This research project focuses on the characterization of the role of small RNAs and their associated Argonaute proteins in transcriptional and post-transcriptional regulation of germline gene expression. Using the nematode C. elegans, we have recently showed that one of the germline-expressed Argonaute protein, CSR-1, promotes germline transcription. However, CSR-1 also possess an endonucleolytic activity that might participate in post-transcriptional silencing. Therefore, two possible functions of the protein might regulate the germline transcriptome. 1) CSR-1 promotes specific germline transcription programs in the nucleus, and 2) negatively regulates expression of target transcripts in the cytoplasm. To gain mechanistic insights into these two functions, we aim to use RNA-seq, sRNA-seq, ChIP-seq, GRO-seq, Ribo-seq, RIP-seq, iCLIP in wild type worms, knock out and catalytic inactive mutants of CSR-1 protein at different times of germline development.

The canonical (“textbook”) process of DNA homology search and recognition is initiated by DNA double-strand breaks and is mediated by the universally conserved recombinases of the RecA family. Using the phenomenon “Repeat Induced Point mutation” (RIP) in N. crassa as a model system, we have previously revealed the existence of another way to search for DNA homology, which does not require RecA proteins and which apparently operates on intact DNA double helices. This pathway can be extremely efficient, as it allows some fungi to detect the presence of only two gene-sized DNA repeats in the genome. Our current work on Meiotic Silencing by Unpaired DNA (MSUD) has shown that the same recombination-independent pathway may also be involved in the early steps of homologous chromosome pairing in meiosis, thus emerging as a conserved, perhaps fundamental mechanism of DNA homology search and recognition. We are now interested in further investigating this mechanism, using RIP and MSUD as two complementary recombination-independent processes. Specifically for this project, we are interested in identifying molecular genetic features that associate with (or even trigger) the production of small RNAs during MSUD in N. crassa.

Streptococcus agalactiae (GBS) is a commensal gram-positive bacteria which asymptomatically colonize the genital and intestinal tract of healthy women. However, GBS is the leading cause of bacterial invasive infections in newborns in developed countries. The ability of GBS to succeed both as a commensal and a pathogen relies on a highly dynamic regulation of colonization and virulence related genes. The major regulator identified to date is the two-component system CovSR (Control of Virulence Sensor and Regulator). The transcription of almost 15% of the genome is dependent on CovR, but the genes directly regulated by CovR and the regulation of CovR-DNA binding by CovR-phosphorylation are ill-defined. To characterize the genome-wide CovR binding sites, we performed chromatin immunoprecipitation and sequencing (ChIP-Seq). Technically, we developed an epitope-tagged and functional form of CovR expressed under an inducible promoter. Quantitative PCR on ChIP samples (ChIP-qPCR) and small-scale footprint experiments revealed an enrichment of binding regions on specific promoters whose transcription are CovR-dependent. Sequencing (ChIP-seq) has been done to 1) characterize the landscape of CovR binding sites along the chromosome and to reveal the function of genes directly regulated by CovR; 2) to decipher the mechanism of regulation by performing the same experiment in strains with different level of CovR phosphorylation; and 3) to unravel an evolutionary strategy of genetic rewiring leading to the emergence of hypervirulent GBS strain by comparing the CovR direct regulon and the evolution of promoter sequences in different clinical stains.

Dans notre laboratoire nous nous intéressons à la conception et construction de banques d’anticorps simple chaine issus du répertoire immunitaire des alpagas. Ces banques immunes, sont constituées d’un million de séquences ADN différentes codant pour les domaines variables des anticorps simple chaine spécifiques d’une protéine cible donnée, plus communément appelés domaines VHH ou Nanobodies®. Nous souhaitons à présent caractériser ces banques crées au laboratoire par séquençage ADN à haut débit (NGS Séquence MiSeq). Cette méthode conduit à la création de fichiers composés de millions de séquences ADN nécessitant d’être traitées et analysées par la suite.

This project to characterize defective viral genomes of shrimp pathogens i.e. Yellow head virus (YHV) from next-generation sequencing of viral RNAs data by using DI-tector or similar tools. NGS data are already available for analysis. We are looking for someone interested in virology in general and more precisely the complexity of viral genomes.

The ViroScreen workflow is a bioinformatic pipeline dedicated the the analysis of NGS metagenomic data, especially in the field of virology (virome analysis). The aim of the project is to implement this bioinformatic workflow, developed with Corinne Maufrais (Bioinformatics and Biostatistics Hub), in Galaxy, to make it easly accessible to the scientific community, in easy-to-use way.

In the TSARA project, we aim at developing and validating a molecular platform (Dual Index Targeted Amplicon Deep Sequencing on Illumina iSeq) and a dedicated bioinformatics pipeline for targeted high throughput sequencing of genetic loci related to antimalarial drug resistance. The iSeq Illumina platform, proposed here, is designed for dual indexing of samples, consisting of a 3’ and 5’ individual barcode, which allow the user to connect every sequence generated to a specific sample (up to 384 samples). The development and the validation of this novel molecular tool will be performed at IP Paris first by using DNA extracted from P. falciparum reference strains (3D7, Dd2 and culture-adapted Cambodian strains) (WP1) and second, by using P. falciparum DBS samples collected from two malaria endemic areas (Central African Republic and Cameroon) (WP2). In the WP3, we will host and train staff from IP Bangui and CP Cameroon to this new approach. Our final objective is that Dual Index Targeted Amplicon Deep Sequencing on iSeq along with minimal bioinformatics infrastructure operated in countries that are endemic for malaria to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy.

In the TSARA project, we aim at developing and validating a molecular platform (Dual Index Targeted Amplicon Deep Sequencing on Illumina iSeq) and a dedicated bioinformatics pipeline for targeted high throughput sequencing of genetic loci related to antimalarial drug resistance. The iSeq Illumina platform, proposed here, is designed for dual indexing of samples, consisting of a 3’ and 5’ individual barcode, which allows the user to connect every sequence generated to a specific sample (up to 384 samples). The development and the validation of this novel molecular tool will be performed at IP Paris first by using DNA extracted from P. falciparum reference strains (3D7, Dd2 and culture-adapted Cambodian strains) (WP1) and second, by using P. falciparum DBS samples collected from two malaria-endemic areas (The central African Republic and Cameroon) (WP2). In the WP3, we will host and train staff from IP Bangui and CP Cameroon to this new approach. Our final objective is that Dual Index Targeted Amplicon Deep Sequencing on iSeq along with minimal bioinformatics infrastructure operated in countries that are endemic for malaria to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure the continued success of the malaria treatment policy

The Milieu Intérieur program aims to understand the genetic and environmental determinants of normal healthy immune responses. One component of this program involves analysis of the nasopharyngeal mucosal immune variation. At upper respiratory tract level, nasal-associated lymphoid tissue is strategically located to potential respiratory pathogens; in this context, B cell activation induces specific secretory IgA while T cells/ILCs produce cytokines. One factor that has been poorly studied and needs to be integrated in respiratory mucosal immune variation is the microbiome. Under normal conditions, the relationship between the nasopharyngeal microbiomes (bacterial, fungal, viral) and the host is symbiotic with many physiologic benefits. On the other hand, there is increasing evidence that suggest that the nasopharyngeal microbiome composition plays an important role in the pathogenesis of bacterial and viral acute respiratory tract infections including COVID-19. For example, even in healthy individuals potentially pathogenic bacteria and virus can also be found embedded in the community of nasopharynx commensals. Still, we have little information on the characterization of “healthy” nasopharyngeal microbiomes. Previously we have established methods to characterize bacterial nasopharynx microbiota by 16S rRNA sequencing and assays are in place to analyze secretory antibodies, interferons (IFNs) and cytokines concentrations in nasopharyngeal secretions of healthy individuals and disease patients. Our capacity to quantitate antibodies, IFNs and cytokines has allowed us to determine biomarkers in nasopharyngeal secretions that can distinguish clinical outcomes in different diseases. New evidence suggests that non-bacterial microorganisms such as viruses and fungi could be critical in modulating immunity in the host. We have performed shotgun metagenomic sequencing of nasal swab RNA from 12 healthy and disease individuals. We would like to request bioinformatic assistance to analyze the sequencing datasets in this pilot study in order to assess potential RNA viromes (assembly, identification, clustering, and taxonomy). Successful identification of known viruses will provide proof of concept for this approach which can then be applied to well-characterized normal control and disease cohorts.