ERC FUNDED PROJECTS

For many years, the ubiquitin-26S proteasome degradation pathway was considered the primary route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by a ubiquitin-independent mechanism mediated by the core 20S proteasome itself. Although initially believed to be limited to rare exceptions, degradation by the 20S proteasome is now understood to have a wide range of substrates, many of which are key regulatory proteins. Despite its importance, little is known about the mechanisms that control 20S proteasomal degradation, unlike the extensive knowledge acquired over the years concerning degradation by the 26S proteasome. Our overall aim is to reveal the multiple regulatory levels that coordinate the 20S proteasome degradation route. To achieve this goal we will carry out a comprehensive research program characterizing three distinct levels of 20S proteasome regulation: Intra-molecular regulation- Revealing the intrinsic molecular switch that activates the latent 20S proteasome. Inter-molecular regulation- Identifying novel proteins that bind the 20S proteasome to regulate its activity and characterizing their mechanism of function. Cellular regulatory networks- Unraveling the cellular cues and multiple pathways that influence 20S proteasome activity using a novel systematic and unbiased screening approach. Our experimental strategy involves the combination of biochemical approaches with native mass spectrometry, cross-linking and fluorescence measurements, complemented by cell biology analyses and high-throughput screening. Such a multidisciplinary approach, integrating in vitro and in vivo findings, will likely provide the much needed knowledge on the 20S proteasome degradation route. When completed, we anticipate that this work will be part of a new paradigm – no longer perceiving the 20S proteasome mediated degradation as a simple and passive event but rather a tightly regulated and coordinated process.

1 500 000 €

Start date: 2015-04-01, End date: 2020-03-31

This proposal brings together two fields in biology, namely the emerging field of phase-separated assemblies in cell biology and state-of-the-art cellular cryo-electron tomography, to advance our understanding on a fundamental, yet illusive, question: the molecular organization of the cytoplasm. Eukaryotes organize their biochemical reactions into functionally distinct compartments. Intriguingly, many, if not most, cellular compartments are not membrane enclosed. Rather, they assemble dynamically by phase separation, typically triggered upon a specific event. Despite significant progress on reconstituting such liquid-like assemblies in vitro, we lack information as to whether these compartments in vivo are indeed amorphous liquids, or whether they exhibit structural features such as gels or fibers. My recent work on sample preparation of cells for cryo-electron tomography, including cryo-focused ion beam thinning, guided by 3D correlative fluorescence microscopy, shows that we can now prepare site-specific ‘electron-transparent windows’ in suitable eukaryotic systems, which allow direct examination of structural features of cellular compartments in their cellular context. Here, we will use these techniques to elucidate the structural principles and cytoplasmic environment driving the dynamic assembly of two phase-separated compartments: Stress granules, which are RNA bodies that form rapidly in the cytoplasm upon cellular stress, and centrosomes, which are sites of microtubule nucleation. We will combine these studies with a quantitative description of the crowded nature of cytoplasm and of its local variations, to provide a direct readout of the impact of excluded volume on molecular assembly in living cells. Taken together, these studies will provide fundamental insights into the structural basis by which cells form biochemical compartments.

1 228 125 €

Start date: 2018-02-01, End date: 2023-01-31

Chromosomal DNA is continuously subjected to exogenous and endogenous damaging insults. In the presence of DNA damage cells activate a multi-faceted checkpoint response that delays cell cycle progression and promotes DNA repair. Failures in this response lead to genomic instability, the main feature of cancer cells. Several cancer-prone human syndromes including the Ataxia teleangiectasia (A-T), the A-T Like Disorder (ATLD) and the Seckel Syndrome reflect defects in the specific genes of the DNA damage response such as ATM, MRE11 and ATR. DNA damage response pathways are poorly understood at biochemical level in vertebrate organisms. We have established a cell-free system based on Xenopus laevis egg extract to study molecular events underlying DNA damage response. This is the first in vitro system that recapitulates different aspects of the DNA damage response in vertebrates. Using this system we propose to study the biochemistry of the ATM, ATR and the Mre11 complex dependent DNA damage response. In particular we will: 1) Dissect the signal transduction pathway that senses DNA damage and promotes cell cycle arrest and DNA damage repair; 2) Analyze at molecular level the role of ATM, ATR, Mre11 in chromosomal DNA replication and mitosis during normal and stressful conditions; 3) Identify substrates of the ATM and ATR dependent DNA damage response using an innovative screening procedure.

1 000 000 €

Start date: 2008-07-01, End date: 2013-06-30

Many Gram-positive (pathogenic) bacteria are dependent on the uptake of vitamins from the environment or from the infected host. We have recently discovered the long-elusive family of membrane protein complexes catalyzing such transport. The vitamin transporters have an unprecedented modular architecture consisting of a single multipurpose energizing module (the Energy Coupling Factor, ECF) and multiple exchangeable membrane proteins responsible for substrate recognition (S-components). The S-components have characteristics of ion-gradient driven transporters (secondary active transporters), whereas the energizing modules are related to ATP-binding cassette (ABC) transporters (primary active transporters). The aim of the proposal is threefold: First, we will address the question how properties of primary and secondary transporters are combined in ECF transporters to obtain a novel transport mechanism. Second, we will study the fundamental and unresolved question how protein-protein recognition takes place in the hydrophobic environment of the lipid bilayer. The modular nature of the ECF proteins offers a natural system to study the driving forces used for membrane protein interaction. Third, we will assess whether the ECF transport systems could become targets for antibacterial drugs. ECF transporters are found exclusively in prokaryotes, and their activity is often essential for viability of Gram-positive pathogens. Thus they could turn out to be an Achilles’ heel for the organisms. Structural and mechanistic studies (X-ray crystallography, microscopy, spectroscopy and biochemistry) will reveal how the different transport modes are combined in a single protein complex, how transport is energized and catalyzed, and how protein-protein recognition takes place. Microbiological screens will be developed to search for compounds that inhibit prokaryote-specific steps of the mechanism of ECF transporters.

1 500 000 €

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

The proposed work concerns the theoretical and numerical development of robust and adaptive methodologies for broadband imaging in clutter. The word clutter expresses our uncertainty on the wave speed of the propagation medium. Our results are expected to have a strong impact in a wide range of applications, including underwater acoustics, exploration geophysics and ultrasound non-destructive testing. Our machinery is coherent interferometry (CINT), a state-of-the-art statistically stable imaging methodology, highly suitable for the development of imaging methods in clutter. We aim to extend CINT along two complementary directions: novel types of applications, and further mathematical and numerical development so as to assess and extend its range of applicability. CINT is designed for imaging with partially coherent array data recorded in richly scattering media. It uses statistical smoothing techniques to obtain results that are independent of the clutter realization. Quantifying the amount of smoothing needed is difficult, especially when there is no a priori knowledge about the propagation medium. We intend to address this question by coupling the imaging process with the estimation of the medium's large scale features. Our algorithms rely on the residual coherence in the data. When the coherent signal is too weak, the CINT results are unsatisfactory. We propose two ways for enhancing the resolution of CINT: filter the data prior to imaging (noise reduction) and waveform design (optimize the source distribution). Finally, we propose to extend the applicability of our imaging-in-clutter methodologies by investigating the possibility of utilizing ambient noise sources to perform passive sensor imaging, as well as by studying the imaging problem in random waveguides.

690 000 €

Start date: 2010-06-01, End date: 2015-11-30

I propose to establish a research group to develop completely new tools in order to solve three important problems on the relationships between automorphic forms and Galois representations, which lie at the heart of the Langlands program. The first is to prove Serre’s conjecture for real quadratic fields. I will use automorphic induction to transfer the problem to U(4) over the rational numbers, where I will use automorphy lifting theorems and results on the weight part of Serre's conjecture that I established in my earlier work to reduce the problem to proving results in small weight and level. I will prove these base cases via integral p-adic Hodge theory and discriminant bounds. The second is to develop a geometric theory of moduli spaces of mod p and p-adic Galois representations, and to use it to establish the Breuil–Mézard conjecture in arbitrary dimension, by reinterpreting the conjecture in geometric terms. This will transform the subject by building the first connections between the p-adic Langlands program and the geometric Langlands program, providing an entirely new world of techniques for number theorists. As a consequence of the Breuil-Mézard conjecture, I will be able to deduce far stronger automorphy lifting theorems (in arbitrary dimension) than those currently available. The third is to completely determine the reduction mod p of certain two-dimensional crystalline representations, and as an application prove a strengthened version of the Gouvêa–Mazur conjecture. I will do this by means of explicit computations with the p-adic local Langlands correspondence for GL_2(Q_p), as well as by improving existing arguments which prove multiplicity one theorems via automorphy lifting theorems. This work will show that the existence of counterexamples to the Gouvêa-Mazur conjecture is due to a purely local phenomenon, and that when this local obstruction vanishes, far stronger conjectures of Buzzard on the slopes of the U_p operator hold.

1 131 339 €

Start date: 2012-10-01, End date: 2017-09-30

Host cytokines, chemokines and type I IFNs are critical effectors of the innate immune response to viral and bacterial pathogens. Several classes of germ-line encoded pattern recognition receptors have been identified, which sense non-self nucleic acids and trigger these responses. Recently NLRP-3, a member of the NOD-like receptor (NLR) family, has been shown to sense endogenous danger signals, environmental insults and the DNA viruses adenovirus and HSV. Activation of NLRP-3 induces the formation of a large multiprotein complex in cells termed inflammasome , which controls the activity of pro-caspase-1 and the maturation of pro-IL-1² and pro-IL18 into their active forms. NLRP-3, however, does not regulate these responses to double stranded cytosolic DNA. We identified the cytosolic protein AIM2 as the missing receptor for cytosolic DNA. AIM2 contains a HIN200 domain, which binds to DNA and a pyrin domain, which associates with the adapter molecule ASC to activate both NF-ºB and caspase-1. Knock down of AIM2 down-regulates caspase-1-mediated IL-1² responses following DNA stimulation or vaccinia virus infection. Collectively, these observations demonstrate that AIM2 forms an inflammasome with the DNA ligand and ASC to activate caspase-1. Our underlying hypothesis for this proposal is that AIM2 plays a central role in host-defence to cytosolic microbial pathogens and also in DNA-triggered autoimmunity. The goals of this research proposal are to further characterize the DNA ligand for AIM2, to explore the molecular mechanisms of AIM2 activation, to define the contribution of AIM2 to host-defence against viral and bacterial pathogens and to assess its function in nucleic acid triggered autoimmune disease. The characterization of AIM2 and its role in innate immunity could open new avenues in the advancement of immunotherapy and treatment of autoimmune disease.

1 727 920 €

Start date: 2009-12-01, End date: 2014-11-30

Currently, more than 30% of all Europeans suffer from one or more allergic disorder but treatment is still mostly symptomatic due to a lack of understanding the underlying causality. Allergies are caused by type 2 immune responses triggered by recognition of harmless antigens. Both genetic and environmental factors have been proposed to favour allergic predisposition and both factors have a huge impact on the symbiotic microbiota and the intestinal immune system. Recently we and others showed that the transcription factor ROR(γt) seems to play a key role in mucosal tolerance in the gut and also regulates intestinal type 2 immune responses. Based on these results I postulate two major events in the gut for the development of an allergy in the lifetime of an individual: First, a failure to establish mucosal tolerance or anergy constitutes a necessity for the outbreak of allergic symptoms and allergic disease. Second, a certain ‘core’ microbiome or pathway of the intestinal microbiota predispose certain individuals for the later development of allergic disorders. Therefore, I will address the following aims: 1) Influence of ROR(γt) on mucosal tolerance induction and allergic disorders 2) Elucidate the T cell receptor repertoire of intestinal Th2 and ROR(γt)+ Tregs and assess the role of alternative NFκB pathway for induction of mucosal tolerance 3) Identification of ‘core’ microbiome signatures or metabolic pathways that favour allergic predisposition ALLERGUT will provide ground-breaking knowledge on molecular mechanisms of the failure of mucosal tolerance in the gut and will prove if the resident ROR(γt)+ T(reg) cells can function as a mechanistic starting point for molecular intervention strategies on the background of the hygiene hypothesis. The vision of ALLERGUT is to diagnose mucosal disbalance, prevent and treat allergic disorders even before outbreak and thereby promote Public Health initiative for better living.

1 498 175 €

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

We are concerned with localization properties of solutions to hyperbolic PDEs, especially problems with a geometric component: how do boundaries and heterogeneous media influence spreading and concentration of solutions. While our first focus is on wave and Schrödinger equations on manifolds with boundary, strong connections exist with phase space localization for (clusters of) eigenfunctions, which are of independent interest. Motivations come from nonlinear dispersive models (in physically relevant settings), properties of eigenfunctions in quantum chaos (related to both physics of optic fiber design as well as number theoretic questions), or harmonic analysis on manifolds. Waves propagation in real life physics occur in media which are neither homogeneous or spatially infinity. The birth of radar/sonar technologies (and the raise of computed tomography) greatly motivated numerous developments in microlocal analysis and the linear theory. Only recently toy nonlinear models have been studied on a curved background, sometimes compact or rough. Understanding how to extend such tools, dealing with wave dispersion or focusing, will allow us to significantly progress in our mathematical understanding of physically relevant models. There, boundaries appear naturally and most earlier developments related to propagation of singularities in this context have limited scope with respect to crucial dispersive effects. Despite great progress over the last decade, driven by the study of quasilinear equations, our knowledge is still very limited. Going beyond this recent activity requires new tools whose development is at the heart of this proposal, including good approximate solutions (parametrices) going over arbitrarily large numbers of caustics, sharp pointwise bounds on Green functions, development of efficient wave packets methods, quantitative refinements of propagation of singularities (with direct applications in control theory), only to name a few important ones.

1 293 763 €

Start date: 2018-02-01, End date: 2023-01-31

The analysis of geometric and functional inequalities naturally leads to consider the extremal cases, thus looking for optimal sets, or optimal functions, or optimal constants. The most classical examples are the (different versions of the) isoperimetric inequality and the Sobolev-like inequalities. Much is known about equality cases and best constants, but there are still many questions which seem quite natural but yet have no answer. For instance, it is not known, even in the 2-dimensional space, the answer of a question by Brezis: which set, among those with a given volume, has the biggest Sobolev-Poincaré constant for p=1? This is a very natural problem, and it appears reasonable that the optimal set should be the ball, but this has never been proved. The interest in problems like this relies not only in the extreme simplicity of the questions and in their classical flavour, but also in the new ideas and techniques which are needed to provide the answers. The main techniques that we aim to use are fine arguments of symmetrization, geometric constructions and tools from mass transportation (which is well known to be deeply connected with functional inequalities). These are the basic tools that we already used to reach, in last years, many results in a specific direction, namely the search of sharp quantitative inequalities. Our first result, together with Fusco and Maggi, showed what follows. Everybody knows that the set which minimizes the perimeter with given volume is the ball. But is it true that a set which almost minimizes the perimeter must be close to a ball? The question had been posed in the 1920's and many partial result appeared in the years. In our paper (Ann. of Math., 2007) we proved the sharp result. Many other results of this kind were obtained in last two years.

540 000 €

Start date: 2010-08-01, End date: 2015-07-31

This is a proposal for research at the interface of analytic number theory, automorphic forms and algebraic geometry. Motivated by fundamental conjectures in number theory, classical problems will be investigated in higher order situations: general number fields, automorphic forms on higher rank groups, the arithmetic of algebraic varieties of higher degree. In particular, I want to focus on - computation of moments of L-function of degree 3 and higher with applications to subconvexity and/or non-vanishing, as well as subconvexity for multiple L-functions; - bounds for sup-norms of cusp forms on various spaces and equidistribution of Hecke correspondences; - automorphic forms on higher rank groups and general number fields, in particular new bounds towards the Ramanujan conjecture; - a proof of Manin's conjecture for a certain class of singular algebraic varieties. The underlying methods are closely related; for example, rational points on algebraic varieties will be counted by a multiple L-series technique.

1 004 000 €

Start date: 2010-10-01, End date: 2015-09-30

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

"Archaea constitute the third domain of life and are believed to be close to the origin of life. They comprise a diverse group of micro-organisms that combine bacterial and eukaryotic features, but also employ many novel mechanisms. They possess a unique cell envelope with a cytoplasmic membrane of ether lipids surrounded by a proteinaceous S-layer and various cell appendages such as flagella, pili and more unusual structures. Studies have shown that the archaeal flagellum is an unique structure as it functionally resembles the bacterial flagellum, but structurally it is a simple type IV pilus. Moreover, we have shown that this type IV pilus can rotate. Therefore I propose to name the archaeal flagellum, the archaellum, as it is fundamentally different from the bacterial flagellum. In this proposal I aim to understand the assembly and mechanism of rotation of the archaellum of the thermocacidophilic crenarchaen Sulfolobus acidocaldarius by using biochemical, genetic and biophysical methods. The main milestons are: - Biochemical and structural characterization of all archaellum subunits - To understand the assembly pathway of the archaellum and the interactions of its different subunits - To understand how rotation of the filament is achieved and which subunits are important for this movement This work will identify a new, relatively simple motor complex that has evolved from primordial type IV pili assembly machineries and therefore uncover general principles of macromolecular assemblies at cellular surfaces and a novel mechanism to generate mechanical force that can be translated into movement."

1 464 317 €

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

ATM is the protein kinase that is mutated in the hereditary autosomal recessive disease ataxia telangiectasia (A-T). A-T patients display immune deficiencies, cancer predisposition and radiosensitivity. The molecular role of ATM is to respond to DNA damage by phosphorylating its substrates, thereby promoting repair of damage or arresting the cell cycle. Following the induction of double-strand breaks (DSBs), the NBS1 protein is required for activation of ATM. But ATM can also be activated in the absence of DNA damage. Treatment of cultured cells with hypotonic stress leads to the activation of ATM, presumably due to changes in chromatin structure. We have recently described a second ATM cofactor, ATMIN (ATM INteractor). ATMIN is dispensable for DSBs-induced ATM signalling, but ATM activation following hypotonic stress is mediated by ATMIN. While the biological role of ATM activation by DSBs and NBS1 is well established, the significance, if any, of ATM activation by ATMIN and changes in chromatin was up to now completely enigmatic. ATM is required for class switch recombination (CSR) and the suppression of translocations in B cells. In order to determine whether ATMIN is required for any of the physiological functions of ATM, we generated a conditional knock-out mouse model for ATMIN. ATM signaling was dramatically reduced following osmotic stress in ATMIN-mutant B cells. ATMIN deficiency led to impaired CSR, and consequently ATMIN-mutant mice developed B cell lymphomas. Thus ablation of ATMIN resulted in a severe defect in ATM function. Our data strongly argue for the existence of a second NBS1-independent mode of ATM activation that is physiologically relevant. While a large amount of scientific effort has gone into characterising ATM signaling triggered by DSBs, essentially nothing is known about NBS1-independent ATM signaling. The experiments outlined in this proposal have the aim to identify and understand the molecular pathway of ATMIN-dependent ATM signaling.

1 499 881 €

Start date: 2012-02-01, End date: 2018-01-31

Early life immune balance is essential for survival and establishment of healthy immunity in later life. We aim to define how age-dependent regulation of dendritic cell (DC) development contributes to this crucial immune balance. DCs are versatile controllers of immunity that in neonates are qualitatively distinct from adults. Why such age-dependent differences exist is unclear but newborn DCs are considered underdeveloped and functionally immature. Using ontogenetic tracing of conventional DC precursors, I have found a previously unappreciated developmental heterogeneity of DCs that is particularly prominent in young mice. Preliminary data indicate that distinct waves of DC poiesis contribute to the functional differences between neonatal and adult DCs. I hypothesize that the neonatal DC compartment is not immature but rather that DC poiesis is developmentally regulated to create essential age-dependent immune balance. Further, I have identified a unique situation in early life to address a fundamental biological question, namely to what extent cellular function is pre-programmed by developmental origin (nature) versus environmental factors (nurture). In this proposal, we will first use novel models to fate map the origin of the DC compartment with age. We will then define to what extent cellular origin determines age-dependent functions of DCs in immunity. Using innovative comparative gene expression profiling and integrative epigenomic analysis the cell intrinsic mechanisms regulating the age-dependent functions of DCs will be characterized. Because environmental factors in utero and after birth critically influence immune balance, we will finally define the impact of maternal infection and metabolic disease, as well as early microbial encounter on DC poiesis. Characterizing how developmentally regulated DC poiesis shapes the unique features of early life immunity will provide novel insights into immune development that are vital to advance vaccine strategies.

1 500 000 €

Start date: 2017-06-01, End date: 2022-05-31

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

The aim of this transdisciplinary project is to develop and analyse multiscale mathematical models of pattern formation in multicellular systems controlled by the dynamics of intracellular signalling pathways and cell-to-cell communication and to develop new mathematical methods for the modelling of such complex processes. This aim will be achieved through a close collaboration with experimental groups and comprehensive analytical investigations of the mathematical problems arising in the modelling of these biological processes. The mathematical methods and techniques to be employed will be the analysis of systems of partial differential equations, asymptotic analysis, as well as methods of dynamical systems. These techniques will be used to formulate the models and to study the spatio-temporal behaviour of solutions, especially stability and dependence on characteristic scales, geometry, initial data and key parameters. Advanced numerical methods will be applied to simulate the models. This comprehensive methodology goes beyond the state-of-the-art, since usually the analyses are limited to a single aspect of model behaviour. Groundbreaking impacts envisioned are threefold: (i) The project will contribute to the understanding of mechanisms of structure formation in the developmental process, in the context of recently discovered signalling pathways. In addition, some of the factors and mechanisms playing a role in developmental processes, such as Wnt signalling, are implicated in carcinogenesis, for instance colon and lung cancer. (ii) Accurate quantitative and predictive mathematical models of cell proliferation and differentiation are important for the control of tumour growth and tissue egeneration; (iii) Qualitative analysis of multiscale mathematical models of biological phenomena generates challenging mathematical problems and, therefore, the project will lead to the development of new mathematical theories and tools.

750 000 €

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

Immune systems are highly variable, but the sources of this variation are poorly understood. Genetic variation only explains a minor fraction of this, and we are unable to accurately predict the risk of immune mediated disease or severe infection in any given individual. I recently found that immune cells and proteins in healthy twins vary because of non-heritable influences (infections, vaccines, microbiota etc.), with only minor influences from heritable factors (Brodin, et al, Cell 2015). When and how such environmental influences shape our immune system is now important to understand. Birth represents the most transformational change in environment during the life of any individual. I propose, that environmental influences at birth, and during the first months of life could be particularly influential by imprinting on the regulatory mechanisms forming in the developing immune system. Adaptive changes in immune cell frequencies and functional states induced by early-life exposures could determine both the immune competence of the newborn, but potentially also its long-term trajectory towards immunological health or disease. Here, I propose a study of 1000 newborn children, followed longitudinally during their first 1000 days of life. By monitoring immune profiles and recording many environmental influences, we hope to understand how early life exposures can influence human immune system development. We have established a new assay based on Mass Cytometry and necessary data analysis tools (Brodin, et al, PNAS 2014), to simultaneously monitor the frequencies, phenotypes and functional states of more than 200 blood immune cell populations from only 100 microliters of blood. By monitoring environmental influences at regular follow-up visits, by questionnaires, serum measurements of infection, and gut microbiome sequencing, we aim to provide the most comprehensive analysis to date of immune system development in newborn children.

1 422 339 €

Start date: 2016-07-01, End date: 2021-06-30

Transcription in single cells is a stochastic process that arises from the random collision of molecules, resulting in heterogeneity in gene expression in cell populations. This heterogeneity in gene expression influences cell fate decisions and disease progression. Interestingly, gene expression variability is not the same for every gene: noise can vary by several orders of magnitude across transcriptomes. The reason for this transcript-specific behavior is that genes are not transcribed in a continuous fashion, but can show transcriptional bursting, with periods of gene activity followed by periods of inactivity. The noisiness of a gene can be tuned by changing the duration and the rate of switching between periods of activity and inactivity. Even though transcriptional bursting is conserved from bacteria to yeast to human cells, the origin and regulators of bursting remain largely unknown. Here, I will use cutting-edge single-molecule RNA imaging techniques to directly observe and measure transcriptional bursting in living yeast cells. First, bursting properties will be quantified at different endogenous and mutated genes to evaluate the contribution of cis-regulatory promoter elements on bursting. Second, the role of trans-regulatory complexes will be characterized by dynamic depletion or gene-specific targeting of transcription regulatory proteins and observing changes in RNA synthesis in real-time. Third, I will develop a new technology to visualize the binding dynamics of single transcription factor molecules at the transcription site, so that the stability of upstream regulatory factors and the RNA output can directly be compared in the same cell. Finally, I will examine the phenotypic effect of different bursting patterns on organismal fitness. Overall, these approaches will reveal how bursting is regulated at the molecular level and how different bursting patterns affect the heterogeneity and fitness of the organism.

1 950 775 €

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

My proposal aims to take advantage of my ground-breaking finding that it is possible to mature, or activate, the [FeFe] hydrogenase enzyme (HydA) using synthetic mimics of its catalytic [2Fe] cofactor. (Berggren et al, Nature, 2013) We will now explore the chemistry and (bio-)technological potential of the enzyme using an interdisciplinary approach ranging from in vivo biochemical studies all the way to synthetic model chemistry. Hydrogenases catalyse the interconversion between protons and H2 with remarkable efficiency. Consequently, they are intensively studied as alternatives to Pt-catalysts for these reactions, and are arguably of high (bio-) technological importance in the light of a future “hydrogen society”. The project involves the preparation of novel “artificial” hydrogenases with the primary aim of designing spectroscopic model systems via modification(s) of the organometallic [2Fe] subsite. In parallel we will prepare in vitro loaded forms of the maturase HydF and study its interaction with apo-HydA in order to further elucidate the maturation process of HydA. Moreover we will develop the techniques necessary for in vivo application of the artificial activation concept, thereby paving the way for a multitude of studies including the reactivity of artificial hydrogenases inside a living cell, but also e.g. gain-of-function studies in combination with metabolomics and proteomics. Inspired by our work on the artificial maturation system we will also draw from our knowledge of Nature’s [FeS] cluster proteins in order to prepare a novel class of “miniaturized hydrogenases” combining synthetic [4Fe4S] binding oligopeptides with [2Fe] cofactor model compounds. Our interdisciplinary approach is particularly appealing as it not only provides further insight into hydrogenase chemistry and the maturation of metalloproteins, but also involves the development of novel tools and concepts applicable to the wider field of bioinorganic chemistry.

1 494 880 €

Start date: 2017-02-01, End date: 2022-01-31

Recent advances have shown that therapeutic manipulations of key cell-cell interactions can have dramatic clinical outcomes. Most notable are several early successes in cancer immunotherapy that target the tumor-T cell interface. However, these successes were only partial. This is likely because the few known interactions are just a few pieces of a much larger puzzle, involving additional signaling molecules and cell types. Dendritic cells (DCs), play critical roles in the induction/suppression of T cells. At early cancer stages, DCs capture tumor antigens and present them to T cells. However, in advanced cancers, the tumor microenvironment (TME) disrupts the crosstalk between DCs and T cells. We will take a multi-step approach to explore how the TME imposes a suppressive effect on DCs and how to reverse this hazardous effect. First, we will use single cell RNA-seq to search for genes in aggressive human and mouse ovarian tumors that are highly expressed in advanced tumors compared to early tumors and that encode molecules that suppress DC activity. Second, we will design a set of CRISPR screens to find genes that are expressed in DCs and regulate the transfer of the suppressive signals. The screens will be performed in the presence of suppressive molecules to mimic the TME and are expected to uncover many key genes in DCs biology. We will develop a new strategy to find synergistic combinations of genes to target (named Perturb-comb), thereby reversing the effect of local tumor immunosuppressive signals. Lastly, we will examine the effect of modified DCs on T cell activation and proliferation in-vivo, and on tumor growth. We expect to find: (1) Signaling molecules in the TME that affect the immune system. (2) New cytokines and cell surface receptors that are expressed in DCs and signal to T cells. (3) New key regulators in DC biology and their mechanisms. (4) Combinations of genes to target in DCs that reverse the TME’s hazardous effects.

1 500 000 €

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

Cancer is the result of a multi-step process requiring the accumulation of mutations in several genes. For most cancers, the target cells of oncogenic mutations are unknown. Adult stem cells (SCs) might be the initial target cells as they self-renew for extended periods of time, providing increased opportunity to accumulate the mutations required for cancer formation. Certain cancers contain cells characteristics of SC with high self-renewal capacities and the ability to reform the parental tumor upon transplantation. However, whether the initial oncogenic mutations arise in normal stem cells or in more differentiated cells that re-acquire stem cell-like properties remains to be determined. The demonstration that SCs are the target cells of the initial transforming events and that cancers contain cells with SC characteristics await the development of tools allowing for the isolation and characterization of normal adult SCs. In most epithelia from which cancers naturally arise, such tools are not yet available. We have recently developed novel methods to specifically mark and isolate multipotent epidermal slow-cycling SCs, making it now possible to determine the role of SC during epithelial cancer formation. In this project, we will use mice epidermis as a model to define the role of SC in epithelial cancer initiation and growth. Specifically, we will determine whether epithelial SCs are the initial target cells of oncogenic mutations during skin cancer formation, whether oncogenic mutations lead preferentially to skin cancer when they arise in SC rather than in more committed cells and whether cancer stem cells contribute to epithelial tumor growth and relapse after therapy.

1 600 000 €

Start date: 2008-07-01, End date: 2013-12-31

According to string theory, coherent sheaves on three-dimensional Calabi-Yau spaces encode fundamental properties of the universe. On the other hand, they have a purely mathematical definition. We will develop and use the new field of categorified Donaldson-Thomas (DT) theory, which counts these objects. Via the powerful perspective of noncommutative algebraic geometry, this theory has found application in recent years in a wide variety of contexts, far from classical algebraic geometry. Categorification has proved tremendously powerful across mathematics, for example the entire subject of algebraic topology was started by the categorification of Betti numbers. The categorification of DT theory leads to the replacement of the numbers of DT theory by vector spaces, of which these numbers are the dimensions. In the area of categorified DT theory we have been able to prove fundamental conjectures upgrading the famous wall crossing formula and integrality conjecture in noncommutative algebraic geometry. The first three projects involve applications of the resulting new subject: 1. Complete the categorification of quantum cluster algebras, proving the strong positivity conjecture. 2. Use cohomological DT theory to prove the outstanding conjectures in the nonabelian Hodge theory of Riemann surfaces, and the subject of Higgs bundles. 3. Prove the comparison conjecture, realising the study of Yangian quantum groups and the geometric representation theory around them as a special case of DT theory. The final objective involves coming full circle, and applying our recent advances in noncommutative DT theory to the original theory that united string theory with algebraic geometry: 4. Develop a generalised theory of categorified DT theory extending our results in noncommutative DT theory, proving the integrality conjecture for categories of coherent sheaves on Calabi-Yau 3-folds.

1 239 435 €

Start date: 2017-11-01, End date: 2022-10-31

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