malmquist, N.A. (2016). Plasmodium falciparum PfSET7: enzymatic characterization and cellular localization of a novel protein methyltransferase in sporozoite, liver and erythrocytic stage parasites. Scientific Reports 6.

Scientific or technical background required for work program A background in molecular biology or in pharmacology would be appreciated.


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Title of the work program 20

Study of the Mechanism Underlying the Persistant Activation of the Pro-apoptotic Factor c-Jun N-terminal Kinase (JNK) by Pathogenic Strains of Neisseria meningitidis Description of the work program

Neisseria meningitidis is an exclusive human bacterium. Acquisition of this bacterium often leads to asymptomatic colonization (carriage) and rarely results in invasive disease (mainly septicaemia and meningitis) associated with strains that belong to a small number of genetic lineages referred to as hyper-invasive clonal complexes. Among these groupings, isolates that belong to the clonal complex ST-11 (cc11) positively correlated with fatal outcome of the disease and higher virulence in mice. We have previously shown that epithelial cells infected with cc11 isolates, but not asymptomatic isolates, exhibited features of apoptosis in later steps of infection. Apoptotic cell death promoted by cc11 isolates resulted from the nuclear cleavage of NF-κB mediated by a secreted form of the meningococcal IgA protease. Cleavage of NF-κB leads to a sustained activation of the pro-apoptotic factor c-Jun N-terminal Kinase (JNK) whereas transiently activated in cells infected with asymptomatic carriage isolates. Nevertheless, the mechanism underlying the sustained activation of JNK remains unknown. This project aims to explore the signalling pathway linking IgA protease-mediated cleavage of NF-kB and persistant activation of JNK. This work which relies on the use of meningococcal clinical isolates and mutants will be performed within the Invasive Bacterial Infections Unit at the Institut Pasteur. The implantation of the French National Reference Centre for Meningococci in our unit offers a good opportunity to bridge fundamental research to clinical science and human health. Furthermore, human cell lines established for meningococcal infection and several molecular and cell biology approaches will be involved. The access to different platform facilities within the institute will be therefore advantageous to conduct this work. Tutor/supervisor

First name, Last name Ala-Eddine DEGHMANE Phone +33 1 44 38 95 90 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):


1. Deghmane A-E, Veckerlé C, Giorgini D, Hong E, Ruckly C, Taha M-K. Differential modulation of TNF-alpha-induced apoptosis by Neisseria meningitidis. PLoS pathogens. 2009;5:e1000405.

2. Deghmane AE, El Kafsi H, Giorgini D, Abaza A, Taha MK. Late repression of NF-kappaB activity by invasive but not non-invasive meningococcal isolates is required to display apoptosis of epithelial cells. PLoS Pathog. 2011;7(12):e1002403. PMCID: 3228807.


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3. Besbes A, Le Goff S, Antunes A, Terrade A, Hong E, Giorgini D, et al. Hyperinvasive Meningococci Induce Intra-nuclear Cleavage of the NF-kappaB Protein p65/RelA by Meningococcal IgA Protease. PLoS Pathog. 2015;11(8):e1005078.

4. Antunes A, Derkaoui M, Terrade A, Denizon M, Deghmane AE, Deutscher J, et al. The Phosphocarrier Protein HPr Contributes to Meningococcal Survival during Infection. PLoS One. 2016;11(9):e0162434.

5. Derkaoui M, Antunes A, Poncet S, Nait Abdallah J, Joyet P, Maze A, et al. The phosphocarrier protein HPr of Neisseria meningitidis interacts with the transcription regulator CrgA and its deletion affects capsule production, cell adhesion, and virulence. Mol Microbiol. 2016;100(5):788-807.

6. Narni-Mancinelli E, Gauthier L, Baratin M, Guia S, Fenis A, Deghmane AE, et al. Complement factor P is a ligand for the natural killer cell-activating receptor NKp46. Sci Immunol. 2017;2(10).

7. Guiddir T, Gros M, Hong E, Terrade A, Denizon M, Deghmane AE, et al. Unusual Initial Abdominal Presentations of Invasive Meningococcal Disease. Clin Infect Dis. 2018;67(8):1220-7

Scientific or technical background required for work program We are looking for excellent applicants with motivated personality, strong communication

skills and fluent in English. A solid background in microbiology and cell biology is essential.

The candidate is expected to take own initiatives as well as to collaborate with colleagues.


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Title of the work program 21 Motility of skin-dwelling African trypanosomes Description of the work program Trypanosoma brucei gambiense causes chronic Human African Trypanosomiasis. These extra-cellular protist parasites are transmitted by tsetse flies and proliferate in the host blood. We have recently demonstrated that the skin was a major yet overlooked anatomical reservoir for trypanosomes that may thwart control programs. This project is part of a larger programme that aims at understanding the biological significance of skin parasites, especially how they impact our current view of transmission, diagnosis and treatment. Here, we will especially unravel the importance of motility for the development of skin-dwelling parasites. The infectious process occurring in the skin during the chronic phase of sleeping sickness has largely been overlooked so far. First, we will use parasites constitutively expressing a cytosolic photo-convertible marker (Dendra2) to assess the parasite proliferation and spreading in skin explants and mouse models. Second, we will test the ability of motility mutants (LC1-/-) to invade the dermis of mice. In case skin-dwelling parasites were seen to disseminate in the skin, their motility will be characterized by intravital tracking. Parasites will also be extracted from the skin and challenged to ex vivo tracking experiments in various in vitro media to verify whether their motility is more adapted to the viscous environment of the skin, and to evaluate their specific tropism to particular skin elements. This will allow us to illustrate a potential phenotypic adaptation of trypanosomes to the skin, especially in terms of motility.

Tutor/supervisor

First name, Last name Brice Rotureau and Christelle Travaillé

Phone E-mail [email protected] and

[email protected]


https://research.pasteur.fr/en/team/group-brice-rotureau/


1.Rotureau B, Van Den Abbeele J. Through the dark continent: African trypanosome development in the tsetse fly. Frontiers in cellular and infection microbiology. 2013;3:53. Epub 2013/09/26. doi: 10.3389/fcimb.2013.00053. PubMed PMID: 24066283; PubMed Central PMCID: PMC3776139.


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2.Buscher P, Cecchi G, Jamonneau V, Priotto G. Human African trypanosomiasis. Lancet. 2017. doi: 10.1016/S0140-6736(17)31510-6. PubMed PMID: 28673422.

3.Capewell P, Cren-Travaille C, Marchesi F, Johnston P, Clucas C, Benson RA, et al. The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes. eLife. 2016;5. doi:10.7554/eLife.17716. PubMed PMID: 27653219; PubMed Central PMCID: PMCPMC5065312.

4.Calvo-Alvarez E, Cren-Travaille C, Crouzols A, Rotureau B. A new chimeric triple reporter fusion protein as a tool for in vitro and in vivo multimodal imaging to monitor the development of African trypanosomes and Leishmania parasites. Infect Genet Evol. 2018. Epub 2018/01/18. doi: 10.1016/j.meegid.2018.01.011. PubMed PMID: 29339220.

Scientific or technical background required for work program Skills in parasitology, cell culture, molecular biology and / or in vivo imaging


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Title of the work program 22 Role of ROS/RNS in Dengue pathogenesis Description of the work program Dengue infection is caused by one of the 4 serotypes of the Dengue virus. Symptoms are variable: from asymptomatic to mild fever (DF), Haemorrhagic fever (DHF) or Shock syndrome (DSS) that can lead to death. No drugs exist to cure Dengue infection. A vaccine is now available, however, its efficacy is controversial and will require further development. Close monitoring of dengue-infected patients with fever is the only way to ensure that patients receive proper care when most needed (i.e., in the cases of DHF or DSS). Using human plasma samples from a Cambodian cohort of Dengue-infected patients with fever arriving at the hospital, our preliminary data showed a significant decrease of ROS/RNS species in DSS patients. There is an inverse correlation between the severity of the symptoms and the remaining ROS/RNS species. Our project aims to pursue 2 lines of research:

1) Use ROS/RNS as a biomarker of Dengue disease severity to set up a prognostic kit: Among patients with confirmed Dengue fever arriving at the hospital, this biomarker could be used to identify patients at high risk of developing the severe forms of Dengue disease and that will need closer monitoring.

2) Characterize the role of ROS/RNS species in human Dengue pathogenesis. This work program is scheduled for 6-10 months, depending on the proficiency of the student.

Tutor/supervisor

First name, Last name Marie NGUYEN – de BERNON Phone 01 45 68 89 62 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://research.pasteur.fr/en/member/marie-nguyen-de-bernon/


1. Oliveira M, Lert-Itthiporn W, Cavadas B, Fernandes V, Chuansumrit A, Anunciação O, Casademont I, Koeth F, Penova M, Tangnararatchakit K, Khor CC, Paul R, Malasit P, Matsuda F, Simon-Lorière E, Suriyaphol P, Pereira L, Sakuntabhai A (2018). Joint ancestry and association test indicate two distinct pathogenic pathways involved in classical dengue fever and dengue shock syndrome. PLoS Negl Trop Dis 02:e0006202


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2. Simon-Lorière E, Duong V, Tawfik A, Ung S, Ly S, Casadémont I, Prot M, Courtejoie N, Bleakley K, Buchy P, Tarantola A, Dussart P, Cantaert T, Sakuntabhai A (2017). Increased adaptive immune responses and proper feedback regulation protect against clinical dengue. Sci Transl Med 9(405)

Scientific or technical background required for work program Students that are curiosity-driven, open-minded and with (some) previous lab skills will be

preferred.


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Title of the work program 23 Role of stromal cells in immunity at barrier surfaces Description of the work program Our lab has previously shown that mesenchymal stromal populations have essential roles in inflammation (JI 2009), tissue repair (Nature Medicine 2012) and maintenance of the intestinal stem cell niche (PNAS 2017). In this project, we will investigate the role of stromal cells in the regulation of immune responses at barrier surfaces (such as skin, intestine). To that aim, we will analyze WT and mutant conditional knock-out mice for candidate molecules that have an essential role in the stromal crosstalk with immune cells. Using mouse models of injury/inflammation, we will measure the quality and intensity of the immune response in WT and mutant mice (by FACS), visualize the stromal crosstalk with immune cells (by confocal microscopy) and investigate the molecular mechanism underlying stroma-immune crosstalk (by transcriptomics). Altogether this project will allow us to determine the impact of stromal cells in immunity at barrier surfaces. Tutor/supervisor

First name, Last name Lucie PEDUTO Phone +33 1 44 38 94 27 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://research.pasteur.fr/en/team/group-lucie-peduto/

Selected publications or patents of the Research Group offering the work program Di Carlo, S.E. & Peduto, L. The perivascular origin of pathological fibroblasts. J Clin Invest 128, 54-63

(2018) Peduto L, Sansonetti PJ, Nigro G. Intestinal stem cells and their niche at homeostasis and under stress.

Book chapter in “Advances in Stem Cells and their niche”. Elsevier, doi.org/10.1016 /bs.asn.2017.12.002 (2018)

Emgard J, Kammoun H, Garcia-Cassani B, Chesne J., Parigi SM, Jacob JM, Cheng HW, Evren E, Das S, Czarnewski P, Sleiers N, Melo-Gonzales F, Kvedaraite E, Svensson M., Scandella E, Hepworth MR, Huber S, Ludewig B, Peduto L, Villablanca EJ, Veiga-Fernandes H, Pereira JP, Flavell RA, Willinger T. Oxysterol sensing through GPR183 (EBI2) controls the lymphoid tissue-inducing function of innate lymphoid cells and colonic inflammation. Immunity, 48(1):120-132.e8 (2018)

Stzepourginski I, Nigro G, Jacob JM, Dulauroy S, Sansonetti PJ, Eberl G, Peduto L. CD34+ mesenchymal cells are a major component of the intestinal stem cells niche at homeostasis and after injury. PNAS, 114(4): E506-E513 (2017)


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Dulauroy, S., Di Carlo, S.E., Langa, F., Eberl, G. & Peduto, L. Lineage tracing and genetic ablation of ADAM12(+) perivascular cells identify a major source of profibrotic cells during acute tissue injury. Nature Medicine 18, 1262-1270 (2012)

Schilte C, Couderc T, Chretien F, Sourisseau M, Gangneux N, Guivel-Benhassine F, Kraxner A, Tschopp J, Higgs S, Michault A, Arenzana-Seisdedos F, Colonna M, Peduto L, Schwartz O, Lecuit M, and Albert ML. Type I IFN controls chikungunya virus via its action on non-hematopoietic cells. J Exp Med, 207 (2): 429-442 (2010)

Peduto L, Dulauroy S, Lochner M, Spaeth G, Morales MA, Cumano A, and Eberl G. Inflammation recapitulates the ontogeny of lymphoid stromal cells. J Immunol, 182 (9): 5789-5799 (2009)

Scientific or technical background required for work program To achieve this project, the candidate will use different experimental approaches including

multiparametric FACS after isolation from tissues, transcriptomics (qPCR arrays or RNAseq),

and confocal microscopy to visualize the stromal crosstalk within the tissue

microenvironment. Highly motivated and creative candidates with a strong interest in

immunology /stromal biology are strongly encouraged to apply. Previous experience with

FACS, transcriptomics or confocal microscopy would be a plus, but is not required.


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Title of the work program 24 Defining the mechanism of secretion of CNF1 toxin from pathogenic Escherichia coli Description of the work program The Cytotoxic Necrotizing Factor-1 (CNF1) toxin is particularly prevalent in pathogenic strains of Escherichia coli of the phylogroup B2 that reside in the normal gut microbiota and are causative agents of both intra- and extra-intestinal infections. CNF1 is a deamidase that is considered as an environmental factor catalyzing a “somatic” gain-of-function mutation on Rho GTPases. Small Rho GTPases are essential signaling hubs controlling a variety of critical cell functions including actin cytoskeleton and membrane dynamics, cell-cell and -extracellular matrix adhesion, reactive oxygen species production, as well as cell autonomous or systemic innate defenses against pathogens. Importantly, study of CNF1 toxin by our group has been instrumental in deciphering a cellular system that guards the flux of active Rho GTPases by mean of ubiquitin and proteasome systems (UPS)-mediated regulation of their stability and how this host regulation is subverted by bacteria to invade cells. We also question how chronical exposure of the host to CNF1-producing Escherichia coli could contribute to late onset or pejorative evolution of colorectal cancer. Despite large advances in these area, the mechanism of CNF1 toxin secretion by uropathogenic strains of E.coli is still unknown. Indeed, although CNF1 is produced during bacteria interaction with the host the conditions of optimum secretion are not defined. By conducting cell biology approaches coupled to genetic engineering of bacterial strains we aim to better define the conditions of CNF1 secretion during host cell infection. Both physiological and physical conditions will be tested comprising pH (urine, gastro-intestinal tract), oxygen concentration, iron, particular metabolic conditions of host cells. Based on bioinformatics, structure modeling, and genome analyzes data acquired in the lab, the student will also generate mutants of defined candidate genes using lambda red recombination and site-directed mutagenesis techniques, and test them for their secretory phenotype. Defining the condition of maximal secretion of CNF1 toxin will be instrumental in better establishing the precise steps of Toxin function during infection. Tutor/supervisor

First name, Last name Amel Mettouchi Phone +33 1 45 68 83 09 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://research.pasteur.fr/fr/team/bacterial-toxins/


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Selected publications or patents of the Research Group offering the work program CNF1-like deamidase domains: common Lego bricks among cancer-promoting immunomodulatory bacterial virulence factors. Ho M, Mettouchi A, Wilson BA, Lemichez E. Pathog Dis. 2018 Jul 1;76(5). Escherichia coliα-hemolysin counteracts the anti-virulence innate immune response triggered by the Rho GTPase activating toxin CNF1 during bacteremia. Diabate M, Munro P, Garcia E, Jacquel A, Michel G, Obba S, Goncalves D, Luci C, Marchetti S, Demon D, Degos C, Bechah Y, Mege JL, Lamkanfi M, Auberger P, Gorvel JP, Stuart LM, Landraud L, Lemichez E, Boyer L. PLoS Pathog. 2015 Mar 17;11(3):e1004732. The E3 ubiquitin-ligase HACE1 catalyzes the ubiquitylation of active Rac1. Torrino S, Visvikis O, Doye A, Boyer L, Stefani C, Munro P, Bertoglio J, Gacon G, Mettouchi A, Lemichez E. Dev Cell. 2011 Nov 15;21(5):959-65. Escherichia coli producing CNF1 toxin hijacks Tollip to trigger Rac1-dependent cell invasion. Visvikis O, Boyer L, Torrino S, Doye A, Lemonnier M, Lorès P, Rolando M, Flatau G, Mettouchi A, Bouvard D, Veiga E, Gacon G, Cossart P, Lemichez E. Traffic. 2011 May;12(5):579-90. CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. Doye A, Mettouchi A, Bossis G, Clément R, Buisson-Touati C, Flatau G, Gagnoux L, Piechaczyk M, Boquet P, Lemichez E. Cell. 2002 Nov 15;111(4):553-64. Scientific or technical background required for work program Basic knowledge in Microbiology.

Skills in basic Molecular Biology techniques (PCR, Cloning), immunofluorescence and simple

eukaryotic cell culture practices will be welcome, but the candidate will be trained on all

techniques in the team.


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Title of the work program 25 Visualizing and quantifying gene transfer in live bacteria during antibiotic treatment. Description of the work program When bacteria are exposed to low doses of antibiotics, they are able to quickly modify their genetic content to enhance their survival against the antibiotic, leading to the development of resistant bacteria1,2. In order to slow down the emergence and the spread of antibiotic resistance, a growing public health concern, one main challenge is to gain knowledge on the mechanisms of genetic variability across scales from a single cell to mixed bacterial populations. In the Mazel laboratory that I newly joined, we focus on how bacterial genomes acquire and maintain their plasticity3,4. More specifically we investigate the mechanisms by which bacteria transfer resistance genes, using Escherichia coli and Vibrio cholerae as model organisms. I put recent efforts to understand the role of membrane vesicles in gene transfer. Membrane vesicles are small vesicles of lipids that bud off of the bacterial outer membrane and embed a large variety of molecules (including DNA). We find them particularly attractive because of their unique feature of long-distance intercellular transport of cellular material that gives cells a chance to generate long-distance horizontal gene transfer (HGT). Yet, whether such transport possibly results in horizontal gene transfer events that contribute to genome plasticity has not been addressed quantitatively in living bacteria. By combining molecular biology tools to fluorescence microscopy and microfluidic techniques, we aim at answering the following questions in which the student will be directly involved during his/her stay at Pasteur:

1- Can the gene transfer via the membrane vesicles be visualized in real time in living bacteria?

We will take advantage of the Green Fluorescent Protein (GFP) gene to determine the transfer rates between a donor and a recipient cell under the microscope. In parallel, DNA transfer rates will be determined genetically.

2- Are these vesicles dynamic? How are they produced? How do they attach to a target cell? How fast can they deliver their content to a recipient cell? Tracking single vesicle behavior in real time with high resolution imaging will allow us to tackle these questions.

3- Individuals in a bacterial population are not equal and respond differently to

antibiotic stress. Such variability between bacteria cells is named phenotypic heterogeneity. Do these small vesicles contribute to generate phenotypic heterogeneity in a bacterial population? We will monitor the effect of membrane vesicles on the expression levels of selected stress response genes in a bacterial


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population growing in a microfluidic device designed to encapsulate a few bacteria in thousands microhabitats5.

Altogether, we expect these results to shed light on a general mechanism to exchange non-specialized genetic material between individuals in bacterial populations and thus, to increase knowledge to fight back against resistant microorganisms.

Tutor/supervisor

First name, Last name Julia Bos Phone +33 1 45 68 8597

E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://www.researchgate.net/profile/Julia_Bos/info https://scholar.google.com/citations?user=goQsc6UAAAAJ&hl=en

Selected publications or patents of the Research Group offering the work program 1-Emergence of antibiotic resistance from multinucleated bacterial filaments J Bos, Q Zhang, S Vyawahare, E Rogers, SM Rosenberg, RH Austin. Proceedings of the National Academy of Sciences. 2015; 112 (1), 178-183. 2-Multiple Pathways of Genome Plasticity Leading to Development of Antibiotic Resistance. Baharoglu Z, Garriss G, Mazel D. Antibiotics (Basel). 2013 ;2(2):288-315. Review. 3-Genomic Plasticity of Vibrio cholerae. Escudero JA, Mazel D. Int Microbiol. 2017;20(3):138-148. Review. 4- Integrons: agents of bacterial evolution. Mazel D. Nat Rev Microbiol. 2006 ;4(8):608-20. Review. 5-Universal microfluidic platform for bioassays in anchored droplets. Amselem G, Guermonprez C, Drogue B, Michelin S, Baroud CN. Lab Chip. 2016;16(21):4200-4211.

Scientific or technical background required for work program As our experiments are conducted both under the microscope (epifluorescence) and at the

bench, students with traditional (micro)biology, molecular biology, optics and biophysics skills

are encouraged to apply.


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Title of the work program 26 Deciphering the mechanisms underlying DNA double strand break-induced complex genome rearrangements Description of the work program

DNA double-strand breaks (DSBs) are toxic cellular lesions that must be efficiently repaired to maintain genome stability and prevent cancer. The two main DSB repair pathways are homologous recombination (HR) and nonhomologous end joining (NHEJ), with pathway choice being critical to preserve DNA integrity upon chromosomal breakage. Aberrant repair of chromosome breaks can generate a dicentric chromosome which causes the chromosome to become highly unstable during subsequent cell divisions, a phenomenon known as breakage-fusion-bridge.

In lymphocytes, antigen receptor diversity is generated through unique cut-and-paste recombination mechanisms, which shuffle pre-existing DNA elements to create an almost unlimited repertoire of functional genes encoding for antigen receptor molecules. These processes rely on complex interplays between DNA damage response and repair proteins and, as such, represent a unique paradigm to study the regulation of DNA repair pathways and their dysfunctions leading to chromosomal instability.

In this project, we will use DNA damage-inducible B cell lines in which critical genes have been removed with CRISPR/Cas9 genome editing to analyze at the cellular level the function of specific DNA damage response and repair proteins in promoting/suppressing complex genome rearrangements. Tutor/supervisor

First name, Last name Ludovic, Deriano Phone +33 (0)1 44 38 93 55 E-mail [email protected] Profile on http://www.researchgate.net/ Yes


Dev H, et al. The SHLD1/2 protein complex promotes non-homologous end-joining and counteracts homologous recombination in BRCA1-deficient cells. Nature Cell Biology. 2018 Jul 18.

Lenden Hasse H, et al. Generation and CRISPR/Cas9 editing of transformed progenitor B cells as a pseudo-physiological system to study DNA repair gene function in V(D)J recombination. J Immunol Methods. 2017 Sep 4.

Lescale C, et al. Specific Roles of XRCC4 Paralogs PAXX and XLF during V(D)J Recombination. Cell Reports. 2016 Sep 13.

L. Deriano and D.B. Roth. Modernizing the Nonhomologous End Joining Repertoire: Alternative and Classical NHEJ Share The Stage. Annual Review of Genetics. 2013 Nov 23.


Knowledge in Genetics, Immunology, Molecular and Cellular Biology are desirable. Experience in cell culture, cytogenetics, microscopy and/or molecular biology techniques would be beneficial.


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Title of the work program 27 Deciphering the tubulin code in trypanosomes Description of the work program Microtubules are one of the main components of the cell cytoskeleton. Tubulin post-translational modifications confer different properties to microtubules in what is now defined as the tubulin code. The addition of one or more glutamates forming a side chain is one of the most abundant modifications, especially represented in cilia and flagella. Trypanosoma brucei is an ideal model to study the tubulin code since their cytoskeleton relies mostly on microtubules and the tubulin code is less complex, facilitating interpretation. Our group has identified several enzymes potentially involved in glutamylation and the first knockout studies revealed unexpected contributions to this process. The aim of this project will be to investigate the contribution of other enzymes to the establishment and the function of the tubulin code using reverse genetics (knockout, in situ tagging, CRISPR-Cas9) and imaging approaches (in vivo imaging, super-resolution microscopy). The work is part of a multi-disciplinary project involving partners with expertise in mass spectrometry and tubulin post-translational modifications.

Tutor/supervisor

First name, Last name Philippe Bastin/Serge Bonnefoy Phone 0140613835/3834 E-mail [email protected]/[email protected] Profile on http://www.researchgate.net/ (if applicable):

https://www.researchgate.net/profile/Philippe_Bastin https://www.researchgate.net/profile/Serge_Bonnefoy https://research.pasteur.fr/en/team/trypanosome-cell-biology/


1. Bertiaux, E., Mallet, A., Fort, C., Blisnick, T., Bonnefoy, S., Jung, J., Lemos, M., Marco, S., Vaughan, S., Trepout, S., Tinevez, J.Y. and Bastin, P. (2018). Bidirectional intraflagellar transport is restricted to two sets of microtubule doublets in the trypanosome flagellum. J Cell Biol. DOI: 10.1083/jcb.201805030 | Published October 1, 2018.

2. Bertiaux, E., Morga, B., Blisnick, T., Rotureau, B., and Bastin, P. (2018). Control of flagellum length by a grow-and-lock model. Curr. Biol., in press.

3. Bonnefoy, S., Watson, C.M., Kernohan, K.D., Lemos, M., Hutchinson, S., Poulter, J.A., Crinnion, L.A., O'Callaghan, C., Hirst, R.A., Rutman, A., et al. Bastin, P. and Sheridan, E. (2018). Biallelic Mutations in LRRC56 encoding a protein associated with intraflagellar transport, cause mucociliary clearance and laterality defects. Am. J. Human Genet., in press.


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4. Vincensini, L., Blisnick, T., Bertiaux, E., Hutchinson, S., Georgikou, C., Ooi, C.P., and Bastin, P. (2018). Flagellar incorporation of proteins follows at least two different routes in trypanosomes. Biol Cell 110, 33-47.

5. Trepout, S., Tassin, A.M., Marco, S., and Bastin, P. (2017). STEM tomography analysis of the trypanosome transition zone. J Struct Biol. 202, 51-60.

6. Fort, C., Bonnefoy, S., Kohl, L., and Bastin, P. (2016). Intraflagellar transport is required for the maintenance of the trypanosome flagellum composition but not its length. J Cell Sci 129, 3026-3041.

7. Subota, I., Julkowska, D., Vincensini, L., Reeg, N., Buisson, J., Blisnick, T., Huet, D., Perrot, S., Santi-Rocca, J., Duchateau, M., et al. (2014). Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localization and dynamics. Molecular & cellular proteomics : MCP 13, 1769-1786.

8. Blisnick, T., Buisson, J., Absalon, S., Marie, A., Cayet, N., and Bastin, P. (2014). The intraflagellar transport dynein complex of trypanosomes is made of a heterodimer of dynein heavy chains and of light and intermediate chains of distinct functions. Mol Biol Cell 25, 2620-2633.

9. Huet, D., Blisnick, T., Perrot, S., and Bastin, P. (2014). The GTPase IFT27 is involved in both anterograde andretrograde intraflagellar transport. eLife 2014;10.7554/eLife.02419.

Scientific or technical background required for work program Scientific background: Good knowledge in cell biology

Technical background: Some expertise in cell culture and/or light microscopy would be

desirable.


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Title of the work program 28 Design of TCR transfer vectors for HIV immunotherapy Description of the work program Rare cases of spontaneous control of HIV infection reveal that the human immune system has the capacity to mount an efficient antiviral response against HIV. Patients who contain HIV replication in the absence of therapy, called HIV Controllers, show signs of particularly efficient T cell responses, and maintain full CD4 helper function in the long term. We obtained recent evidence that CD4+ T cells of these rare patients preferentially express a particular set of shared T cell receptors (TCRs) directed at HIV capsid. These shared or "public" TCRs were found to detect Gag antigen with unusually high affinity. When transferred into healthy donor CD4+ T cells, the Gag-specific TCRs conferred properties characteristic of HIV Controller CD4+ T cell responses, including high antigen sensitivity and the capacity to produce multiple cytokines simultaneously. Of note, these TCRs were of sufficiently high affinity to also function in CD8+ T cells, in the absence of the CD4 coreceptor. Thus, both CD4+ and CD8+ T cells could be retargeted to the most conserved region of HIV-1 capsid by TCR transfer.

We propose to apply these findings to the field of HIV immunotherapy, by developing TCR transfer vectors that could be used to confer efficient antiviral responses against HIV. The main objective of this project will be to improve TCR lentivectors by inserting mutations that facilitate the pairing of the two transferred TCR chains, which should limit the formation of mixed TCRs that incorporate endogenous TCR chains. The student will also insert a suicide gene downstream of the TCR to increases the safety of the vector. The lentivector constructs will then be tested for their capacity to express the TCRs of interest by flow cytometry with TCR-specific antibodies and MHC II tetramers. The lentivectors will then be tested in primary T cells for their capacity to confer Gag-specific recognition, using intracellular cytokine staining. The cytotoxic function of TCR-transduced T cells will be analyzed by measuring the elimination of Gag-expressing target cells. The long-term goal will be to use these optimized TCR vectors in a humanized mouse model, to test their capacity at eliminating HIV-infected cells in vivo, and evaluate their potential as immunotherapeutic tools to restore efficient immune responses in HIV-infected patients.

Tutor/supervisor

First name, Last name Lisa Chakrabarti Phone +33 1 45 68 89 45 E-mail [email protected]


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https://www.researchgate.net/profile/Lisa_Chakrabarti


- Vingert B., Perez-Patrigeon S., Jeannin P., Lambotte O., Boufassa F., Lemaître F., Kwok W.W., Theodorou I., Delfraissy J.F., Thèze J., and Chakrabarti L.A. ; for the ANRS EP36 HIV Controllers Study Group. (2010) HIV Controllers CD4+ T cells respond to minimal amounts of Gag antigen due to high TCR avidity. PLoS Pathogens 6(2): e1000780

- Vingert B., Benati D., Lambotte O., de Truchis P., Slama L., Jeannin P., Galperin M., Perez-Patrigeon S., Boufassa F., Kwok W. W., Lemaitre F., Delfraissy J. F., Theze J., and Chakrabarti L. A. (2012) HIV Controllers maintain a population of highly efficient Th1 effector cells in contrast to patients treated in the long term. Journal of Virology 86: 10661-10674.

- Lissina A., Chakrabarti L.A., Takiguchi M., and Appay V. (2016) TCR clonotypes: molecular determinants of T cell efficacy against HIV. Current Opinion in Virology 16:77-85.

- Benati D., Galperin M., Lambotte O., Gras S., Lim A., Mukhopadhyay M., Nouël A., Campbell K.A., Lemercier B., Claireaux M., Hendou S., Lechat P., De Truchis P., Boufassa F., Rossjohn J., Delfraissy J.F., Arenzana-Seisdedos F., and Chakrabarti L.A. (2016) Public TCRs confer high-avidity CD4 responses to HIV Controllers. Journal of Clinical Investigation doi: 10.1172/JCI83792

- Mukhopadhyay M, Galperin M, Patgaonkar M, Vasan S, Ho DD, Nouël A, Claireaux M., Benati D, Lambotte O, Huang Y, and Chakrabarti LA (2017, in press) DNA vaccination by electroporation amplifies broadly cross-restricted public TCR clonotypes shared with HIV controllers. Journal of Immunology

- Claireaux M., Galperin M., Benati D., Nouël A., Mukhopadhyay M., Klingler J., de Truchis P., Zucman D., Hendou S., Boufassa F., Moog C., Lambotte O., and Chakrabarti L.A. (2018). High frequency of HIV-specific circulating follicular helper T cells associates with preserved memory B cell responses in HIV controllers. mBio 2018 May 8;9(3). pii: 9:e00317-18.

- Galperin M.*, Farenc C.*, Mukhopadhyay M., Jayasinghe D., Decroos A., Benati D., Tan L.L., Ciacchi L., Reid H.H., Rossjohn J.**, Chakrabarti L.A.**, and Gras S.** (2018) CD4+ T cell mediated HLA class II cross-restriction in HIV controllers. Science Immunology. 2018 Jun 8;3(24). pii: eaat0687.

Scientific or technical background required for work program We are looking for a highly motivated candidate with knowledge in the fields of Virology and/or Immunology, and good communication skills in spoken and written English. Prior expertise in viral vector engineering and flow cytometry will be a plus.


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Title of the work program 29 Cell size regulation in a fungal pathogen Description of the work program Cryptococcus are an encapsulated basidiomycete yeasts responsible for more than 300,000 deaths per year and principally infects immunocompromised patients (1). This fungus lives in the environment and can be isolated from the soil, decaying wood or bird’s droppings. As such it has to cope with different stresses and predators including worms and amebae and has for that developed specific features, the most prominent ones being its polysaccharide capsule and its ability to synthesize melanin. Associated with its ability to grow at 37°C, these virulence factors make these fungi deadly pathogens. Cellular morphology has been also related to virulence in these yeasts. Filamentation (2) but more importantly cell size alterations have been associated with virulence. Thus, whereas the normal size of this yeast cell is usually comprised between 5-10 µm, very large cells (up to 80 µm in diameter) named Titan-cells have been observed during model infections in the lungs (3). In addition, very small yeast cells have been observed in vivo and in vitro (4, 5). The proposed subject aims to understand how the cells size is regulated. We have identified a growth condition inducing the production of very small cells. The questions we would like to answer concern the signal triggering the modification of the size in this condition and the mechanism by which it is transduced. For this last part, anticipating that some kinase transduction pathways should be involved, a kinase mutant strain collection will be screened (6). For this project, microbiology, biochemistry and genetic analyses will be performed.

Tutor/supervisor

First name, Last name Guilhem Janbon Phone + 33 (0)1 45 68 83 56 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://www.researchgate.net/profile/Guilhem_Janbon

Selected publications or patents of the Research Group offering the work program 1. Goebels C., Thonn A., Gonsalez-Hilarion S., Rolland O., Moyrand F., Beilharz T. H. &

Janbon, G. (2013) Introns regulate gene expression in Cryptococcus neoformans in a Pab2p dependent pathway. PLoS Genetics 9, e1003686. (Recommended by F1000).

2. Janbon G., Ormerod K.L., Paulet D., Byrnes III, E.J., Yadav Y., Chatterjee G., Mullapudi N., Hon C.H, Billmyre R.B., Brunel F., Bahn Y.S., Chen W., Chen Y., Chow E.W.L., Coppée J-Y., Floyd-Averette A., Gaillardin C., Gerik K.J., Goldberg J., Gonzalez-Hilarion S., Gujja S., Hamlin J.L., Hsueh Y.P., Ianiri G., Jones S., Kodira C.D., Kozubowski L., Lam W., Marra M., Mesner L.D., Mieczkowski P.A., Moyrand F., Nielsen K., Proux C., Rossignol T., Schein J.E., Sun S., Wollschlaeger C. Wood I.A., Zeng Q., Neuvéglise C., Newlon C.S., Perfect J.R., Lodge, J.K., Idnurm A., Stajich J.E.


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Kronstad, J.W., Sanyal K. Heitman J., Fraser J.A., Cuomo C.A. & F.S. Dietrich (2014) Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation PLoS Genetics 10, e1004261. (Recommended by F1000)

3. Wollschlaeger C., Trevijano-Contador N., Wang X., Legrand M., Zaragoza O., Heitman J., & Janbon, G. (2014) Distinct and redundant roles of exonucleases in Cryptococcus neoformans: Implications for virulence and mating. Fungal Gen. Biol. 73, 20-28.

4. Jung K.W., Yang D.H., Maeng S., Lee K.T., So Y.S., Hong J., Choi J.Y., Byun H.J., Kim H., Bang S., Song M.H., Lee J.W., Kim M.S., Kim S.Y., Ji J.H., Park G., Kwon H., Cha S., Meyers G., Wang L.L., Jang J., Janbon G., Adedoyin G., Kim T., Averette A.K., Heitman J., Cheong E., Lee Y.H., Lee Y.W. & Bahn Y.S. (2015) Systematic functional profiling of transcription factor networks in Cryptococcus neoformans. Nature Com. 7, 6757 ((Recommended by F1000).

5. Gonzalez-Hilarion S, Paulet D, Lee K-T, Hon C-C, Lechat P, Mogensen E, Moyrand F, Proux C, Barboux R, Bussotti G, Hwang J, Coppée J-Y, Bahn Y-S & Janbon G (2016) Intron retention-dependent gene regulation in Cryptococcus neoformans. Scientific Reports 6, 32252.

6. Bonnet, A., Grosso, AR., Elkaoutari, E., Coleno, E. Presle,A., Sridhara, S.C., Janbon, G., Géli, V., de Almeida, S.F., & Palancade, B. (2017) Introns protect eukaryotic genomes from transcription-associated genetic instability. Mol Cell. 67, 608-621.

7. Janbon G (2018). Intron in Cryptococcus. Memórias do Instituto Oswaldo Cruz 113, e170519.

Scientific or technical background required for work program 1. Kwon-Chung KJ, Fraser JA, Doering TL, Wang Z, Janbon G, Idnurm A, Bahn YS.

Cryptococcus neoformans and Cryptococcus gattii, the etiologic agents of cryptococcosis. Cold Spring Harb Perspect Med. 2014;4:a019760.

2. Wang L, Zhai B, Lin X. The Link between Morphotype Transition and Virulence in Cryptococcus neoformans. PLoS Pathog. 2012;8(6):e1002765. doi: 10.1371/journal.ppat.1002765. PubMed PMID: PMC3380952.

3. Zaragoza O, Nielsen K. Titan cells in Cryptococcus neoformans: Cells with a giant impact. Curr Opinion Microbiol. 2013;16(4):409-13. doi: 10.1016/j.mib.2013.03.006. PubMed PMID: PMC3723695.

4. Neilson JB, Fromtling RA, Bulmer GS. Cryptococcus neoformans: size range of infectious particles from aerosolized soil. Infect Immun. 1977;17(3):634-8. PubMed PMID: PMC421174.

5. Denham ST, Verma S, Reynolds RC, Worne CL, Daugherty JM, Lane TE, Brown JCS. Regulated Release of Cryptococcal Polysaccharide Drives Virulence and Suppresses Immune Cell Infiltration into the Central Nervous System. Infect Immun. 2018;86(3):e00662-17. doi: 10.1128/IAI.00662-17. PubMed PMID: PMC5820953.

6. Lee K-T, So Y-S, Yang D-H, Jung K-W, Choi J, Lee D-G, Kwon H, Jang J, Wang LL, Cha S, Meyers GL, Jeong E, Jin J-H, Lee Y, Hong J, Bang S, Ji J-H, Park G, Byun H-J, Park SW, Park Y-M, Adedoyin G, Kim T, Averette AF, Choi J-S, Heitman J, Cheong E, Lee Y-H, Bahn Y-S. Systematic functional analysis of kinases in the fungal pathogen Cryptococcus neoformans. Nature Communications. 2016;7:12766. doi: 10.1038/ncomms12766. PubMed PMID: PMC5052723.


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Title of the work program 30 Characterization of DNA motion in living cells and its influence on transcriptional activity Description of the work program Scientific context of the project The dynamic organization of the genome in time and space plays a crucial role in the functional specification of a cell. In particular the interplay between multiple distant enhancers and their target gene promoters has critical mechanistic consequences on gene activity patterns during cell differentiation and development. Understanding and being able to control and engineer these processes will be a crucial factor in the human health sector, including curing cancer, controlling developmental defects, and engineering tissues and organs. Short abstract of the project My laboratory has begun to shine light (literally!) on these processes and visualized for the first time in living cells the interaction between an enhancer and its target promoter and how that interaction influences transcription kinetics. The current project is aimed at computationally characterizing the dynamics and the motion of fluorescent DNA loci that represent transcription sites. Students will get processed imaging data, computationally identify DNA loci, compute mean square displacements, perform a fluctuation analysis, and test simple mathematical models with these analyzed data sets. Scientific question of the project What are the spatial trajectories of DNA loci representing promoters, enhancers, and individual transcription sites in living cells? What are the constraints and confinements of these trajectories and how do they influence transcriptional activity? Answers to these questions will lead to a more fundamental understanding of the impact of space and time on transcription and thus to a new angle for how to control and interfere with transcription programs. Methodologies used for the project Students will be handed 4d imaging data, in which cell nuclei on the surface of developing embryos a visible. The movement of multiple DNA foci (labeled by various colors) will be characterized using Matlab or any other language the student feels most comfortable with.

Tutor/supervisor

First name, Last name Thomas, Gregor Phone 0140613692 E-mail [email protected] Webpage https://research.pasteur.fr/en/team/physics-of-

biological-functions/


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• Zoller B, Little SC, Gregor T (2018). Diverse spatial expression patterns emerge from

unified kinetics of transcriptional bursting. Cell 175(3)—online October 18. • Chen H, Levo M, Barinov L, Fujioka M, Jaynes JB, Gregor T (2018). Dynamic

interplay between enhancer-promoter topology and gene activity, Nature Genetics 5(9): 1296—1303.

• Gregor T, Garcia HG, Little SC (2014). The embryo as a laboratory: quantifying transcription in Drosophila. Trends in Genetics 30(8): 364–375.

• Garcia HG, Tikhonov M, Lin A, Gregor T (2013). Quantitative imaging of transcription in living Drosophila embryos links polymerase activity to patterning. Current Biology 23(21): 2140–2145.

Scientific or technical background required for work program The ideal candidate has a strong interest for collaborative and interdisciplinary research and to

bridge quantitative and live sciences. A background in mathematics, computer science and/or

the physical sciences is a plus. Prior training in biology is not necessary but encouraged.

Technically all candidates must have prior programming experience, ideally (but not

necessarily) in Matlab and/or Python.


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Title of the work program 31 Establishing a mouse model system for identification of a novel class of “insecticides” to control disease transmitting mosquitoes Description of the work program With the increasing threat of arbovirus epidemics, it is urgent to find novel efficient means to target the mosquito vectors. The basic question of this project is to demonstrate that a filaria mouse model could be used to investigate a mosquito killing effect when mosquitoes bite infected and filaria clearing-drug treated mice. The student will be involved in the production of filaria infection using the mouse model established at the MNHN in Paris, and assessing mortality of mosquitoes feeding on those mice. Technical skills will cover: mouse blood smear staining and PCR for filaria detection in mice over time, mosquito feeding on mice and mortality recording, and statistical analysis. Technicians will assist for mosquito production covering three genera: Aedes, Anopheles and Culex. Once the model is validated, artificial feeding of mosquitoes will be performed to initiate the identification of the blood component responsible for the mosquito killing effect. Metabolomic analysis will then ensue. The student will be hosted in C. Bourgouin group, member of the Functional Genetics of Infectious Diseases Unit directed by Anavaj Sakuntabhai. The Unit belongs to the Genome and Genetics department that will offer broad interaction with many PhD students and postdocs, as well as with junior and senior scientists of the department Tutor/supervisor

First name, Last name Catherine Bourgouin, Functional Genetics Phone +33-1 45 68 82 24 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable): Catherine_Bourgouin

http://www.researcherid.com/rid/F-1253-2014


Antiviral immunity of Anopheles gambiae is highly compartmentalized, with distinct roles for RNA interference and gut microbiota: Carissimo, Guillaume; Pondeville, Emilie; McFarlane, Melanie; et al. Proceedings of the National Academy of Sciences of the United States of America Volume: 112 Issue: 2 Pages: E176-E185 Published: JAN 13 2015

Transgenic Anopheles stephensi coexpressing single-chain antibodies resist Plasmodium falciparum development Isaacs, Alison T.; Jasinskiene, Nijole; Tretiakov, Mikhail; et al. Proceedings of the National Academy of Sciences of the United States of America Volume: 109 Issue: 28 Pages: E1922-E1930 Published: JUL 10 2012


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Fine Pathogen Discrimination within the APL1 Gene Family Protects Anopheles gambiae against Human and Rodent Malaria Species. Mitri, Christian; Jacques, Jean-Claude; Thiery, Isabelle; et al. Source: Plos Pathogens Volume: 5 Issue: 9 Published: SEP 2009

Mosquito-based transmission blocking vaccines for interrupting Plasmodium development Lavazec, Catherine; Bourgouin, Catherine. Microbes and Infection Volume: 10 Issue: 8 Pages: 845-849 Published: JUL 2008

Characterization of the genes encoding the hemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis. Bourgouin, C; Klier, A; Rapoport, G. Molecular and General Genetics Volume: 205 Issue: 3 Pages: 390-397 Published: 1986

Scientific or technical background required for work program With the increasing threat of arbovirus epidemics, it is urgent to find novel efficient means to target the mosquito vectors. It was shown that drug treatment of human population infected with filariasis induced a long lasting-killing effect of Aedes mosquitoes biting the treated patients. We hypothesized that the killing molecules or mechanisms arose from the interaction between the filaria, the drug and the human immune system. We want to investigate this mechanism using a robust mouse model of filaria, that recapitulates very closely human immune response to filaria. We expect that this animal model provides similar long-lasting killing effect of mosquitoes. For that project mice will be infected with Litomosoides sigmodontis, a mouse model of filaria. Once development of micro-filaria, the stage that infect arthropod, is evidenced by blood smears and PCR, mice will be treated with different drugs to clear filaria or mock injected. An additional control of non-infected mice treated with drug single dose will be produced From 5 days following the drug treatment up to 3 months, batches of 50 mosquitoes will be fed on each group of mice, each week. Mosquito mortality will be recorded 24h after feeding and then every other day until all mosquitoes die, based on 30 days life expectancy for untreated mosquitoes. We will first investigate the killing effect on Aedes mosquitoes (arbovirus vectors) and extend the experiments to Culex (arbovirus vectors) and Anopheles (malaria vectors). We will use cohorts of 10 mice per treatment as not all will develop microfilaria. The non-infected mice treated with drugs is intended to complement the infected, non-treated infected control, for monitoring the drug effect per se, but also to foresee death of infected untreated mice over time. Filaria for infecting mice will be produced at the MNHN (Museum National d’Histoire Naturelle) in collaboration with Dr Coralie Martin who is expert in filaria mouse model systems and transported to inoculate mice at the Institut Pasteur. We strongly believe that this project and approach should leads to a novel molecule or mechanism that latter can be translate as an innovative “insecticide” to controlling urban mosquito populations.


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Title of the work program 32 Studying the Function of Tunneling Nanotubes in the Developing Cerebellum Description of the work program The way in which cells interact with each other is a topic that has fascinated biologists for many centuries. Our group is interested in a novel type of cell-to-cell interaction that has recently been characterized as a direct connection between cells. This connection is known as a Tunneling Nanotube, or TNT. TNTs are direct anatomical connections between cells found in many cell types and contexts. Unlike other filamentous bridges (e.g. filopodia), TNTs connect the cytoplasm of distant cells. TNTs vary in diameter and can extend up to over 100 µm in length. These dynamic structures selectively transfer cellular cargo such as cytoplasmic molecules, plasma membrane components, vesicles, and organelles. In addition, TNTs have been shown to be “hijacked” by various pathogens such as bacteria, viruses, and prions in order to transfer onto other cells. The necessity for the transfer of cargo is a distinguishing feature of TNTs; similar membranous structures such as neutrophil membrane tethers play a role in adhesion and cell migration but not in cell-to-cell communication, for example. TNTs’ function in transferring cargo between cells may therefore be relevant to understand essential biological processes such as development, immune response, cancer, tissue regeneration, and electrical signal transmission. In our group, we spent the last decade setting up the tools to identify and characterize TNTs in culture and have demonstrated that they play an important role in the intercellular transfer of amyloidogenic proteins involved in neurodegenerative diseases. We have also shown that TNTs form between primary neurons and astrocytes in vitro, and proposed the notion that TNTs may play a major role in the spreading of neurodegenerative diseases in the brain. In spite of the wealth of knowledge we have acquired of TNTs in culture however, the field still lacks evidence showing that these structures exist in vivo. In order to tackle this question, we partnered with the laboratory of Dr. Jeff Lichtman, a group at Harvard University interested in unraveling the brain connectome by using Serial Sectioning Scanning Electron Microscopy. By using this technique, which the Lichtman lab coupled with other custom-built tools such as an automatic tape-collecting lathe ultramicrotome, they have been capable of imaging slices of brain tissue at the nanometer resolution. After sectioning and acquiring the region of interest, the images are then aligned and given to us for manually analysis using a custom-built software named VAST. At the moment, we have been segmenting granule cells in a small region of the cerebellar vermal lobule VIII of a wild-type P3 mouse. The block is made up of ~1700 electron micrograph slices cut at 30 nm each, making a total volume of 51,000 nm (51 µm) in thickness. In this dataset, we have one confirmed connection of 1,400 nm in length and 700 nm in diameter. Inside this cylindrical tube, we observed small vesicles and what appears to be an array of


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parallel fibers, presumably actin. In addition to the P3 dataset, we also have a P7 volume made up of ~2500 slices, or 75,000nm (75um) in thickness. Using Matlab and 3ds Max, we create reconstructions of our segmented cells for better 3D visualization and characterization. Processed reconstructions are then visualized in video format using Adobe Premiere. During the student’s time in our lab, s/he will learn how to segment granule cells in the datasets described above and look for TNT-like connections. In addition, s/he will carry out immunofluorescence experiments on post-fixed brain sections harvested from mice at different stages in development and test the expression of TNT-associated proteins using various antibody labeling protocols. S/he will be trained on the use of a Zeiss LSM confocal microscope which s/he will use to acquire images of the immunolabeled sections. By the end of the internship, the student will have gained both computational and practical skills. Some of the computational tools the student will use are: VAST, 3ds Max, ImageJ/Fiji and ICY. Among wet lab practices: immunofluorescent labeling and confocal microscopy.

Tutor/supervisor

First name, Last name Chiara Zurzolo Phone +33 (0)1 45 68 82 77 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

First name, Last name Diego Cordero Phone +33 (0)1 44 38 95 54 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

https://research.pasteur.fr/en/member/diego-cordero-cervantes/

Selected publications or patents of the Research Group offering the work program Gousset K, Schiff E, Langevin C, Marijanovic Z, Caputo A, Browman DT, Chenouard N, de Chaumont F, Martino A, Enninga J, Olivo-Marin JC, Männel D, Zurzolo C, Prions hijack tunnelling nanotubes for intercellular spread, Nat. Cell Biol. 2009 Mar;11(3):328-36. Abounit S, Zurzolo C, Wiring through tunneling nanotubes–from electrical signals to organelle transfer, J. Cell. Sci. 2012 Mar;125(Pt 5):1089-98. Costanzo M, Abounit S, Marzo L, Danckaert A, Chamoun Z, Roux P, Zurzolo C, Transfer of polyglutamine aggregates in neuronal cells occurs in tunneling nanotubes, J. Cell. Sci. 2013 Aug;126(Pt 16):3678-85.


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Zhu S, Victoria GS, Marzo L, Ghosh R, Zurzolo C, Prion aggregates transfer through tunneling nanotubes in endocytic vesicles, Prion 2015 Mar;9(2):125-35. Abounit S, Bousset L, Loria F, Zhu S, de Chaumont F, Pieri L, Olivo-Marin JC, Melki R, Zurzolo C, Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes, EMBO J. 2016 Oct;35(19):2120-2138. Delage E, Cervantes DC, Pénard E, Schmitt C, Syan S, Disanza A, Scita G, Zurzolo C, Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes, Sci Rep 2016 Dec;6:39632. Abounit S, Wu JW, Duff K, Victoria GS, Zurzolo C, Tunneling nanotubes: A possible highway in the spreading of tau and other prion-like proteins in neurodegenerative diseases, Prion 2016 Sep;10(5):344-351. Loria F, Vargas JY, Bousset L, Syan S, Salles A, Melki R, Zurzolo C, α-Synuclein transfer between neurons and astrocytes indicates that astrocytes play a role in degradation rather than in spreading. Acta Neuropathol. 2017 Nov;134(5):789-808. Victoria GS, Zurzolo C, The spread of prion-like proteins by lysosomes and tunneling nanotubes: Implications for neurodegenerative diseases. J. of Cell Biology. 2017 Sep 4;216(9):2633-2644.

Scientific or technical background required for work program Previous experience in cell biology, neuroscience, or graphic illustration, is preferred but not

mandatory. We are seeking motivated candidates who share our passion for science, are

enthusiastic about learning new techniques, and are interested in combining multidisciplinary

approaches at the forefront of neurobiology.


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Title of the work program 33 Studies of a disease-associated TYK2 variant Description of the work program The complex etiology of auto-immune diseases (AID) results from combinations of genetic, epigenetic and environmental factors that impact on the immune response and on individual susceptibility. Genome-wide association studies have identified hundreds of AID susceptibility genetic variants (single-nucleotide polymorphisms or SNPs). The identification of causal variants and their functional characterization should help in uncovering pathogenic mechanisms and inform on therapeutical intervention strategies. For many years our laboratory has been interested in TYK2, a protein tyrosine kinase of the JAK family. TYK2 is involved in signaling pathways initiated by several cytokines that regulate the immune response (type I and type III IFNs, IL-6, IL-12, IL-23, IL-22 and IL-10). Several SNP in the TYK2 locus have been implicated in AID, suggesting that TYK2 is a source of inter-individual variation and a common risk factor for immune-related complex diseases. We previously reported on the study of two disease-associated TYK2 coding variants. We now focus on a common SNP located proximal to an intron-exon boundary. Software-assisted analysis predicts that this nucleotide substitution could destroy an exonic splicing regulatory element. We have obtained preliminary data suggesting that this SNP could impact splicing of a critical exon. Moreover, by searching the public eQTL database GTEx (Genotype Tissue expression project) we found a correlation between this variant and TYK2 expression levels in some organs or tissues, like adrenal gland and skin. The primary goal of this project is to demonstrate the causality of this SNP, linking this nucleotide change to a splicing event affecting TYK2 transcript expression. CRISPR-Cas9-mediated modification will be used to introduce the single nucleotide change in a uniform genetic background. This will be done in 293T cells and H295R cells derived from adrenal gland, where the SNP correlates with TYK2 expression with the highest effect size. This will allow the monitoring of TYK2 expression under the regulation of cell type-specific splicing factors in control and modified cells. The student will then analyze the functional consequence on signaling and transcriptional responses to type I interferon and other cytokines, notably IL-6. This study is expected to provide new knowledge on the TYK2 genotype-phenotype relationship and on cytokine-driven pathogenic mechanisms where TYK2 may be involved.

• Li Z, Gakovic M, Ragimbeau J, Eloranta ML, Rönnblom L, Michel F and S. Pellegrini. 2013. Two rare disease-associated Tyk2 variants are catalytically impaired but signaling competent. J Immunol 190:2335-44

• Diogo D, Bastarache L, Liao KP, Graham RR, Fulton RS, et al. 2015. TYK2 protein-coding variants protect against rheumatoid arthritis and autoimmunity, with no evidence of major pleiotropic effects on non-autoimmune complex traits. PLoS One 10:e0122271


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• Dendrou CA, Cortes A, Shipman L, Evans HG, Attfield KE, et al. 2016. Resolving TYK2 locus genotype-to-phenotype differences in autoimmunity. Sci Transl Med 8:363ra149

Tutor/supervisor

First name, Last name Sandra, PELLEGRINI Phone +33140613305 E-mail [email protected] Profile on: https://orcid.org/0000-0001-5837-7589

Lab websites:https://research.pasteur.fr/en/member/sandra-pellegrini/ Selected publications or patents of the Research Group offering the work program 1. Li Z, Gakovic M, Ragimbeau J, Eloranta ML, Rönnblom L, Michel F, and S. Pellegrini. 2013. Two

rare disease-associated Tyk2 variants are catalytically impaired but signaling competent. J Immunol. 190:2335-44

2. Zhang X, Bogunovic D, Payelle-Brogard B, Francois-Newton V, Speer S, Yuan C, Volpi S, Li Z, Sanal O, Mansouri D, et al. García-Sastre A, Abel L, Lebon P, Notarangelo L, Boisson-Dupuis S, Crow YJ, Casanova J-L and Pellegrini S. 2015. Human intracellular ISG15 prevents IFN-α/β over-amplification and auto-inflammation. Nature, 517:89

3. Wilmes S, Beutel O, Li Z, Francois-Newton V, Richter CP, Janning D, Kroll C, Hanhart P, Hötte K, You C, Uzé G, Pellegrini S, Piehler J. 2015. Receptor dimerization dynamics as regulatory valve for plasticity of type I interferon signalling. J Cell Biol. 209:579

4. Meuwissen MEC, Schot R, Buta S, Oudesluijs G, Tinschert S, Speer SD, Li Z et al, Crow YJ, Verheijen FW, Pellegrini S, Bogunovic D and Mancini GMS. 2016. Human USP18 deficiency underlies type 1 interferonopathy leading to severe pseudo-TORCH syndrome. J Exp Med 213:1163

5. Speer SD, Z Li, S Buta, B Payelle-Brogard, F Vigant, TJ Gardner, M Hermann, J Duehr, O Sanal, N Mansouri, P Tabarsi, D Mansouri, V Francois-Newton, C F Daussy, MR Rodriguez, DJ Lenschow, Al Freiberg, D Tortorella, B Lee, A García-Sastre, S Pellegrini and D Bogunovic. 2016. ISG15 deficiency and increased viral resistance in humans but not mice. Nature Commun, 7:11496

6. Govender U, Corre B, Bourdache Y, Pellegrini S, Michel F. 2017. Type I interferon-enhanced IL-10 expression in human CD4 T cells is regulated by STAT3, STAT2, and BATF transcription factors. J Leukoc Biol. 101:1181

7. Arimoto KI, Löchte S, Stoner SA, Burkart C, Zhang Y, Miyauchi S, Wilmes S, Fan JB, Heinisch JJ, Li Z, Yan M, Pellegrini S, Colland F, Piehler J, Zhang DE. 2017. STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling. Nat Struct Mol Biol. 24:279-289

Scientific or technical background required for work program The student will be committed and motivated. She/he will have a good theoretical background and prior technical experience in cellular and molecular biology and be familiar with signaling pathways. A good knowledge in human genetics and basic immunology is recommended.


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Title of the work program 34 Actin-activated bacterial nucleotidyl cyclase toxins Description of the work program Bacterial adenylate cyclase toxins are potent virulence factors that synthesize cAMP to modulate or disable the function of the host cell. Pseudomonas aeruginosa, an opportunistic human pathogen that causes severe acute infections in immunocompromised individuals and is a major cause of chronic infections in cystic fibrosis patients, encodes an adenylate cyclase toxin, called ExoY [1]. Together with 3 other proteins (ExoS, ExoT, and ExoU), ExoY is injected directly into the host cell utilizing the type III secretion system (T3SS) where they contribute to virulence of the pathogen [for review see [2, 3]]. Recent results show that substrate specificity of ExoY is not restricted to ATP as ExoY was shown to promote the intracellular accumulation of cAMP and cGMP, cCMP as well as cUMP. In order to prevent detrimental effects resulting from the catalytic activity of ExoY inside the bacterial host, the protein is kept inactive inside the bacterial cell and acquires catalytic activity only after its delivery to the eukaryotic host cell through its interaction with a eukaryotic cofactor. We recently identified actin as said cofactor in our laboratory [4]. Actin is ubiquitously and abundantly present in eukaryotic cells and as such an appropriate indicator for the arrival of the bacterial toxin in the infected host. We also showed that the ExoY-like adenylate cyclase from Vibrio nigripulchritudo, despite its only distant relatedness to P. aeruginosa ExoY, is also activated by actin. These results suggest the presence of a group of actin-activated nucleotidyl cyclases (AA-NC). The two AA-NCs that we characterized, show important differences concerning their activation mechanism and substrate specificity. To obtain further inside into the group of AA-NCs, the proposed project envisages the characterization of an ExoY-like protein from the genus Proteus. This genus harbors species (P. vulgaris, P. mirabilis, P. penneri), which are opportunistic human pathogens often responsible for urinary tract infections.

During work on this project, methods of molecular biology, biochemistry and bacteriology will be employed in our laboratory. References: 1. Yahr, T. L., Vallis, A. J., Hancock, M. K., Barbieri, J. T. & Frank, D. W. (1998) ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system, Proc Natl Acad Sci U S A. 95, 13899-904. 2. Engel, J. & Balachandran, P. (2009) Role of Pseudomonas aeruginosa type III effectors in disease, Curr Opin Microbiol. 12, 61-6. 3. Hauser, A. R. (2009) The type III secretion system of Pseudomonas aeruginosa: infection by injection, Nat Rev Microbiol. 7, 654-65. 4. Belyy, A., Raoux-Barbot, D., Saveanu, C., Namane, A., Ogryzko, V., Worpenberg, L., David, V., Henriot, V., Fellous, S., Merrifield, C., Assayag, E., Ladant, D., Renault, L. & Mechold, U. (2016) Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins, Nat Commun. 7, 13582.


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Keywords: bacterial toxins, nucleotidyl cyclase, cNMP, actin, ExoY

Tutor/supervisor

First name, Last name Undine MECHOLD Phone 33-140613870 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):


• Belyy, A., Raoux-Barbot, D., Saveanu, C., Namane, A., Ogryzko, V., Worpenberg, L., David, V., Henriot, V., Fellous, S., Merrifield, C., Assayag, E., Ladant, D., Renault, L. & Mechold, U. (2016) Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins, Nat Commun. 7, 13582

Scientific or technical background required for work program The candidate should have a solid knowledge in molecular biology and biochemistry,

good English language skills, should be rigorous and organized with the ability to take

initiative and should be eager to learn new methods.


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Title of the work program 1 International IP French Guyane Manipulating the mosquito microbiota Description of the work program The microbiota of Aedes mosquitoes impacts their capacity to transmit diseases, notably inducing an immune response and affecting mosquito fitness. The study of the tripartite interaction between the mosquito, its microbiota and pathogens is thus regarded with high interest as a source of new transmission blocking strategies. However, the microbiota composition of laboratory-reared mosquitoes is extremely different from that of field-collected ones. In order to study the role of the microbiota in mosquitoes with a more relevant microbiota composition, new experimental set ups are needed. In this context, this project will study host-microbe and microbe-microbe interactions in gnotobiotic mosquitoes, i.e. mosquitoes with a known microbiota composition. These will by obtained by re-colonising germ-free individuals with bacterial strains isolated from field mosquitoes. During the internship the student will: A) contribute to the isolation of bacteria from the gut of field mosquitoes (field collections, mosquito dissection, microbiology). B) combine some bacteria isolated from field mosquitoes to assess their optimal in vitro growth conditions and possible inter-bacteria interactions (promotion or competition). C) introduce the selected bacteria in germ-free recipient mosquitoes and determine the stability of the manipulated microbiota in both sugar-fed and blood-fed mosquitoes. D) determine the effects of the manipulated microbiota on mosquito fitness (development, survival, fecundity, fertility). This project will be performed on a secondary campus of Institut Pasteur, at Institut Pasteur de la Guyane in French Guiana.

Tutor/supervisor

First name, Last name Mathilde, Gendrin Phone +594 (0) 594 29 26 18 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

Selected publications or patents of the Research Group offering the work program • The tripartite interactions between the mosquito, its microbiota and Plasmodium, Romoli O, Gendrin M, Parasites and Vectors (2018) 11: 200, doi: 10.1186/s13071-018-2784-x


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• Larval diet affects mosquito development and permissiveness to Plasmodium infection, Linenberg I, Christophides GK, Gendrin M, Scientific Reports (2016) 6:38230 • Seasonality and locality affect the diversity of Anopheles gambiae and Anopheles coluzzii midgut microbiota from Ghana, Akorli J, Gendrin M, Pels NAP, Yeboah-Manu D, Christophides GK, Wilson MD, PLOS ONE (2016) 11(6): e015752 • Antibiotics in ingested human blood affect the mosquito microbiota and capacity to transmit malaria, Gendrin M, Rodgers FH, Yerbanga RS, Ouédraogo JB, Basáñez MG, Cohuet A, Christophides GK, Nature Communications (2015) 6: 5921

Scientific or technical background required for work program Experience working in sterile conditions is preferred and general background in microbiology

and host-microbe relationships is required. Reading the most recent literature on the subject

will be required before the start of the internship.


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Title of the work program 2 international IP Cote d‘Ivoire Novel Phages for the Biocontrol of bacterial infections in environmental ecosystems in tropical countries. Description of the work program The program is subdivided in 3 steps:

1. Isolation and characterization of new phages from different sources. 2. Application of phages in several matrices and in food products 3. Implementation of phagotherapy

Tutor/supervisor

First name, Last name Solange Kakou Ngazoa Phone 00225 08240453 E-mail [email protected] Profile on http://www.researchgate.net/ (if applicable):

Pr Kakou Ngazoa Solange, Maitre de recherche Pasteur Institute Cote d’Ivoire, Abidjan Plateforme de Biologie Moléculaire www.pasteur.ci


§ Ngazoa-Kakou S, Philippe C, Tremblay DM, Loignon S, Koudou A, Abole A, Ngolo Coulibaly D, Kan Kouassi S, Kouamé Sina M, Aoussi S, Dosso M, Moineau S. 2018. Complete genome sequence of Ebrios, a novel T7virus isolated from the Ebrie Lagoon in Abidjan, Côte d’Ivoire. A Society of Microbiology. Genome Announc. 6:e00280-18. https://doi.org/10.1128/genomeA.00280-18.

§ Kakou Ngazoa ES, Kouya D, Koudou A. et al. 2017 New identification of phages in water and in fishes from lagoon Ebrié and their application for multidrug strains in Abidjan, West Africa. World Journal of Pharmaceutical Research, , Vol. 6, issue 9, 84-89

Scientific or technical background required for work program Master II or PhD students (no undergraduate) Experience in microbiology/viroloy methods (sampling, culture) and molecular biology Self autonomy for execution of the activities.

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