ERC FUNDED PROJECTS

Plants typically release large quantities of volatiles in response to attack by herbivores or pathogens. I may claim to have contributed to various breakthroughs in this research field, including the discovery that the volatile blends induced by different attackers are astonishingly specific, resulting in characteristic, readily distinguishable odour blends. Using maize as our model plant, I wish to take several leaps forward in our understanding of this signal specificity and use this knowledge to develop sensors for the real-time detection of crop pests and diseases. For this, three interconnected work-packages will aim to: • Develop chemical analytical techniques and statistical models to decipher the odorous vocabulary of plants, and to create a complete inventory of “odour-prints” for a wide range of herbivore-plant and pathogen-plant combinations, including simultaneous infestations. • Develop and optimize nano-mechanical sensors for the detection of specific plant volatile mixtures. For this, we will initially adapt a prototype sensor that has been successfully developed for the detection of cancer-related volatiles in human breath. • Genetically manipulate maize plants to release a unique blend of root-produced volatiles upon herbivory. For this, we will engineer gene cassettes that combine recently identified P450 (CYP) genes from poplar with inducible, root-specific promoters from maize. This will result in maize plants that, in response to pest attack, release easy-to-detect aldoximes and nitriles from their roots. In short, by investigating and manipulating the specificity of inducible odour blends we will generate the necessary knowhow to develop a novel odour-detection device. The envisioned sensor technology will permit real-time monitoring of the pests and enable farmers to apply crop protection treatments at the right time and in the right place.

2 498 086 €

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

The project outlined here addresses the fundamental question how the brain encodes and controls behavior. While we have a reasonable understanding of the role of entire brain areas in such processes, and of mechanisms at the molecular and synaptic levels, there is a big gap in our knowledge of how behavior is controlled at the level of defined neuronal circuits. In natural environments, chances for survival depend on learning about possible aversive and appetitive outcomes and on the appropriate behavioral responses. Most studies addressing the underlying mechanisms at the level of neuronal circuits have focused on aversive learning, such as in Pavlovian fear conditioning. Understanding how activity in defined neuronal circuits mediates appetitive learning, as well as how these circuitries are shared and interact with aversive learning circuits, is a central question in the neuroscience of learning and memory and the focus of this grant application. Using a multidisciplinary approach in mice, combining behavioral, in vivo and in vitro electrophysiological, imaging, optogenetic and state-of-the-art viral circuit tracing techniques, we aim at dissecting the neuronal circuitry of appetitive Pavlovian conditioning with a focus on the amygdala, a key brain region important for both aversive and appetitive learning. Ultimately, elucidating these mechanisms at the level of defined neurons and circuits is fundamental not only for an understanding of memory processes in the brain in general, but also to inform a mechanistic approach to psychiatric conditions associated with amygdala dysfunction and dysregulated emotional responses including anxiety and mood disorders.

2 497 200 €

Start date: 2016-01-01, End date: 2020-12-31

T cell engineering with chimeric antigen receptors has opened the door to effective immunotherapy. CARs are fusion genes encoding receptors whose extracellular domain comprises a single chain variable fragment (scFv) antibody that binds to a tumour surface epitope, while the intracellular domain comprises the signalling module of CD3ζ along with powerful costimulatory domains (e.g. CD28 and/or 4-1BB). CARs are a major breakthrough, since they allow bypassing HLA restrictions or loss, and they can incorporate potent costimulatory signals tailored to optimize T cell function. However, solid tumours present challenges, since they are often genetically unstable, and the tumour microenvironment impedes T cell function. The tumour vasculature is a much more stable and accessible target, and its disruption has catastrophic consequences for tumours. Nevertheless, the lack of affinity reagents has impeded progress in this area. The objectives of this proposal are to develop the first potent and safe tumour vascular-disrupting tumour immunotherapy using scFv’s and CARs uniquely available in my laboratory. I propose to use these innovative CARs to understand for the first time the molecular mechanisms underlying the interactions between anti-vascular CAR-T cells and tumour endothelium, and exploit them to maximize tumour vascular destruction. I also intend to employ innovative engineering approaches to minimize the chance of reactivity against normal vasculature. Lastly, I propose to manipulate the tumour damage mechanisms ensuing anti-vascular therapy, to maximize tumour rejection through immunomodulation. We are poised to elucidate critical interactions between tumour endothelium and anti-vascular T cells, and bring to bear cancer therapy of unparalleled power. The impact of this work could be transforming, given the applicability of tumour-vascular disruption across most common tumour types.

2 500 000 €

Start date: 2013-08-01, End date: 2018-07-31

The objective of this project is to overcome current limitations for antibody production that are inherent to the extant immune system of vertebrates. This will be done by creating an all-in-one artificial/synthetic counterpart based exclusively on prokaryotic parts, devices and modules. To this end, ARISYS will exploit design concepts, construction hierarchies and standardization notions that stem from contemporary Synthetic Biology for the assembly and validation of (what we believe is) the most complex artificial biological system ventured thus far. This all-bacterial immune-like system will not only simplify and make affordable the manipulations necessary for antibody generation, but will also permit the application of such binders by themselves or displayed on bacterial cells to biotechnological challenges well beyond therapeutic and health-related uses. The work plan involves the assembly and validation of autonomous functional modules for [i] displaying antibody/affibody (AB) scaffolds attached to the surface of bacterial cells, [ii] conditional diversification of target-binding sequences of the ABs, [iii] contact-dependent activation of gene expression, [iv] reversible bi-stable switches, and [v] clonal selection and amplification of improved binders. These modules composed of stand-alone parts and bearing well defined input/output functions, will be assembled in the genomic chassis of streamlined Escherichia coli and Pseudomonas putida strains. The resulting molecular network will make the ABs expressed and displayed on the cell surface to proceed spontaneously (or at the user's decision) through subsequent cycles of affinity and specificity maturation towards antigens or other targets presented to the bacterial population. In this way, a single, easy-to-handle (albeit heavily engineered) strain will govern all operations that are typically scattered in a multitude of separate methods and apparatuses for AB production.

2 422 271 €

Start date: 2013-05-01, End date: 2019-04-30

The prognosis of patients with metastatic pancreatic cancer (PDAC) is very poor. Recent studies have started to elucidate the genetic landscape of this disease to show that PDAC is a genetically complex, unstable, and heterogeneous cancer. However, in-depth analysis of individual patient genomes couple with personalize Avatar mouse models is providing highly effective therapeutic opportunities for the individual patient. Thus, metastatic PDAC appears a candidate disease to implement a genomics-base, personalized treatment approach. In this project, we will conduct an open label, multicenter, randomized phase III study in patients with standard of care resistant metastatic pancreatic cancer aiming to test the hypothesis that an integrated personalized treatment approach improves survival compare to a conventional treatment. Patients randomized to the personalize treatment arm will undergo a biopsy of a metastatic lesion to perform a targeted genome analysis using next generation sequencing. In addition, we will generate a personalize Avatar mouse model from the same patient. We will employ sophisticated bioinformatic analysis as well as mining of drug response-genetic databases to select, for each individual patient, candidate therapeutic targets that will be experimentally tested in the patient´s Avatar model to select the most effective regimen that will ultimately applied to the patient. In addition, based on the genomic data, we will design an individualized monitoring plan for each patient using BEAMing technology to monitor circulating levels of mutated genes. Furthermore, with a discovery goal, we will perform in depth genomic analysis of metastatic PDAC lesions in this cohort of clinically well-annotated patients with Avatar mouse models for therapeutic validation. Overall we expect this work will contribute to our understanding of PDAC and will favourably impact the treatment of this dismal cancer.

2 498 688 €

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

Asymmetric cell division is a key mechanism for the generation of cell diversity in eukaryotes. During this process, a polarized mother cell divides into non-equivalent daughters. These may differentially inherit fate determinants, irreparable damages or age determinants. Our aim is to decipher the mechanisms governing the individualization of daughters from each other. In the past ten years, our studies identified several lateral diffusion barriers located in the plasma membrane and the endoplasmic reticulum of budding yeast. These barriers all restrict molecular exchanges between the mother cell and its bud, and thereby compartmentalize the cell already long before its division. They play key roles in the asymmetric segregation of various factors. On one side, they help maintain polarized factors into the bud. Thereby, they reinforce cell polarity and sequester daughter-specific fate determinants into the bud. On the other side they prevent aging factors of the mother from entering the bud. Hence, they play key roles in the rejuvenation of the bud, in the aging of the mother, and in the differentiation of mother and daughter from each other. Recently, we accumulated evidence that some of these barriers are subject to regulation, such as to help modulate the longevity of the mother cell in response to environmental signals. Our data also suggest that barriers help the mother cell keep traces of its life history, thereby contributing to its individuation and adaption to the environment. In this project, we will address the following questions: 1 How are these barriers assembled, functioning, and regulated? 2 What type of differentiation processes are they involved in? 3 Are they conserved in other eukaryotes, and what are their functions outside of budding yeast? These studies will shed light into the principles underlying and linking aging, rejuvenation and differentiation.

2 200 000 €

Start date: 2010-05-01, End date: 2015-04-30

BIOFORCE has a highly ambitious applied objective: to create transgenic cereal plants that will provide a near-complete micronutrient complement (vitamins A, C, E, folate and essential minerals Ca, Fe, Se and Zn) for malnourished people in the developing world, as well as built-in resistance to insects and parasitic weeds. This in itself represents a striking advance over current efforts to address food insecurity using applied biotechnology in the developing world. We will also address fundamental mechanistic aspects of multi-gene/pathway engineering through transcriptome and metabolome profiling. Fundamental science and applied objectives will be achieved through the application of an exciting novel technology (combinatorial genetic transformation) developed and patented by my research group. This allows the simultaneous transfer of an unlimited number of transgenes into plants followed by library-based selection of plants with appropriate genotypes and phenotypes. All transgenes integrate into one locus ensuring expression stability over multiple generations. This proposal represents a new line of research in my laboratory, founded on incremental advances in the elucidation of transgene integration mechanisms in plants over the past two and a half decades. In addition to scientific issues, BIOFORCE address challenges such as intellectual property, regulatory and biosafety issues and crucially how the fruits of our work will be taken up through philanthropic initiatives in the developing world while creating exploitable opportunities elsewhere. BIOFORCE is comprehensive and it provides a complete package that stands to make an unprecedented contribution to food security in the developing world, while at the same time generating new knowledge to streamline and simplify multiplex gene transfer and the simultaneous modification of multiple complex plant metabolic pathways

2 290 046 €

Start date: 2009-04-01, End date: 2014-03-31

Traffic-related air pollution is an important environmental problem that may affect neurodevelopment. Ultrafine particles (UFP) translocate to the brains of experimental animals resulting in local proinflammatory overexpression. As the basic elements for thinking are acquired by developing brains during infancy and childhood, susceptibility may be elevated in early life. We postulate that traffic-related air pollution (particularly UFPs and metals/hydrocarbons content) impairs neurodevelopment in part via effects on frontal lobe maturation, likely increasing attention-deficit/hyperactivity disorder (ADHD). BREATHE objectives are to develop valid methods to measure children's personal UFP exposure and to develop valid neuroimaging methods to assess correlations between neurobehavior, neurostructural alterations and particle deposition in order to reveal how traffic pollution affects children¿s exposure to key contaminants and brain development, and identify susceptible subgroups. We have conducted general population birth cohort studies providing preliminary evidence of residential air pollution effects on prenatal growth and mental development. We aim to demonstrate short and long-term effects on neurodevelopment using innovative epidemiological methods interfaced with environmental chemistry and neuroimaging following 4000 children from 40 schools with contrasting high/low traffic exposure in six linked components involving: repeated psychometric tests, UFP exposure assessment using personal, school and home measurements, gene-environment interactions on inflammation, detoxification pathways and ADHD genome-wide-associated genes, neuroimaging (magnetic resonance imaging/spectroscopy) in ADHD/non-ADHD children, integrative causal modeling using mathematics, and replication in 2900 children with neurodevelopment followed from pregnancy. We believe the expected results will have worldwide global planning and policy implications.

2 499 230 €

Start date: 2011-08-01, End date: 2016-07-31

The frontier in cancer therapy of orchestrating the immune system to attack tumors is producing unprecedented survival benefit in some patients. The corollary is lack of efficacy both in ostensibly responsive tumor types as well as others that are mostly non-responsive. The basis lies in pre-existing and adaptive resistance mechanisms that circumvent induction of tumor-reactive cytotoxic T cells (CTLs) capable of infiltrating solid tumors and eliminating cancer cells. A priori, cancers induced by expression of human papillomavirus oncogenes should be responsive to immunotherapy: these cancers encode immunogenic neo-antigens – the oncoproteins E6/7 – necessary for their manifestation. Rather, such tumors are poorly responsive to immunotherapies. Results from my lab and others using mouse models of HPV-induced cancer have established an actionable hypothesis: during tumorigenesis, such tumors erect multiple barriers to the induction, infiltration, and killing of cancer cells by tumor antigen-reactive CTLs. These include overarching systemic antigen-nonspecific immunosuppression mediated by expanded populations of myeloid cells in spleen and lymph nodes, complemented by immune response-impairing barriers operative in the tumor microenvironment. A spectrum of models will probe these barriers, genetically and pharmacologically, establishing their functional importance, alone and in concert. A major focus will be on how oncogene-expressing keratinocytes elicit a marked expansion of immunosuppressive myeloid cells in spleen and lymph nodes, and how these myeloid cells in turn inhibit development and activation of CD8 T cells and antigen-presenting dendritic cells. Then we’ll assess the therapeutic potential of barrier-breaking strategies combined with immuno-stimulatory modalities. This project will deliver new knowledge about multi-faceted barriers to immunotherapy in these refractory cancers, helping lay the groundwork for efficacious immunotherapy.

2 500 000 €

Start date: 2020-01-01, End date: 2024-12-31

Bacterial biofilms are the primary cause of chronic infections and of resulting infection relapses. To be able to interfere with bacterial persistence it is vital to understand the molecular details of biofilm formation and to define how motile planktonic cells transit into surface-grown communities. The nucleotide second messenger cyclic di-guanosinemonophosphate (cdGMP) has emerged as a central regulatory factor governing bacterial surface adaptation and biofilm formation. Although cdGMP signaling may well represent the Achilles heel of bacterial communities, cdGMP networks in bacterial pathogens are exquisitely complex and an integrated cellular system to uncover the details of cdGMP dynamics is missing. To quantitatively describe cdGMP signaling we propose to exploit Caulobacter crescentus, an organism with a simple bimodal life-style that integrates the sessile-motile switch into its asymmetric division cycle. We aim to: 1) identify the role and regulation of all diguanylate cyclases and phosphodiesterases that contribute to the asymmetric cellular program with the goal to model the temporal and spatial distribution of cdGMP during development; 2) identify and characterize cdGMP effectors, their downstream targets and cellular pathways; 3) elucidate how cdGMP coordinates cell differentiation with cell growth and propagation; 4) unravel the role of cdGMP as an allosteric regulator in mechanosensation and in rapid adaptation of bacteria to growth on surfaces; 5) develop novel tools to quantitatively describe cdGMP network dynamics as the basis for mathematical modeling that provides the predictive power to experimentally test and refine important network parameters. We propose a multidisciplinary research program at the forefront of bacterial signal transduction that will provide the molecular and conceptual framework for a rapidly growing research field of second messenger signaling in pathogenic bacteria.

2 496 000 €

Start date: 2013-05-01, End date: 2018-04-30

"Our research group has worked over the years at the interface between cancer and ageing, with a strong emphasis on mouse models. More recently, we became interested in cellular reprogramming because we hypothesized that understanding cellular plasticity could yield new insights into cancer and ageing. Indeed, during the previous ERC Advanced Grant, we made relevant contributions to the fields of cellular reprogramming (Nature 2013), cellular senescence (Cell 2013), cancer (Cancer Cell 2012), and ageing (Cell Metabolism 2012). Now, we take advantage of our diverse background and integrate the above processes. Our unifying hypothesis is that cellular plasticity lies at the basis of tissue regeneration (“adaptive cellular plasticity”), as well as at the origin of cancer (“maladaptive gain of cellular plasticity”) and ageing (“maladaptive loss of cellular plasticity”). A key experimental system will be our “reprogrammable mice” (with inducible expression of the four Yamanaka factors), which we regard as a tool to induce cellular plasticity in vivo. The project is divided as follows: Objective #1 – Cellular plasticity and cancer: role of tumour suppressors in in vivo de-differentiation and reprogramming / impact of transient de-differentiation on tumour initiation / lineage tracing of Oct4 to determine whether a transient pluripotent-state occurs during cancer. Objective #2 – Cellular plasticity in tissue regeneration and ageing: impact of transient de-differentiation on tissue regeneration / contribution of the damage-induced microenvironment to tissue regeneration / impact of transient de-differentiation on ageing. Objective #3: New frontiers in cellular plasticity: chemical manipulation of cellular plasticity in vivo / new states of pluripotency / characterization of in vivo induced pluripotency and its unique properties. We anticipate that the completion of this project will yield new fundamental insights into cancer, regeneration and ageing."

2 488 850 €

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

"Centrioles are critical for the formation of cilia, flagella and centrosomes, as well as for human health. The mechanisms governing centriole formation constitute a long-standing question in cell biology. We will pursue an innovative multidisciplinary research program to gain further insight into these mechanisms, using human cells, C. elegans and Trichonympha as model systems. This program is expected to also have a major impact by contributing a novel cell free assay to the field, thus paving the way towards making synthetic centrioles. Six specific aims will be pursued: 1) Deciphering HsSAS-6/STIL distribution and dynamics. We will use super-resolution microscopy, molecular counting, photoconversion and FCS to further characterize these two key components required for centriole formation in human cells. 2) The SAS-6 ring model as a tool to redirect centriole organization. Utilizing predictions from the SAS-6 ring model, we will assay the consequences for centrioles and cilia of altering the diameter and symmetry of the structure. 3) Determining the architecture of C. elegans centrioles. We will conduct molecular counting and cryo-ET of purified C. elegans centrioles to determine if they contain a spiral or a cartwheel, as well as identify SAS-6-interacting components. 4) Comprehensive 3D map and proteomics of Trichonympha centriole. We will obtain a ~35 Å 3D map of the complete T. agilis centriole, perform proteomic analysis to identify its constituents and test their function using RNAi. 5) Regulation of cartwheel height and centriole length. We will explore whether cartwheel height is set by SAS-6 proteins and perform screens in human cells to identify novel components regulating cartwheel height and centriole length. 6) Novel cell free assay for cartwheel assembly and centriole formation. Using SAS-6 proteins on a lipid monolayer as starting point, we will develop and utilize a cell-free assay to reconstitute cartwheel assembly and centriole format"

2 499 270 €

Start date: 2014-04-01, End date: 2019-03-31

"DNA in higher organisms is organized in a nucleoprotein complex called chromatin. The structure of chromatin is responsible for compacting DNA to fit within the nucleus and for governing its access in nuclear processes. Epigenetic information is encoded chiefly via chromatin modifications. Readout of the genetic code depends on chromatin remodeling, a process actively altering chromatin structure. An understanding of the hierarchical structure of chromatin and of structurally based, remodeling mechanisms will have enormous impact for developments in medicine. Following our high resolution structure of the nucleosome core particle, the fundamental repeating unit of chromatin, we have endeavored to determine the structure of the chromatin fiber. We showed with our X-ray structure of a tetranucleosome how nucleosomes could be organized in the fiber. Further progress has been limited by structural polymorphism and crystal disorder, but new evidence on the in vivo spacing of nucleosomes in chromatin should stimulate more advances. Part A of this application describes how we would apply these new findings to our cryo-electron microscopy study of the chromatin fiber and to our crystallographic study of a tetranucleosome containing linker histone. Recently, my laboratory succeeded in providing the first structurally based mechanism for nucleosome spacing by a chromatin remodeling factor. We combined the X-ray structure of ISW1a(ATPase) bound to DNA with cryo-EM structures of the factor bound to two different nucleosomes to build a model showing how this remodeler uses a dinucleosome, not a mononucleosome, as its substrate. Our results from a functional assay using ISW1a further justified this model. Part B of this application describes how we would proceed to the relevant cryo-EM and X-ray structures incorporating dinucleosomes. Our recombinant ISW1a allows us to study in addition the interaction of the ATPase domain with nucleosome substrates."

2 500 000 €

Start date: 2013-01-01, End date: 2017-12-31

Organismal development requires proper timing of events such as cell fate choices, but the mechanisms that control temporal patterning remain poorly understood. In particular, we know little of the cyclical timers, or ‘clocks’, that control recurring events such as vertebrate segmentation or nematode molting. Furthermore, it is unknown how cyclical timers are coordinated with the global, or linear, timing of development, e.g. to ensure an appropriate number of cyclical repeats. We propose to elucidate the components, wiring, and properties of a prototypic developmental clock by studying developmental timing in the roundworm C. elegans. We build on our recent discovery that nearly 20% of the worm’s transcriptome oscillates during larval development – an apparent manifestation of a clock that times the various recurring events that encompass each larval stage. Our aims are i) to identify components of this clock using genetic screens, ii) to gain insight into the system’s architecture and properties by employing specific perturbations such as food deprivation, and iii) to understand the coupling of this cyclic clock to the linear heterochronic timer through genetic manipulations. To achieve our ambitious goals, we will develop tools for mRNA sequencing of individual worms and for their developmental tracking and microchamber-based imaging. These important advances will increase temporal resolution, enhance signal-to-noise ratio, and achieve live tracking of oscillations in vivo. Our combination of genetic, genomic, imaging, and computational approaches will provide a detailed understanding of this clock, and biological timing mechanisms in general. As heterochronic genes and rhythmic gene expression are also important for controlling stem cell fates, we foresee that the results gained will additionally reveal regulatory mechanisms of stem cells, thus advancing our fundamental understanding of animal development and future applications in regenerative medicine.

2 358 625 €

Start date: 2017-10-01, End date: 2022-09-30

Over many years, our research group has explored the complex relationship between cancer and ageing. As part of this work, we have generated mouse models of protease deficiency which are protected from cancer but exhibit accelerated ageing. Further studies with these mice have allowed us to unveil novel mechanisms of both normal and pathological ageing, to discover two new human progeroid syndromes, and to develop therapies for the Hutchinson-Gilford progeria syndrome, now in clinical trials. We have also integrated data from many laboratories to first define The hallmarks of ageing and the current possibilities for Metabolic control of longevity. Now, we propose to leverage our extensive experience in this field to further explore the relative relevance of cell-intrinsic and -extrinsic mechanisms of ageing. Our central hypothesis is that ageing derives from the combination of both systemic and cell-autonomous deficiencies which lead to the characteristic loss of fitness associated with this process. Accordingly, it is necessary to integrate multiple approaches to understand the mechanisms underlying ageing. This integrative and multidisciplinary project is organized around three major aims: 1) to characterize critical cell-intrinsic alterations which drive ageing; 2) to investigate ageing as a systemic process; and 3) to design intervention strategies aimed at expanding longevity. To fully address these objectives, we will use both hypothesis-driven and unbiased approaches, including next-generation sequencing, genome editing, and cell reprogramming. We will also perform in vivo experiments with mouse models of premature ageing, genomic and metagenomic studies with short- and long-lived organisms, and functional analyses with human samples from both progeria patients and centenarians. The information derived from this project will provide new insights into the molecular mechanisms of ageing and may lead to discover new opportunities to extend human healthspan.

2 456 250 €

Start date: 2017-09-01, End date: 2022-08-31

The recent anthropogenic global warming makes a detailed understanding of coupling processes between climate and biogeochemical cycles of pressing importance. The atmospheric archive of air bubbles enclosed in polar ice cores provides the only direct record of greenhouse gas changes in the past, and the key to understanding the related changes in biogeochemical cycles and climate/greenhouse gas feedbacks. Crucial questions about greenhouse gas variability on very short (decadal) and very long (orbital) time scales still remain open. To answer these questions, the ice core community has proposed new drilling projects with the goal of nearly doubling the time span of the available ice core record to the last 1.5 million years and of covering the entire Holocene greenhouse gas record in unprecedented decadal resolution. These goals have one thing in common: due to glacier flow most of this record will only be found in a very thin layer in the bottom-most ice of the cores. Completely new analytical approaches are needed to unlock the atmospheric archive in this ice in order to gain high-resolution, high-precision measurements, while at the same time drastically reducing sample consumption compared to established techniques. The deepSLice project will make such a step change in ice core analytics by developing a novel coupled Continuous Sublimation Extraction-Quantum Cascade Laser Spectrometer system. It will allow us to simultaneously measure CO2, CH4 and N2O concentrations as well as the isotopic composition of CO2 on air samples of only 1-2 ml at standard pressure and temperature, reducing the required sample size by one order of magnitude. This non-destructive analysis will make it also possible for the complete air sample to be recollected after analysis and used for other measurements. This method will be applied to existing and new ice cores in order to study past changes in greenhouse gases and the underlying biogeochemical cycles in unparalleled detail.

2 255 788 €

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

Chronic pain syndromes are to a large extent due to maladaptive plastic changes in the CNS. A CNS area particularly relevant for such changes is the spinal dorsal horn, where inputs from nociceptive and non-nociceptive fibers undergo their first synaptic integration. This area harbors a sophisticated network of interneurons, which function as a gate-control unit for incoming sensory signals. Several different types of interneurons can be distinguished based e.g. on their neurotransmitter and neuropeptide content. Despite more than 40 years of research, our knowledge about the integration of these neurons in dorsal horn circuits and their contribution to sensory processing is still very limited. This proposal aims at a comprehensive characterization of the dorsal horn neuronal network under normal conditions and in chronic pain states with a focus on inhibitory interneurons. A genome-wide analysis of the gene expression profile shall be made from defined dorsal horn interneurons genetically tagged with fluorescent markers and isolated by fluorescence activated cell sorting. A functional characterization of the connectivity of these neurons in spinal cord slices and of their role in in vivo sensory processing shall be achieved with optogenetic tools (channelrhodopsin-2), which permit activation of these neurons with light. Finally, behavioral analyses shall be made in mice after diphteria toxin-mediated ablation of defined interneuron types. All three approaches shall be applied to naïve mice and to mice with inflammatory or neuropathic pain. The results from these studies will improve our understanding of the malfunctioning of sensory processing in chronic pain states and will provide the basis for novel approaches to the prevention or reversal of chronic pain states.

2 467 000 €

Start date: 2010-05-01, End date: 2016-04-30

Virtually every species of insect harbors facultative bacterial endosymbionts that are transmitted from females to their offspring, often in the egg cytoplasm. These symbionts play crucial roles in the biology of their hosts. Many manipulate host reproduction in order to spread within host populations. Others increase the fitness of their hosts under certain conditions. For example, increasing tolerance to heat or protecting their hosts against natural enemies. Over the past decade, our understanding of insect endosymbionts has shifted from seeing them as fascinating oddities to being ubiquitous and central to the biology of their hosts, including many of high economic and medical importance. However, in spite of growing interest in endosymbionts, very little is known about the molecular mechanisms underlying most endosymbiont-insect interactions. For instance, the basis of the main phenotypes caused by endosymbionts, including diverse reproductive manipulations or symbiont-protective immunity, remains largely enigmatic. The goal of the present application is to fill this gap by dissecting the interaction between Drosophila and its native endosymbiont Spiroplasma poulsonii. This project will use a broad range of approaches ranging from molecular genetic to genomics to dissect the molecular mechanisms underlying key features of the symbiosis, including vertical transmission, male killing, regulation of symbiont growth, and symbiont-mediated protection against parasitic wasps. We believe that the fundamental knowledge generated on the Drosophila-Spiroplasma interaction will serve as a paradigm for other endosymbiont-insect interactions that are less amenable to genetic studies.

1 963 926 €

Start date: 2014-05-01, End date: 2019-04-30

"Integration of large scale genetic and epigenetic analysis needs to be coupled with well defined biological hypotheses that can be experimentally tested. This project is aimed at developing a novel integrated approach to understand genetic and epigenetic predisposition to cancer with skin as model system. The Caucasian (West European) and Asian (East Asian) populations differ substantially in their predisposition to skin cancer, specifically Squamous Cell Carcinoma (SCC). The underlying mechanisms are poorly understood. As in other organs, skin SCC results from changes in both epithelial and mesenchymal compartments. We will be focusing on two key gene regulatory networks of cells of the two compartments (keratinocytes and dermal fibroblasts), with a key role in skin SCC. The ""keratinocyte network"" has Notch/p53/p63 as key nodes, while the ""dermal fibroblast network"" had Notch and AP1 family members. We will pursue two main goals : 1) We will test the hypothesis that a linkage can be established between specific genetic and epigenetic marks in the Caucasian versus Asian populations and differences in expression and function of ""keratinocyte and/or dermal fibroblast network genes"". 2) We will test the hypothesis that keratinocytes and/or dermal fibroblasts of Caucasian versus Asian individuals differ in their tumor yielding capability, and that these differences in cancer forming capability are due to differences in either ""keratinocyte or dermal fibroblast network genes"". The applicant is a world leader in epithelial signaling and cancer biology, and is heading interdisciplinary research efforts that bridge the basic and clinical sciences. Together with his bioinformatician and clinician collaborators, he is in an excellent position to attain the high goals of the proposal. The approach has not been attempted before, is only possible within the frame of an advanced ERC grant, and has substantial basic as well as translational/clinical implications."

2 495 425 €

Start date: 2014-02-01, End date: 2020-01-31

A hallmark of cancer is tumor cell heterogeneity, which results from combinations of multiple genetic and epigenetic alterations within an individual tumor. In contrast, we have recently discovered that most human colorectal cancers (CRCs) are composed of mixtures of phenotypically distinct tumor cells organized into a stem cell hierarchy that displays a striking resemblance to the healthy colonic epithelium. We showed that long-term regeneration potential of tumor cells is largely influenced by the position that they occupy within the tumor's hierarchy. To analyze the organization of CRCs without the constraints imposed by tumor cell transplantation experiments, we have developed a method that allows for the first time tracking and manipulating the fate of specific cell populations in whole human tumors. This technology is based on editing the genomes of primary human CRCs cultured in the form of tumor organoids using Zinc-Finger Nucleases to knock-in either lineage tracing or cell ablation alleles in genes that define colorectal cancer stem cells (CRC-SCs) or differentiated-like tumor cells. Edited tumor organoids generate CRCs in mice that reproduce the tumor of origin while carrying the desired genetic modifications. This technological advance opens the gate to perform classical genetic and developmental analysis in human tumors. We will exploit this advantage to address fundamental questions about the cell heterogeneity and organization of human CRCs that cannot be tackled through currently existing experimental approaches such as: Are CRC-SCs the only tumor cell population with long term regenerating potential? Can we cure CRC with anti-CRC-SC specific therapies? Will tumor cell plasticity contribute to the regeneration of the CRC-SC pool after therapy? Do quiescent-SCs regenerate CRC tumors after standard chemotherapy? Can we identify these cells? How do common genetic alterations in CRC influence the CRC hierarchy? Do they affect the stem cell phenotype?

2 499 405 €

Start date: 2014-04-01, End date: 2019-03-31

The majority of animals show an external bilateral symmetry, precluding the observation of multiple internal left-right (L/R) asymmetries which are fundamental for organ packaging and function. A prominent molecular pathway converging on and downstream of the Pitx2 transcription factor confers left-handed information in the left side of the embryo, with players expressed on the right ensuring that the left determinants are excluded. Therefore, conferring or excluding left identity in left and right hand sides, respectively, drives L/R asymmetry. Some indications suggest that a program actively specifying right–handed information could exist on the right. Our recent findings support this view. In a screening for novel regulators of the epithelial to mesenchymal transition (EMT), we have identified a transcription factor, EMT2, which similarly to well known factor Snail, it is an EMT inducer. The EMT is crucial for the development of tissues during embryonic development and for the progression of carcinomas to the invasive state. Strikingly, again as Snail, in addition to promote EMT, the EMT2 factor is predominantly expressed on the right side and may operate instructing L/R identity on the right-hand side of the embryo. With this background, our knowledge of the EMT and a series of genome-wide high-throughput approaches and a comprehensive functional analysis using the chick, the fish and the mouse as model systems we propose to reveal the putative molecular pathways conveying right-handed information and to reveal commonalities between L/R pathways and the EMT. In the long run, we aim at better understanding human pathologies that involve these morphogenetic and cellular processes, including pathological situs conditions (i.e. altered organ positioning) and cancer progression.

2 460 000 €

Start date: 2013-05-01, End date: 2018-12-31

In recent years, my laboratory, as well as others, have established the observation that epigenetic disruption, particularly in the DNA methylation and histone modification patterns, contributes to the initiation and progression of human tumors (Esteller, Nat Rev Genet 2007; Esteller, N Engl J Med 2008; Esteller, Nat Rev Biotech, In Press, 2010). Even more recently, it has been recognized that microRNAs, small non-coding RNAs that are thought to regulate gene expression by sequence-specific base pairing in mRNA targets, also play a key role in the biology of the cell, and that they can also have an impact in the development of many diseases, including cancer (le Sage and Agami, 2006; Blenkiron and Miska, 2007). However, there is little understanding about epigenetic modifications that might regulate the activity of microRNAs and other non-coding RNAs (ncRNAs), such as long non-coding RNAs (lncRNAs), Piwi-interacting RNAs (piRNAs), small-interfering RNAs (siRNAs), transcribed ultraconserved regions (T-UCRs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), long interspersed ncRNAs (lincRNAs), promoter-associated RNAs (PASRs and PALRs) and terminator-associated sRNAs (TASRs) (Calin et al., 2007; Mercer, et al., 2009; Ghildiyal & Zamore, 2009; Jacquier, 2009). Our ignorance in this respect is even more significant if we consider these questions in the domain of cancer. Making best use of our expertise in several of these fields, my group will tackle the study of the epigenetic modifications that regulate ncRNA expression and how the DNA methylation and histone modifications profiles of these loci might become distorted in human cancer. These findings could have profound consequences not only in the understading of tumor biology, but in the design of better molecular staging, diagnosis and treatments of human malignancies.

2 497 240 €

Start date: 2011-04-01, End date: 2016-03-31

Evolution of animal morphology relies on changes in developmental programs that control body plans and organ shape. Such changes are thought to arise form alteration of the expression of functionally conserved developmental genes and their vast downstream networks. Although this hypothesis has a profound impact on the way we view animal evolution, final proof is still lacking. The hypothesis calls for evolution to take place mainly through modifications of cis-regulatory elements (CREs) controlling gene expression. However, these genomic regions are precisely those that we understand the least and, until recently, basic knowledge on how regulatory information is organized in the 3D genome or how to spatio-temporally assign CREs to their target genes was unknown. The advent of next generation sequencing-based tools has made possible to identify genome-wide CREs and reveal how they are organized in the 3D genome. But this new knowledge has been largely ignored by most hypotheses on the evolution of gene expression, development and animal morphology. These new high-throughput methods have been mainly restricted to selected model organisms, and due to the lack of sequence conservation of CREs across lineages, we still have very limited information about the impact of CREs on animal morphology evolution. By integrating in a systematic and phylogenetically driven manner the contribution of CREs and their 3D organization to animal morphology at different evolutionary scales, we will for the first time link evolution, regulatory information, genome 3D architecture and morphology. We will apply this strategy to study animal morphology along the evolution of deuterostome body plans, the generation of fin morphological diversity in vertebrates, and the recent phenotypic changes in fish adapted to cave environments. Our proposal will make ground-breaking advances in our understanding of the global principles underlying the evolution of cis-regulatory DNA and animal form.

2 499 514 €

Start date: 2017-09-01, End date: 2022-08-31

The ubiquitous FIC domain catalyzes post-translational modifications (PTMs) of target proteins; i.e. adenylylation (=AMPylation) and, more rarely, uridylylation and phosphocholination. Fic proteins are thought to play critical roles in intrinsic signaling processes of prokaryotes and eukaryotes; however, a subset encoded by bacterial pathogens is translocated via dedicated secretion systems into the cytoplasm of mammalian host cells. Some of these host-targeted Fic proteins modify small GTPases leading to collapse of the actin cytoskeleton and other drastic cellular changes. Recently, we described a large set of functionally diverse homologues in pathogens of the genus Bartonella that are required for their “stealth attack” strategy and persistent course of infection [1, 2]. Our preliminary functional analysis of some of these host-targeted Fic proteins of Bartonella demonstrated adenylylation activity towards novel host targets (e.g. tubulin and vimentin). Moreover, in addition to the canonical adenylylation activity they may also display a competing kinase activity resulting from altered ATP binding to the FIC active site. Finally, we described a conserved mechanism of FIC active site auto- inhibition that is relieved by a single amino acid exchange [1], thus facilitating functional analysis of any Fic protein of interest. Despite this recent progress only a few Fic proteins have been functionally characterized to date; our understanding of the functional plasticity of the FIC domain in mediating diverse target PTMs and their specific roles in infection thus remains limited. In this project, we aim to study the vast repertoire of host-targeted Fic proteins of Bartonella to: 1) identify novel target proteins and types of PTMs; 2) study their physiological consequences and molecular mechanisms of action; and 3) analyze structure-function relationships critical for FIC-mediated PTMs and infer from these data determinants of target specificity, type of PTM and mode of regulation. At the forefront of infection biology research, this project is ground-breaking as (i) we will identify a plethora of novel host target PTMs that are critical for a “stealth attack” infection strategy and thus will open new avenues for investigating fundamental mechanisms of persistent infection; and (ii), we will unveil the molecular basis of the remarkable functional versatility of the structurally conserved FIC domain.

1 699 858 €

Start date: 2014-02-01, End date: 2019-01-31