ERC FUNDED PROJECTS

This project investigates the two-way relationship between spatio-temporal genome organization and coordinated gene regulation, through an approach at the interface between physics, computer science and biology. In the nucleus, preferred positions are observed from chromosomes to single genes, in relation to normal and pathological cellular states. Evidence indicates a complex spatio-temporal coupling between co-regulated genes: e.g. certain genes cluster spatially when responding to similar factors and transcriptional noise patterns suggest domain-wide mechanisms. Yet, no individual experiment allows probing transcriptional coordination in 4 dimensions (FISH, live locus tracking, Hi-C...). Interpreting such data also critically requires theory (stochastic processes, statistical physics…). A lack of appropriate experimental/analytical approaches is impairing our understanding of the 4D genome. Our proposal combines cutting-edge single-molecule imaging, signal-theory data analysis and physical modeling to study how genes coordinate in space and time in a single nucleus. Our objectives are to understand (a) competition/recycling of shared resources between genes within subnuclear compartments, (b) how enhancers communicate with genes domain-wide, and (c) the role of local conformational dynamics and supercoiling in gene co-regulation. Our organizing hypothesis is that, by acting on their microenvironment, genes shape their co-expression with other genes. Building upon my expertise, we will use dual-color MS2/PP7 RNA labeling to visualize for the first time transcription and motion of pairs of hormone-responsive genes in real time. With our innovative signal analysis tools, we will extract spatio-temporal signatures of underlying processes, which we will investigate with stochastic modeling and validate through experimental perturbations. We expect to uncover how the functional organization of the linear genome relates to its physical properties and dynamics in 4D.

1 499 750 €

Start date: 2018-04-01, End date: 2023-03-31

The main objective of this research project is to contribute at bridging the gap between the two main branches of financial theory, namely corporate finance and asset pricing. It is motivated by the conviction that these two aspects of financial activity should and can be analyzed within a unified framework. This research will borrow from these two approaches in order to construct theoretical models that allow one to analyze the design and issuance of financial securities, as well as the dynamics of their valuations. Unlike asset pricing, which takes as given the price of the fundamentals, the goal is to derive security price processes from a precise description of firm’s operations and internal frictions. Regarding the latter, and in line with traditional corporate finance theory, the analysis will emphasize the role of agency costs within the firm for the design of its securities. But the analysis will be pushed one step further by studying the impact of these agency costs on key financial variables such as stock and bond prices, leverage, book-to-market ratios, default risk, or the holding of liquidities by firms. One of the contributions of this research project is to show how these variables are interrelated when firms and investors agree upon optimal financial arrangements. The final objective is to derive a rich set of testable asset pricing implications that would eventually be brought to the data.

1 000 000 €

Start date: 2008-11-01, End date: 2014-10-31

The human kind is witnessing an escalation of obesity-related health problems such as cardiovascular diseases and type 2 diabetes. A recent groundbreaking study revealed adiponectin receptors (ADIPOR) as key targets for treating such obesity-related diseases. Indeed, the modulation of this integral membrane protein by small molecules agonists ameliorates diabetes and prolongs lifespan of genetically obese rodent model. Despite these exciting results and the importance of ADIPOR in human physiology, there is a complete lack of knowledge of ADIPOR mechanisms of action and pharmacology. This is mainly due to the challenges associated with the characterization of membrane protein structure and function. To fill this gap of knowledge and based on my extensive experience in membrane protein biology, I propose here to characterize the the proximal signaling pathways associated with ADIPOR activation as well as the molecular and structural mechanisms of ADIPOR activation. We will develop an innovative integrated strategy combining state-of-the-art molecular and structural pharmacology approaches including 1) molecular analyses of ADIPOR network of interaction using resonance energy transfer measurement in living cells and a proteomic analysis and 2) structural analyses of ADIPOR and signaling complexes using biophysics and X-ray crystallography. Our data will have a major impact on drug discovery for treating obesity-related diseases as it will enable the application of structure-based drug design and in silico screening for the molecular control of ADIPOR activity. The proposed high-risk endeavor of obtaining structural data on these atypical membrane signaling complexes is a new direction both for my career and for the field of adiponectin biology; the exceptionally high gain from these studies fully justifies the risks; the feasibility of this project is supported by my recent success in membrane protein pharmacology, biochemistry, biophysics and crystallography.

1 989 518 €

Start date: 2015-07-01, End date: 2020-06-30

Brain and spinal cord diseases affect 38% of the European population and cost over 800 billion € annually; representing by far the largest health challenge. ALS is a prevalent neurological disease caused by motor neuron death with an invariably fatal outcome. I contributed to ALS research with the groundbreaking discovery of TDP-43 mutations, functionally characterized these mutations in the first vertebrate model and demonstrated a genetic interaction with another major ALS gene FUS. Emerging evidence indicates that four major causative factors in ALS, C9orf72, TDP-43, FUS & SQSTM1, genetically interact and could function in common cellular mechanisms. Here, I will develop zebrafish transgenic lines for all four genes, using state of the art genomic editing tools to combine simultaneous gene knockout and expression of the mutant alleles. Using these innovative disease models I will study the functional interactions amongst these four genes and their converging effect on key ALS pathogenic mechanisms: autophagy degradation, stress granule formation and RNA regulation. These studies will permit to pinpoint the molecular cascades that underlie ALS-related neurodegeneration. We will further expand the current ALS network by proposing and validating novel genetic interactors, which will be further screened for disease-causing variants and as pathological markers in patient samples. The power of zebrafish as a vertebrate model amenable to high-content phenotype-based screens will enable discovery of bioactive compounds that are neuroprotective in multiple animal models of disease. This project will increase the fundamental understanding of the relevance of C9orf72, TDP-43, FUS and SQSTM1 by developing animal models to characterize common pathophysiological mechanisms. Furthermore, I will uncover novel genetic, disease-related and pharmacological modifiers to extend the ALS network that will facilitate development of therapeutic strategies for neurodegenerative disorders

2 000 000 €

Start date: 2017-04-01, End date: 2022-03-31

DNA repair pathways evolved as an intricate network that senses DNA damage and resolves it in order to minimise genetic lesions and thus preventing tumour formation. Gaining in recognition the last few years, the alternative end-joining (alt-EJ) DNA repair pathway was recently shown to be up-regulated and required for cancer cell viability in the absence of homologous recombination-mediated repair (HR). Despite this integral role, the alt-EJ repair pathway remains poorly characterised in humans. As such, its molecular composition, regulation and crosstalk with HR and other repair pathways remain elusive. Additionally, the contribution of the alt-EJ pathway to tumour progression as well as the identification of a mutational signature associated with the use of alt-EJ has not yet been investigated. Moreover, the clinical relevance of developing small-molecule inhibitors targeting players in the alt-EJ pathway, such as the polymerase Pol Theta (Polθ), is of importance as current anticancer drug treatments have shown limited effectiveness in achieving cancer remission in patients with HR-deficient (HRD) tumours. Here, we propose a novel, multidisciplinary approach that aims to characterise the players and mechanisms of action involved in the utilisation of alt-EJ in cancer. This understanding will better elucidate the changing interplay between different DNA repair pathways, thus shedding light on whether and how the use of alt-EJ contributes to the pathogenic history and survival of HRD tumours, eventually paving the way for the development of novel anticancer therapeutics. For all the abovementioned reasons, we are convinced this project will have important implications in: 1) elucidating critical interconnections between DNA repair pathways, 2) improving the basic understanding of the composition, regulation and function of the alt-EJ pathway, and 3) facilitating the development of new synthetic lethality-based chemotherapeutics for the treatment of HRD tumours.

1 498 750 €

Start date: 2017-07-01, End date: 2022-06-30

Malaria parasite infection in humans has been called “the strongest known force for evolutionary selection in the recent history of the human genome”, and I hypothesize that a similar statement may apply to the mosquito vector, which is the definitive host of the malaria parasite. We previously discovered efficient malaria-resistance mechanisms in natural populations of the African malaria vector, Anopheles gambiae. Aim 1 of the proposed project will implement a novel genetic mapping design to systematically survey the mosquito population for common and rare genetic variants of strong effect against the human malaria parasite, Plasmodium falciparum. A product of the mapping design will be living mosquito families carrying the resistance loci. Aim 2 will use the segregating families to functionally dissect the underlying molecular mechanisms controlled by the loci, including determination of the pathogen specificity spectra of the host-defense traits. Aim 3 targets arbovirus transmission, where Anopheles mosquitoes transmit human malaria but not arboviruses such as Dengue and Chikungunya, even though the two mosquitoes bite the same people and are exposed to the same pathogens, often in malaria-arbovirus co-infections. We will use deep-sequencing to detect processing of the arbovirus dsRNA intermediates of replication produced by the RNAi pathway of the mosquitoes. The results will reveal important new information about differences in the efficiency and quality of the RNAi response between mosquitoes, which is likely to underlie at least part of the host specificity of arbovirus transmission. The 3 Aims will make significant contributions to understanding malaria and arbovirus transmission, major global public health problems, will aid the development of a next generation of vector surveillance and control tools, and will produce a definitive description of the major genetic factors influencing host-pathogen interactions in mosquito immunity.

2 307 800 €

Start date: 2013-03-01, End date: 2018-02-28

RNA interference (RNAi) is a conserved sequence-specific, gene-silencing mechanism that is induced by double-stranded RNA (dsRNA). One of the functions of this pathway is the defense against parasitic nucleic acids: transposons and viruses. Previous results demonstrated that viral infections in Drosophila melanogaster are fought by an antiviral RNAi response and that components of the endocytic pathway are required for dsRNA entry to initiate the RNAi response. Recently we have shown that infected insect cells spread a systemic silencing signal that elicits a protective RNAi-dependent immunity throughout the organism. This suggests that the cell-autonomous RNAi response is insufficient to control a viral infection and that flies also rely on systemic immune response to fight against such infections. As a junior group leader, I will study the mechanisms that mediate the RNAi-based antiviral response in insects. By combining biochemical, cellular, molecular and genomic approaches, both in vivo and in cell culture, I will analyze the mechanisms underlying viral tropism, systemic propagation of the antiviral signal and the basis of the persistence of the antiviral state. Furthermore, I will examine whether the dsRNA-uptake pathway is conserved in mosquitoes and its relationship with viral immunity in that host. This comprehensive approach will tackle how this nucleic acid-based immunity works in insects to generate an anti-viral stage. A better understanding of the role of RNA silencing in insects during virus infection will allow the exploitation of this pathway for improvement of public health related problems such as arbovirus infection and disease.

1 900 000 €

Start date: 2009-10-01, End date: 2014-12-31

Arboviruses infect millions of people each year, however, mechanisms that drive viral emergence and maintenance remain largely unknown. A combination of host factors (e.g., human mobility), mosquito factors (e.g., abundance) and viral factors (e.g., transmissibility) interconnect to drive spread. Further, for endemic arboviruses, complex patterns of population immunity, built up over many years, appear key to the emergence of particular lineages. To disentangle the contribution of these different drivers, we need detailed data from the same pathogen system over a long time period from the same location. In addition, we need new methods, which can integrate these different data sources and allow appropriate mechanistic inferences. In this project, I will use the most globally prevalent arbovirus, dengue virus, as a case study. I will focus on Thailand where all four dengue serotypes have circulated endemically for decades and excellent long-term data and isolates exist, to address two fundamental questions: i) How do population-level patterns of immunity evolve over time and what is their impact on strain dynamics? I will use mechanistic models applied to historic serotype-specific case data to reconstruct the evolving immune profile of the population and explore the impact of immunity on viral diversity using sequences from archived isolates from each year over a 50-year period. ii) How do human behaviors, vector densities interact with immunity to dictate spread? I will work with geolocated full genome sequences from across Thailand and use detailed data on how people move, their contact patterns, their immunity profiles and mosquito distributions to study competing hypotheses of how arboviruses spread. I will compare the key drivers of dengue spread with that found for outbreaks of Zika and chikungunya. This proposal addresses fundamental questions about the mechanisms that drive arboviral emergence and spread that will be relevant across disease systems.

1 499 896 €

Start date: 2019-01-01, End date: 2023-12-31

Brain information processing is commonly thought to be a neuronal performance. However recent data point to a key role of astrocytes in brain development, activity and pathology. Indeed astrocytes are now viewed as crucial elements of the brain circuitry that control synapse formation, maturation, activity and elimination. How do astrocytes exert such control is matter of intense research, as they are now known to participate in critical developmental periods as well as in psychiatric disorders involving synapse alterations. Thus unraveling how astrocytes control synaptic circuit formation and maturation is crucial, not only for our understanding of brain development, but also for identifying novel therapeutic targets. We recently found that connexin 30 (Cx30), an astroglial gap junction subunit expressed postnatally, tunes synaptic activity via an unprecedented non-channel function setting the proximity of glial processes to synaptic clefts, essential for synaptic glutamate clearance efficacy. Our work not only reveals Cx30 as a key determinant of glial synapse coverage, but also extends the classical model of neuroglial interactions in which astrocytes are generally considered as extrasynaptic elements indirectly regulating neurotransmission. Yet the molecular mechanisms involved in such control, its dynamic regulation by activity and impact in a native developmental context are unknown. We will now address these important questions, focusing on the involvement of this novel astroglial function in wiring developing synaptic circuits. Thus using a multidisciplinary approach we will investigate: 1) the molecular and cellular mechanisms underlying Cx30 regulation of synaptic function 2) the activity-dependent dynamics of Cx30 function at synapses 3) a role for Cx30 in wiring synaptic circuits during critical developmental periods This ambitious project will provide essential knowledge on the molecular mechanisms underlying astroglial control of synaptic circuits.

2 000 000 €

Start date: 2016-10-01, End date: 2021-09-30

B cells are the main actors of successful vaccines, and their protective capacity relies on several subsets with innate-like and memory properties that fulfill different effector functions. In the present project, we wish to develop approaches in both mice and humans, to confront the similarities and the differences of their B cell responses. The three aims proposed are: 1) To study the different B cell subsets and TFH cells engaged in a memory response through the use of a new mouse reporter line allowing their irreversible labeling (inducible Cre recombinase under the control of the Bcl6 gene): this will be performed in different conditions of TH1 vs. TH2 polarization, as well as during a chronic viral infection, in which virus-specific antibodies have been shown to be required to control the disease (in collaboration with D. Pinschewer, Basel) 2) To study whether the lifelong persistence of B cell memory, as occurs for memory B cells against smallpox that we can obtain at high purity from aged donor's spleens, corresponds to a specific transcriptional program at the miRNA, lncRNA or mRNA level, as well as a specific cell homeostasis 3) To discriminate the specific effector function of human marginal zone and IgM memory B cells in, respectively, T-independent and T-dependent responses, as well as their specific differentiation/diversification pathway. The general goal is to delineate the regulatory pathways leading to the activation and persistence of the different B cell subsets, allowing for a better understanding of the conditions leading to their pathological or beneficial mobilization.

2 098 750 €

Start date: 2016-09-01, End date: 2021-08-31

In bacteria, the though external cell wall and the intracellular actin-like (MreB) cytoskeleton are major determinants of cell shape. The biosynthetic pathways and chemical composition of the cell wall, a three dimensional polymer network that is one of the most prominent targets for antibiotics, are well understood. However, despite decades of study, little is known about the complex cell wall ultrastructure and the molecular mechanisms that control cell wall morphogenesis in time and space. In rod-shaped bacteria, MreB homologues assemble into dynamic structures thought to control shape by serving as organizers for the movement and assembly of macromolecular machineries that effect sidewall elongation. However, the mechanistic details used by the MreB cytoskeleton to fulfil this role remain to be elucidated. Furthermore, development of high-resolution microscopy techniques has led to new breakthroughs this year, published by our lab and others, which are shaking the model developed over the last decade and re-questioning the MreB “actin cytoskeleton” designation. The aim of this project is to combine powerful genetic, biochemical, genomic and systems biology approaches available in the model bacterium Bacillus subtilis with modern high-resolution light microscopic techniques to study the dynamics and mechanistic details of the MreB cytoskeleton and of CW assembly. Parameters measured by the different approaches will be combined to quantitatively describe the features of bacterial cell morphogenesis.

1 650 050 €

Start date: 2013-02-01, End date: 2019-01-31

Regulation of gene expression plays a key role in the ability of bacteria to rapidly adapt to changing environments and to colonize extremely diverse habitats. The relatively recent discovery of a plethora of small regulatory RNAs and the beginning of their characterization has unravelled new aspects of bacterial gene expression. First, the expression of many bacterial genes responds to a complex network of both transcriptional and post-transcriptional regulators. However, the properties of the resulting regulatory circuits on the dynamics of gene expression and in the bacterial adaptive response have been poorly addressed so far. In a first part of this project, we will tackle this question by characterizing the circuits that are formed between two widespread classes of bacterial regulators, the sRNAs and the two-component systems, which act at the post-transcriptional and the transcriptional level, respectively. The study of sRNAs also led to major breakthroughs regarding the basic mechanisms of gene expression. In particular, we recently showed that repressor sRNAs can target activating stem-loop structures located within the coding region of mRNAs that promote translation initiation, in striking contrast with the previously recognized inhibitory role of mRNA structures in translation. The second objective of this project is thus to draw an unprecedented map of non-canonical translation initiation events and their regulation by sRNAs. Overall, this project will greatly improve our understanding of how bacteria can so rapidly and successfully adapt to many different environments, and in the long term, provide clues towards the development of anti-bacterial strategies.

1 999 754 €

Start date: 2019-09-01, End date: 2024-08-31

The functioning of a single cell or organism is governed by the laws of chemistry and physics. The bridge from biology to chemistry and physics is provided by structural biology: to understand the functioning of a cell, it is necessary to know the atomic structure of macromolecular assemblies, which may contain hundreds of components. To characterise the structures of the increasingly large and often flexible complexes, high resolution structure determination (as was possible for example for the ribosome) will likely stay the exception, and multiple sources of structural data at multiple resolutions are employed. Integrating these data into one consistent picture poses particular difficulties, since data are much more sparse than in high resolution methods, and the data sets from heterogeneous sources are of highly different and unknown quality and may be mutually inconsistent, and that data are in general averaged over large ensembles and long times. Molecular modelling, a crucial element of any structure determination, plays an even more important role in these multi-scale and multi-technique approaches, not only to obtain structures from the data, but also to evaluate their reliability. This proposal is to develop a consistent framework for this highly complex data integration problem, principally based on Bayesian probability theory. Appropriate models for the major types data types used in hybrid approaches will be developed, as well as representations to include structural knowledge for the components of the complexes, at multiple scales. The new methods will be applied to a series of problems with increasing complexity, going from the determination of protein complexes with high resolution information, over low resolution structures based on protein-protein interaction data such as the nuclear pore, to the genome organisation in the nucleus.

2 130 212 €

Start date: 2012-05-01, End date: 2017-04-30

Diabetes has become one of the most widespread metabolic disorders with epidemic dimensions affecting almost 6% of the world’s population. Despite modern treatments, the life expectancy of patients with Type 1 diabetes remains reduced as compared to healthy subjects. There is therefore a need for alternative therapies. Towards this aim, using the mouse, we recently demonstrated that the in vivo forced expression of a single factor in pancreatic alpha-cells is sufficient to induce a continuous regeneration of alpha-cells and their subsequent conversion into beta-like cells, such converted cells being capable of reversing the consequences of chemically-induced diabetes in vivo (Collombat et al. Cell, 2009). The PI and his team therefore propose to further decipher the mechanisms involved in this alpha-cell-mediated beta-cell regeneration process and determine whether this approach may be applied to adult animals and whether it would efficiently reverse Type 1 diabetes. Furthermore, a major effort will be made to verify whether our findings could be translated to human. Specifically, we will use a tri-partite approach to address the following issues: (1) Can the in vivo alpha-cell-mediated beta-cell regeneration be induced in adults mice? What would be the genetic determinants involved? (2) Can alpha-cell-mediated beta-cell regeneration reverse diabetes in the NOD Type 1 diabetes mouse model? (3) Can adult human alpha-cells be converted into beta-like cells? Together, these ambitious objectives will most certainly allow us to gain new insight into the mechanisms defining the identity and the reprogramming capabilities of mouse and human endocrine cells and may thereby open new avenues for the treatment of diabetes. Similarly, the determination of the molecular triggers implicated in the beta-cell regeneration observed in our diabetic mice may lead to exciting new findings, including the identification of “drugable” targets of importance for human diabetic patients.

1 500 000 €

Start date: 2012-01-01, End date: 2016-12-31

Bacterial biofilm formation is a paramount developmental process in both Gram-positive and Gram-negative species and in many pathogens has been associated with processes of horizontal gene transfer, antibiotic resistance development and pathogen persistence. Bacterial biofilms are collaborative sessile macrocolonies embedded in complex extracellular matrix that secures both mechanical resistance and a medium for intercellular exchange. Biogenesis platforms for the secretion of biofilm matrix components - many of which controlled directly or indirectly by the intracellular second messenger c-di-GMP - are important determinants for biofilm formation and bacterial disease, and therefore present compelling targets for the development of novel therapeutics. During my Ph.D. and post-doctoral work I studied the structure and function of c-di-GMP-sensing protein factors controling extracellular matrix production by DNA-binding at the transcription initiation level or by inside-out signalling mechanisms at the cell envelope, as well as membrane exporters involved directly in downstream matrix component secretion. Here, I propose to apply my expertise in microbiology, protein science and structural biology to study the structure and function of exopolysaccharide secretion systems in Gram-negative species. Using Pseudomonas aeruginosa, Vibrio spp. and Escherichia coli as model organisms, my team will aim to reveal the global architecture and individual building components of several expolysaccharide-producing protein megacomplexes. We will combine X-ray crystallography, biophysical and biochemical assays, electron microscopy and in cellulo functional studies to provide a comprehensive view of extracellular matrix production that spans the different resolution levels and presents molecular blueprints for the development of novel anti-infectives. Over the last year I have laid the foundation of these studies and have demonstrated the overall feasibility of the project.

1 499 901 €

Start date: 2018-08-01, End date: 2023-07-31

Biodiversity loss is one of the greatest environmental challenges of our time. There is mounting evidence that biodiversity increases the stability of ecosystem functions and services, suggesting that it may be critical to the sustainability of ecosystems and human societies in the face of environmental changes. Classical ecological theory, however, has focused on measures of stability that cannot explain and predict these stabilizing effects, especially in spatial systems. The goal of BIOSTASES is to develop a coherent body of new theory on the stability of ecosystems and coupled social–ecological systems and its relationships with biodiversity at multiple spatial scales that can better inform empirical research. BIOSTASES will reach this goal through four complementary objectives. First, it will propose a mathematical framework focused on temporal variability as an empirically relevant measure of stability, and use this framework to build robust early warning signals for critical transitions. Second, it will use dynamical metacommunity models to explore a wide range of novel questions related to ecosystem stability and diversity–stability relationships across scales. Third, it will study the stability of complex meta-ecosystems to provide new perspectives on the stability of food webs and on synergies and trade-offs between multiple ecosystem services across space. Fourth, it will develop novel theory to study the long-term dynamics and sustainability of coupled social–ecological systems. BIOSTASES proposes an ambitious innovative research programme that will provide new perspectives on the stability and sustainability of ecological and coupled social–ecological systems in the face of environmental changes. It will contribute to bridging the gaps between theoretical and empirical ecology and between ecology and social sciences, and to developing new approaches in biodiversity conservation, landscape management, and sustainable development.

2 092 644 €

Start date: 2015-09-01, End date: 2020-08-31

My lab is interested in the development of the tissue that gives rise to vertebrae and skeletal muscles called the paraxial mesoderm. A striking feature of this tissue is its segmental organization and we have made major contributions to the understanding of the molecular control of the segmentation process. We identified a molecular oscillator associated to the rhythmic production of somites and proposed a model for vertebrate segmentation based on the integration of a rhythmic signaling pulse gated spatially by a system of traveling FGF and Wnt signaling gradients. We are also studying the differentiation of paraxial mesoderm precursors into the muscle, cartilage and dermis lineages. Our work identified the Wnt, FGF and Notch pathways as playing a prominent role in the patterning and differentiation of paraxial mesoderm. In this application, we largely focus on the molecular control of paraxial mesoderm development. Using microarray and high throughput sequencing-based approaches and bioinformatics, we will characterize the transcriptional network acting downstream of Wnt, FGF and Notch in the presomitic mesoderm (PSM). We will also use genetic and pharmacological approaches utilizing real-time imaging reporters to characterize the pacemaker of the segmentation clock in vivo, and also in vitro using differentiated embryonic stem cells. We further propose to characterize in detail a novel RA-dependent pathway that we identified and which controls the somite left-right symmetry. Our work is expected to have a strong impact in the field of congenital spine anomalies, currently an understudied biomedical problem, and will be of utility in elucidating the etiology and eventual prevention of these disorders. This work is also expected to further our understanding of the Notch, Wnt, FGF and RA signalling pathways which are involved in segmentation and in the establishment of the vertebrate body plan, and which play important roles in a wide array of human diseases.

2 500 000 €

Start date: 2010-04-01, End date: 2015-03-31

We and others have recently delineated the molecular architecture of a new feedback pathway in brain synapses, which operates as a synaptic circuit breaker. This pathway is supposed to use a group of lipid messengers as retrograde synaptic signals, the so-called endocannabinoids. Although heterogeneous in their chemical structures, these molecules along with the psychoactive compound in cannabis are thought to target the same effector in the brain, the CB1 receptor. However, the molecular catalog of these bioactive lipids and their metabolic enzymes has been expanding rapidly by recent advances in lipidomics and proteomics raising the possibility that these lipids may also serve novel, yet unidentified physiological functions. Thus, the overall aim of our research program is to define the molecular and anatomical organization of these endocannabinoid-mediated pathways and to determine their functional significance. In the present proposal, we will focus on understanding how these novel pathways regulate synaptic and extrasynaptic signaling in hippocampal neurons. Using combination of lipidomic, genetic and high-resolution anatomical approaches, we will identify distinct chemical species of endocannabinoids and will show how their metabolic enzymes are segregated into different subcellular compartments in cell type- and synapse-specific manner. Subsequently, we will use genetically encoded gain-of-function, loss-of-function and reporter constructs in imaging experiments and electrophysiological recordings to gain insights into the diverse tasks that these new pathways serve in synaptic transmission and extrasynaptic signal processing. Our proposed experiments will reveal fundamental principles of intercellular and intracellular endocannabinoid signaling in the brain.

1 638 000 €

Start date: 2009-11-01, End date: 2014-10-31

The brain is an extraordinary complex assembly of neuronal and glial cells that underpins cognitive functions. How adequate numbers of these cells are generated by neural stem cells in embryonic and early postnatal development and how they distribute and interconnect within brain tissue is still debated. In particular, the potentialities of individual neural stem cells, their potential heterogeneity and the mechanisms regulating their function are still poorly characterized in situ; likewise, the clonal architecture of mature brain tissue and its influence on neural circuitry are only partially explored. Deciphering these aspects is essential to link neural circuit development, structure and function, and to understand the aetiology of neurodevelopmental disorders. We have recently established transgenic strategies to simultaneously track the lineage of multiple individual neural stem cells in the intact developing brain and experimentally perturb their development. We will use these approaches in combination with recent large-volume imaging methods for high-throughput analysis of individual neural and glial clones in the mouse cortex. This will allow us to assay neural progenitor potentialities and equivalence, characterize developmental changes occurring in the neurogenic niche, describe the clonal organization of the mature cortex and study its link with neural connectivity. To decipher intrinsic and extrinsic mechanisms regulating neural progenitor activity and understand how they produce appropriate numbers of cells, we will assay the outcome of functional perturbations targeting key steps of neural development, introduced in precursors or in their local environment. These experiments will reveal how neural stem cell output might be regulated by cell interactions and intercellular signals. This multidisciplinary project will set the basis for quantitative analysis of brain development with single-cell resolution in normal and pathological conditions.

1 929 713 €

Start date: 2015-07-01, End date: 2020-06-30

Intercellular communication is critical for multicellularity. It coordinates the activities within individual cells to support the function of an organism as a whole. Plants have developed remarkable cellular machines -the Plasmodesmata (PD) pores- which interconnect every single cell within the plant body, establishing direct membrane and cytoplasmic continuity, a situation unique to plants. PD are indispensable for plant life. They control the flux of molecules between cells and are decisive for development, environmental adaptation and defence signalling. However, how PD integrate signalling to coordinate responses at a multicellular level remains unclear. A striking feature of PD organisation, setting them apart from animal cell junctions, is a strand of endoplasmic reticulum (ER) running through the pore, tethered extremely tight (~10nm) to the plasma membrane (PM) by unidentified “spokes”. To date, the function of ER-PM contacts at PD remains a complete enigma. We don’t know how and why the two organelles come together at PD cellular junctions. I recently proposed that ER-PM tethering is in fact central to PD function. In this project I will investigate the question of how integrated cellular responses benefit from organelle cross-talk at PD. The project integrates proteomic/bioinformatic approaches, biophysical/modelling methods and ultra-high resolution 3D imaging into molecular cell biology of plant cell-to-cell communication and will, for the first time, directly address the mechanism and function of ER-PM contacts at PD. We will pursue three complementary objectives to attain our goal: 1) Identify the mechanisms of PD membrane-tethering at the molecular level 2) Elucidate the dynamics and 3D architecture of ER-PM contact sites at PD 3) Uncover the function of ER-PM apposition for plant intercellular communication. Overall, the project will pioneer a radically new perspective on PD-mediated cell-to-cell communication, a fundamental aspect of plant biology

1 999 840 €

Start date: 2018-06-01, End date: 2023-05-31

Synaptic scaffolding molecules control the localization and the abundance of neurotransmitter receptors at the synapse, a key parameter to shape synaptic transfer function. Most characterized synaptic scaffolds are intracellular, yet a growing number of secreted proteins appear to organize the synapse from the outside of the cell. We recently demonstrated in C. elegans that an evolutionarily conserved protein secreted by motoneurons specifies the excitatory versus inhibitory identity of the postsynaptic domains at neuromuscular synapses. We propose to use this system as a genetically tractable paradigm to perform a comprehensive characterization of this unforeseen synaptic organization. Specifically, this project will pursue 4 complementary aims: 1) Identify and characterize a comprehensive set of genes that organize and control the formation and maintenance of these scaffolds through a series of genetic screens based on the direct visualization of fluorescent acetylcholine and GABA receptors in living animals. 2) Solve the spatial synaptic organization of these scaffolds at a nanoscale resolution using super-resolutive and correlative light and electron microscopy, and analyze their dynamic behavior in vivo by implementing Single Particle Tracking imaging in living worms. 3) Decipher the role of the synaptomatrix in the organization of synaptic extracellular scaffolds and evaluate its functional contribution at the physiological and molecular levels using a candidate gene strategy and innovative imaging. 4) Analyze the formation and decline of these scaffolds at the lifetime scale and evaluate the role of synaptic activity and aging in these processes by taking advantage of the possibility to follow identified synapses over the entire life of C. elegans. Using powerful genetics in combination with cutting-edge in vivo imaging and electrophysiology, we anticipate to identify new genes and new mechanisms at work to regulate normal and pathological synaptic function.

2 492 750 €

Start date: 2016-10-01, End date: 2021-09-30

This proposal aims to investigate the role of cytosolic DNA sensing pathways in CD4 T cell differentiation. Cellular host defense to pathogens relies on the detection of pathogen-associated molecular patterns including deoxyribonucleic acid (DNA), which can be recognized by host myeloid cells through Toll-like receptor (TLR) 9 binding. Recent evidence however suggests that innate immune cells can also perceive cytoplasmic DNA from infectious or autologous origin through cytosolic DNA sensors triggering TLR9-independent signaling. Activation of cytosolic DNA sensor-dependent signaling pathways has been clearly shown to trigger innate immune responses to microbial and host DNA, but the contribution of cytosolic DNA sensors to the differentiation of CD4 T cells, an essential event for shaping adaptive immune responses, has not been documented. This proposal aims to fill this current knowledge gap. We aim to decipher the molecular series of transcriptional events triggered by DNA in CD4 T cells that ultimately result in altered T cell differentiation. This aim will be addressed by combining in vitro and in vivo approaches such as advanced gene expression analysis of CD4 T cells and use of transgenic and gene-deficient mice. Structure activity relationship and biophysical studies will also be performed to unravel novel immunomodulators able to affect CD4 T cell differentiation.

1 500 000 €

Start date: 2016-08-01, End date: 2021-07-31

The eukaryotic genome is packaged into large-scale chromatin structures that occupy distinct domains in the nucleus and this organization is now seen as a key contributor to genome functions. Two key functions of the genome can take advantage of nuclear organization: regulated gene expression and the propagation of a stable genome. To understand these fundamental processes, we have chosen to use yeast as a model system that allows genetics, molecular biology and advanced live microscopy approaches to be combined. Budding yeast have been very powerful to demonstrate that gene position can play an active role in regulating gene expression. Distinct subcompartments dedicated to either gene silencing or activation of specific genes are positioned at the nuclear periphery. To gain insight into the mechanisms underlying this sub-compartmentalization, we will address three complementary issues: - What are the mechanisms involved in the establishment and maintenance of silent nuclear compartments? - How and why are some activated genes recruited to the nuclear periphery? - What are the relationships between repressive and activating nuclear compartments? Concerning the maintenance of genome integrity, recent advances in yeast highlight the importance of nuclear architecture. However, how nuclear organization influences the formation and processing of DNA lesions remain poorly understood. We will focus on two main questions: - How and where in the nucleus are double strand breaks recognized, processed, and repaired? - Where do breaks or gaps resulting from replicative stress at 'fragile sites' arise in the nucleus and how does nuclear organization influence their stability? We hope to gain a better understanding of the mechanisms presiding nuclear organization and its importance for genome functions. These mechanisms are likely to be conserved and will be subsequently tested in higher eukaryotic cells.

1 000 000 €

Start date: 2008-09-01, End date: 2014-05-31

Faithful chromosome segregation in all eukaryotes relies on centromeres, the chromosomal sites that recruit kinetochore proteins and mediate spindle attachment during cell division. Fundamental to centromere function is a histone H3 variant, CenH3, that initiates kinetochore assembly on centromeric DNA. CenH3 is conserved throughout most eukaryotes; its deletion is lethal in all organisms tested. These findings established the paradigm that CenH3 is an absolute requirement for centromere function. My recent findings undermined this paradigm of CenH3 essentiality. I showed that CenH3 was lost independently in four lineages of insects. These losses are concomitant with dramatic changes in their centromeric architecture, in which each lineage independently transitioned from monocentromeres (where microtubules attach to a single chromosomal region) to holocentromeres (where microtubules attach along the entire length of the chromosome). Here, I aim to characterize this unique CenH3-deficient chromosome segregation pathway. Using proteomic and genomic approaches in lepidopteran cell lines, I will determine the mechanism of CenH3-independent kinetochore assembly that led to the establishment of their holocentric architecture. Using comparative genomic approaches, I will determine whether this kinetochore assembly pathway has recurrently evolved over the course of 400 million years of evolution and its impact on the chromosome segregation machinery. My discovery of CenH3 loss in holocentric insects establishes a new class of centromeres. My research will reveal how CenH3 that is essential in most other eukaryotes, could have become dispensable in holocentric insects. Since the evolution of this CenH3-independent chromosome segregation pathway is associated with the independent rises of holocentric architectures, my research will also provide the first insights into the transition from a monocentromere to a holocentromere.

1 497 500 €

Start date: 2018-04-01, End date: 2023-03-31

Cell polarity is fundamental to many aspects of cell and developmental biology and it is implicated in differentiation, proliferation and morphogenesis in both unicellular and multi-cellular organisms. We study the mechanisms that regulate cell polarity during both asymmetric cell division and epithelial cell polarization in Drosophila. To understand these fundamental processes, we are currently using two complementary approaches. Firstly, we are coupling genetic tools to state of the art time-lapse microscopy to genetically dissect the mechanisms of cortical cell polarization and mitotic spindle orientation. Secondly, we are introducing two innovative inter-disciplinary methodologies into the fields of cell and developmental biology: 1) single molecule imaging during asymmetric cell division, to unravel the mechanism of polarized protein distribution within the cell; 2) multi-scale tensor analysis of epithelial tissues to describe and understand how epithelial tissues grow, acquire and maintain their shape and organization during development. Using both conventional and innovative methodologies, our goals over the next four years are to better understand how molecules and protein complexes move and are activated at different locations within the cell and how cell polarization impacts on cell identities and on epithelial tissue growth and morphogenesis. Since the mechanisms underlying cell polarization are conserved throughout evolution, the proposed experiments will improve our understanding of these processes not only in Drosophila, but in all animals.

1 159 000 €

Start date: 2008-09-01, End date: 2013-08-31

Cellular respiration provides energy to power essential processes of life. Respiratory complexes are macromolecular batteries coupling electron flow through a wire of metal clusters and cofactors with proton transfer across the inner membrane of mitochondria and bacteria. Waste products of these cellular factories are reactive oxygen species causing ageing and diseases. Assembly and maturation mechanisms of respiratory complexes remain enigmatic because of their membrane location, multisubunit composition and cofactor insertion. E. coli Complex I, one of the largest membrane proteins, composed of 14 conserved subunits with 9 Fe/S clusters and a flavin, is a minimal model for its 45-subunit human homologue. When proton pumping by respiratory complexes is affected, bacteria become resistant to antibiotics requiring proton gradient for uptake. Based on the latest genetic data, we realize that the huge E. coli macromolecular cage, the structure of which we recently solved by cryo-electron microscopy (cryoEM), in conjunction with a novel protein cofactor, is a specific chaperone for Fe/S cluster biogenesis and assembly of respiratory complexes. This integrated multidisciplinary project combines cryoEM and other structural, biophysical and spectroscopic techniques, to uncover the functional mechanism of this emerging chaperone. The structural plasticity of the chaperone fuelled by ATP hydrolysis, and its interaction with Fe/S cluster biogenesis systems and the main respiratory complexes as a function of stresses, will be scrutinized to gain quasiatomic insights into the way the chaperone operates on its substrates. A novel technology for synergetic in situ investigation of protein complexes in the bacterial cytoplasm by optical imaging, state-of-the-art cryogenic correlative light and electron microscopy, and subtomogram analysis, will be developed and used to obtain snapshots of the chaperone-substrate interactions in the cellular context.

1 999 956 €

Start date: 2015-10-01, End date: 2020-09-30

Neuromodulators such as acetylcholine and dopamine are able to rapidly reprogram neuronal information processing and dynamically change brain states. Degeneration or dysfunction of cholinergic and dopaminergic neurons can lead to neuropsychiatric conditions like schizophrenia and addiction or cognitive diseases such as Alzheimer’s. Neuromodulatory systems control overlapping cognitive processes and often have similar modes of action; therefore it is important to reveal cooperation and competition between different systems to understand their unique contributions to cognitive functions like learning, memory and attention. This is only possible by direct comparison, which necessitates monitoring multiple neuromodulatory systems under identical experimental conditions. Moreover, simultaneous recording of different neuromodulatory cell types goes beyond phenomenological description of similarities and differences by revealing the underlying correlation structure at the level of action potential timing. However, such data allowing direct comparison of neuromodulatory actions are still sparse. As a first step to bridge this gap, I propose to elucidate the unique versus complementary roles of two “classical” neuromodulatory systems, the cholinergic and dopaminergic projection system implicated in various cognitive functions including associative learning and plasticity. First, we will record optogenetically identified cholinergic and dopaminergic neurons simultaneously using chronic extracellular recording in mice undergoing classical and operant conditioning. Second, we will determine the postsynaptic impact of cholinergic and dopaminergic neurons by manipulating them both separately and simultaneously while recording consequential changes in cortical neuronal activity and learning behaviour. These experiments will reveal how major neuromodulatory systems interact to mediate similar or different aspects of the same cognitive functions.

1 499 463 €

Start date: 2017-05-01, End date: 2022-04-30

How variations in whole chromosome number impact organism homeostasis remains an open question. Variations to the normal euploid genome content are frequently found in healthy animals and are thought to contribute with phenotypic variability in adverse situations. Yet they are also at the basis of several human diseases, including neuro-developmental disorders and cancer. Our preliminary data shows that physiological aneuploidy can be identified in certain cells during development. Moreover, we have observed that when induced through mutations, non-euploid cells are effectively eliminated from the cycling population. A quantitative view of the frequency of non-euploid karyotypes and the mechanisms underlying their genesis is lacking in the literature. Further, the tissue specific responses at play to eliminate non-euploid cells, when induced through mutations are not understood. The objectives of this proposal are to quantitatively assess the occurrence of physiological chromosome number variations gaining insight into mechanisms involved in generating it. Additionally, we will identify the tissue-specific pathways involved in maintaining organism homeostasis through the elimination of non-euploid cells. We will use a novel genetic approach to monitor individual chromosome loss at the level of the entire organism, combine it with quantitative methods and state-of-the art-microscopy, and focus on two model organisms - Drosophila and mouse - during development and adulthood. We predict that the findings resulting from this proposal will significantly impact the fields of cell, developmental and animal physiology, generating novel concepts that will bridge the existing gaps in the field, and expand our understanding of the links between karyotype variations, animal development and disease establishment.

2 000 000 €

Start date: 2017-07-01, End date: 2022-06-30

The immune repertoire of healthy individuals contains a fraction of antibodies (Abs) that are able to bind with high affinity various endogenous or exogenous low molecular weight compounds, including cofactors essential for the aerobic life, such as riboflavin, heme and ATP. Despite identification of cofactor-binding Abs as a constituent of normal immune repertoires, their fundamental characteristics and have not been systematically investigated. Thus, we do not know the origin, prevalence and physiopathological significance of cofactor-binding Abs. Moreover, the molecular mechanisms of interaction of cofactors with Abs are ill defined. Different proteins use cofactors to extend the chemistry intrinsic to the amino acid sequence of their polypeptide chain(s). Thus, one can speculate that the alliance of Abs with low molecular weight compounds results in the emergence of untypical properties of Abs and offers a strategy for designing a new generation of therapeutic Abs. Moreover, cofactor-binding Abs may be used for delivery of cytotoxic compounds to particular sites in the body, or for scavenging pro-inflammatory compounds. The principal goal of the present proposal is to gain a basic understanding on the fraction of cofactor-binding Abs in immune repertoires and to use this knowledge for the rational design of novel classes of therapeutic Abs. In this project, we will address the following questions: 1) understand the origin and prevalence of cofactor-binding Abs in immune repertoires; 2) characterize the molecular mechanisms of interaction of cofactors with Abs; 3) Understand the physiopathological roles of cofactor-binding Abs, and 4) use cofactor binding for the development of novel types of therapeutic Abs. A comprehensive understanding of various aspects of cofactor-binding Abs should lead to advances in fundamental understanding and in the development of innovative therapeutic and diagnostic tools.

1 255 000 €

Start date: 2016-09-01, End date: 2021-08-31

The application's unifying theme is cognition. Any decision reflects the information that comes to the decision-maker's awareness at the moment of making the decision. In turn, this information is the stochastic outcome of a sequence of more or less conscious choices and of awareness manipulation by third parties. The three parts of this application all are concerned with two factors of limited awareness (cognitive costs and motivated beliefs) and with the application of imperfect cognition to economics. The various projects can be subsumed into three themes, each with different subprojects: 1. Self-serving beliefs, laws, norms and taboos (expressive function of the law, taboos, dignity and contracts). 2. Cognition, markets, and contracts (mechanism design under costly cognition, directing attention in markets and politics). 3. Cognition and individual decision-making (foundations of some non-standard preferences). The methodology for this research will be that of formal economic modeling and welfare analysis, enriched with important insights from psychology and sociology. It will also include experimental (laboratory) investigations. The output will first take the form of a series of articles in economics journals, as well as, for the research described in Part 1, a book to disseminate the research to broader, multidisciplinary and non-specialized audiences.

1 910 400 €

Start date: 2010-04-01, End date: 2016-03-31

Early diagnostics based on multiple biomarkers is key in numerous diseases, yet current technologies for multiplexed detection are complicated and expensive. Living cells detect and process various environmental signals in parallel and can self-replicate, presenting an attractive platform for scalable and affordable autonomous diagnostic devices. In this project, I will apply my expertise in synthetic biology, the rational engineering of biological systems, to build cell-based biosensors for multiplexed diagnosis using the non-pathogenic bacterium Bacillus subtilis. In a first research line, I will conceive a scalable detection machinery by engineering chimeric receptors detecting extracellular biomarkers via sensing domains derived from antibodies. In a second research line, I will implement bio-molecular computing systems operating within and across bacterial cells to perform multiplexed biomarkers analysis. I will deploy in B. subtilis biomolecular logic gates and will engineer specific cell-cell communication systems to perform distributed multicellular computation in a bacterial consortia. My project is highly interdisciplinary and is at the cross-roads of genetic engineering, structural biology, biophysics, modeling, and clinics. On foundational point of view, I will make several breakthrough contributions to synthetic biology: (i) Advancing engineering frameworks for the Gram-positive model, B. subtilis. (ii) Pushing the limits of custom-ligand detection by engineered cells (iii) Exploring the frontiers of man-made biological computers. On an applied point of view, I plan to deliver a first prototype for the urinary diagnostic of diabetic nephropathy, a major complication of diabetes. Because of the modular design principles applied, my sensing platform will be reusable to diagnose other pathologies as well as for applications requiring custom-detection and bio-molecular computation like targeted therapy, drug delivery, or environmental monitoring.

1 500 000 €

Start date: 2015-05-01, End date: 2020-04-30

Despite its widespread occurrence across the tree of life, many questions still remain unanswered concerning the fascinating phenomenon of convergent evolution. Ant-eating mammals constitute a textbook example of morphological convergence with at least five independent origins in placentals (armadillos, anteaters, aardvarks, pangolins, and aardwolves). The large extent of convergent morphological evolution, the importance of molecular convergence, and the role of the host microbiome in diet adaptation are currently gaining acceptance. However, large-scale comparative studies combining morphology, host genomics, and metagenomics of the associated microbiome are still lacking. In the ConvergeAnt project, we propose taking advantage of the unique set of convergently evolved characters associated with the ant-eating diet to investigate the molecular mechanisms underlying phenotypical adaptation. By using state-of-the art phenotyping methods based on X-ray micro-computed tomography and Illumina sequencing technologies we will combine morphometric, genomic, and metagenomic approaches to evaluate the extent of convergent evolution in the skull of myrmecophagous placentals, in their genomes, and in their associated oral and gut microbiomes. With this ambitious research proposal, we aim at providing answers to longstanding but fundamental evolutionary questions pertaining to the mechanisms of convergent evolution. The ConvergeAnt project will be the first of its kind to apply such an integrative approach to investigate the complex interplay between the mammalian genome and its associated microbiome in a classical case of adaptive convergence driven by a highly specialized diet.

1 880 570 €

Start date: 2016-09-01, End date: 2021-08-31

In the mammalian brain, the neocortex is the site where sensory information is integrated into complex cognitive functions. This is accomplished by the activity of both principal glutamatergic neurons and locally-projecting inhibitory GABAergic interneurons, interconnected in complex networks. Inhibitory neurons play several key roles in neocortical function. For example, they shape sensory receptive fields and drive several high frequency network oscillations. On the other hand, defects in their function can lead to devastating diseases, such as epilepsy and schizophrenia. Cortical interneurons represent a highly heterogeneous cell population. Understanding the specific role of each interneuron subtype within cortical microcircuits is still a crucial open question. We have examined properties of two major functional interneuron subclasses in neocortical layer V: fast-spiking (FS) and low-threshold spiking (LTS) cells. Our previous data indicate that each group expresses a novel form of self inhibition, namely autaptic inhibitory transmission in FS cells and an endocannabinoid-mediated slow self inhibition in LTS interneurons. In this proposal we will address three major questions relevant to self-inhibition of neocortical interneurons: 1) What is the role of FS cell autapses in coordinating fast network synchrony? 2) What are the molecular mechanisms underlying autaptic asynchronous release, prolonging FS cell self-inhibition by several seconds, and what is its relevance during physiological and pathological network activities? 3) What are the induction mechanisms, the molecular players involved and the functional roles within cortical microcircuits of the endocannabinoid-mediated long-lasting self-inhibition in LTS interneurons? Results of these experiments will lead to a better understanding of GABAergic interneuron regulation of neocortical excitability, relevant to both normal and pathological cortical function.

996 000 €

Start date: 2008-10-01, End date: 2014-03-31

Developing tissues have a remarkable plasticity illustrated by their capacity to regenerate and form normal organs despite strong perturbations. This requires the adjustment of single cell behaviour to their neighbours and to tissue scale parameters. The modulation of cell growth and proliferation was suggested to be driven by mechanical inputs, however the mechanisms adjusting cell death are not well known. Recently it was shown that epithelial cells could be eliminated by spontaneous live-cell delamination following an increase of cell density. Studying cell delamination in the midline region of the Drosophila pupal notum, we confirmed that local tissue crowding is necessary and sufficient to drive cell elimination and found that Caspase 3 activation precedes and is required for cell delamination. This suggested that a yet unknown pathway is responsible for crowding sensing and activation of caspase, which does not involve already known mechanical sensing pathways. Moreover, we showed that fast growing clones in the notum could induce neighbouring cell elimination through crowding-induced death. This suggested that crowding-induced death could promote tissue invasion by pretumoural cells. Here we will combine genetics, quantitative live imaging, statistics, laser perturbations and modelling to study crowding-induced death in Drosophila in order to: 1) find single cell deformations responsible for caspase activation; 2) find new pathways responsible for density sensing and apoptosis induction; 3) test their contribution to adult tissue homeostasis, morphogenesis and cell elimination coordination; 4) study the role of crowding induced death during competition between different cell types and tissue invasion 5) Explore theoretically the conditions required for efficient space competition between two cell populations. This project will provide essential information for the understanding of epithelial homeostasis, mechanotransduction and tissue invasion by tumoural cells

1 489 147 €

Start date: 2018-01-01, End date: 2022-12-31

CRISPR-Cas loci are the adaptive immune system of archaea and bacteria. They can capture pieces of invading DNA and use this information to degrade target DNA through the action of RNA-guided nucleases. The consequences of DNA cleavage by Cas nucleases, i.e. how breaks are processed and whether they can be repaired, remains to be investigated. A better understanding of the interplay between DNA repair and CRISPR-Cas is critical both to shed light on the evolution and biology of these fascinating systems and for the development of biotechnological tools based on Cas nucleases. CRISPR systems have indeed become a popular tool to edit Eukaryotic genomes. The strategies employed take advantage of different DNA repair pathways to introduce mutations upon DNA cleavage. In bacteria however, the introduction of breaks by Cas nucleases in the chromosome has been described to kill the cell. Preliminary data indicates that this might not always be the case and that some DNA repair pathways could compete with CRISPR immunity allowing cells to survive. Using a combination of bioinformatics and genetics approaches we will investigate the interplay between CRISPR and DNA repair in bacteria with a particular focus on the widely used CRISPR-Cas9 system. The knowledge gained from this study will then help us develop novel tools for bacterial genome engineering. In particular we will introduce a NHEJ pathway in E.coli making it possible to perform CRISPR knockout screens. Finally using CRISPR libraries and multiplexed targeting, we will generate for the first time all combinations of pair-wise gene knockouts in an organism, a task that for now remains elusive, even for large consortiums and with the use of automation. This will enable to decipher genome-scale genetic interaction networks, an important step for our understanding of bacteria as a system.

1 499 763 €

Start date: 2016-03-01, End date: 2021-02-28

In the European Union, 15 million people have an unruptured intracranial aneurysm (IA) that may rupture one day and lead to subarachnoid haemorrhage (SAH). The IA rupture event is ominous and lingers as a clinical quandary. No safe and effective non-invasive therapies have, as of yet, been identified and implemented in clinical practice mainly because of a lack of knowledge of the underlying mechanisms. Increasing evidence points to inflammation as one of the leading factors in the pathogenesis of IA. Intrasaccular clot formation is a common feature of IA occurring unruptured and ruptured IA. In addition to forming clots, activated platelets support leukocyte recruitment. Interestingly, platelets also prevent local hemorrhage in inflammatory situations independently of their ability to form a platelet plug. We hypothesize that the role of platelet may evolve throughout the development of IA: initially playing a protective role of in the maintenance of vascular integrity in response to inflammation and contributing later to intrasaccular thrombus formation. What are the platelet signaling pathways and responses involved and to what extent do they contribute to the disease and the rupture event? To answer these questions, we designed an interdisciplinary proposal, which gathers biophysical, pharmacological, and in-vivo approaches, with the following objectives: I) To investigate platelet functions from patients diagnosed with intracranial aneurysm at the sites of aneurysm sac. II) To delineate platelet mechanisms and responses in a cutting-edge technology of a 3D reconstruction of IA that will take into account the hemodynamic shear stress. III) To test in a preclinical mouse model of IA efficient anti-platelet therapies and define a therapeutic window to intervene on platelet activation. The proposed project will yield new insights in IA disease and in life science, from cell biology to the discovery of potential new targets in cardiovascular medicine.

1 498 618 €

Start date: 2018-06-01, End date: 2023-05-31

T cell cross priming (the initiation of CD8+ T cell responses to antigen that are not expressed by dendritic cells, DCs) requires the phagocytosis of antigens by DCs and their presentation on MHC class I molecules, a process referred to as “cross presentation”. Here, we propose a series of integrated approaches to address the most fundamental mechanisms of cross presentation and explore the use of this process for translational purposes in human cancer. This proposal will pursue three main objectives: 1) To analyze the mechanisms of control of antigen cross presentation and phagocytic functions in DCs. We will use genome wide screens and conditional KO mice, associated to quantitative assays for phagosomal functions and cross presentation, to investigate the molecular mechanisms of cross presentation in vitro and in vivo. 2) To study the epigenetic programing of cross presentation during the ontogeny of mouse DC subpopulations. We will define a “cross presentation gene signature” that will be validated by systematic gene silencing in vitro and we will analyze the epigenetic basis of control of cross presentation-related genes developing DCs. 3) To investigate the regulation of cross presentation in human primary DCs and to develop translational approaches in cancer. We will study cross presentation and phagosome functions in primary human DC subpopulations and its regulation by innate receptors for the development of original immunomodulation and vaccination strategies. We will explore the use of DCs cross presentation abilities in solid tumor infiltrating DCs and their use for prognosis in cancer. The results of this project will unravel fundamental mechanisms of phagocytosis and its control by innate signals in mice and humans. The proposal also aims at defining new possible strategies for cancer treatment and prognosis.

2 500 000 €

Start date: 2014-09-01, End date: 2019-08-31

The hippocampus is essential for building episodic memories. Coding of locations, contexts or events in the hippocampus is based on the correlated activity of neuronal ensembles; however, the mechanisms promoting the recruitment of individual neurons into information-coding ensembles are poorly understood. In particular, the recurrent synaptic network of pyramidal cells (PCs) in the hippocampal CA3 area, receiving external inputs from the entorhinal cortex and the dentate gyrus, is thought to be essential for associative memory. Current models of the associative functions of CA3 are mainly based on plasticity of these synaptic connections. Recent work by us and others however suggests that active, voltage-dependent properties of CA3PC dendrites may also promote ensemble functions. Dendritic voltage-dependent ion channels allow nonlinear amplification of spatiotemporally correlated synaptic inputs (such as those produced by ensemble activity) and can even generate local dendritic spikes, which may elicit specific action potential patterns and induce synaptic plasticity. Furthermore, dendritic processing may be modulated by activity-dependent regulation of dendritic ion channels. However, still little is known about the active properties of CA3PC dendrites and their functions during spatial coding or memory tasks. The general aim of my research program is to understand the cellular mechanisms that underlie the formation of hippocampal memory-coding neuronal ensembles. Specifically, we will test the hypothesis that active input integration by dendrites of individual CA3PCs plays an important role in their recruitment into specific context-coding ensembles. By combining in vitro (patch-clamp electrophysiology and two-photon (2P) microscopy in slices) and in vivo (2P imaging and activity-dependent labelling in behaving rodents) approaches, we will provide an in-depth understanding of the dendritic components contributing to the generation of the CA3 ensemble code.

1 990 314 €

Start date: 2018-06-01, End date: 2023-05-31

The principles of sensory perception are still a large experimental and theoretical puzzle. A strong difficulty is that perception emerges from networks of recurrently connected brain areas whose activity and function are poorly approximated by current generic mathematical models. These models also fail to explain many of the fundamental structures effortlessly identified by the brain (shapes, objects, auditory or tactile categories). I here propose to establish a new approach combining high-throughput population recoding methods with a tailored theoretical framework to derive computational principles operating throughout sensory systems and leading to biologically structured perception. This approach follows on the recent mathematical proposal, suggested by Deep Machine Learning methods, that complex perceptual objects emerge through series of simple nonlinear operations combining increasingly complex sensory features along the sensory pathways. Starting with the mouse auditory system as a model pathway, we will recursively extract, with model-free methods, the main nonlinear sensory features encoded in genetically tagged output and local neurons at different processing stages, using optical and electrophysiological high density recording techniques in awake animals. The role of these features in perception will be identified with behavioural assays. Specific intra- and interareal feedback connections, typically not included in Deep Leaning models, will be opto- and chemogenetically perturbed to assess their contribution to precise nonlinearities of the system and their role in the emergence of complex perceptual structures. Based on these structural, functional and perturbation data, a new generation of well-constrained and predictive sensory processing models will be built, serving as a platform to extract general computational principles missing to link neural activity to perception and to fuel artificial neural networks technologies.

1 983 886 €

Start date: 2018-09-01, End date: 2023-08-31

Diatoms are major contributors of primary production in the ocean and participate in carbon sequestration over geologically relevant timescales. As key components of the Earth’s carbon cycle and marine food webs we need to understand the eco-evolutionary underpinnings of their ecological success to forecast their fate in a future ocean impacted by anthropogenic change. Genomes and epigenomes from model diatoms, as well as hundreds of transcriptomes from multiple species, have revealed genetic and epigenetic processes regulating gene expression in response to changing environments. The Tara Oceans survey has in parallel generated resources to explore diatom abundance, diversity and gene expression in the world’s ocean in widely contrasting conditions. DIATOMIC will build on these resources to understand how evolutionary and ecological processes combine to influence diatom adaptations to their environment at unprecedented spatiotemporal scales. To examine these processes over timescales relevant to current climate change, DIATOMIC includes the pioneering exploration of ancient diatom DNA from the sub-seafloor to reveal the genetic and epigenetic bases of speciation and adaptation that have impacted their ecological success during the last 100,000 years, when Earth experienced major climatological events and an increase in anthropogenic impacts. As a model for exploring eco-evolutionary processes in the past and contemporary ocean we will focus primarily on Chaetoceros because this diatom genus is ancient, ubiquitous, abundant and contributes significantly to carbon export. Key findings will be additionally supported by lab-based studies using the diatom Phaeodactylum for which exemplar molecular tools exist. Specifically, the project will address: 1. What molecular features characterize genome evolution in diatoms? 2. Which processes determine diatom metapopulation structure? 3. What can ancient DNA tell us about diatom adaptations to environmental change in the past?

2 495 753 €

Start date: 2019-11-01, End date: 2024-10-31

"NK cells are innate lymphocytes that play a role in the early response against intracellular pathogens and against tumors. Several NK cell subsets have been described in peripheral organs that correspond to discrete stages of in vivo maturation. How NK cells differentiate from early precursors and what are the specific functions of each NK cell subset are unresolved issues. Here, we propose a three-aim program to address these questions. First, we want to revisit the partition of the NK cell population that is currently based on surface markers of undefined function by looking at the expression of transcription factors (TF) essential for NK cell development and maturation, such as T-bet and Eomes, using novel TF reporter mice. This strategy should also allow us to identify very early steps of NK cell development (NK cell progenitors) that remain ill defined. Second, we will try and identify molecular mechanisms that induce transition between NK cell maturation stages. For this we will take advantage of a previous gene profiling analysis that pointed at several pathways and TF that were highly regulated during NK cell maturation. The role of these pathways and TF in the differentiation of NK cells will be measured using a novel Cre/lox system allowing NK-specific gene deletion. Detailed analysis of mouse mutants will be used to delineate the role of selected genes and pathways in NK cell differentiation. Third, we will compare patterns of migration, cytokine secretion, in vivo cytotoxicity and global gene expression by individual NK cell subsets during an airway infection by Influenza to get insight on the specific functions of NK cell subsets during immune responses. Altogether, the results of this study should provide developmental, molecular and functional evidences to support the physiological relevance of NK cell subsets. This may improve strategies that aim at manipulating NK cell function for the benefit of patients with cancer or chronic infectious diseases"

1 340 757 €

Start date: 2012-01-01, End date: 2017-12-31

The molecular mechanisms that serve to couple DNA replication, chromosome segregation and cell division are largely unknown in bacteria. This led a considerable interest to the study of Escherichia coli FtsK, an essential cell division protein that assembles into DNA-pumps to transfer chromosomal DNA between the two daughter cell compartments during septation. Indeed, our recent work suggests that FtsK might regulate the late stages of septation to ensure DNA is fully cleared from the septum before it is allowed to close. This would be the first example of a cell cycle checkpoint in bacteria. FtsK-mediated DNA transfer is required in 15% of the cells at each generation in E. coli, in which it serves to promote the resolution of topological problems arising from the circularity of the chromosome by Xer recombination. However, the FtsK checkpoint could be a more general feature of the bacterial cell cycle since FtsK is highly conserved among eubacteria, including species that do not possess a Xer system. Indeed, preliminary results from the lab indicate that DNA transfer by FtsK is required independently of Xer recombination in Vibrio cholerae. To confirm the existence and the generality of the FtsK checkpoint in bacteria, we will determine the different situations that lead to a requirement for FtsK-mediated DNA transfer by studying chromosome segregation and cell division in V. cholerae. In parallel, we will apply new fluorescent microscopy tools to follow the progression of cell division and chromosome segregation in single live bacterial cells. PALM will notably serve to probe the structure of the FtsK DNA-pumps at a high spatial resolution, FRET will be used to determine their timing of assembly and their interactions with the other cell division proteins, and TIRF will serve to follow in real time their activity with respect to the progression of chromosome dimer resolution, chromosome segregation, and septum closure.

1 565 938 €

Start date: 2012-02-01, End date: 2017-01-31

The impairment of central nervous system (CNS) leads to irreversible loss of vital functions because, unlike young neurons, mature neurons are not able to regenerate. Thus, understanding the detailed mechanisms of axonal growth and repair remains one of the greatest challenges of neurobiology and for society. If the extrinsic factors fail to reach levels required for regeneration, manipulating intrinsic pathways has shown promising results. Particularly, my work demonstrated that the simultaneous activation of mTOR, JAK/STAT and c-myc pathways allows exceptional regeneration with axons close to their targets. However it also exacerbates previously described phenomenon of misguidance with potential aberrant circuit formation. My unique model opens up the possibility to explore these fundamental questions of CNS regeneration. I propose to address the yet unexplored problem of the guidance of regenerating axons in adults in order to promote the formation of a functional new circuit after injury. Indeed what are the modalities of guidance in the adult? Are axons still responsive to developmental guidance cues and are they still expressed? Can regenerative axons form connections with their targets and are these connections functional? To answer these critical questions, I will use the combination of state of the art biochemistry, imaging, and electrophysiology in an in-vivo and ex-vivo model of the visual system to 1) Understand axon guidance in mature system in order to properly drive regenerative axons to their brain targets and avoid aberrant projections, and 2) Analyze the formation of a functional optic nerve circuit after injury. Altogether, these results will generate major breakthroughs in a fundamental but uncovered mechanism of axon guidance during regeneration and the functionality of de novo formed circuits. They will open up new ways for innovative therapeutic development after CNS trauma but also to the large spectrum of neurodegenerative diseases.

1 499 410 €

Start date: 2018-03-01, End date: 2023-02-28

This proposal is centred on the development of small molecule probes derived from DNA damaging agents to identify their genomic targets using a novel unbiased approach. Although, several genotoxic drugs have been used for decades to treat cancers, the exact mechanisms by which they operate are not fully understood. It is established that these compounds interfere with the processes of transcription and replication, thereby promoting genomic instability and cell death. However, there is as yet no genome-wide map of the exact location of sites that are putative targets for these drugs in vivo. This information is critical to understand and rationalize cellular responses to genotoxic agents. Here, we propose to develop an innovative discovery- based methodology that will combine click chemistry in situ, affinity pull-down techniques and high throughput DNA sequencing (Drug-Seq), to identify the genomic interactome of DNA damaging drugs in order to elucidate their cellular activity at the molecular level. Two independent lines of enquiry will be followed. Firstly, we will establish the genomic interacting landscape of landmark drugs including etoposide, camptothecin and cisplatin using Drug-Seq. Secondly, we will perform regular chromatin immuno- precipitation sequencing (ChIP-Seq) of selected proteins linked to the cellular response of interest to validate Drug-Seq and further identify druggable genomic sites. An important aim of this proposal is to establish a universal methodology to decipher small molecule/genome interactions in vivo that trigger a particular response in disease-relevant models. We also seek to interrogate the role of chromatin in regulating drug/genome interactions and to define whether it is possible to act on the epigenome to modulate the activity and specificity of these drugs. Collectively, we anticipate our study will lay down the foundation for personalized medicine with the implementation of rational rather than empirical clinical protocols.

1 999 900 €

Start date: 2015-09-01, End date: 2020-08-31

Activity-dependent plasticity of synaptic transmission together with refinement of neural circuits connectivity are amongst the core mechanisms underlying learning and memory. While there is already extensive knowledge on some of the mechanisms of synaptic plasticity, fundamental questions remain on the dynamics of the underlying molecular events and the functional roles of various forms of synaptic plasticity in information processing, learning and behavior. We previously uncovered basic features of glutamate receptor movements and their role in excitatory synaptic transmission. Our new ground-breaking objectives are: 1) to uncover, in a physiological context, the dynamic mechanisms through which synapses modulate their strength in response to neuronal activity by integrating on different space and time scales the properties of receptor traffic pathways and associated stabilization mechanisms, 2) to use our knowledge and innovative tools to interfere with these trafficking mechanisms in order to decipher the specific roles of different forms of synaptic plasticity in given brain functions and behavioral tasks. For this aim, I lead a team of neurobiologists, physicists and chemists with a collaborative record of accomplishment. We will combine imaging, cellular neurobiology, physiology and behavior to probe the mechanisms and roles of different forms of synaptic plasticity. New in tissue high-resolution imaging combined with innovative molecular reporters and electrophysiology will allow analysis of receptor traffic during short and long-term synaptic plasticity in physiological conditions. We will probe the interplay between activity-dependent changes in synaptic strength and circuit function with new photo-activable modifiers of receptor traffic with an unprecedented time and space resolution. Use of these tools in vivo will allow identifying the roles of synaptic plasticity in sensory information processing and the various phases of spatial memory formation.

2 499 505 €

Start date: 2019-02-01, End date: 2024-01-31

This project studies dynamic mechanisms. By “dynamic mechanisms”, we mean policies to which a principal (e.g., a seller, an employer, or a regulator) can commit to induce the agents (e.g., buyers, employees, or regulated firms) to take the desired actions over time. Several components of the project are envisaged: - Competition in dynamic mechanisms. o I propose a competitive setting in which agents (e.g., buyers or workers) learn about the offers of different principals over time. Agents may receive more than one offer at a time, leading to direct competition between mechanisms. Received offers are agents’ private information, permitting strategic delay of acceptance (for instance, an agent may want to wait to evaluate new offers that received in the future). - Robust predictions for a rich class of stochastic processes. o We study optimal dynamic mechanisms for agents whose preferences evolve stochastically with time. We develop an approach to partially characterizing these mechanisms which (unlike virtually all of the existing literature) does not depend on ad-hoc restrictions on the stochastic process for preferences. - Efficient bilateral trade with budget balance: dynamic arrival of traders o I study bilateral trade with budget balance, when traders (i) arrive over time, and (ii) have preferences which evolve stochastically with time. The project aims at an impossibility result in this setting: contrary to the existing literature which does not account for dynamic arrivals, budget-balanced efficient trade is typically impossible, even for very patient traders. - Pre-event ticket sales and complementary investments o We provide a rationale for the early allocation of capacity to customers for events such as flights and concerts based on customers’ demand for pre-event complementary investments (such as booking a hotel or a babysitter). We examine efficient and profit-maximizing mechanisms.

1 321 625 €

Start date: 2017-01-01, End date: 2021-12-31

The DOHaD (Developmental Origins of Health and Disease) hypothesis states that, because of developmental plasticity, in utero and early postnatal stressors can increase chronic disease risk in childhood and later. Alterations of epigenetic marks, impacting on gene expression, are one mechanism that could explain such long-term impact. These hypotheses have so far been tested mostly in animals, and little for atmospheric pollutants, for which animal evidence is scarce. We aim to characterize the impact of environmental exposures on childhood health. Our focus is on two families of pollutants with a highly prevalent and controllable exposure in humans: atmospheric pollutants and specific high-volume non-persistent chemicals (Bisphenol A, other phenols and phthalates). These pollutants are archetypal of modern life pollutants challenging environmental health research. We will set up a new type of mother-child cohort with early recruitment in pregnancy, intense follow-up (including geolocalisation of subjects with GPS combined with fine-scale air pollution modelling), personal exposure monitoring, repeated collection of biological samples. Transcriptomic analysis, non-invasive clinical examinations (Doppler and ultrasound imaging, ECG, early postnatal evaluation of lung function) will bring clues regarding target functions. This observational approach in humans will be supplemented by an animal experiment aiming at characterizing the impact of in utero exposure to traffic-related atmospheric pollutants on foetal development and health in adulthood, and characterizing target functions and organs more finely than the human study can allow. E-DOHaD spans over the whole range of environmental health disciplines, with epidemiologic and toxicologic studies being conducted in parallel to ease comparability and results synthesis. E-DOHaD is expected to have far-reaching implications in environmental health research and for public health.

1 499 870 €

Start date: 2013-02-01, End date: 2018-01-31

Studies concerning the mechanism of DNA replication have advanced our understanding of genetic transmission through multiple cell cycles. Recent work has shed light on possible means to ensure the stable transmission of information beyond just DNA and the concept of epigenetic inheritance has emerged. Considering chromatin-based information, key candidates have arisen as epigenetic marks including DNA and histone modifications, histone variants, non-histone chromatin proteins, nuclear RNA as well as higher-order chromatin organization. Thus, understanding the dynamics and stability of these marks following disruptive events during replication and repair and throughout the cell cycle becomes of critical importance for the maintenance of any given chromatin state. To approach these issues, we propose to study the maintenance of heterochromatin at centromeres, key chromosomal regions for the proper chromosome segregation. Our current goal is to access to the sophisticated mechanisms that have evolved in order to facilitate inheritance of epigenetic marks not only at the replication fork, but also at other stages of the cell cycle, during repair and development. Beyond inheritance of DNA methylation, understanding how inheritance of histone variants and their modifications can be controlled either coupled or not coupled to DNA replication will be a major focus of this project. Our studies will build on the expertise and tools developed over the years in a strategy that integrates molecular, cellular, and biochemical approaches. This will be combined with the use of new technologies to monitor cell cycle (Fucci), protein dynamics (SNAP-Tagging) together with single molecule analysis involving DNA and chromatin combing. We wish to define a possible framework for an understanding of both the stability and reversibility of epigenetic marks and their dynamics at centromeres. These lessons may teach us general principles of inheritance of epigenetic states.

2 490 483 €

Start date: 2010-06-01, End date: 2015-12-31

This project offers a theoretical and empirical investigation of matching markets. Matching is, broadly speaking, the study of complementarities, which explains the formation of coalitions. Matching models are found in many applied fields within Economics: Labour Economics, Family Economics, Consumer theory of differentiated goods (hedonic models), Trade, etc. Desirable properties of these coalitions, such as stability, lead to testable implications of the surplus that individuals generate in a match, allowing for structural estimation of matching models. The goal of this proposal is to expand the frontiers of the theory of matching to design a very general and highly flexible model of matching that will lend itself to estimation and thus lead to empirical findings in various fields of Economics. Based on promising work initiated by the PI, this proposal seeks to bridge the gap between the theory and the empirics of matching markets that was traditionally observed in this literature. Particular focus will be given to situations where stable outcomes may not exist (such as unipartite, or one-to-many matching models), frictions, taxes. In these cases, a thorough investigation is carried on what solution concept should be used, and what are the testable implications. Applications will be given to various empirical issues or policy relevant questions such as: - The nature of the complementarities between senior and junior employees within teams, - The role played by the marriage market in the problem of rural depletion in China, - The impact of CEO risk aversion on assignment to firms, and on the CEO compensation package, - The pricing of attributes of French wines.

1 119 000 €

Start date: 2013-01-01, End date: 2018-09-30