ERC FUNDED PROJECTS

The goals are 1. To develop a scale assessing the diversity of active ageing with four dimensions that are ability (what people can do), activity (what people do do), ambition (what are the valued activities that people want to do), and autonomy (how satisfied people are with the opportunity to do valued activities); 2. To examine health and physical and psychological functioning as the determinants and social and build environment, resilience and personal skills as modifiers of active ageing; 3. To develop a multicomponent sustainable intervention aiming to promote active ageing (methods: counselling, information technology, help from volunteers); 4. To test the feasibility and effectiveness on the intervention; and 5. To study cohort effects on the phenotypes on the pathway to active ageing. “If You Can Measure It, You Can Change It.” Active ageing assessment needs conceptual progress, which I propose to do. A quantifiable scale will be developed that captures the diversity of active ageing stemming from the WHO definition of active ageing as the process of optimizing opportunities for health and participation in the society for all people in line with their needs, goals and capacities as they age. I will collect cross-sectional data (N=1000, ages 75, 80 and 85 years) and model the pathway to active ageing with state-of-the art statistical methods. By doing this I will create novel knowledge on preconditions for active ageing. The collected cohort data will be compared to a pre-existing cohort data that was collected 25 years ago to obtain knowledge about changes over time in functioning of older people. A randomized controlled trial (N=200) will be conducted to assess the effectiveness of the envisioned intervention promoting active ageing through participation. The project will regenerate ageing research by launching a novel scale, by training young scientists, by creating new concepts and theory development and by producing evidence for active ageing promotion

2 044 364 €

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

Despite the discovery of amyloidosis more than a century ago, the molecular and cellular mechanisms of these devastating human disorders remain obscure. In addition to their involvement in disease, amyloid fibrils perform physiological functions, whilst others have potentials as biomaterials. To realise their use in nanotechnology and to enable the development of amyloid therapies, there is an urgent need to understand the molecular pathways of amyloid assembly and to determine how amyloid fibrils interact with cells and cellular components. The challenges lie in the transient nature and low population of aggregating species and the panoply of amyloid fibril structures. This molecular complexity renders identification of the culprits of amyloid disease impossible to achieve using traditional methods. Here I propose a series of exciting experiments that aim to cast new light on the molecular and cellular mechanisms of amyloidosis by exploiting approaches capable of imaging individual protein molecules or single protein fibrils in vitro and in living cells. The proposal builds on new data from our laboratory that have shown that amyloid fibrils (disease-associated, functional and created from de novo designed sequences) kill cells by a mechanism that depends on fibril length and on cellular uptake. Specifically, I will (i) use single molecule fluorescence and non-covalent mass spectrometry and to determine why short fibril samples disrupt biological membranes more than their longer counterparts and electron tomography to determine, for the first time, the structural properties of cytotoxic fibril ends; (ii) develop single molecule force spectroscopy to probe the interactions between amyloid precursors, fibrils and cellular membranes; and (iii) develop cell biological assays to discover the biological mechanism(s) of amyloid-induced cell death and high resolution imaging and electron tomography to visualise amyloid fibrils in the act of killing living cells.

2 498 465 €

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

Argonaute nucleases are key players of the eukaryotic RNA interference (RNAi) system. Using small RNA guides, these Argonaute (Ago) proteins specifically target complementary RNA molecules, resulting in regulation of a wide range of crucial processes, including chromosome organization, gene expression and anti-virus defence. Since 2010, my research team has studied closely-related prokaryotic Argonaute (pAgo) variants. This has revealed spectacular mechanistic variations: several thermophilic pAgos catalyse DNA-guided cleavage of double stranded DNA, but only at elevated temperatures. Interestingly, a recently discovered mesophilic Argonaute (CbAgo) can generate double strand DNA breaks at moderate temperatures, providing an excellent basis for this ARGO project. In addition, genome analysis has revealed many distantly-related Argonaute variants, often with unique domain architectures. Hence, the currently known Argonaute homologs are just the tip of the iceberg, and the stage is set for making a big leap in the exploration of the Argonaute family. Initially we will dissect the molecular basis of functional and mechanistic features of uncharacterized natural Argonaute variants, both in eukaryotes (the presence of an Ago-like subunit in the Mediator complex, strongly suggests a regulatory role of an elusive non-coding RNA ligand) and in prokaryotes (selected Ago variants possess distinct domains indicating novel functionalities). After their thorough biochemical characterization, I aim at engineering the functionality of the aforementioned CbAgo through an integrated rational & random approach, i.e. by tinkering of domains, and by an unprecedented in vitro laboratory evolution approach. Eventually, natural & synthetic Argonautes will be selected for their exploitation, and used for developing original genome editing applications (from silencing to base editing). Embarking on this ambitious ARGO expedition will lead us to many exciting discoveries.

2 177 158 €

Start date: 2019-07-01, End date: 2024-06-30

DNA double-strand breaks are perhaps the most harmful DNA damages and result in carcinogenic chromosome aberrations. Cells protect their genome by activating a complex signaling and repair network, collectively denoted DNA damage response (DDR). A key initial step of the DDR is the activation of the 360 kDa checkpoint kinase ATM (ataxia telangiectasia mutated) by the multifunctional DSB repair factor Mre11-Rad50-Nbs1 (MRN). MRN senses and tethers DSBs, processes DSBs for further resection, and recruits and activates ATM to trigger the DDR. A mechanistic basis for the activities of the core DDR sensor MRN has not been established, despite intense research over the past decade. Our recent breakthroughs on structures of core Mre11-Rad50 and Mre11-Nbs1 complexes enable us now address three central questions to finally clarify the mechanism of MRN in the DDR: - How does MRN interact with DNA or DNA ends in an ATP dependent manner? - How do MRN and associated factors such as CtIP process blocked DNA ends? - How do MRN and DNA activate ATM? We will employ an innovative structural biology hybrid methods approach by combining X-ray crystallography, electron microscopy and small angle scattering with crosslink mass spectrometry and combine the structure-oriented techniques with validating in vitro and in vivo functional studies. The anticipated outcome will clarify the structural mechanism of one of the most important but enigmatic molecular machineries in maintaining genome stability and also help understand the molecular defects associated with several prominent cancer predisposition and neurodegenerative disorders.

2 498 019 €

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

In the last forty years, researchers in the Field of Migration and Ethnic Studies looked at the integration of migrants and their descendants. Concepts, methodological tools and theoretical frameworks have been developed to measure and predict integration outcomes both across different ethnic groups and in comparison with people of native descent. But are we also looking into the actual integration of the receiving group of native ‘white’ descent in city contexts where they have become a numerical minority themselves? In cities like Amsterdam, now only one in three youngsters under age fifteen is of native descent. This situation, referred to as a majority-minority context, is a new phenomenon in Western Europe and it presents itself as one of the most important societal and psychological transformations of our time. I argue that the field of migration and ethnic studies is stagnating because of the one-sided focus on migrants and their children. This is even more urgent given the increased ant-immigrant vote. These pressing scientific and societal reasons pushed me to develop the project BAM (Becoming A Minority). The project will be executed in three harbor cities, Rotterdam, Antwerp and Malmö, and three service sector cities, Amsterdam, Frankfurt and Vienna. BAM consists of 5 subprojects: (1) A meta-analysis of secondary data on people of native ‘white’ descent in the six research sites; (2) A newly developed survey for the target group; (3) An analysis of critical circumstances of encounter that trigger either positive or rather negative responses to increased ethnic diversity (4) Experimental diversity labs to test under which circumstances people will change their attitudes or their actions towards increased ethnic diversity; (5) The formulation of a new theory of integration that includes the changed position of the group of native ‘white’ descent as an important actor.

2 499 714 €

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

The biomembrane is a prerequisite of life. It enables the cell to maintain a controlled environment and to establish electrochemical gradients as rapidly accessible energy stores. Biomembranes also provide scaffold for organisation and spatial definition of signal transmission in the cell. Crystal structures of membrane proteins are determined with an increasing pace. Along with functional studies integral studies of individual membrane proteins are now widely implemented. The BIOMEMOS proposal goes a step further and approaches the function of the biomembrane at the higher level of membrane protein complexes. Through a combination of X-ray crystallography, electrophysiology, general biochemistry, biophysics and bioinformatics and including also the application of single-particle cryo-EM and small-angle X-ray scattering, the structure and function of membrane protein complexes of key importance in life will be investigated. The specific targets for investigation in this proposal include: 1) higher-order complexes of P-type ATPase pumps such as signalling complexes of Na+,K+-ATPase, and 2) development of methods for structural studies of membrane protein complexes Based on my unique track record in structural studies of large, difficult structures (ribosomes and membrane proteins) in the setting of a thriving research community in structural biology and biomembrane research in Aarhus provides a critical momentum for a long-term activity. The activity will take advantage of the new possibilities offered by synchrotron sources in Europe. Furthermore, a single-particle cryo-EM research group formed on my initiative in Aarhus, and a well-established small-angle X-ray scattering community provides for an optimal setting through multiple cues in structural biology and functional studies

2 444 180 €

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

"Transcriptional regulation of protein coding genes in eukaryotic cells requires a complex interplay of sequence-specific DNA-binding factors, co-activators, general transcription factors (GTFs), RNA polymerase II and the epigenetic status of target sequences. Nuclear transcription complexes function as large multiprotein assemblies and are often composed of functional modules. The regulated decision-making that exists in cells governing the assembly and the allocation of factors to different transcription complexes to regulate distinct gene expression pathways is not yet understood. To tackle this fundamental question, we will systematically analyse the regulated biogenesis of transcription complexes from their sites of translation in the cytoplasm, through their assembly intermediates and nuclear import, to their site of action in the nucleus. The project will have four main Aims to decipher the biogenesis of transcription complexes: I) Investigate their co-translation-driven assembly II) Determine their cytoplasmic intermediates and factors required for their assembly pathways III) Uncover their nuclear import IV) Understand at the single molecule level their nuclear assembly, dynamics and action at target genes To carry out these aims we propose a combination of multidisciplinary and cutting edge approaches, out of which some of them will be high-risk taking, while others will utilize methods routinely run by the group. The project builds on several complementary expertise and knowledge either already existing in the group or that will be implemented during the project. At the end of the proposed project we will obtain novel results extensively describing the different steps of the regulatory mechanisms that control the assembly and the consequent gene regulatory function of transcription complexes. Thus, we anticipate that the results of our research will have a major impact on the field and will lead to a new paradigm for contemporary metazoan transcription."

2 500 000 €

Start date: 2014-01-01, End date: 2018-12-31

"In recent years, we have made significant contributions to the understanding of the tumour suppressors p53, p16INK4a, and ARF, particularly in relation with cellular senescence and aging. The current project is motivated by two hypothesis: 1) that the INK4/ARF locus is a sensor of epigenetic damage and this is at the basis of its activation by oncogenes and aging; and, 2) that the accumulation of cellular damage and stress is at the basis of both cancer and aging, and consequently ""anti-damage genes"", such as tumour suppressors, simultaneously counteract both cancer and aging. With regard to the INK4/ARF locus, the project includes: 1.1) the generation of null mice for the Regulatory Domain (RD) thought to be essential for the proper regulation of the locus; 1.2) the study of the INK4/ARF anti-sense transcription and its importance for the assembly of Polycomb repressive complexes; 1.3) the generation of mice carrying the human INK4/ARF locus to analyze, among other aspects, whether the known differences between the human and murine loci are ""locus autonomous""; and, 1.4) to analyze the INK4/ARF locus in the process of epigenetic reprogramming both from ES cells to differentiated cells and, conversely, from differentiated cells to induced-pluripotent stem (iPS) cells. With regard to the impact of ""anti-damage genes"" on cancer and aging, the project includes: 2.1) the analysis of the aging of super-INK4/ARF mice and super-p53 mice; 2.2) we have generated super-PTEN mice and we will examine whether PTEN not only confers cancer resistance but also anti-aging activity; and, finally, 2.3) we have generated super-SIRT1 mice, which is among the best-characterized anti-aging genes in non-mammalian model systems (where it is named Sir2) involved in protection from metabolic damage, and we will study the cancer and aging of these mice. Together, this project will significantly advance our understanding of the molecular mechanisms underlying cancer and aging."

2 000 000 €

Start date: 2009-04-01, End date: 2015-03-31

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

A central question in chromatin biology is how to organize the genome and mark specific regions with histone variants. Understanding how to establish and maintain, but also change chromatin states is a fundamental challenge. Histone chaperones, escort factors that regulate the supply, loading, and degradation of histone variants, are key in their placement at specific chromatin landmarks and bridge organization from nucleosomes to higher order structures. A series of studies have underlined chaperone-variant partner selectivity in multicellular organisms, yet recently, dosage imbalances in natural and pathological contexts highlight plasticity in these interactions. Considering known changes in histone dosage during development, one should evaluate chaperone function not as fixed modules, but as a dynamic circuitry that adapts to cellular needs during the cell cycle, replication and repair, differentiation, development and pathology. Here we propose to decipher the mechanisms enabling adaptability to natural and experimentally induced changes in the dosage of histone chaperones and variants over time. To follow new and old proteins, and control dosage, we will engineer cellular and animal models and exploit quantitative readout methods using mass spectrometry, imaging, and single-cell approaches. We will evaluate with an unprecedented level of detail the impact on i) soluble histone complexes and ii) specific chromatin landmarks (centromere, telomeres, heterochromatin and regulatory elements) and their crosstalk. We will apply this to determine the impact of these parameters during distinct developmental transitions, such as ES cell differentiation and T cell commitment in mice. We aim to define general principles for variants in nuclear organization and dynamic changes during the cell cycle/repair and in differentiation and unravel locus specific-roles of chaperones as architects and bricklayers of the genome, in designing and building specific nuclear domains.

2 499 697 €

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

A PubMed search for ‘epigenetic’ identifies nearly 35,000 entries, yet the molecular mechanisms by which chromatin modification and gene expression patterns are actually inherited during chromosome replication — mechanisms which lie at the heart of epigenetic inheritance of gene expression — are still largely uncharacterised. Understanding these mechanisms would be greatly aided if we could reconstitute the replication of chromosomes with purified proteins. The past few years have seen great progress in understanding eukaryotic DNA replication through the use of cell-free replication systems and reconstitution of individual steps in replication with purified proteins and naked DNA. We will use these in vitro replication systems together with both established and novel chromatin assembly systems to understand: a) how chromatin influences replication origin choice and timing, b) how nucleosomes on parental chromosomes are disrupted during replication and are distributed to daughter chromatids, and c) how chromatin states and gene expression patterns are re-established after passage of the replication fork. We will begin with simple, defined templates to learn basic principles, and we will use this knowledge to reconstitute genome-wide replication patterns. The experimental plan will exploit our well-characterised yeast systems, and where feasible explore these questions with human proteins. Our work will help explain how epigenetic inheritance works at a molecular level, and will complement work in vivo by many others. It will also underpin our long-term research goals aimed at making functional chromosomes from purified, defined components to understand how DNA replication interacts with gene expression, DNA repair and chromosome segregation.

1 983 019 €

Start date: 2015-11-01, End date: 2020-10-31

The complement system is a regulatory pathway in mammalian plasma that enables the host to recognize and clear invading pathogens and altered host cells, while protecting healthy host tissue. This regulatory system consists of ~30 large multi-domain plasma and cell-surface proteins, that act in concert through an interplay of proteolysis and complex formations on target membranes. We study the molecular events on membranes that ensure initiation and amplification of the response, protection of host cells and activation of immune responses leading to cell lysis, phagocytosis and B-cell stimulation. In the past few years, we have resolved the structural details of the large complement proteins involved in the central, aspecific labelling and amplification step; with recent data we revealed the structural basis of the assembly and activity of the protease complex associated with this step. These insights into the central aspecific reaction, and the experiences gained on working with these large multi-domain proteins and complexes, give us an excellent starting point to addres the questions of specificity, protection and activation of immune cells. The goal of the proposal is to elucidate the multivalent molecular mechanisms of recognition, regulation and immune cell activation of the complement system on target membranes. We will use protein crystallography and electron microscopy to study the interactions and conformational changes involved in protein complex formation, and (single-molecule) fluorescence to resolve the multivalent molecular events, the conformational states and transitions that occur on the membrane. The combined data will provide mechanistic insights into the specifity of immune clearance by the complement system. Understanding the molecular mechanisms of complement activation and regulation will be instrumental in developing more potent therapeutics to control infections, prevent tissue damage and fight tumours by immunotherapies.

1 700 000 €

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

Food safety and security are high priority issues throughout Europe at present, the subject of intense government concern, public interest, media speculation and academic scrutiny. With few exceptions, academic research on food has been fragmented with too little interaction between food scientists, health researchers and social scientists. This application builds on the success of a recently completed research programme (Changing Families, Changing Food, 2005-8) which brought together an inter-disciplinary team of over 40 researchers from the food, health and social sciences to address the complex relationships between families and food which lie at the heart of current concerns about food safety and public health. The current proposal aims to take forward the findings of that programme regarding the socially embedded nature of contemporary food choice and to make a step change in our understanding of contemporary consumer anxiety through a focused and concerted programme of research on the political and moral economies of food. The project focuses on consumer anxieties about food at a range of geographic scales, from the global scale of international food markets to the domestic scale of individual households. By taking a whole chain approach -- examining food production and consumption at all points along the chain from farm to fork -- the findings of our research will enable a major advance in our understanding of contemporary anxieties around food, with tangible effects on public health (including the reduction of obesity, diabetes and coronary heart disease).

1 684 460 €

Start date: 2009-01-01, End date: 2012-12-31

Cryopreservation practices are an essential dimension of contemporary life sciences. They make possible the freezing and storage of cells, tissues and other organic materials at very low temperatures and the subsequent thawing of these at a future date without apparent loss of vitality. Although cryotechnologies are fundamental to reproductive technologies, regenerative medicine, transplantation surgery and conservation biology, they have largely escaped scholarly attention in science and technology studies, anthropology and sociology. CRYOSOCIETIES explores the crucial role of cryopreservation in affecting temporalities and the concept of life. The project is based on the thesis that in contemporary societies, cryopreservation practices bring into existence a new form of life: “suspended life”. “Suspended life” enables vital processes to be kept in a liminal state in which biological substances are neither fully alive nor dead. CRYOSOCIETIES generates profound empirical knowledge about the creation of “suspended life” through three ethnographic studies that investigate various sites of cryopreservation. A fourth subproject develops a complex theoretical framework in order to grasp the temporal and spatial regimes of the different cryopractices. CRYOSOCIETIES breaks analytical ground in three important ways. First, the project provides the first systematic and comprehensive empirical study of “suspended life” and deepens our knowledge of how cryopreservation works in different settings. Secondly, it undertakes pioneering work on cryopreservation practices in Europe, generating novel ways of understanding how “suspended life” is assembled, negotiated and mobilised in European societies. Thirdly, CRYOSOCIETIES develops an innovative methodological and theoretical framework in order to address the relationality and materiality of cryopreservation practices and to explore the concept of vitality and the politics of life in the 21st century.

2 497 587 €

Start date: 2019-04-01, End date: 2024-03-31

Specificity in the ubiquitin-proteasome system is largely conferred by ubiquitin E3 ligases (E3s). Cullin-RING ligases (CRLs), constituting ~30% of all E3s in humans, mediate the ubiquitination of ~20% of the proteins degraded by the proteasome. CRLs are divided into seven families based on their cullin constituent. Each cullin binds a RING domain protein, and a vast repertoire of adaptor/substrate receptor modules, collectively creating more than 200 distinct CRLs. All CRLs are regulated by the COP9 signalosome (CSN), an eight-protein isopeptidase that removes the covalently attached activator, NEDD8, from the cullin. Independent of NEDD8 cleavage, CSN forms protective complexes with CRLs, which prevents destructive auto-ubiquitination. The integrity of the CSN-CRL system is crucially important for the normal cell physiology. Based on our previous work on CRL structures (Fischer, et al., Nature 2014; Fischer, et al., Cell 2011) and that of isolated CSN (Lingaraju et al., Nature 2014), We now intend to provide the underlying molecular mechanism of CRL regulation by CSN. Structural insights at atomic resolution, combined with in vitro and in vivo functional studies are designed to reveal (i) how the signalosome deneddylates and maintains the bound ligases in an inactive state, (ii) how the multiple CSN subunits bind to structurally diverse CRLs, and (iii) how CSN is itself subject to regulation by post-translational modifications or additional further factors. The ERC funding would allow my lab to pursue an ambitious interdisciplinary approach combining X-ray crystallography, cryo-electron microscopy, biochemistry and cell biology. This is expected to provide a unique molecular understanding of CSN action. Beyond ubiquitination, insight into this >13- subunit CSN-CRL assembly will allow examining general principles of multi-subunit complex action and reveal how the numerous, often essential, subunits contribute to complex function.

2 200 677 €

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

We pioneered an initially highly controversial connection between DNA damage and (accelerated) aging. In the previous ERC grant ‘DamAge’ we reached the stage that (segmental) aging in DNA repair-deficient mice can be largely controlled. The severity of the repair defect determines the rate of segmental aging; the repair pathways affected influence which organs age fast; conditional repair mutants allow targeting accelerated aging to any organ. Importantly, we found that dietary restriction (DR), the only universal intervention known to delay aging, extends remaining life- and healthspan in progeroid Ercc1Δ/- mutants by 200% (see Vermeij et al., Nature 2016 and fig.2). Also Xpg-/- progeroid repair mice strongly benefit from DR, generalizing this finding. The prominent Alzheimer- and Parkinson-like neurodegeneration is even retarded up to 30-fold(!) disclosing powerful untapped reserves unleashed by 30% less food, with enormous clinical potential. Also we discovered that in accelerated and normal aging gene expression declines due to accumulating stochastic transcription-blocking lesions and that DR counteracts genomic dysfunction. In Dam2Age we will focus on the cross-talk between DNA damage, aging and DR with emphasis on the relevance for normal aging, elucidate underlying mechanisms and use our unique -for DR research superior- mouse models and a variety of novel assays to search for effective nutritional-pharmacological DR mimetics. This serves as a stepping stone towards potent universal therapy for a range of repair-deficient progeroid syndromes and prevention of many aging-related diseases, most urgently dementia’s, to promote sustained health.

2 251 719 €

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

During its duplication, DNA, the carrier of our genetic information, is particularly vulnerable to decay, and the capacity of cells to deal with replication stress has been recognised as a major factor protecting us from genome instability and cancer. A major pathway that allows cells to overcome or bypass DNA lesions during replication is activated by posttranslational modifications of the sliding clamp protein PCNA. Whereas monoubiquitylation of PCNA allows mutagenic translesion synthesis by damage-tolerant DNA polymerases, polyubiquitylation is required for an error-free pathway that involves template switching to the undamaged sister chromatid, involving a recombination-like mechanism. Hence, damage bypass contributes to genome maintenance, but can itself be a source of genomic instability. It is therefore not surprising that PRR is a highly regulated process whose activity is limited to the appropriate situations by stringent control mechanisms. The proposed project aims at understanding DNA damage bypass in its cellular context. Using a combination of new and established technology, we will address the role of chromatin dynamics in the reaction, its spatial and temporal control in relation to genome replication, and its coordination with other PCNA-dependent processes in the cell. To this end, we will establish technology to isolate and analyse the composition of damage bypass tracts, develop and implement novel methods to induce lesions and image damage processing in live cells, and exploit a spectrum of biochemical and biophysical techniques to investigate the role of PCNA as a molecular tool-belt in the coordination of its interaction partners. In combination, these approaches will give important insight into how the replication of damaged DNA is managed with high efficiency and accuracy within the cell.

2 476 388 €

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

"The degradation of mature mRNAs has emerged as a key step in the regulation of eukaryotic gene expression. Modulation of the half-life of mRNAs via their degradation is a powerful and versatile mechanism to swiftly alter the expression of proteins in response to changes in physiological conditions. The decay of mRNAs is performed by a set of macromolecular complexes that act in a sequential and coordinated manner, progressively eroding the ends of the transcript until its degradation is complete. These macromolecular assemblies contain only a few catalytically active subunits and a large number of regulatory components. How and why the activities are regulated within the architecture of the complexes is largely unknown. Also unclear are the mechanisms with which the complexes communicate with each other and/or with the changing composition of the nucleic acid. In this project, we will reconstitute the key protein complexes in mRNA decay from recombinant proteins in vitro. Specifically, we will focus on the evolutionary conserved deadenylation, decapping and exosome-Ski complexes. The reconstituted complexes will be used for structural studies to derive atomic models of the holoenzymes using a combination of X-ray crystallography and cryoelectron microscopy. In parallel to obtaining static views of the individual steps in the pathway, we will establish the assays to study how information from one processing step is passed on to the next in a dynamic manner. We will address the basis for the timing and interrelationship of the conserved enzymatic machineries and the influence of the mRNP composition on their activity. Our final goal is to recapitulate the complex behavior of the mRNA decay pathway in vitro. Our lab has extensive experience in biochemical reconstitution of protein complexes, in vitro biochemical assays and X-ray crystallography. In the next five years, we plan to implement cryoelectron microscopy within the scope of this proposal."

2 499 344 €

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

Our genetic material is continually subjected to damage, either from endogenous sources such as reactive oxygen species, produced as by-products of oxidative metabolism, from the breakdown of replication forks during cell growth, or by agents in the environment such as ionising radiation or carcinogenic chemicals. To cope with DNA damage, cells employ elaborate and effective repair processes that specifically recognise a wide variety of lesions in DNA. These repair systems are essential for the maintenance of genome integrity. Unfortunately, some individuals are genetically predisposed to crippling diseases or cancers that are the direct result of mutations in genes involved in the DNA damage response. For several years our work has been at the forefront of basic biological research in the area of DNA repair, and in particular we have made significant contributions to the understanding of inheritable diseases such as breast cancer, Fanconi anemia, and the neurodegenerative disorder Ataxia with Oculomotor Apraxia (AOA). The focus of this ERC proposal is: (i) to determine the mechanism of action and high-resolution structure of the BRCA2 tumour suppressor, and to provide a detailed picture of the interplay between BRCA2, PALB2, RAD51AP1 and the RAD51 paralogs, in terms of RAD51 filament assembly, using biochemical, electron microscopic and cell biological approaches, (ii) to determine the biological role of a unique structure-selective tri-nuclease complex (SLX1-SLX4-MUS81-EME1-XPF-ERCC1), with particular emphasis on its roles in DNA crosslink repair and Fanconi anemia, and (iii) to understand the actions of Senataxin, which is defective in AOA2, in protecting against genome instability in neuronal cells. These three distinct and yet inter-related areas of the research programme will provide an improved understanding of basic mechanisms of DNA repair and thereby underpin future therapeutic developments that will help individuals afflicted with these diseases.

2 203 153 €

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

Nicotinic acetylcholine receptors (nAChRs) mediate neuronal synaptic transmission and modulation. They contribute to higher brain functions such as cognition and reward and are important drug targets. Recent studies have revealed that these acetylcholine-gated ion channels display an unanticipated conformational plasticity, adopting multiple allosteric states that shape the time course of their electrophysiological response. To date, a single nAChR structure has been solved at high resolution, and our understanding of the conformational transitions remains so far elusive. To address this challenge, we propose to develop a top-down approach starting from the study of the conformational transitions of nAChRs functionally expressed in cells, and then dissecting the molecular mechanisms on purified proteins. In WP1, we will develop an innovative fluorescence quenching approach to follow the protein motions concomitant with channel opening at the cell membrane. In WP2, we will further exploit this technique on purified proteins, to study the role/requirement of lipids, and their pharmacological crosstalk with allosteric modulators acting at the transmembrane domain. In WP3, the gained knowledge will open original routes to solve 3D structures of nAChRs, in novel conformations and in complex with allosteric modulators. The research will be centered on the major brain nAChRs, primarily the homomeric α7 and also the heteromeric α4β2 nAChRs that are major physiological players and key potential therapeutic targets. This multidisciplinary project combines electrophysiology, fluorescence, pharmacology, membrane protein biochemistry and structural biology, together with in silico modeling, molecular dynamics and ligand docking. The results will provide fundamental insights into the allosteric mechanisms underlying both nAChR function and its modulation by allosteric modulators that hold promises in therapeutics.

2 282 105 €

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

This project aims to reformulate and generalize standard theories of human health and mortality. It proposes new formal models and a systematic agenda to empirically test hypotheses that link developmental biology, epigenetics and adult human illness, disability and mortality. We seek to break new ground developing innovative formal models for illnesses and mortality, testing new hypotheses about the evolution of human health and, to the extent permitted by findings, reformulating standard theories to make them applicable to a less restrictive segment of populations than they are now. Over the past two decades there has been massive growth of research on the nature of delayed adult effects of conditions experienced in early life. This field of research is known as the Developmental Origins of Adult Health and Disease (DOHaD). Increasing evidence suggests that the mechanisms that are implicated are epigenetic and constitute an evolved adaptation selected over thousands of years to improve fitness in changing landscapes. The emergence of DOHaD is as close as we will ever come to a paradigmatic shift in the study of human health, disability and mortality. The most tantalizing possibility is that advances in our understanding of epigenetic mechanisms will shed light on pathways linking early exposures and delayed adult health thus fundamentally transforming our understanding of human illnesses and, in one fell swoop, bridge population health, epigenetics, and developmental and evolutionary biology. The overarching goal of this project is to contribute to this nascent area of study by (a) proposing new formal demographic models of health, disability and mortality; (b) empirically testing DOHaD predictions with population data; (c) testing a microsimulation model to verify DOHaD predictions about two conditions, obesity and Type 2 Diabetes, and (d) assessing the adult health, disability and mortality toll implicated by relations between early conditions, obesity and T2D.

2 852 655 €

Start date: 2019-03-01, End date: 2024-02-29

Our body constantly encounters pathogens or malignant transformation. Consequently, the adaptive immune system is in place to eliminate infected or cancerous cells. Specific immune reactions are triggered by selected peptide epitopes presented on major histocompatibility complex class I (MHC-I) molecules, which are scanned by cytotoxic T lymphocytes. Intracellular transport, loading, and editing of antigenic peptides onto MHC-I are coordinated by a highly dynamic multisubunit peptide-loading complex (PLC) in the ER membrane. This multitasking machinery orchestrates the translocation of proteasomal degradation products into the ER as well as the loading and proofreading of MHC-I molecules. Sampling of myriads of different peptide/MHC-I allomorphs requires a precisely coordinated quality control network in a single macromolecular assembly, including the transporter associated with antigen processing TAP1/2, the MHC-I heterodimer, the oxidoreductase ERp57, and the ER chaperones tapasin and calreticulin. Proofreading by MHC-I editing complexes guarantees that only very stable peptide/MHC-I complexes are released to the cell surface. This proposal aims to gain a holistic understanding of the PLC and MHC-I proofreading complexes, which are essential for cellular immunity. We strive to elucidate the mechanistic basis of the antigen translocation complex TAP as well as the MHC-I chaperone complexes within the PLC. This high-risk/high-gain project will define the inner working of the PLC, which constitutes the central machinery of immune surveillance in health and diseases. The results will provide detailed insights into the architecture and dynamics of the PLC and will ultimately pave the way for unraveling general principles of intracellular membrane-embedded multiprotein assemblies in the human body. Furthermore, we will deliver a detailed understanding of mechanisms at work in viral immune evasion.

2 181 250 €

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

Structure determination of G protein-coupled receptors (GPCRs) has been exceedingly successful over the last 5 years due to the development of complimentary generic methodologies that will now allow the structure determination of virtually any GPCR. However, these technologies address only two aspects of the process, namely the stability of the receptors during purification and the ability to form well-diffracting crystals. The strategies also apply only to GPCRs and not transporters or ion channels. The recent successes have been of GPCRs that are expressed in either yeasts or in insect cells using the baculovirus expression system, but many membrane proteins are expressed poorly in these systems or may be expressed in a misfolded non-functional form. A second issue with the future structure determination of GPCRs is the lack of generic technologies to allow the crystallisation of arrestin-GPCR and G protein-GPCR complexes. Although one G protein GPCR complex has been crystallised this was exceedingly diffciult and resulted in poor resolution of the GPCR component of the complex. We believe that it is possible to thermostabilise both arrestin and heterotrimeric G proteins, which will allow a simplified strategy for the crystallisation and structure determination of GPCR complexes. This is based on the development of the strategy of conformational thermostabilisation of GPCRs developed in our lab that has resulted in the structure determination of 3 different GPCRs bound to either antagonists, partial agonists, full agonists and/or biased agonists. The aims are: 1. The development of generic methodology for the production of eukaryotic membrane proteins in mammalian cells. 2. The development of a thermostable functional arrestin mutant 3. Structures of β1-adrenoceptor, adenosine A2A receptor and angiotensin receptor bound to a G protein and arrestin 4. Understanding the role of each amino acid residue in the activation process of GPCRs through saturation mutagenes

2 378 162 €

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

"The Environmental Justice Atlas (www.ejatlas.org) is a global database built by us, drawing on activist and academic knowledge. It maps 1500 conflicts. To improve geographical and thematic coverage it will grow to 3000 by 2019. It systematizes conflicts across 100+ fields documenting the commodities at stake, the actors involved, impacts, forms of mobilizations and outcomes allowing analyses that will lead to a general theory of ecological distribution conflicts. We shall research the links between changes in social metabolism and resource extraction conflicts at the “commodity frontiers”. Also other questions in political ecology and social movement theory such as the effectiveness of direct action by grassroots protesters compared to institutional forms of contention. Does the involvement of different actors, e.g. indigenous groups, relate to different conflict outcomes? How often does the IUCN ally itself to ""the environmentalism of the poor""? Do mobilizations and outcomes vary across sectors (mining, hydroelectric dams, waste incinerators) according to project differences in economic and biophysical dimensions, environmental and health risks? Are conflicts on point resources (mining, oil extraction) regularly different from conflicts in agriculture? Can we track networked resistances against Western companies, compared to those from China or other countries? Resistance to environmental damage has brought into being many local and some international EJOs pushing for alternative social transformations. We shall study the Vocabulary of Environmental Justice they deploy: climate justice, water justice, food sovereignty, biopiracy, sacrifice zones, and other terms specific to countries: Chinese “cancer villages”, Indian “sand mafias”, Brazilian “green deserts” (eucalyptus plantations). Finally, are there signs of an alliance between the Global Environmental Justice Movement and the small European movement for “prosperity without growth”, décroissance, Post-Wachstum?"

1 910 811 €

Start date: 2016-06-01, End date: 2021-05-31

Polycomb-mediated epigenetic regulation of gene expression is central to development and environmental plasticity in most eukaryotes. Polycomb Repressive Complex 2 (PRC2) is targeted to genomic sites, known as nucleation regions or Polycomb Response elements, and switches those targets to an epigenetically silenced state. But what constitutes the switching mechanism is unknown. Core epigenetic switching mechanisms have proven difficult to elucidate due to the complex molecular feedbacks involved. We will exploit a well-characterized gene system, Arabidopsis FLC, to address a central question – what are the core events that constitute a Polycomb switch? Our hypothesis is that the epigenetic switch involves stochastic conformationally-induced oligomerization, generating an ordered protein assembly of PRC2 accessory proteins and PRC2, that is then robustly distributed onto both daughter strands during DNA replication through self-templating feedback mechanisms. We will determine the local chromatin features that promote the epigenetic switch independently at each allele (i.e., in cis). We will also dissect the involvement of DNA replication in the transition from metastable to long-term epigenetic silencing, associated with the Polycomb complex spreading across the body of the locus. This interdisciplinary proposal combines molecular genetics/biology, computational biology, with structural biology, achieved through close working relationships with Prof. Martin Howard (John Innes Centre), Dr Mariann Bienz (MRC Laboratory of Molecular Biology, Cambridge) and Dr Julian Sale, (MRC Laboratory of Molecular Biology, Cambridge). This blue-sky programme aims to provide important new concepts in Polycomb-mediated epigenetic switching mechanisms, important for the whole epigenetics field.

2 101 325 €

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

The ribosome is a large cellular organelle that plays a central role in the process of protein synthesis in all organisms. Currently, structural information at atomic resolution exists only for bacterial ribosomes and some of their functional complexes. Eukaryotic ribosomes are larger and significantly more complex than their bacterial counterparts. They consist of two unequal subunits with a combined molecular weight of approximately 4 million Daltons and contain 70-80 different protein molecules and four different RNAs. Currently the only structural information on eukaryotic ribosomes is available from cryo electron microscopic reconstructions in the nanometer resolution range, which is insufficient to derive information about the function of the eukaryotic ribosome at the atomic level. The aim of this proposal is to use X-ray crystallography to obtain structural and functional information on the eukaryotic ribosome and its functional complexes at high resolution. The key targets of the structural work will be: i) the structure of the small ribosomal subunit, ii) the structure of the large ribosomal subunit, and iii) structures of complexes involved in the initiation of protein synthesis. Besides the obvious fundamental importance of this research for understanding protein synthesis in eukaryotes the proposed studies will also be the prerequisite for understanding the structural basis of the regulation of protein synthesis in normal cells and how it is perturbed in various diseases. Finally, comparing the structures of bacterial and eukaryotic ribosomes is important for understanding the specificity of various clinically used antibiotics for the bacterial ribosome.

2 446 725 €

Start date: 2010-07-01, End date: 2015-06-30

"Regulation and fidelity of gene expression is fundamental to the differentiation and maintenance of all living organisms. While historically attention has been focused on the process of transcriptional activation, we predict that RNA turnover pathways are equally important for gene expression regulation. This has been implied for selected protein-coding RNAs (mRNAs) but is virtually unexplored for non-protein-coding RNAs (ncRNAs). The intention of the EURECA proposal is to establish cutting-edge research to characterize mammalian nuclear RNA turnover; its factor utility, substrate specificity and regulatory capacity. We foresee that RNA turnover is at the core of gene expression regulation - forming intricate connection to RNA productive systems – thus, being centrally placed to determine RNA fate. EURECA seeks to dramatically improve our understanding of cellular decision processes impacting RNA levels and to establish models for how regulated RNA turnover helps control key biological processes. The realization that the number of ncRNA producing genes was previously grossly underestimated foretells that ncRNA regulation will impact on most aspects of cell biology. Consistently, aberrant ncRNA levels correlate with human disease phenotypes and RNA turnover complexes are linked to disease biology. Still, solid models for how ncRNA turnover regulate biological processes in higher eukaryotes are not available. Moreover, which ncRNAs retain function and which are merely transcriptional by-products remain a major challenge to sort out. The circumstances and kinetics of ncRNA turnover are therefore important to delineate as these will ultimately relate to the likelihood of molecular function. A fundamental challenge here is to also discern which protein complements of non-coding ribonucleoprotein particles (ncRNPs) are (in)compatible with function. Balancing single transcript/factor analysis with high-throughput methodology, EURECA will address these questions."

2 497 960 €

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

"The overall aim of this research programme is to uncover how family life courses influence health and well-being in later adulthood, whether family related strengths or disadvantages relevant to health offset or compound socio-economic sources of disadvantage, and the extent to which these associations are influenced by societal factors. An important element will be to consider the role of intergenerational influences, including support flows. The geographical focus will be on Europe and the methodological focus on the advanced quantitative analysis of large scale longitudinal data sets. These data sets, chosen for their complementary strengths, will include both country specific and cross national sources. Three major interlinked strands of work will be undertaken. These will focus on 1) Impacts of parenting and partnership histories on health and mortality in mid and later life. 2) Intergenerational support exchanges: demographic, cultural and policy influences and effects on health of both providers and receivers. 3) An over arching theme to be addressed in the above strands and consolidated in the third is how investments in family and social networks are related to socio-economic disparities in later life health and mortality. The programme is will bring together perspectives from a range of disciplines to address issues of great relevance to current policy challenges in Europe. It is challenging because of the problem of dealing with issues of health selection and possible bias arising from various kinds of missing data which will require methodological care and innovation. Results will contribute to the development of theory, the development of methods and provide substantive knowledge relevant to the health and well-being of older Europeans."

1 423 110 €

Start date: 2013-07-01, End date: 2018-06-30

One of the important consequences of the Second Demographic Transition has been the increasing complexity of families. The aim of this project is to study how rising family complexity has affected two fundamental aspects of intergenerational relationships: reproduction and solidarity. Theoretically, family complexity is distinguished into four dimensions: (a) the length, timing and nature of exposure to the child, (c) biological relatedness to the child, and (c) characteristics of parent-parent ties (triadic effects), (d) characteristics of the wider family network. Using insights from several disciplines, I develop a common theoretical framework for understanding intergenerational reproduction and solidarity. To test the theory, an innovative multiactor survey is developed with an oversampling strategy in which for each adult child, information is collected on all parent figures, and for each parent, information on all adult children. In addition, register data are used to analyze one aspect of reproduction in a dynamic fashion (educational reproduction) and vignette data are used to analyze one aspect of solidarity in more depth (norms prescribing solidarity). By studying reproduction and solidarity as outcomes, I shift the traditional focus from examining how the SDT has affected individual well-being, to the question of how the SDT has affected relationships. In doing so, I analyze a new problem in demography and sociology and contribute to classic debates about population ageing and social inequality. Theoretically, the study of family complexity yields unique opportunities to test ideas about the nature of intergenerational relationships and will shed new light on the traditional dichotomy of social vis-à-vis biological bases of intergenerational relationships. Methodological innovation is made by developing solutions for well-known problems of multiactor data, thereby strengthening the theoretical relevance of survey data for the social sciences.

2 499 533 €

Start date: 2015-11-01, End date: 2020-10-31

An impressive variety of motile and morphogenetic processes are driven by site-directed polarized asssembly of actin filaments. In the past ten years, breathtaking advances coming from cell biology, cell biophysics, and biochemistry have brought insight into the molecular bases for production of force and movement by site-directed actin polymerization. Yet, we do not know, with the detail sufficient to understand how force is produced, by which molecular mechanisms the filaments are nucleated or created by branching. We do not know by which elementary steps insertional polymerization of barbed ends of filaments against the membrane is performed by different protein machineries, nor how these machineries work in a coordinated fashion. Here we propose a multiscale and interdisciplinary approach of the mechanisms used by the major actin nucleators to organize the motile response of actin. The elementary reactions involved in the processive walk of formin at the growing barbed ends of filaments and the role of ATP hydrolysis in force production will be analyzed by a combination of biochemical solution studies and physical methods using functionalized GUVs and optical tweezers. The multifunctionality of WH2 domains involved in actin sequestration, filament nucleation severing and processive elongation will be similarly examined in an interdisciplinary perspective from structural biology at atomic resolution to physics at the mesoscopic scale. Biochemical and structural methods and single molecule measurements (TIRFM) will shed light into the elementary steps and structural mechanism of filament branching. Biomimetic assays with functionalized GUVs associated with biophysical methods like FRAP or fluorescence correlation spectroscopy will elucidate how different filament initiating machineries segregate in the membrane as a consequence of their interactions with growing filaments and function in a coordinated fashion during actin-based motility.

2 434 195 €

Start date: 2010-05-01, End date: 2015-10-31

This interdisciplinary project (combining social and earth sciences) addresses a key gap in the knowledge of global assessments concerning the likely consequences of future climate change on future human wellbeing. More information about the determinants of future adaptive capacity is necessary for setting policy priorities today: Should the significant funds allocated for adaptation be invested in enhancing existing infrastructure or currently practiced agricultural strategies (some of which may not be tenable under future climates), or should they invest alternatively in enhancing human empowerment through education and health which in consequence will enable affected societies to better cope with whatever challenges the future will bring? This study is expected to bring significant progress in this difficult multidisciplinary, yet highly relevant, field through a combination of: (a) New global science-based, long-term projections of human capital (population by age, sex and level of education) as a key element of adaptive capacity; (b) Three empirical multi-national studies on key factors involved in past vulnerability and adaptations to the Sahelian drought, Hurricane Mitch and the Asian tsunami; (c) Three prospective case studies assessing future adaptive capacity for the Phuket region, Mauritius and the Nicobar islands; (d) All held together and put into perspective by the elaboration of a new demographic theory of long-term social change with predictive power. This rather complex project structure is necessary for reaching generalizable and useful results. All components have been designed to complement each other to maximize the chances of achieving path-breaking and at the same time tangible results in this highly complex, multidisciplinary field. All components of the study will build on previous work of IIASA and Wolfgang Lutz and hence minimize the need to acquire additional experience for the case study sites or for the methodology used.

2 438 402 €

Start date: 2009-03-01, End date: 2014-07-31

"GLOBAL-RURAL aims to advance our understanding of the workings and impact of globalization in rural regions through the development and application of new conceptual and methodological approaches. Globalization has a pervasive influence in transforming rural economies and societies, with implications for the major societal challenges of environmental change and resource security. However, in comparison to studies of the global city, relatively little research has focused on the ‘global countryside’, and existing research lacks integration. GLOBAL-RURAL will develop an integrated perspective by drawing on relational analysis (and particularly the approaches of ‘assemblage theory’ and ‘countertopography’) to focus on the actual mechanics by which rural localities are ‘re-made’ through engagement with globalization processes, examining the mediating effect of national and regional context and the opportunity for local interventions. The research will be organized through five work packages. WP1 will develop the methodological application of assemblage theory to analysing the global countryside, informed by case studies in 6 countries. WP2 will combine GIS analysis of quantitative and qualitative data to produce new narratives and visualisations of globalization processes, impacts and responses. WP3 will focus on mundane, ‘everyday globalization’ in a Welsh small town, using a countertopographic methodology. WP4 will apply the assemblage methodology developed in WP1 to analysing the differential global engagement of rural localities in Brazil, China and Tanzania. WP5 will apply the methodology to examine conflicts around renewable energy schemes, mining and water projects and industrial agriculture in rural areas, and the implications for strategies to address global challenges. A sixth work package, WP6, will identify the policy applications of the research, and disseminate research findings to academic and non-academic users."

2 263 107 €

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

"This project aims at a socio-historical study of the transition between the two regimes of knowledge and action, which have characterized the government of health after World War II: the regime of international public health, dominating during the first decades of the postwar era, which was centered on eradication policies, nation-states and international UN organizations; the present regime of global health, which emerged in the 1980s and is centered on risk management and chronic diseases, market-driven regulations, and private-public alliances. The project seeks to understand this transition in terms of globalization processes, looking at the making of knowledge, the production and commercialization of health goods, the implementation of public health programs, and routine medical work. It will focus on four fields of investigations: tuberculosis, mental health, traditional medicine and medical genetics in order to understand how categories, standardized treatment regimens, industrial products, management tools or specific specialties have become elements in the global government of health. The project associates historical and anthropological investigations of practices in both international and local sites with strong interests in: a) the changing roles of WHO; b) the developments taking place in non-Western countries, India in the first place. The expected benefits of this research strategy are: a) to take into account social worlds including laboratories, hospitals, enterprises, public health institutions and international organizations; b) to approach the global as something translated in and emerging from local practices and local knowledge; c) to explore different levels of circulations beyond the classical question of North-South transfers; d) to deepen our understanding of the transition from the political and economical order of the Cold War into a neo-liberal and multi-centric age of uncertainty."

2 307 432 €

Start date: 2014-08-01, End date: 2019-07-31

The development of new approaches to dissect the diverse roles for carbohydrates in living cells is a major challenge for modern cell biology. The huge diversity of carbohydrates is reflected in a multiplicity of function; in addition to acting as energy sources, carbohydrates play major roles in structure, signalling and epigenetics. The work programme will build upon the applicant’s excellence in the mechanistic and structural enzymology of carbohydrate-active enzymes to tackle the key challenges of modern cellular glycobiology. Our vision is to provide fundamental structural and mechanistic-dissection of key proteins and their complexes and to use these as the foundation to deliver enzyme inhibitors as tools to probe the cellular function of specific glycans. The programme’s three strands will each scale a major pinnacle of carbohydrate biochemistry. Strand 1 will focus on mammalian glycosidases involved in glycocerebroside metabolism and genetic disease. We will unlock new 3-D information for glycocerebrosidase 2 (GBA2) and use these together with GBA1 to design and exploit novel and specific enzyme inhibitors as mechanistic and cellular probes, novel chaperones and imaging agents. Strand 2 will focus on the key endoplasmic reticulum enzyme endomannosidase, both its mechanistic novelty and its exploitation to perturb cellular glycans to unlock its biological roles and deliver compounds for anti-viral therapeutics. Strand 3 will probe the modification and elaboration of specific human N-glycans and their role in cell surface receptor biology. It will focus on the GlcNAc transferase V catalysed formation of polylactosamine epitopes and their regulation of growth factor signalling at the cell surface both in health and cancerous tissues. GlycoPOISE will both answer cardinal structural and chemical mechanistic questions in the enzymology of glycobiology and inform strategies for the observation and inhibition of carbohydrate-active enzymes and their exploitation

2 500 000 €

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

NMR spectroscopy detects in a unique way with atomic resolution biomolecular dynamics in the previously hidden time range between approximately 5 nano- to 50 microseconds (ns-ms time range). The detection of this motion happens in equilibrium under physiological conditions without the need for a triggering reaction. On the example of ubiquitin, this dynamics was found by us to be important for molecular recognition between proteins implying conformational selection rather than induced fit. Only free solution ensembles including this dynamics accessed the full conformational heterogeneity of structures in recognition complexes. Molecular dynamics analysis suggests high correlation of these ns-ms dynamical modes. Here, we propose to establish with NMR experimentally the correlated nature of the ns-ms dynamics, to describe ensembles reflecting ns-ms and sub-ns dynamics by separating the time scales. In this context, using temperature jump-infra-red spectroscopy and solid state NMR we want to determine the time scale of the ns to ms motion more precisely. Since the ns-ms time scale is slower than diffusion, dynamics on this time scale could be a mechanism of regulating or limiting the kinetics of molecular association and recognition. Therefore, we want to determine on-rates by NMR spectroscopy and want to explore whether mutants that do not affect the binding interface but will affect the dynamics modulate the on-rates. This would allow the control of binding kinetics and explore the influence of ns-¼s dynamics on protein-protein recognition on the long run also for membrane proteins. In addition specificity for drug interactions could be increased addressing extremal conformations present in the ns-¼s ensembles for homologous proteins with otherwise very similar average structures at interaction interfaces. If the proposal is successful this would open up new opportunities for drug design and design of protein-protein interactions.

2 212 000 €

Start date: 2009-07-01, End date: 2014-06-30

Nucleosomes, ~147 base pairs of DNA wrapped around an histone protein octamer, package and protect nuclear DNA but also carry important biological information. The position and composition of nucleosomes along chromosomal DNA is a key element of defining the state and identity of a cell. Chromatin remodellers are ATP dependent molecular machines that position, move or edit nucleosomes in a genome wide manner. Collectively, they shape the nucleosome landscape and play central roles in the maintenance and differentiation of cells, but also in pathological transformations. INO80, a megadalton large remodeller consisting of 15 or more subunits, is involved in replication, gene expression and DNA repair. It models chromatin by positioning barrier nucleosomes around nucleosome free regions, editing nucleosomes and generating nucleosome arrays. However, structural mechanisms for INO80 and other remodelling machines are poorly understood due to their complexity. To provide a comprehensive mechanistic framework, to understand how INO80 senses nucleosome free regions to position barrier nucleosomes and how it generates arrays or senses DNA breaks, I propose a challenging but ground-breaking endeavour using a combination of cryo-EM and functional approaches. We address structures of fungal and human INO80 complexes at promoter regions, on di-nucleosomes and at DNA ends and develop quantitative positioning assays to reveal common and distinct features of shaping chromatin in different species. We also explore cryo-EM as tool towards revealing distinct steps the chemo-mechanical remodelling reactions. The proposed research will help derive fundamental molecular principles underlying the modelling of the nucleosome landscape.

2 201 875 €

Start date: 2019-10-01, End date: 2024-09-30

INZI aims to analyse the complex interplay of actors, policies and projects that have shaped research into and control of Human African Trypanosomiasis (HAT) until the present day. Research has mainly been steered from outside of Africa, firstly by colonial authorities and latterly by an array of agencies, foundations and international organisations. Despite this, investment in research and control measures has declined and fragmented across Africa. This project seeks to examine, in proper historical context and from a systematic perspective, the evolution of Africa’s HAT research apparatus, to gain insight into the relationship between science and development, and build our understanding of how science can work better for development. INZI will generate a panoptic, integrated analysis of the evolving HAT global assemblage in order to extend our knowledge of 1) The evolving relationship between the organisation of science and the development of material technologies in developing country contexts; 2) The relationship between policy and practice in mediating particular scientific and technological trajectories; and 3) The nature of innovation, what it means in a developing country context, and how it may be promoted. This will significantly advance our understanding of how science is practiced in developing countries, how technologies emerge, and ultimately how science and technological innovation can be organised to ensure development is transformational, not unobtainable. Empirical research will be focused around five ‘research strands’ that each reflects a key modality or dimension of HAT research and control. These strands are: 1) Institutions; 2) Markets; 3) Partnerships; 4) Systems, and 5) Locations. Alongside the development of these five research strands, and in constant interaction with them, a series of connective analytical activities will be designed to truly integrate analysis of ‘micro-level processes’ and ‘macro-structures and forces’.

1 539 786 €

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

Equal partition of the genetic material to the daughter cells in mitosis requires the accurate anchorage of the mother cell s chromosomes to spindle microtubules. This process takes place at kinetochores, complex scaffolds containing ~100 different proteins. Conceptually, kinetochores can be viewed as performing four distinct but highly integrated functions: 1) they bind centromeric chromatin at a specialized protein-DNA interface; 2) they build a dynamic microtubule-binding interface that is tightly linked to the centromere-binding interface; 3) they correct erroneous microtubule attachments; 4) they synchronize the progression of the cell cycle oscillator with the progression of the microtubule-kinetochore attachment process. In mammals, all four functions are essential, and their abrogation has untenable consequences for normal cell life. Conversely, their partial impairment has been implicated in chromosome instability and in the development of cancer and an array of genetic diseases. Our goal is to be able to map the kinetochore functions schematized above to the as yet largely uncharacterized architecture of the kinetochore and to unravel the elements of feedback control that allow their integration. By using a combination of structural and functional methods, we have made several recent important contributions to the field of kinetochore biology. In this application, we propose to take our efforts to a new level of complexity that will allow us to gain an integrated view of how kinetochores bind microtubules, how they correct improper attachment, and how they coordinate microtubule attachment with cell cycle progression. Our approach rests on strong experience in biochemical reconstitution and structural analysis, and is complemented by the introduction of methods to assess and model the dynamic responses of kinetochores to their variable environment.

2 500 000 €

Start date: 2009-03-01, End date: 2014-09-30

Ubiquitin (Ub) is a small modifier that labels proteins in a highly specific manner. Like phosphorylation, modification of proteins by Ub is prevalent in the majority of cellular processes. An increasing number of distinct functions have been assigned to different types of ubiquitin modifications (monoUb and different Lys-linked chains). Moreover, aberrations in the ubiquitin system underlie many disease states, including cancer, inflammatory, immune and metabolic disorders as well as neurodegeneration. The most recently described physiological ubiquitin modification is the linear ubiquitin chain, in which ubiquitin monomers are conjugated via Met-Gly linkages. We have found that linear ubiquitin chains bind specifically to the NEMO adaptor molecule, an event critical for the proper regulation of NF-ºB signaling (Rahighi, 2009). Here we propose to use a multidisciplinary strategy to study the role of linear ubiquitination in the NF-ºB pathway, autophagy, apoptosis and DNA repair and how these changes can impact on disease states such as inflammation and cancer development. Scientific objectives are: " Characterize the components of linear ubiquitination: E3 ligases, specific substrates and domains recognizing linear ubiquitin chains " Elucidate the in vivo role of linear ubiquitination in the regulation of the NF-ºB pathway, apoptosis and DNA repair. " Reveal the molecular basis for the connections between linear ubiquitination and selective autophagy " Identify elements in the linear ubiquitin network as potential drug targets " Generate transgenic mouse models of inflammatory diseases and cancer " Develop system and computational biology approaches to assess the global role of linear ubiquitination in cellular proteome

2 440 560 €

Start date: 2010-06-01, End date: 2015-05-31

Alternative splicing of messenger RNA precursors is a prevalent form of gene regulation that greatly expands the coding capacity and regulatory opportunities of higher eukaryotic genomes. It contributes to cell differentiation and pluripotency and its deregulation promotes cancer progression, as evidenced by the frequent occurrence of cancer-associated mutations in splicing factors, which are also targets of anti-tumor drugs. Despite its prevalence and relevance, the underlying mechanisms of regulation remain poorly understood. This proposal aims to develop and apply systematic approaches that can allow us to carry out the equivalent of genetic analysis of splicing regulation in cancer and pluripotent cells. These technologies can help to unweave the complex network of functional interactions within the spliceosome and of the spliceosome with regulatory factors, exhaustively map the contribution of regulatory sequences and be used to investigate, with unprecedented detail, mechanisms of regulation for essentially any regulator or alternative splicing event operating in a particular cell line. Such approaches can offer a unique opportunity to address key unresolved mechanistic questions, including the molecular basis for positional effects of splicing regulatory factors (RNA Maps), the regulatory potential of the core spliceosome and the integration of alternative splicing with other cell regulatory programs. We will combine these approaches with biochemical and cellular assays to investigate detailed mechanisms of regulation relevant for the control of cell proliferation and/or pluripotency in cancer and induced pluripotent stem (iPS) cells. Progress in this area can contribute to reveal the molecular logic governing a key layer of gene regulation and has the potential to discover novel factors and regulatory circuits that trigger or modulate cell growth, differentiation and cancer progression.

2 159 574 €

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

"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

Self-assembly is a hallmark of Biology. We are far from a complete understanding of this natural assembly, which in turn limits our ability to mimic biological construction in the bioengineering of tuneable synthetic systems. This proposal addresses the major challenge of membrane protein folding. Here, I intend to make a step change to my pioneering biophysical studies and investigate co-translational membrane protein folding. A central feature will be the creation of synthetic systems to probe key events in co-translational folding, as the protein folds in the membrane whilst emerging from the ribosome. An ambitious target is to make these systems tuneable, which will provide a new tool for the fabrication of membrane proteins and artificial cells in Synthetic Biology. The assembly of a tuneable artificial module that affords control over membrane protein synthesis is unprecedented. I focus on the ubiquitous superfamily of major facilitator proteins, namely the best studied family member, lactose permease, LacY. This proposal has state of the art biophysical mechanistic studies at its core, which interleave into Cell and Synthetic Biology. There are two themes: Theme 1. Determination of fundamental membrane protein folding parameters: folding transition states and lipid control Phi-value analysis will be used to probe the folding transition state of LacY; the first such analysis of a multi-domain membrane protein. Lipid parameters that control LacY folding will be quantified, including bilayer asymmetry using novel droplet interface bilayer methods. Theme 2: construction of tuneable synthetic co-translational folding systems Engineered ribosomes and translocon insertion machinery will be incorporated and LacY folding will be controlled. Translation will be regulated or halted using mutant ribosomes, arrest sequences, altered codon usage and controlling tRNA addition. Trapped LacY folding intermediates will be studied using biophysical methods.

2 312 389 €

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

Extensive study of the p53 protein has resulted in a detailed understanding of its role in tumour suppression, information that is being used to develop small molecule modulators of p53 that are presently under evaluation for cancer therapy. However, it has recently become clear that p53 also plays roles in aspects of human health and disease extending beyond cancer - although most of these are poorly understood. We therefore propose to investigate some of these non-cancer functions of p53, with an emphasis on the role of p53 in the regulation of metabolism, extending to an analysis of whether p53 contributes to pathologies such as diabetes and obesity. This is a pioneering project that brings p53 research into new areas, yet builds on the solid platform of existing knowledge about the regulation and function of p53. State of the art genomic, proteomic, metabolomic and imaging analyses will be used to identify the roles of p53 in the response to metabolic stress caused by nutrient deficiency or excess, and investigate how these activities balance cell survival and cell death. Models will be developed to address how these functions of p53 relate to the control of metabolism and disease in vivo. Understanding how the cellular response to metabolic stress is controlled, and identifying a role for p53 in the regulation of metabolic homeostasis has enormous potential to influence the diagnosis and treatment of disease. The proposal will help to define the role of p53 in the development of diabetes and obesity, and lay the groundwork for the investigation of a role for p53 in other pathologies, such as neurodegenerative disease, cardiovascular disease and liver disease. These studies will therefore have far-reaching impact on some of the most prominent health threats in the developed world.

2 437 814 €

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

"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

Mitochondrial cristae biogenesis is an enigma ever since the first imaging of mitochondria, the ‘powerhouses’ of eukaryotic cells, by electron microscopy in the 1950s. The mitochondrial cristae, dynamic and structurally conserved invaginations of the mitochondrial inner membrane, are essential for respiratory ATP generation. Thereby, the form and function of the mitochondrial inner membrane are deeply intertwined. Indeed, irregular or disturbed cristae morphologies are believed to cause numerous human diseases, including neurodegeneration, cardiomyopathies, metabolic disorders and cancer. Previous approaches to study cristae biogenesis have relied primarily on the use of 2D electron microscopy and biochemistry to analyse mutant cells defective in cristae formation. Based on striking pilot experiments, we propose to study cristae biogenesis by a radically different approach. We will induce synchronous cristae development in gene-edited cell lines initially defective in cristae formation. We will then follow de novo cristae biogenesis over time by combining a series of enabling approaches, including live cell and MINFLUX super-resolution microscopy, 3D (cryo) electron microscopy, label-free (SWATH) mass spectrometry, and single molecule counting. These technologies have just emerged in the last few years, and thus this proposal would not have been possible a few years ago. The primary aim of this proposal is to establish a deep, comprehensive and quantitative understanding of cristae biogenesis in human cells. Using theses insights, we will also investigate the effects of mutations in mitochondrial proteins associated with human diseases on cristae biogenesis. Altogether, if successful, the outcome will represent a paradigm shift in our knowledge of how mitochondrial ultrastructure in healthy and diseased cells is generated and maintained. Our findings might spark innovative and novel strategies for the treatment of devastating human mitopathies.

2 286 248 €

Start date: 2019-10-01, End date: 2024-09-30

Contemporary antislavery campaigners invoke the history of Atlantic World slavery to highlight the plight of 48 million people today living in exceptionally harsh circumstances, described as ‘modern slavery’. Yet in a world where many are oppressed and exploited, the lines between ‘modern slavery’ and other forms of drudgery, exclusion, and domination, are not easily drawn. Critics argue that the dominant discourse of ‘modern slavery’ relies upon a highly selective vision of injustice and suffering, and fails to consider or challenge the structural inequalities and systems of domination (race, caste, class, gender, age, nationality) that routinely restrict rights and freedoms. The proposed project retains a concern with the continuing significance of Atlantic World history, but upturns conventional discourse by interrogating the problem of freedom – as opposed to slavery - in the contemporary world. Through fieldwork in Brazil, Ghana, Italy, Portugal and the UK with groups that appear in dominant discourse as at risk of ‘modern slavery’, its key aims are: i) to revisit histories of marronage and other strategies by which enslaved and newly emancipated people sought to move closer to freedom in the Atlantic World historically, and ask what light they can shed on the perception, pursuit and practice of freedom by marginalized and rightless people in the Atlantic World today; ii) to use insights from this dialogue between past and present to contribute to theoretical debates on freedom, and its relation to agency, honour, gender, age, race, mobility, property, and personhood; iii) to work with research participants to co-produce counter-narratives to conventional antislavery stories of ‘modern slavery’, and, by communicating them through performance as well as text, encourage more nuanced popular and political debate on the contemporary meaning and practice of freedom.

1 847 596 €

Start date: 2018-10-01, End date: 2023-09-30

The hypoxic response in humans is regulated by enzymes that catalyse the post-translational hydroxylation of hypoxia inducible factor (HIF). Prolyl-hydroxylation signals for HIF-alpha degradation whilst asparaginyl-hydroxylation blocks the transcriptional activity of HIF. The absolute requirement of the HIF hydroxylases for oxygen enables them to act as hypoxia sensors. The overall goal of the proposed programme is to capitalize on recent advances in this field arising from the joint Schofield and Ratcliffe laboratories, in a multidisciplinary chemistry-biology approach aimed at opening new horizons both for the basic molecular understanding, and for the therapeutic manipulation, of the human transcriptional response to hypoxia. The specific objectives of the proposed programme will be pursued via defined and syngergistic work packages and include (i) To develop and apply state-of-the-art methods for monitoring oxygen-dependant hydroxylation within cells that will enable us to examine the role of the hydroxylases as signal integration points for redox factors; (ii) To define the existence and nature of the structural and kinetic features that underpin the physiological function of HIF hydroxylases in oxygen homeostasis; (iii) To define the extent and biological roles of post-translational hydroxylation in human cells; (iv) To develop novel templates for selective inhibition and activation of individual human HIF hydroxylases. We will follow a multidisciplinary approach ranging from kinetic and high-resolution structural analyses on the hydroxylases to studies in animal cells. We aim that the results will not only be of use in ongoing pharmaceutical attempts to modulate the natural hypoxic response for the treatment of ischemic disease and cancer, but will serve as a paradigm for biomedicinal analyses of signalling systems.

3 000 000 €

Start date: 2009-03-01, End date: 2014-02-28

Through evolution, cells have developed the exquisite ability to sense, transduce and integrate mechanical and biochemical signals (i.e. mechanobiology) to generate appropriate responses. These key events are rooted at the molecular and nanoscale levels, a size regime difficult to access, hindering our progress towards mechanistic understanding of mechanobiology. Recent evidence from my Lab (and others) shows that the lateral nanoscale organisation of mechanosensitive membrane receptors and signalling molecules is crucial for cell function. Yet, current models of mechanosensing are based on force-induced molecular conformations, completely overlooking the chief role of mechanical forces on the nanoscale spatiotemporal organisation of the plasma membrane. The GOAL of NANO-MEMEC is to provide mechanistic understanding on the role of mechanical stimuli in the spatiotemporal nanoarchitecture of adhesion signalling platforms at the cell membrane. To overcome the technical challenges of probing these processes at the relevant spatiotemporal scales, I will exploit cuttingedge biophysical tools exclusively developed in my Lab that combine super-resolution optical nanoscopy and single molecule dynamics in conjunction with simultaneous mechanical stimulation of living cells. Using this integrated approach, I will: First: dissect mechanical and biochemical coupling of membrane mechanosensing at the nanoscale. Second: visualise the coordinated recruitment of integrin-associated signalling proteins in response to force, i.e., mechanotransduction. Third: test how force-induced spatiotemporal membrane remodelling influences the migratory capacity of immune cells, i.e., mechanoresponse. NANO-MEMEC conveys a new fundamental concept to the field of mechanobiology: the roles of mechanical stimuli in the dynamic remodelling of membrane nanocompartments, modulating signal transduction and ultimately affecting cell response, opening new-fangled research avenues in the years to come.

2 212 063 €

Start date: 2018-12-01, End date: 2023-11-30

Post-translational modification by ubiquitin and ubiquitin-like proteins (UBLs) is a major eukaryotic regulatory mechanism. Nonetheless, we have little understanding of the detailed mechanisms by which most E3 ligases mark specific targets with monoubiquitin, multiple ubiquitins or specific polyubiquitin chains, or of how UBL modifications transform the functions of their targets. This proposal addresses both problems. First, we will discover the structural mechanisms by which the UBL NEDD8 (58% identical to ubiquitin) activates numerous distinct functions of its targets, which are cullin subunits of cullin-RING E3 ubiquitin ligases (CRLs). Second, we will take a tour-de-force structural, biochemical, and molecular cell biological approach to determine how NEDD8-activated E3 ligases regulate their substrates. Because CRLs form nearly half of all E3 ligases, and as we recently discovered, neddylated CRLs act in part by activating monoubiquitylation by another family of E3 ligases (Ariadne-family RBR E3s), the proposed studies will establish paradigms for a major fraction of ubiquitylating enzymes. To achieve these goals, we will devise novel chemical biology tools to capture fleeting assemblies that typically only occur during chemical reactions, and visualize structures of neddylated CRLs “in action” by cryo EM. We will generate a resource of novel reagents that detect, label, and affinity purify activated forms of E3 ligases to temporally track their interactions during pathways they regulate in cells. And we will define the mechanisms and structures of a class of atypical, disease-associated giant E3 ligases whose domains and interacting partners are so peculiar that their activities remain elusive. Overall, we will comprehensively define how a UBL directly regulates its targets, and how two major E3 ligase families mediate regulation.

2 193 871 €

Start date: 2018-10-01, End date: 2023-09-30