Expertise

Hub members Have many expertise, covering most of the fields in bioinformatics and biostatistics. You'll find below a non-exhaustive list of these expertise

Search by keywords | Search by organisms

Searched keyword : Differential analysis

Related people (0)

Sorry nobody has this skill yet...

Related projects (60)

Mapping the cell surface signature of the developing mouse heart

Cell surface protein signatures have been successful to discriminate hematopoietic progenitor populations allowing major advances in understanding blood cell production, to define pathways in hematologic malignancies and to foster new therapeutic approaches. Limited knowledge on the phenotype of cells that participate in heart formation impairs our understanding of progenitors of the cardiac cell lineages and their eventual persistence in the adult organ. As a consequence, therapies to restore heart function after injury have been unsuccessful. A number of membrane proteins have been identified on cardiomyocytes; on cardiac fibroblasts; and on endothelial cells, however a multi-parametric analysis of the phenotype of the different cardiac cell compartments along development is still missing. We combined multi-parametric flow cytometry with transcriptional characterization, based on well-known gene expression patterns, to describe major cardiac cell-subsets. The expression of CD24, CD54, Sca-1 and CD90 allowed defining cardiac populations in the non-hematopoietic and non-endothelial cell fraction by flow cytometry. Transcriptional profiling of the sorted populations enabled the identification of cardiomyocytes, in the CD24+ population, while differential expression of CD54, Sca-1 and CD90 defined four cardiac stromal compartments. The identified subsets exhibited specific distributions in three analyzed regions (atria, auriculo-ventricular junction and ventricles). We have thus identified a panel of surface markers, some of which novel in the cardiac context, that allowed assigning surface signatures to different cellular fractions by their unique transcriptional profiles. This work is the foundation for comprehensive studies on the role of different cell fractions by their unique transcriptional profiles.



Project status : Closed

Characterisation of skeletal muscle stem cell properties in distinct physiological states

Stem cells are defined by their is their capacity for self-renewal and differentiation. Some adult tissues maintain a reservoir of stem cells, that generally reside within specialized microenvironments, known as stem cell niches, that regulate their behaviour. Skeletal muscle stem (satellite) cells are quiescent in homeostatic conditions in adults, and they are activated after muscle injury, when they re-enter the cell cycle, proliferate and differentiate into myoblasts, which will then fuse to form new muscle fibers. Satellite cells express the paired/homeodomain gene Pax7, which plays a critical role in satellite cell maintenance postnatally. Numerous experiments have shown that the skeletal muscle stem cell population is heterogeneous, therefore like many other stem cell systems, characterising the stem cell states is a major objective. In our laboratory, a reversible dormant cell state was identified, correspondent to a Pax7Hi quiescent subpopulation (top 10% of the Pax7-nGFP+ cells isolated from the transgenic mouse model Tg:Pax7-nGFP) with a lower metabolic activity and longer lag for the first cell division compared to Pax7Lo cells [1]. Muscle stem cells that survive for extended periods post-mortem are also dormant, suggesting that this property, in addition to anoxia [2] contributes to their viability. Therefore, different physiological states are associated with distinct cell states of muscle stem cells. Metabolism could play a critical role in dictating whether a cell remains quiescent, proliferates or differentiates. Stem cell metabolic plasticity in homeostasis and differentiation, as well as during cell reprogramming, is well described in different cell systems. However, unanswered questions remain regarding the metabolic regulation of satellite cell biology and skeletal muscle regeneration. In this project, we will investigate the behaviour of muscle stem cells in distinct physiological states, especially post-mortem and aging.



Project status : Closed

A long-term mission for an assigned CIH-embedded bioinformatician to provide bioinformatic support to the CIH community

The Center for Human Immunology (CIH) supports researchers involved in translational research projects by providing access to 16 different cutting edge technologies. Currently, the CIH hosts over 60 scientific projects coming from 8 departments of the Institut Pastuer and 5 external teams. In order to respond to the growing needs of these projects in the area of single cell analysis, the CIH has introduced a significant number of single-cell/single-molecule technologies over the past 2-3 years. These new technologies, such as the Personal Genome Machine (PGM) and Ion Proton sequencers, iSCAN microarray scanner, Nanostring technology for transcriptomics profiling and real-time PCR machine BioMark, give rise to large datasets with high dimensionality. Such trend, in terms of data complexity, is also true for flow cytometry technologies (currently reaching over 20 parameters per cell). The exploration of this data is generally beyond the scope of scientists involved in translational research projects. In order to maximize the research outcomes obtained from the analysis of these rich datasets, and to ensure that the full potential of our technologies can be served to the users of the CIH, we would require a proximity bioinformatics support. A CIH-embedded bioinformatician would: 1) design and implement standard analysis pipelines for each of the data-rich technologies of the CIH; 2) provide regular ‘bioinformatics clinics’ to allow scientists the possibility to customize standard pipelines to their specific needs; 3) run trainings on the ‘R software’ platform and other data analysis tools (such as Qlucore) of interest for the CIH users. The objective would be to empower the users to run exploratory analysis by themselves, and to teach good practices in terms of data management and data analysis.    



Project status : In Progress

RNAseq analysis-gene ontology enrichment Clostridium tetani



Project status : Closed

Study of the early pathogenesis during Lassa fever in cynomolgus monkeys and its correlation with the outcome

Because of their increasing incidence, dramatic severity, lack of treatment or vaccine, complicated diagnosis, misreading of the pathogenesis, and need for a maximum containment, Viral Hemorrhagic Fevers (VHF) constitute a major public health problem. There is therefore an urgent need to further study VHF to understand the pathogenesis of the severe disease and the host responses involved in their control or in the dramatic damages. Among VHF, Lassa fever (LF) is probably the most worrying one because of its endemicity and the large number of cases. LF is caused by the Old-World arenavirus Lassa virus (LASV). It is endemic to West Africa and is responsible for 300,000 cases and 5,000 to 6,000 deaths each year. We propose here to study the pathogenesis of VHF by using LF in cynomolgus monkeys as a paradigm, with a particular emphasis on the very early events. The viral tropism, pathophysiological mechanisms, and immune responses will be studied during the course of infection, including the incubation period. Powerful approaches will be used to (1) identify early biological markers of infection, to be able to confirm infection and isolate patients; (2) determine the viral tropism and dynamics during the course of infection to understand the natural history of virus into its host. (3) characterize the early pathogenic events that lead to the severe hemorrhagic syndrome to fully understand the pathophysiogenesis of VHF and identify new therapeutic targets. (4) identify the immune responses involved in the control of infection or in the fatal outcome, to reveal the involvement of immunopathological mechanisms and help to design a vaccine approach. This ambitious and unprecedented project will allow to develop therapeutic and prophylactic approaches but also to identify early biological markers of infection and improve the early diagnosis to optimize the management of outbreaks in the field and increase the survival rate in patients.



Project status : In Progress

Exploring immunological mechanisms of human graft-verus-host disease after hematopoietic stem cell transplantation

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for many hematologic malignancies. The main therapeutic benefit derives both from the ability to treat patients with intensive chemotherapy and from a potent graft-versus-leukemia (GVL) effect mediated by donor T lymphocytes. Unfortunately, in some patients, donor T cells also attack host normal tissues, giving rise to graft-versus-host disease (GVHD). GVHD prevalence is between 40-80% depending on patient and transplantation characteristics and GVHD remains the main cause of non-relapse morbidity and mortality. Despite the advances in the field of HSCT and GVHD prophylaxis, disease processes in humans remain poorly understood, and the lack of biomarkers for the early diagnosis and prognosis of GVHD contributes to the high mortality of the disease. The objective of the study is to investigate the cellular and molecular mechanisms involved in the immune reconstitution after transplantation and to explore the mechanisms of acute GVHD. For three independent cohorts of donor-recipient pairs, blood samples were collected from the all the donors before transplantation and for the respective recipients either at GVHD onset or at the Day 30 or Day 90 for recipients that did not develop GVHD. Donors and recipients’ samples were analyzed using different approaches: spectral flow cytometry to investigate the cellular correlates of immune reconstitution after HSCT and of GVHD onset, gene expression analysis by NanoString technology to assess the molecular profile of immune cell populations important for GVHD development (CD4+ T cells, CD8+ T cells, NK cells and monocytes) as well as a metabolomics profiling of serum samples using mass spectrometry.



Project status : Closed

Defining Shigella-targeting of human lamina propria mononuclear cells using CyTOF technology

Invasion of human intestinal epithelial cells by Shigella flexneri is secondary to the delivery of bacterial effectors into the host cell cytoplasm via a type III secretion system (T3SS). By using a beta-lactamase reporter tool we observed that in contrast to the epithelium, human lymphocytes are mainly targeted by injection of T3SS effectors not resulting in subsequent cell invasion (Pinaud et al., 2017). Furthermore, we observed that the targeting process, in form of successful injection of effectors into the host cell, is dependent on glycan-glycan interactions between bacterial and host cell surfaces rendering the targeting process to be dependent on the activation state of the host cell (Belotserkovsky et al., 2018). CyTOF technology is a research tool used for phenotypic analysis of complex cell population allowing for the simultaneous labelling of up to 40 different surface and intracellular marker without issues of compensation as present in regular flow cytometry (van Unen et al., 2016). Using CyTOF technology and the beta-lactamase reporter tool, we will perform a detailed analysis of Shigella targeting in a complex cell population using human lamina propria mononuclear cells (LPMCs), isolated from human colon explants. Analysis will address the question if specific cellular subsets are preferentially targeted in the intestinal environment and if this differs from targeting of peripheral blood mononuclear cells (PBMCs) diverging in their immune phenotypes and cellular activation.



Project status : Closed

Transcriptomics of Anopheles – Plasmodium vivax interactions towards identification of malaria transmission blocking targets

Despite the worldwide importance of malaria due to Plasmodium vivax, there is currently almost no data on the molecular responses of the Anopheles mosquito vectors to this parasite species. Understanding these responses will contribute to identify relevant strategies to interrupt the transmission of P. vivax by targeting the mosquito vector. Such approaches are urgently needed, as P. vivax is difficult to target on the long term in humans as a consequence of the hypnozoite stage that is responsible for relapses. This project will investigate the molecular response of Anopheles arabiensis, member of the Anopheles gambiae complex, to P. vivax from experimental infections performed in Madagascar where both mosquito and parasite are present. This is a unique situation that will capitalize on the strong knowledge and tools available for An. gambiae sensu lato. The response will be compared to the one triggered by P. falciparum, also present in Madagascar. From mosquitoes infected in a field setting, a transcriptomic (RNAseq) approach will be used to identify common and unique pathways to both parasite species. These analyses will further contribute to identify targets for interrupting transmission of each or both parasites simultaneously. However, a pilot study performed with the PF transcriptomics & epigenomics revealed that the current release of the An. arabiensis genome is poorly annotated. Therefore, to be able to make sens of the RNAseq analyses per se, a strong support from the C3BI Hub for bioinformatics and biostatistics is critical.



Project status : Closed

Identification d’une mémoire épigénomique à Streptococcus pneumoniae

Les modifications de la chromatine, au niveau de l’ADN ou des histones, jouent un rôle fondamental dans la régulation de l’expression des gènes chez les eucaryotes, en contrôlant l’accès de la machinerie transcriptionnelle aux séquences promotrices. Des études récentes ont mis en évidence que modifier la chromatine est l’un des moyens par lesquels les bactéries pathogènes interfèrent avec le programme transcriptionnel des cellules hôtes. Cependant, les mécanismes moléculaires sous-jacents sont très peu caractérisés. Ainsi que les rôles de ces modifications pour l’hôte ou pour la bactérie et l'impact à long terme des changements induits chez l’hôte restent mal définis. Les colonisateurs naturels ou les bactéries commensales n'ont pas été étudiés pour leur capacité à modifier les histones de l’hôte. Dans notre projet, nous utilisons le model bactérien, Streptococcus pneumoniae, pour nous focaliser sur les modifications d’histones importantes pour un colonisateur naturel de l’épithélium respiratoire et un pathogène opportuniste redoutable, dans le but de caractériser précisément les modifications des histones qui persistent après la colonisation afin d’évaluer la réponse de la mémoire. Nos résultats préliminaires montrent que cette bactérie induit bien des modifications d’histones qui sont conservés à long terme. Pour identifier les gènes affectés par ces modifications d’histones observées et comprendre leurs roles, nous voulons réaliser du ChIPseq.



Project status : Awaiting Publication

Identifying subpopulation-specific signatures of tuberculosis persistence for enhanced diagnostics.

Microbes are prone to rapid changes and they can either exploit or countervail their variation in a context-dependent manner. To this purpose, both genetic diversity and non-genetic phenotypic variation exist. However, while the overall mutational evolution occurs over lengthy timescales, epigenetic changes take place on a large scale and more rapidly. Collectively this implies that the diversity we observe is profoundly driven by non-genetic variation. This is particularly relevant for the WHO Priority Pathogen Mycobacterium tuberculosis, whose lack of lateral gene transfer and low mutation rate make phenotypic variation an important means of adaptation to stressful conditions. A few studies, including ours, have begun to explore this phenomenon at the single-cell level in M. tuberculosis in axenic and host conditions, which are technically very challenging. This project is based on the assumption that M. tuberculosis can successfully endure harsh environmental conditions thanks to its phenotypic variation. In our view a better understanding of the drivers of phenotypic variation will improve the design and development of original strategies for tuberculosis control. Here we investigate the physiology of M. tuberculosis at the single-cell and subpopulation scale, striving to demystify the bases of phenotypic diversity and the implications for adaptation and persistence. Previously we examined by real-time imaging a fluorescent reporter of ribosomal expression (rRNA-GFP) as a gauge for cellular activity, and found that M. tuberculosis displays phenotypic heterogeneity under optimal growth conditions, which is enhanced in the host, in long-term stationary phase and upon drug exposure. Remarkably we could also detect subpopulations of quiescent bacilli, whose molecular characteristics have yet to be determined, which is the aim of this project. Here we constructed a dual fluorescent reporter of metabolic activity/quiescence in M. tuberculosis, by using our rRNA-GFP reporter as a background strain, further modified with a red fluorescent marker of cellular quiescence. We carried out snapshot microscopy and single-cell analysis during optimal growth conditions as compared with stressful conditions. We found that the cell-activity marker decreases, whereas the cell-quiescence marker is induced under different host-mimetic conditions. We also observed significant intracellular variation during infection assays. Now we envision carrying out a comprehensive analysis of M. tuberculosis phenotypic variation by RNA sequencing. We aim to reveal the molecular differences between subpopulations of bacilli that exhibit discrete metabolic potential, based on their fluorescence output. We have recreated the most interesting conditions on a bulk scale, and sorted active versus quiescent subpopulations, aiming to compare their transcriptional profiles, and to ultimately identify subpopulation-specific biomarkers of persistence towards more accurate diagnostics.



Project status : Awaiting Publication

Determination of the transcriptome controlled by the two-component system BvrR/BvrS using dominant positive and negative BvrR mutants



Project status : Awaiting Publication

Early transcriptional signature of T-cell memory after dengue vaccination

Dengue virus (DENV) induces strong T and B cell responses upon infection. However, there is currently neither vaccine nor specific treatment against DENV, which is spreading worldwide causing 400 million new infections every year, of which 100 million cases are symptomatic, ranging from a self-limiting febrile illness named dengue fever (DF) to more severe life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). One of the major obstacles of dengue vaccine development is the cross-reactivity among antibodies against the different DENV serotypes (designed as DENV1-4) that are 67-75% identical at the amino acid level. Indeed, while a primary infection by one DENV serotype can induce a lifelong immunity against re-infection by the same serotype, subsequent infections by heterologous serotypes increase the risk of developing severe dengue, a phenomenon due to non-neutralizing or sub-neutralizing antibodies and called antibody-dependent enhancement (ADE). To avoid the induction of such enhancing antibodies, and given the identification of CD4+ and CD8+ T cell epitopes from previously dengue virus (DENV)-infected donors, we have designed a minimal DENV antigen (called DENV1-NS, patent EP14305984.8 filled on June 23 2014), which is enriched in conserved and highly antigenic epitopes. Using this minimal DENV1-NS antigen, we have first established in vivo its immunogenicity in transgenic mice expressing HLA class II and class I alleles (with the activation of DENV-specific CD4 and CD8 T cells). We have also shown that a prime-boost DNA immunization of these HLA transgenic mice induces a strong T cell immunity, with a significant protection against DENV1 infection, in the absence of neutralizing or sub-neutralizing anti-DENV antibodies (Roth et al., accepted for publication). Our proposal aims, therefore, to identify early transcriptional signatures correlated with the development of memory CD4 and CD8 T cells in vaccinated animals, which promote enhanced anti-dengue immunity.



Project status : Closed

Proteomic analysis of the intracellular compartments containing Brucella abortus

Brucella abortus is a zoonotic pathogen that affects cattle by inducing abortion. Humans are accidental hosts that acquire the infection through contact with animal fluids from infected animals. The pathogenesis of brucellosis relies in the ability of B. abortus to survive intracellularly. This bacterium enters host cells, evades the lysosomal route and re directs its traffic to the endoplasmic reticulum. The type IV secretion system VirB has been shown to be crucial for the intracellular fate of B. abortus. It has been postulated that through the secretion of bacterial effectors, this system modifies the compartment used by the bacterium to reach the endoplasmic reticulum. We have undertaken a comprehensive proteomic approach to understand the molecular changes induced by B. abortus in this intracellular compartment. Murine macrophages were infected with wild type B. abortus or an isogenic mutant in the type IV secretion system VirB. At 1 and 6 h post infection the cells were disrupted, the compartments were purified using sucrose gradients and after resuspension in triton-containing buffer the bacteria removed by centrifugation. Thus, we obtained fractions representing the compartments and secreted bacterial proteins. These fractions have been analyzed by comprehensive proteomic methods and preliminar analysis indicate that there are consistent differences between the compartments derived from wild type and VirB mutants to believe that we could understand the modulation exerted by the Type IV secretion system. In this project we intend to apply state of the art bioinformatics methods to analyze the already existing proteomic data. We hope to understand in detail the differences in prokaryotic and eukaryotic proteins between compartments derived from wild type and VirB mutants and how the function of the differential proteins might impact the route of the Brucella-containing compartment. These results will allow us to understand how B. abortus directs its intracellular traffic to the endoplasmic reticulum.



Project status : In Progress

Modulation of cellular pathways involved in neuropathology of rabies infection

Viruses have evolved powerful countermeasures to evade host innate immunity, which produces immediate, but non-specific, immune response during infection. Among viruses possessing RNA genomes, the order of negative-single-strand viruses (Mononegavirales) encompasses many human and animal pathogens that cause severe disease, including measles virus, mumps virus and rabies virus. Rabies virus is known for its neurotropic retrograde progression from the site of transmission to brain parenchyma, and towards salivary glands, different organs linked through parasympathetic nervous system. In the cases of human infection, the exhibited symptoms such as hallucination, diplopia, hydrophobia, unsteadiness or paralysis all indicate that there is causality between rabies virus infection and dysfunction of neural activity. However, lack of pathological brain lesions observed at the point of autopsy or noncytolytic propagation devoid of apoptosis suggest that rabies virus possesses mechanisms to evade or delay immune responses and cell death at least for the duration of replication and transmission. Even though the details in molecular perspective of these discoveries are well laid out now, how these proteins work in coordination or if there are hidden components which connect them all together leading toward deterioration of neural cells on the benefit of virus is largely unclear. Moreover, considering the complexity of brain cell composition and how important the neighboring cells are to shape one neuron’s specialization and dependency onto others in homeostasis, which result in the astounding heterogeneity of gene expression, an integrated and holistic approach is mandatory to get a fully comprehensive view of the mechanisms involved. Consequently, we performed an RNASeq analysis in human interneuron cells derived from induced pluripotent stem cells and infected by two recombinant rabies viruses (Tha virus, isolated from a dog in Thailand and Th4M, a less pathogenic virus which is mutated on 4 different residues of the M gene; this virus can no longer escape the NF-KB pathway) in order to obtain transcriptome data by comparison with uninfected cells, and to have an overview of the temporal dynamics of the genes expression.



Project status : Closed

Virulence and natural anti-sense RNA in Entamoeba histolytica, the agent of human amoebiasis

Several evolutionary processes influence microbes virulence, a process ending with damage impacting host survival. Parasites are favored to exploit their hosts prudently to prolong infection in general overcoming the host immune response and avoid killing the host. Parasites also need to use some host resources to reproduce and transmit infections to new hosts. We are interested in parasites affecting humans as Entamoeba histolytica, which is a protozoan parasite and an amitochondriate pathogenic amoeba, which causes amoebiasis (dysentery and liver abscess). In addition to E. histolytica several species infect the human intestine although these do not cause disease and include in most of cases E. dispar and ocassionnally E. moshkovskii. A phylogenetically close Entamoeba, E. invadens infecting snails, is used as cellular model for Entamoeba cyst formation. Supported by the National Agency for Research (ANR-10-GENM-0011) we developed a project to firstly study the phenotype of pathogenic E. histolytica. In collaboration with the Hub we discovered that roughly half of ORFs present anti-sense RNAs (NATs) that map to the 3‘ end of genes. Their nature is modified upon environmental changes. The regulation of NATs is basically governed by genomic sequences within the very short intragenic region of the amoeba compact genome. Secondly, we determined phenotypic differences between Entamoeba species using comparative transcriptomics approaches. The species of Entamoeba infecting humans have common NATs profiles which are different from environmental species. Altogether the data has been compiled in two recently published papers. Previous studies comparing amoebae from the same species but modified for the expression of given virulence factors or exhibiting attenuated virulence as well as from non-pathogenic Entamoeba species indicates: (I) Trophozoites progressively lose their virulence in axenic culture, virulence maintenance requires regular contact with the host. (II) Tissue destruction and survival in target organs rely on a strong adaptive response, achieved by the regulation of the expression of specific parasite genes, encoding potential virulence factors. To further understand the pathogenic process leading to amoebiasis in humans, we are asking on the role of NATs in the adaptative behaviour of E. histolytica. In collaboration with the Hub we plan to attempt to answer this question taking advantage from the tools that we have already developed for the analysis of NATs. Transcriptomes will be analysed from trophozoites growing in culture during several months and at the same time, upon incubation of these trophozoites with human cells.



Project status : In Progress

Transcriptomic comparisons of African trypanosome infections in mouse skin and blood

African trypanosomes are flagellated protist parasites transmitted to mammals by the infectious bite of the tsetse fly. They are responsible for sleeping sickness in humans and nagana in cattle. Trypanosomes first proliferate freely in the blood, and then, about six hours after being inoculated, leave the bloodstream to invade various organs, including the skin, which is an anatomic reservoir for the parasites. Few details are known on the metabolic aspects of the different parasite stages in the blood and in the skin, as well as the immune response of the mammalian host against the different stages of development of dermal parasites. Two strains of trypanosomes (“AnTat1.1E” and “Lister 427”) with different genetic profiles were studied here: one having only one developmental stage named "SL" or slender in reference to their tapered shape in mice, the second having two developmental stages "SL", and "ST" for stumpy in reference to their stocky shape in mice. This will allow us to distinguish the transcriptomic signature of parasites at the ST stage. Mice were sampled 5 days or 4 weeks after experimental infection in order to evaluate how the parasite transcriptome evolves over the course of an infection. In all these conditions, blood and skin samples were taken in order to compare the transcriptomes of blood and dermal parasites on the one hand, and transcriptomes of murine blood and dermal tissues on the other. We will especially scrutinize the metabolic pathways of the two parasite strains in the blood and the skin, as well as the immune response of the host in each compartment, two crucial elements determining the development of the infection.



Project status : In Progress