ERC FUNDED PROJECTS

Biofuel from wood and waste will be a substantial share of our future energy mix. The conversion of lignocellulose to fermentable polysaccharides is the current bottleneck. We propose to use single molecule cut and paste technology to assemble designer cellulosoms and combine enzymes from different species with nanocatalysts.

2 351 450 €

Start date: 2012-03-01, End date: 2018-02-28

"Translation of the genetically encoded information into polypeptides, protein biosynthesis, is a central function executed by ribosomes in all cells. In the case of membrane protein synthesis, integration into the membrane usually occurs co-translationally and requires a ribosome-associated translocon (SecYEG/Sec61). This highly coordinated process is poorly understood, since high-resolution structural information is lacking. Although single particle cryo-electron microscopy (cryo-EM) has given invaluable structural insights for such dynamic ribosomal complexes, the resolution is so far limited to 5-10 Å for asymmetrical particles. Thus, the mechanistic depth and reliability of interpretation has accordingly been limited. Here, I propose to use single particle cryo-EM at improved, molecular resolution of 3-4 Å to study two fundamental ribosome-associated processes: (i) co-translational integration of polytopic membrane proteins and (ii) recycling of the eukaryotic ribosome. First, we will visualize nascent polytopic membrane proteins inserting into the lipid bilayer via the bacterial ribosome-bound SecYEG translocon. Notably, the translocon will be embedded in a lipid environment provided by so-called nanodiscs. Second, we will visualize in a similar approach membrane protein insertion via the YidC insertase, the main alternative translocon. Third, as a novel research direction, we will determine the structure and function of eukaryotic ribosome recycling complexes involving the ABC-ATPase RLI. The results will allow, together with functional biochemical data, an in-depth molecular structure-function analysis of these fundamental ribosome-associated processes. Moreover, reaching molecular resolution for asymmetrical particles by single particle cryo-EM will lift this technology to a level of analytical power approaching X-ray and NMR methods. ERC funding would allow for this highly challenging research to be conducted in an internationally competitive way in Europe."

2 995 640 €

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

Heart failure (HF) affects 15 million Europeans and entails higher mortality and health care costs than cancer. EPLORE addresses this issue by prospective epidemiological research in 4 European countries and by a proof-of-concept clinical trial. WP1 will for the first time at the population level document the incidence and progression of subclinical LV dysfunction and clarify whether asymptomatic LV dysfunction, as picked up by the newest echocardiographic techniques, predicts cardiovascular (CV) outcomes, including HF. WP2 will investigate the contribution of ventricular-arterial coupling disease and mechanical LV dyssynchrony to subclinical LV dysfunction. WP3 will identify a set of urinary polypeptides that signify early LV dysfunction and validate these biomarkers by showing that they predict deterioration of LV function, progression to HF and the incidence of CV complications over and beyond established risk factors. WP3 will also search for novel panels of circulating biomarkers, of which combined measurement will add information (accuracy, sensitivity and specificity) to established biomarkers (e.g., NT-proBNP) and identify genetic variants involved in the progression of LV dysfunction, either causally or as biomarker. WP4 consists of a randomised clinical trial to translate in a high-risk high-gain setting the results of WPs 1-3 into clinical practice and to identify a new treatment modality that potentially slows progression of diastolic LV dysfunction. Dissemination in WP5 will contribute to new guidelines for the prevention and treatment of HF. WP6 includes governance, monitoring research strategies and output, and protection of IPR. In conclusion, EPLORE will advance risk stratification and the early diagnosis of subclinical HF. The project will potentially result into specific treatments for diastolic LV dysfunction and inform guidelines for prevention and treatment of HF. It will benefit 20% of Europeans who currently have subclinical LV dysfunction.

2 391 440 €

Start date: 2012-07-01, End date: 2017-06-30

Humans have evolved intimate symbiotic relationships with a consortium of gut microbes (including fungi) and individual variations in the microbiome influence host health and disease. The fact that fungi are capable of colonizing almost every niche within the human body suggests that they must possess particular immune adaptation mechanisms, the breakdown of which may result in fatal fungal infections and severe fungal diseases. Traditional reductionist approaches of the past have not been sufficient to address these new challenges in the pathogenesis of fungal diseases. Here, I propose an integrated, systems biology approach to understand the role of L-tryptophan (trp) metabolic pathways in multilevel host−fungus interactions. Present in mammals as well as in fungi, pathways of trp metabolic pathways are exploited by the host and the fungal biota for survival and immune adaptation. A variety of indole derivatives, generated through conversion from dietary trp by symbiotic bacteria, activate the aryl hydrocarbon receptor/IL-22 pathway that provides antifungal resistance and tissue repair. Harmful inflammatory responses to fungi are instead tamed by kynurenines generated via the enzyme indoleamine 2,3–dioxygenase (IDO) of the trp pathway. Through high-throughput wet-lab ‘omics’ techniques combined with computational techniques, the project aims at defining the molecular basis of mammalian and fungal IDO activity and a metabolic network linking the metabolic phenotype (metabotype) to immune adaptations and its possible breakdown in experimental and human fungal infections. The project will provide ideal post-graduate training focussed on the development of metabolomics for diagnosis of fungal diseases and optimization of current antifungal therapy and diet that are of relevance to public health care solutions.

2 299 200 €

Start date: 2012-04-01, End date: 2018-03-31

Plant diseases represent one of the most important risks to ensuring global food security and new control strategies for the world’s most serious crop diseases are urgently needed. This project will lead to an unparalleled advance in understanding of the world’s most serious crop disease - rice blast. Each year rice blast disease claims 11-30% of the potential rice harvest, which is enough rice to feed 60 million people. The fungus that causes rice blast disease, Magnaporthe oryzae, is a highly adapted cereal pathogen, which forms specialized infection structures called appressoria that can breach the intact leaf surface and allow the fungus entry to living plant tissue. This project will result in the most comprehensive understanding of the biology of plant infection by a pathogenic fungus. The cross-disciplinary research programme will integrate next generation DNA sequencing, digital transcriptional profiling, and high throughput gene functional analysis, with cell biology and live cell imaging. In this way, the transcriptional networks and gene regulators of fungal virulence will be identified and characterized at a systems level. The project will investigate the nature of the host-pathogen interface using live cell imaging of stable transgenic rice lines expressing fluorescent fusion proteins that define specific sub-cellular domains and will allow the precise mechanism of cellular invasion by the fungus to be determined. The project will also use protein-protein interaction studies to identify the major effectors used by M. oryzae to suppress plant immunity and facilitate proliferation of the fungus within living plant tissue. In parallel, comparative genome analysis will define conserved the full repertoire of effector functions and the transcriptional networks necessary for plant disease. Comprehensive functional analysis of M. oryzae effectors will then be carried out by targeted gene deletion, spatial localization and target identification.

2 498 733 €

Start date: 2012-03-01, End date: 2017-12-31

Oxygenic photosynthesis, that takes place in cyanobacteria algae and plants, provides most of the food and fuel on earth. The light stage of this process is driven by two photosystems. Photosystem II (PSII) that oxidizes water to O2 and 4 H+ and photosystem I (PSI) which in the light provides the most negative redox potential in nature that can drive numerous reactions including CO2 assimilation and hydrogen (H2) production. The structure of most of the complexes involved in oxygenic photosynthesis was solved in several laboratories including our own. Utilizing our plant PSI crystals we were able to generate a light dependent electric potential of up to 100 V. We will develop this system for designing biological based photoelectric devices. Recently, we discovered a marine phage that carries an operon encoding all PSI subunits. Generation, in synechocystis, of a phage-like PSI enabled the mutated complex to accept electrons from additional sources like respiratory cytochromes. This way a novel photorespiration, where PSI can substitute for cytochrome oxidase, is created. The wild type and mutant synechocystis PSI were crystallized and solved, confirming the suggested structural consequences. Moreover, several structural alterations in the mesophilic PSI were recorded. We designed a hydrogen producing bioreactor where the novel photorespiration will enable to utilize the organic material of the cell as an electron source for H2 production. We propose that in conjunction of engineering a Cyanobacterium strain with a temperature sensitive PSII, enhancing rates in its respiratory chain an efficient and sustainable hydrogen production can be achieved.

2 487 000 €

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

"The immune system and pathogens both use membrane pore-forming proteins to penetrate cellular membranes, in order to kill target cells or to allow passage of pathogenic organisms such as malaria parasites, listeria, or toxoplasma. For both fundamental and practical reasons, it is important to understand the biological actions of the ""arms race"" underlying virulence, pathogenesis and immune defense. The key weapon in this membrane attack is a class of proteins that upon activation undergo a dramatic conversion from water-soluble monomers to a large, membrane-inserted assembly. Both the human immune response and microbial pathogenesis rely on membrane disruption by perforin-like proteins for attack and counterattack. This protein superfamily encompasses perforin and complement pore-forming assemblies in the immune system, as well as the more distantly related bacterial cholesterol-dependent cytolysins. With recent advances in electron cryo-microscopy, tomography and correlative fluorescence microscopy, it is now possible to relate the workings of protein machines in model systems such as liposomes to their actions in the cellular context. I wish to capitalize on these technical advances and visualize membrane interactions at the moment the intracellular pathogen Toxoplasma gondii bursts out of its host cell, as well as the delivery of lethal cargo from the cytotoxic lymphocyte to its target cell through the immune synapse. These studies will correlate 3D spatial information at cellular and molecular levels to reveal the operation of dynamic cellular machinery. I have chosen a well-ordered system that can bridge the gulf between cell biology and atomic structure. Innovations in sample preparation combined with state-of the art imaging methods will lead to the molecular definition of a fundamental process in “hostile” communication between cells and will broaden the landscape for drug design for immune disorders and major infectious diseases."

2 311 036 €

Start date: 2012-06-01, End date: 2018-05-31

"The mismatch repair (MMR) system has evolved to correct errors of DNA replication and prevent recombination between non-homologous sequences. Correspondingly, MMR malfunction leads to increased mutation rates and illegitimate recombination, and individuals with inherited MMR gene mutations are predisposed to cancer of the colon and other organs. However, MMR has recently been implicated in processes ranging from DNA damage signaling and drug sensitivity to antibody maturation, some of which contradict and even subvert the classical role of MMR as a guardian of genomic integrity. We suspect that the latter processes are linked to a new, non-canonical MMR (ncMMR) pathway that can be activated outside of the S- and G2 phases of the cell cycle by a variety of lesions and structures. ncMMR lacks strand directionality, involves long stretches of DNA degradation, and our preliminary in vitro evidence suggests that resynthesis of these repair tracts can be mediated not only by high-fidelity, replicative polymerases, but also by error-prone enzymes. In this scenario, ncMMR would actually contribute to mutagenesis. I plan to deploy proteomic, genomic and imaging technologies to identify the components of the ncMMR “mutasome” and to reconstitute the system from purified recombinant components. Furthermore, I wish to study the “action radius” of MMR proteins by characterizing their interactome and analyze its dependence on endogenous states (e.g. cell cycle stages) and exogenous stimuli (e.g. drug treatments) in human and chicken (DT40) cells, and Xenopus laevis egg extracts. I intend to exploit a new system of inducible protein replacement that was recently developed in my laboratory, to stably express MMR, replication, repair and recombination proteins (both wild type and variants). This program should increase our understanding of the pivotal role of MMR in DNA metabolism and its involvement in human disease and cancer."

2 208 084 €

Start date: 2012-04-01, End date: 2016-08-31

In the past three decades, magnetic resonance imaging (MRI) has become a vital tool for clinical diagnosis and research. A major current trend is the introduction of magnets with much more powerful static magnetic fields, including magnets at 7 Tesla (7T) and higher. Advantages of higher magnetic fields include higher signal-to-noise ratios enabling improved spatial and temporal resolution, and new, unique tissue contrasts due to enhanced sensitivity to tissue susceptibility differences. Unfortunately, the radiofrequency (RF) fields used to excite tissue at higher magnetic fields are subject to interference and penetration effects, leading to signal dropouts which vary from subject to subject depending on body habitus. These effects imply that the inherent advantages of 7T often cannot be leveraged to realise practical imaging benefits. A fair evaluation of the diagnostic potential of 7T cannot be achieved, as image quality improvements are handicapped and often counteracted by these unresolved technical hurdles. 7T MRI cannot be considered for routine clinical use or even effectively evaluated for such use until these hurdles have been overcome. Preliminary research indicates that these effects can be addressed by use of parallel transmission strategies. The goal of the proposed project is to develop a highly optimized multi-channel transmit/receive RF coil for body MRI at 7T. This coil should then be used to exploit and manipulate the complex RF field patterns at 7T using parallel transmission approaches. In contrast to previous approaches, a hybrid method including both static and dynamic shimming of the RF field will be investigated. We hypothesise that such an approach would greatly enhance the flexibility of RF manipulation while limiting overall system complexity. It can be conjectured based on the known properties of ultra-high-field MRI that success would have ground-breaking impact on the diagnosis and characterisation of manifold disease processes.

2 099 996 €

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

"Plant diseases represent a significant threat to global food security. One of the most notorious plant pathogens is the Irish potato famine organism Phytophthora infestans. P. infestans, the causal agent of potato and tomato late blight, continues to cost modern agriculture billions of euros annually. The most sustainable strategy to manage late blight is to breed broad-spectrum disease resistance into potato and tomato. However, current disease resistance breeding approaches are slow, inefficient, and have taken little advantage of emerging knowledge of pathogen mechanisms. Resistance genes have been identified, bred, and deployed in agriculture without detailed knowledge of the effectors they are sensing – a ‘blind’ approach. The overall aim of this proposal is to exploit state of the art findings on pathogen effector biology to drive the development of new approaches to breeding disease resistant crops. Our long-term objective is to generate late blight resistant crop varieties. The central hypothesis of the proposed research is that mutations in plant resistance and effector target genes can result in broad-spectrum resistance to late blight. The rationale behind this research is based on exciting preliminary data that demonstrated (i) there is a core set of effectors in P. infestans; (ii) plant proteins targeted by P. infestans effectors are important components of the immune response; and (iii) single amino acid changes in a potato resistance protein can result in expanded effector recognition. To achieve our goal, we plan to pursue the following specific objectives: 1. Understand the molecular details of how three P. infestans effectors modulate plant immunity. 2. Generate synthetic plant resistance genes with expanded recognition of P. infestans effectors. 3. Generate synthetic plant effector targets with modified interaction with P. infestans effectors. 4. Use the synthetic genes to engineer broad-spectrum late blight resistance."

2 496 835 €

Start date: 2012-04-01, End date: 2017-03-31

The human crystalline lens has the capability to dynamically change its shape to focus near and far objects. By age 55, the accommodation capability is lost and optical aids are needed for near vision. Many questions remain open that are critical to understand accommodation, the failure in presbyopia, and the prospects for its correction. Multifocal presbyopic corrections are increasingly used. However, the ideal multifocal pattern, and the optical factors affecting depth-of-focus and adaptation to simultaneous vision remain to be elucidated. The most satisfactory treatment of presbyopia should rely on the restoration of the dynamic and continuous focusing ability of the eye, and this could be achieved in the form of accommodative intraocular lenses (IOLs). Current approaches, relying on potential IOL axial shifts, have proved little effective accommodative amplitude. The project will seek in nature innovative solutions to treat presbyopia. Deeper understanding of the crystalline lens changes with dynamic accommodation and aging will be gained. Novel imaging techniques will be developed and used to assess the dynamic changes of crystalline lens structure, gradient index distribution and microscopic structure of the lens fibers and capsule. In addition, the treatment of presbyopia by multifocal corrections will be explored. Wavefront sensing and optical coherence tomography will be used to understand the bases for the multifocality found in some animal species (as possible inspiration for multifocal patterns), and adaptive optics and visual simulation to understand the reasons for the limited performance of current multifocal treatments, to investigate neural adaptation to the blur in simultaneous vision and to test the proposed new multifocal patterns. Finally, the understanding of the crystalline lens properties and the biomechanics of the implanted IOLs gained in the project will allow to develop a first prototype of crystalline-lens mimicking accommodative IOL.

2 399 548 €

Start date: 2012-05-01, End date: 2018-04-30

Oxidative stress, an excess of radical and other reactive oxygen species (ROS), has been suggested as a major disease mechanism. However, the major clinical trials using anti-oxidants have been failures, even suggesting serious side effects. Here, I propose completely different approaches: First, instead of letting radicals form and then scavenge them we will identify their diseases-relevant sources and prevent their formation or specifically repair the damage caused by ROS. Second, we will differentiate beneficial signalling roles of ROS. In combination, this will result in unprecedented precision and molecular specificity. In 2010, I submitted a somewhat related proposal to the ERC and received a comment as being “too focused on essential hypertension”. This proposal has a much broader focus and impact beyond cardiovascular diseases. In the past months we achieved major breakthroughs by identifying a radical/ROS source (NOX4) as fundamental mechanism in stroke, the fastest growing and soon no 1 cause of death. We are also developing in phase II a radical formation inhibitor for neurotrauma. Moreover, our basic research facilitated the development of drug classes re-activating an oxidatively damaged signalling receptor, now in phase III. Further, we identified angiogenesis as a radical/ROS-dependent and protective (!) signalling event. This proposal is just the beginning: our basic science will open up new fields and leap forward in personalized medicine with groundbreaking technologies and approaches. We will contribute to the diagnosis and early identification of patients at risk and to monitor their successful treatment (in vitro/blood-based); to the localization of disease processes (in vivo/molecular imaging) before the onset of symptoms; and to a new generation of more effective, predictable, and mechanism-based drugs. We also expect to later apply our findings and tools to neurobiology and oncology, where ROS also play physiological and pathological roles.

2 298 000 €

Start date: 2012-08-01, End date: 2018-07-31

"The general aim of REJOIND is to provide proof-of-principle for the in vitro manufacturing of a growing bone, with a bioartificial growth plate as a “driving engine” at its core. To achieve this, we propose a developmental engineering approach, based on the modular design of in vitro processes consisting of sequential units corresponding to in vivo developmental stages. These processes follow a gradual and coordinated progression of tissue growth and cell differentiation that leads to organization of cells into intermediate tissue forms. At every step of the developmental engineering process, computational models will be applied, in order to form a quantitative foundation for every process and to optimize these. After establishment of a manufacturing process of a growth plate, REJOIND will combine this tissue with osteoblasts or articular chondrocytes to build osteochondral tissues or bioartificial joints. Ultimately, REJOIND aims to achieve an autonomous process of in vitro tissue growth allowing guided size expansion. A close interaction between biologists and engineers will make this possible. Pre-clinical applications that will be explored in animal models range from the repair of deep osteochondral defects in a joint surface, to a total joint replacement for small arthritic joints. We expect that a number of these implants will provide a cartilaginous template for bone formation, therefore some will be tested in vivo in appropriate models for healing of long bone defects. In conclusion, REJOIND aims to provide evidence that through the use of developmental engineering, we can build a tissue in vitro, moving the boundary from manufacturing and control at the cellular level to tissue organization and function. This methodology will result in a more reliable in vivo outcome of tissue engineered products, and thus a more predictable and sustainable clinical outcome in the patient."

3 057 673 €

Start date: 2012-09-01, End date: 2017-08-31

A prerequisite for prevention or early diagnosis of a tumour disease is that environmental and/or genetic risk factors are characterized in order to better define risk groups. The present research proposal focus on common malignant tumours, especially breast cancer and malignant melanoma . By combining risk factor studies on endogenous and exogenous environmental, and genetic risk factors and its interaction, the aim is to better characterize strong determinants of risk. The project also aims at better understanding the mechanisms of disease and for different exposures, such as sun exposure and different hormonal exposures, obtain a global assessment of possible positive and negative health and disease effects. The infrastructure includes large population based cohort and case-control studies, pathological and clinical patient information, biobanks from cancer patients and controls, availability of excellent genomic resources and an extensive network of national and international collaborations. Very often it has been possible to work out from a population based perspective. Both for breast cancer and melanoma the research group has identified important genes or modifiers of dominant predisposing genes as well as environmental or constitutional risk factors. Through an extensive international collaboration gene and gene-environemnt interaction studies are undertaken. The ulitmate goal for the new knowledge is to prevent or early diagnose the malignancy. In hereditary cancer and for some of the hormonal exposures successful results already are seen. The applicant has been involved more than 30 years in epidemiological research of cancer, and published more than 400 publications in international journals and fostered a large number of PhD students.

1 200 000 €

Start date: 2012-04-01, End date: 2017-03-31

This project will elucidate the structure and mechanism of ribosome function through the determination of high resolution crystal structures of human ribosome functional complexes. Currently, the only high resolution crystal structures available for the ribosome functional complexes are bacterial ribosomes containing mRNA, tRNAs in E, P and A sites and several translation factors. Recently, we have determined at medium resolution the first eukaryotic ribosome structure. Saccharomyces cerevisaiae ribosomes have been chosen for the first step of the study because they have been extensively investigated in our laboratory by biochemical methods and by x-ray analysis. Step One: We will improve the quality of the crystals and determine the ribosome structure of yeast at atomic resolution (about 3Å resolution). These conditions will be used for an investigation of the ribosome functional complexes in order to better understand the detailed mechanism of the translocation of large molecules (tRNA and mRNA) on the ribosome. Step Two: Ribosomes from human HeLa cells will be investigated via crystallization and x-ray structure determination. Ribosome functional complexes developed on yeast systems will be used as models for the creation and structural investigation of human ribosome complexes. Available biochemical data will provide a framework for the interpretation of structural information which will be obtained. The aims: To determine the atomic resolution crystal structure of the yeast ribosome as a model for all eukaryotic ribosome x-ray studies. To obtain crystals and determine the structure of 80S ribosome from HeLa cells. To determine the structure of the ribosome complexes with mRNA and tRNA in ratcheted and non-ratcheted states in order to describe the mechanism of translocation. To determine structure of the ribosome initiation complex with an internal ribosomal entry site mRNA. To obtain crystals and to determine the structures of 40S initiation complexes.

2 466 000 €

Start date: 2012-04-01, End date: 2017-03-31

The primary aims of this work are tightly connected: (i) development of a bio-inspired “synthetic cell” capable of self-assembling, self-propelling and environmental sensing prepared with reduced molecular complexity compared to living cells (ii) quantitative assessment of the bio-activity of specific cellular components within these “synthetic cells”, leading to better fundamental understanding of their function in living cells (iii) use of these findings for reverse engineering of living cells with tailored adhesive and sensory properties. Integrin based adhesion has been shown to participate in numerous processes in living cells, which sense, via their adhesions, multiple environmental cues, integrate them, and develop a complex, multi-parametric response. However, due to their intrinsic molecular complexity the specific functional roles of different components of the adhesion site are still poorly understood. To address this issue, we will utilize current knowledge of the modular nature of focal adhesions and related integrin-mediated extracellular matrix contacts to develop “synthetic cell” models, consisting of large lipid vesicles, functionalized by transmembrane integrins, various integrin-binding proteins and specific sets of scaffolding and signaling proteins of the adhesion sites. The one-by-one loading of these vesicles by micro-injection with these proteins will allow tight control of the system composition and complexity, and testing of the effect of compositional and environmental variations on the adhesion and signaling features. The “synthetic cells” will be plated on adhesive matrices displaying specific spatial, chemical and mechanical features for testing their chemical and mechanical sensing capabilities. The datasets produced in these experiments will provide a solid basis for reverse engineering perturbations of living cells, in which specific functional pathways will be targeted and/or modified to modulate living cells' functionality.

3 499 799 €

Start date: 2012-05-01, End date: 2018-04-30