ERC FUNDED PROJECTS

Main goal. We aim to understand the puzzling coexistence of antibiotic-resistant and antibiotic-sensitive species in natural soil environments, using novel quantitative experimental techniques and mathematical analysis. The ecological insights gained will be translated into novel treatment strategies for combating antibiotic resistance. Background. Microbial soil ecosystems comprise communities of species interacting through copious secretion of antibiotics and other chemicals. Defence mechanisms, i.e. resistance to antibiotics, are ubiquitous in these wild communities. However, in sharp contrast to clinical settings, resistance does not take over the population. Our hypothesis is that the ecological setting provides natural mechanisms that keep antibiotic resistance in check. We are motivated by our recent finding that specific antibiotic combinations can generate selection against resistance and that soil microbial strains produce compounds that directly target antibiotic resistant mechanisms. Approaches. We will: (1) Isolate natural bacterial species from individual grains of soil, characterize their ability to produce and resist antibiotics and identify the spatial scale for correlations between resistance and production. (2) Systematically measure interactions between species and identify interaction patterns enriched in co-existing communities derived from the same grain of soil. (3) Introducing fluorescently-labelled resistant and sensitive strains into natural soil, we will measure the fitness cost and benefit of antibiotic resistance in situ and identify natural compounds that select against resistance. (4) Test whether such “selection-inverting” compounds can slow evolution of resistance to antibiotics in continuous culture experiments. Conclusions. These findings will provide insights into the ecological processes that keep antibiotic resistance in check, and will suggest novel antimicrobial treatment strategies.

1 900 000 €

Start date: 2012-09-01, End date: 2018-08-31

In the bone-enclosed CNS, increased vascular permeability may cause life-threatening tissue swelling, and/or ischemia and inflammation which compromise tissue repair after trauma or stroke. The brain vasculature possesses several unique features collectively named the blood-brain barrier (BBB) in which passive permeability is almost completely abolished and replaced by a complex of specific transport mechanisms. The BBB is necessary to uphold the specific milieu necessary for neuronal function. Whereas breakdown of the BBB is part of many CNS diseases, including stroke, neuroinflammation, trauma and neurodegenerative disorders, its molecular mechanisms and consequences are unclear and debated. Conversely, the intact BBB is a huge obstacle for drug delivery to the brain. Research on the BBB therefore has two seemingly opposing aims: 1) to seal a damaged BBB and protect the brain from toxic blood products, and 2) to open the BBB “on demand” for drug delivery. A major problem in the BBB field has been the lack of in vivo animal models for molecular and functional studies. So far, available in vitro models are not recapitulating the in vivo BBB. Our recent work on mouse models lacking pericytes, a BBB-associated cell type, demonstrates a specific role for pericytes in the development and regulation of the mammalian BBB. These animal models are the first ones showing a general and significant BBB impairment in adulthood, and as such they provide a unique opportunity to address molecular mechanisms of BBB disruption in disease and in drug transport across the BBB. Importantly, the new models and tools that we have developed allow us to search for relevant druggable mechanisms and molecular targets in the BBB. The long-term goals of this proposal are to develop molecular strategies and tools to open and close the BBB “on demand” for drug delivery to the CNS, and to explore the importance and mechanisms of BBB dysfunction in neurodegenerative diseases and stroke.

2 499 427 €

Start date: 2012-08-01, End date: 2017-07-31

"By taking advantage of great experience in genetic and cardiovascular epidemiology and some of the largest cohorts in the world including 60 000 unique individuals, the applicant aims at (1) improving CVD risk prediction and (2) identifying mechanisms causally related to CVD development in order to provide novel targets for drug discovery and targeted life style interventions for use in primary prevention. In SUBPROJECT 1 we aim at identifying disease causing alleles of loci implicated in CVD by Genome Wide Association Studies (GWAS) and to identify rare alleles with large impact on human CVD. We thus perform whole exome and targeted sequencing in early CVD cases and healthy controls and evaluate all identified variants by relating them to incident CVD in 60.000 individuals. Further, we will create a score of all validated CVD gene variants and test whether such a score improves clinical risk assessment over and above traditional risk factors. In SUBPROJECT 2 we test whether the plasma metabolome- a phenotype representing the product of dietary intake and inherent (e.g. genetic) metabolism- differs between incident CVD cases and controls and between individuals with high and low CVD genetic risk. We further test whether a life style intervention differentially alters the plasma metabolome between individuals with high and low CVD genetic risk. Finally, we will elucidate the mechanisms underlying CVD genetic associations by testing whether myocardial expression of such genes are affected by experimental myocardial infarction (MI) and whether heart function, MI size and the plasma metabolome are affected by adenoviral myocardial CVD gene transfer in rats. In SUBPROJECT 3 we test whether glucose metabolism and CVD risk factors can be ameliorated by suppressing vasopressin (VP) by increased water intake in humans. Finally, we test which of the 3 VP receptors is responsible for adverse glucometabolic VP effects in rats by specific VP receptor pharmacological studies."

1 500 000 €

Start date: 2011-12-01, End date: 2016-11-30

The quest to restore damaged organs is one of the major challenges in medicine. Recent studies in both animals and in humans suggest that the heart has a limited capacity to replenish its own cardiomyocytes (CMs) throughout life, albeit inadequate to compensate for major injuries such as acute myocardial infarction (MI). Most therapeutic research in regenerative cardiogenesis is geared toward stem cell therapy as a means to replace lost CMs associated with ischemic heart disease. Clinical data evaluating the efficacy of cell-based therapy for heart disease are relatively disappointing. This proposal encompasses multidisciplinary and novel approaches to study the molecular and cellular mechanisms that govern the proliferation, differentiation and dedifferentiation of endogenous CMs, combining developmental-, systems- and cell-biology methodologies in vitro and in vivo, in chick, rodent, and human tissue samples. First, we will perform combinatorial perturbations of signaling pathways in chick embryos, focusing primarily on the FGF-ERK pathway, to investigate the molecular switch between cardiac progenitors and CMs (Aim 1). Because adult CMs have limited proliferative capacity, mainly due to mechanical constraints, in Aim 2, we will apply state-of-the-art techniques in cell biology, to determine whether specific mechno-transduction stimuli can prime the proliferation of differentiated CMs. In order to gain deeper insights into the capacity of adult CMs to renew themselves under normal and pathological conditions, in Aim 3, we will employ a novel cell lineage methodology in mouse and human tissue, based on information encoded in genome. Using this methodology, we hope to shed light on the maintenance, renewal and regenerative capacities of adult CMs in vivo. The expected outcome will be a significantly greater understanding of the bidirectional transition from proliferating cardiac progenitors into differentiated CMs, in embryonic and adult hearts.

1 500 000 €

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

Several organisms have evolved specialized ice binding proteins (IBPs) that prevent their body fluids from freezing (antifreeze proteins, AFPs), inhibit recrystallization of ice in frozen tissues, or initiate freezing at moderate supercooling temperatures (ice nucleating proteins, INPs). These proteins have many potential applications in agriculture, food preservation, cryobiology, and biomedical science. The ubiquitous presence of IBPs in such organisms indicates the power of these molecules to enable survival under cold conditions. Despite this key role in nature, however, IBPs have been effectively exploited in only one cryopreservation application, namely, recrystallization inhibition in ice cream. Several terrestrial organisms, including insects, have developed very active forms of AFPs. These hyperactive AFPs (hypAFPs) have not been utilized significantly thus far in cryopreservation techniques. The gap between the obvious potential of IBPs and their actual applications stems from a lack of knowledge regarding the mechanisms by which IBPs interact with ice surfaces and how these proteins can assist in cryoprotection. I propose to investigate the mechanism by which IBPs inhibit ice crystallization and the use of such proteins for cryopreserving cells, tissues, and organisms. My group has a strong record in the study of the interactions between IBPs and ice using novel methods that we have developed, including fluorescence microscopy techniques combined with cooled microfluidic devices. We will investigate the interactions of AFPs with ice and the use of hypAFPs in cryopreservation procedures. This research will contribute to an understanding of the mechanisms by which IBPs act, and apply the acquired knowledge to cryopreservation. The successful implementation of IBPs in cryopreservation would revolutionize the field of cryobiology, with enormous implications for cryopreservation applications in general and the frozen and chilled food industry in particular.

1 500 000 €

Start date: 2011-11-01, End date: 2016-10-31

Up to one third of diabetic patients develop nephropathy, a serious complication of diabetes. Microalbuminuria is the earliest sign of the complication, which may ultimately develop to end-stage renal disease requiring dialysis or a kidney transplant. Insulin resistance and metabolic syndrome are associated with an increased risk for diabetic nephropathy. Interestingly, glomerular epithelial cells or podocytes have recently been shown to be insulin responsive. Further, nephrin, a key structural component of podocytes, is essential for insulin action in these cells. Our novel findings show that adaptor protein CD2AP, an interaction partner of nephrin, associates with regulators of insulin signaling and glucose transport in glomeruli. The results suggest that nephrin and CD2AP are involved, by association with these proteins, in the regulation of insulin signaling and glucose transport in podocytes. We hypothesize that podocytes can develop insulin resistance and that disturbances in insulin response affect podocyte function and contribute to the development of diabetic nephropathy. The aim of this project is to clarify the mechanisms leading to development of insulin resistance in podocytes and to study the association between insulin resistance and the development of diabetic nephropathy. For this we will develop transgenic zebrafish and mouse models by overexpressing/knocking down insulin signaling-associated proteins specifically in podocytes. Further, we aim to identify novel drug leads to treat insulin resistance and diabetic nephropathy by performing high-throughput small molecule library screens on the developed transgenic fish models. The ultimate goal is to find a treatment to combat the early stages of diabetic nephropathy in humans.

2 000 000 €

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

Bacterial cooperation underlies many bacterial traits of practical interest. Many social traits of bacteria are regulated by inter-cellular signalling pathways, generally known as quorum sensing (QS). QS has been proposed as novel target for anti-virulence treatment. To this aim, there is a need to better understand the mechanisms of QS and their social and evolutionary impact. While the basic schemes of a single quorum sensing pathway acting in homogenous conditions are well understood, the system’s level function of QS regulatory networks can only be appreciated by considering the role phenotypic and genetic variability has on shaping the network’s structure and function. Phenotypic variability in complex communities may arise from division of labour between cells and environmental gradients and substantially impact the way cells secrete and interpret QS signals. Genetic variability in QS networks may lead to multiple social relations between cells of different genotypes including cross-talks, interception, manipulation and quenching of signals. This will affect the population structure and performance. The proposed project will study the function of QS signalling in heterogeneous communities. Phenotypic variability and its impact on QS function will be studied in a spatially inhomogeneous cooperating system. Genetic variability will be studied at the macro and micro-scales in a bacterial species showing rapid diversification of their QS networks. Finally, we will rationally design strains with superior ‘cheating’ strategies that can invade and eliminate a cooperative population. Throughout this project, we will use a combination of experimental techniques from microbiology, socio-biology, genetics and microscopy together with mathematical analysis tools from systems biology, population genetics and game theory, to study bacterial cooperation and its dependence on the underlying communication network, social complexity and environmental variation.

1 497 996 €

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

Background: Poor angiogenesis and collateral vessel formation lead to coronary heart disease, claudication, infarctions and amputations while malignant glioma is one of the most aggressive proangiogenic tumors leading to death in a few months. For these diseases either stimulation or blocking, respectively, of angiogenesis may provide novel treatment options. Advancing State-of-the-Art: Our hypothesis is that in ischemia it will be possible to support natural growth of blood vessels with Therapeutic angiogenesis and lymphangiogenesis by using local gene transfer of the new members of vascular endothelial growth factor (VEGF) family and their receptors. New co-receptors, designer mutants and PCR suffling products of VEGFs will be used. New vector technology will be used to achieve long-lasting effects of VEGFs. We aim to develop novel site-specifically integrating, targeted, regulated vectors to precisely express the new VEGFs, their soluble decoy receptors and single-chain therapeutic antibodies (scFv) for pro- and anti-angiogenic purposes. As novel approaches, we have developed metabolically biotinylated lenti- and adenoviruses suitable for targeting and Epigenetherapy where siRNA/miRNAs and short nuclear RNAs regulate endogenous gene expression at the VEGF promoter level via modification of histone code. scFv library for endothelial cells and lentivirus-siRNA library directed to all human and mouse kinases will be screened to identify new mediators of angiogenesis in order to develop next generation pro- and antiangiogenic therapies. Based on our strong track record in Clinical applications, the best new pro- and antiangiogenic approaches will be taken to phase I clinical studies in myocardial ischemia and malignant glioma. Significance: This work should lead to significant advances and new therapies for severe ischemia and malignant glioma. Epigenetherapy and new site-specifically integrating, regulated vectors should be widely applicable in medicine.

2 200 000 €

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

The study of genetic conflict is developing at an almost explosive rate. The recognition that genes or alleles residing in individuals of the two sexes may have conflicting interests is transforming evolutionary biology and, likewise, conflict between genes showing different modes of transmission may fundamentally affect adaptive evolution. The research proposed here will push the frontiers of genetic conflict research and establish new domains. It is aimed at exploring the novel possibility that conflict between mitochondrial and nuclear genes have far-reaching implications for adaptive evolution and at advancing our understanding of the biological consequences of sexual conflict. The project consists of several interrelated parts and will employ insects as model systems. First, I will assess to what extent genetic variation in fitness is sexually antagonistic and what life history traits contribute to sexually antagonistic variation. Second, I will elucidate the genomics of metabolic rate and measure selection on metabolic phenotypes. Third, I will test whether sexually antagonistic epistatic interactions between mitochondrial and nuclear genes generate conflict over metabolic rate. Fourth, I will test the hypothesis that sexual conflict contribute to the evolution of primary and secondary sexual traits. Fifth, I will shed light on the complicated evolutionary interplay between sexual conflict and mating system evolution. I will employ an innovative research strategy, ‘experimental genomics’, in which genomic data is used to guide experimental evolutionary work with distinct genotypes. The research outlined here will collectively provide an unprecedented wealth of information into the role of genetic conflict in several horizons of adaptive evolution, ranging from DNA sequence evolution over life history evolution to speciation, and will set the standard for a new generation of insightful studies aimed at bridging the gap between phenotypic selection and genomics.

2 497 442 €

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

Glucose is key source of nutritional energy and raw material for biosynthetic processes. Maintaining glucose homeostasis requires a regulatory network that functions both in the systemic level through hormonal signaling and locally at the intracellular level. Insulin signalling is the main hormonal mechanism involved in maintaining the levels of circulating glucose through regulation of cellular glucose intake and metabolism. While the signalling pathways mediating the effects of insulin have been thoroughly studied, the transcriptional networks downstream of insulin signalling are not comprehensively understood. In addition to insulin signalling, intracellular glucose sensing mechanisms, including transcription factor complex MondoA/B-Mlx, have recently emerged as important regulators of glucose metabolism. In the proposed project we aim to take a systematic approach to characterize the transcriptional regulators involved in glucose sensing and metabolism in physiological context, using Drosophila as the main model system. We will use several complementary screening strategies, both in vivo and in cell culture, to identify transcription factors regulated by insulin and intracellular glucose. Identified transcription factors will be exposed to a panel of in vivo tests measuring parameters related to glucose and energy metabolism, aiming to identify those transcriptional regulators most essential in maintaining glucose homeostasis. With these factors, we will proceed to in-depth analysis, generating mutant alleles, analysing their metabolic profile and physiologically important target genes as well as functional conservation in mammals. Our aim is to identify and characterize several novel transcriptional regulators involved in glucose metabolism and to achieve a comprehensive overview on how these transcriptional regulators act together to achieve metabolic homeostasis in response to fluctuating dietary glucose intake.

1 496 930 €

Start date: 2012-01-01, End date: 2017-02-28

One of the key challenges in scientific research is to link together our understanding of different levels of biological organisation. This challenge is fundamental to the scientific endeavour: from understand how genes interact to drive the cell, to how cells interact to form organisms, up to how organisms interact to form groups and societies. My own and the research of others has addressed this question in the context of the collective behaviour of animals. Mathematical models of complex systems have been used to successfully predict experimental outcome. Most previous studies are however limited in one important aspect: individuals are treated as identical units. The aim of the proposed research proposed is to investigate features which produce differences within the units. The model systems of our study will be sticklebacks, homing pigeons and house sparrows. Individuals can differ from each other on a range of time scales, from information acquired within the last few minutes, through socially learnt information, to genetically inherited differences. Through a series of experiments on each of the study species, the development of mathematical models which incorporate between individual differences, and novel forms of data analysis, we will begin to understand the role played by individual differences within groups. We will look at the rules of motion for fish and birds; the role of personality in decision-making and how short term information differences improve decision-making accuracy. Achieving the project objectives will greatly enhance our understanding of the relationship between individual animals and the groups they live in, as well as impacting on our understanding of individual differences in other areas of biology.

977 768 €

Start date: 2010-02-01, End date: 2015-01-31

Marine phytoplankton are the basis of marine food webs and are responsible for nearly 50% of the global annual carbon-based primary production. Since phytoplankton exert such a global-scale influence on climate, we are interested in understanding what controls their cell fate during bloom “boom and bust” dynamics. Despite their importance, the molecular basis for their ecological success is still in its infancy. In recent years, the wealth of genomic information from marine microbes, coupled with molecular resources and analytical tools, provide an unprecedented opportunity to address fundamental questions about their unique evolutionary history and ecological role. Nevertheless, there is a critical need to “decode” the genomic resources and translate them into cellular mechanisms, community structure and, eventually, to their role in ecosystem function. This proposed research aims to provide novel insights into the role of a chemical-based “arms race” that mediates and structures the microbial interactions in the marine environment. We will dissect unexplored signaling pathways employed by phytoplankton during sensing and acclimation to changes in their environment. Our overarching objective is to unravel the role of infochemicals and related gene products in regulating phytoplankton surveillance systems in response to environmental stress conditions. We will focus on the three major biotic interactions of key dominant algal groups in the oceans; intercellular signaling in diatoms, host-virus interactions in coccolithophores and predator-prey interactions. We will provide a suite of cellular probes and metabolic biomarkers that will allow in situ detection of chemical signaling and biotic interactions in the oceans and will highlight their role in shaping microbial food webs. Our vision is to provide novel cellular concepts and molecular tools to the link between the intricate mechanisms of cell signaling and stress response, and large biogeochemical cycles.

1 999 648 €

Start date: 2011-11-01, End date: 2016-10-31

The link between inflammation and cancer is now established, yet the underlying molecular mechanisms are unresolved. As tumors progress, they modulate inflammatory cells towards a pro-tumorigenic phenotype. We have shown that inflammatory cells reciprocate by sculpting the parenchymal epithelial cells. I hypothesize that these reciprocal interactions lie at the heart of the link between inflammation and cancer. Hepatocellular carcinoma (HCC), one of the deadliest tumors, is a prototype of inflammation induced cancer. My team will employ a twofold strategy to analyze the changes occuring in inflammatory cells before and after tumors emerge, based on preliminary findings showing that changes in inflammatory cells precede tumorigenesis. First, we will perform comprehensive mapping of the changing inflammatory microenvironment in a mouse model of inflammation induced HCC. We will employ genetic manipulation strategies, coupled to cell isolation techniques to delineate the molecular cues that mediate these changes and then will analyze the functional role of key mediators of these processes in HCC. Microfluidics approaches will give us a highthroughput quantitative view of these heterotypic interactions. The same approaches will be harnessed to identify the interactions that form the liver stem cell niche which dramatically expands in states of chronic inflammation. Second, drawing on our finding that a recurring tumor amplicon drives HCC progression by modulating the microenvironment, we will work towards identifying additional similar amplicons to define additional key effectors of the microenvironment. Of special importance, heterotypic cell interactions that play key roles in both cancer initiation and progression, present ideal therapeutic targets, which are easily accessible and less amenable to mutational selection. Furthermore, the results of our experiments could also have far reaching implications in other inflammatory states and different types of cancer.

1 499 940 €

Start date: 2011-10-01, End date: 2017-09-30

Studies of spatial navigation and neural codes for space have followed two parallel tracks over the last 100 years: One research approach was to study animal navigation in the wild over large spatial scales (kilometers); this approach focused on non-mammalian species and on behavioral studies, with hardly any research on the underlying brain mechanisms. The other approach was to study the navigation of mammals (mostly rats) in mazes and small arenas; this approach revealed 'place cells' in the hippocampus, neurons that become active at specific locations; and 'grid cells' in entorhinal cortex – neurons that respond when the animal passes through the vertices of a hexagonal grid spanning the entire environment. However, it is unknown whether place- and grid-cells are relevant at all to large-scale navigation over kilometers. Thus, there is a large gap between the two parallel approaches to studying spatial memory and navigation – both a conceptual gap, and a gap in spatial scale. Here, we propose to bridge this gap, by recording from place cells and grid cells in a flying mammal – the bat – while it moves in 4 different environments of varying sizes, from centimeters to kilometers. We will conduct both standard (tethered) and wireless neural recordings, and will also pioneer the development of a novel sonar-based virtual reality system for studying large-scale navigation. The same neurons will be recorded across different spatial scales, which will allow comparing various neural-coding schemes. These new setups will allow the first testing for the existence of kilometer-sized hippocampal place-fields and entorhinal grids, in bats navigating through naturalistic virtual landscapes; they will also provide rich information on neural codes for 2-D and 3-D space in the mammalian brain. Our innovative project is expected to provide – for the first time – a true understanding of the brain mechanisms of large-scale, realistic navigation in complex 3-D environments.

1 499 999 €

Start date: 2011-11-01, End date: 2016-10-31

Background Osteoarthritis (OA) is one of the most prevalent disorders of the musculoskeletal system. In OA, articular cartilage degenerates and its structure and mechanical properties change, but monitoring or predicting the progression of OA has not been possible. Magnetic resonance imaging (MRI) is a potential tool for the imaging of joint tissues, estimating cartilage structure and diagnostics of OA, whereas joint loading and estimation of stresses/strains within joint tissues necessitates computational modeling. It would be a major breakthrough if one could develop a technique where, based on MRI and computational modeling, prediction and evaluation of OA progression of a patient under a certain loading condition would be possible. Objectives 1) to combine MRI with computational modeling for the estimation of stresses and possible failure points within human knee joints, and 2) to develop second generation adaptive models of articular cartilage for the prediction of altered tissue structure and composition during OA progression. For the model validation, cartilage structure, composition and biomechanical properties as well as cell responses in situ are characterized. At the end of the project these main aims will be merged 3) to estimate the effect of loading on cartilage degeneration during the progression of OA in a patient-specific manner. Significance Combining MRI information of joint tissues with computational modeling, we develop a tool to evaluate the effect of different interventions on stresses in human joints. By combining this tool with an adaptive model that can estimate the effect of loading on cartilage composition and structure, we hope to be able to predict changes in cartilage properties during OA progression in a patient-specific manner several years ahead. This would help in decision making of clinical treatments and interventions (conservative or surgical) for the prevention or further progression of OA.

1 303 056 €

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

The goal of the proposed research is a comprehensive understanding of how evolutionary potential of pathogen populations interacts with epidemiological dynamics in natural populations. The empirical work will be conducted on the specialist fungal pathogen Podosphaera plantaginis and its host plant Plantago lanceolata in a large network of host populations. I will address the key theories of pathogen evolution, involving life-history trade-offs, competition for resources under multiple infection, and sexual reproduction. This project takes advantage of the exceptional research opportunities offered by the focal study species to test models that have not been validated with respect to realized population dynamics and the persistence of pathogen populations. I have studied the coevolutionary dynamics between P. plantaginis and P. lanceolata for several years. Unique epidemiological data have been collected annually on the occurrence of the pathogen in a network of 4000 host populations since 2001. Recently, I have generated an EST library for the pathogen that allows and facilitates genetic studies. In the planned research, I will combine laboratory experiments with large-scale population surveys, genetic studies and mathematical modelling to achieve the objectives of this proposal. The proposed research has potential for groundbreaking results on pathogen evolution and epidemiology through: i) Simultaneous study of multiple forces driving pathogen evolution and their importance in natural populations, with direct connections to epidemiology. ii) Development of new methodology for the study of obligate parasites like P. plantaginis. iii) Construction of a stochastic, spatially explicit epidemiological model predicting pathogen occurrence from one season to the next with applicability to a wide range of pathogens. iv) Identifying critical life-history stages and mechanisms for virulence evolution yield much needed insights and tools into the battle against disease.

1 498 811 €

Start date: 2011-10-01, End date: 2016-09-30

Aims and significance: The aims of this program are (1) To obtain new understanding of how environmental and life style factors interact with genes to induce immune reactions able to cause the different forms of arthritis that are defined as RA; (2) To use this understanding to develop prevention and targeted therapy for different forms of RA, and to enable efficient and eventually curative therapy. Background: We build on new understanding or RA etiology that has followed from studies on interactions between genes, environment and immunity in different subsets of RA. This has been provided for new detailed studies on specific and eventually disease-inducing autoimmunity in RA. Research program: We will use our infrastructure (longitudinal large cohorts, biobanks genetic information and our molecular immunology laboratory) to investigate (1) how genes and environment interact in causing different forms of RA; (2) how specific immune reactions against post-translationally modified (mainly citrullinated) autoantigens are triggered by environmental agents in specific genetic contexts in different individuals; (3) How these immune reactions target different organs (joints, lungs etc) and eventually cause arthritis in model systems; (4) how the combination of genes, environment and immunity may determine disease course and response to various therapies. Novelty and opportunities to take knowledge of autoimmune disease and RA to a new level: The recent advances in understanding interactions between genes, environment and immunity in RA, provides a striking new opportunity to understand basic features of autoimmunity and autoimmune disease, as well as potentials to prevent and treat RA very early. I believe that the presented program is well positioned to use this opportunity and contribute to a new paradigm for understanding and preventing RA.

2 000 000 €

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

Lack of replicability of scientific discoveries has surfaced too often in recent years, and even reached the attention of the general public. An ignored cause is the inappropriate statistical treatment of two statistical problems: (1) selective inference, manifested in selecting few promising leads following the statistical analysis of the potential many, where ignoring the selection process on estimates, confidence intervals and observed significance; (2) using too optimistic a yardstick of variation with which confidence intervals set and statistical significance of the potential discovery is judged, as a result of ignoring the variability between laboratories and subjects. The first problem becomes more serious as the pool of potential discoveries increases, the second paradoxically becomes more serious as measuring ability improves, which explain why the two problems are more prominent in recent years. Both problems have statistical solutions, but the solutions are not practical as they burden the analysis to a point where the power to discover new findings is exceedingly low. Therefore, unless required by regulating agencies, scientists tend to avoid using these solutions. I propose to develop methods that address such replicablity problems specific to medical research, epidemiology, genomics, brain research, and behavioral neuroscience. The methods include (a) new hierarchical weighted procedures, and model selection methods, that control the false discovery rate in testing; (b) shorter confidence intervals that offer false coverage-statement rate for the selected, both addressing the concern about selective inference; and (c) a compromise between using random effects models for the laboratories and subjects and treating them as fixed, to be aided by multiple laboratory database in behavior genetics and neuroscience. By serving the exact needs of scientists, while avoiding excessive protection, I expect the offered methodologies to become widely adapted.

1 933 200 €

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

Chromatin modifications play a major role in regulating genome function. Perturbations in such modifications can contribute to neoplastic processes. We will focus on a specific chromatin modification: histone H2B monoubiquitylation. The significance of monoubiquitylated H2B (H2Bub) will be studied by manipulating RNF20, the major E3 ubiquitin ligase responsible for H2B ubiquitylation as part of a heteromeric complex with RNF40. In one major line of research, we will assess the biochemistry of RNF20/H2Bub. The effects of RNF20/H2B on gene expression will be explored through identification of proteins that interact with H2Bub and through in vitro incorporation of H2Bub into nucleosomes. Effects of H2Bub on transcription elongation will be studied by a new high resolution ChIP-seq method (NET-seq). Based on recent ChIP-seq data, we will also explore links between H2B and regulation of splicing. Furthermore, we will investigate the regulatory crosstalk between H2Bub and microRNAs. The other major line of research will explore the biology of RNF20/H2Bub, with particular emphasis on cancer-related processes. This will be addressed through a combination of cell culture models and mouse models, including constitutive and conditional RNF20 knockout mice. The contribution of RNF20/H2Bub to various differentiation programs, with particular emphasis on embryonic stem cell differentiation, will also be investigated. In addition, we will study the impact of RNF20/H2Bub on NF-kB activity and on inflammatory responses; this will combine in vitro and in vivo systems, with emphasis on inflammation-related cancer. Finally, we will monitor changes in RNF20, RNF40 and H2Bub status in human tumors and investigate underlying mechanisms.

2 500 000 €

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

We aim to study transcriptomes with single-cell resolution, a long-standing goal in biology, to answer fundamental questions about gene regulation. The main objective concerns gene regulation during in vivo differentiation and in pluripotent cells by studying single-cells from murine preimplantation embryos, a model system with natural single-cell resolution, important biology and medical potential. This would also allow us to explore general regulatory principles of gene expression programs of individual cells. This research program will be accomplished by novel deep sequencing technology of mRNAs (mRNA-Seq) to obtain quantitative, unbiased and genome-wide gene and isoform expression measurements. We are therefore developing new experimental and computational methods for genome-wide analyses of transcriptomes at single-cell resolution. The biological significances of the proposed research are unique insights into early embryonic development. Deep sequencing of transcriptomes will also reveal post-transcriptional gene regulation important for pluripotent cells and identified pluripotency-specific gene and isoform expressions will be important for future stem cell based therapies. The inherit single-cell nature of the model system together with its important biology makes it a model systems exceptionally well suited for a systems biology approach aiming to characterize gene regulation at single-cell resolution. The novel methodology has tremendous potential to enable complete mRNA characterization of individual cells. The deep sequencing approach with state-of-the-art computational analyses is both more quantitative than previous methods and it will give readouts on alternative isoforms generated by alternative promoters, splicing and polyadenylation.

1 654 384 €

Start date: 2010-02-01, End date: 2015-01-31

We have recently shown that EGFR over-expressing tumors can be eradicated by an EGFR homing chemical vector, carrying dsRNA. The vector is PolyInosine/Cytosine (PolyIC) bound to Polyethleneimine-Polyethyleneglycol-EGF (PEI-PEG-EGF, PPE). We have shown that even tumors in which up to 50% of cells do not express EGFR are eradicated, due to the strong tumor-localized bystander effects, which involve the innate immune system. Using this EGFR homing vector we have been able to eradicate EGFR overexpressing tumors by either local or systemic application. Since the success of this strategy seems to be due to the strong bystander effects induced by the internalized PolyIC it is likely that heterogeneous tumors, in which only a portion of the cells harbor the targeted receptor, will be eradicated too, as shown in our preliminary studies (PloS Med, 2006). This strategy actually targets the innate immune system to the tumor. We propose to establish tumors in which decreasing portions of cells over-express EGFR and determine the lowest number of EGFR over-expressing cells that can yield tumor eradication by the lowest dose of PolyIC/PPE. The principle behind the success of the Trojan horse approach is that the targeting moiety, EGF, is tethered to the other components of the vector in such a way that it retains its native EGFR binding properties and its ability to internalize with the receptor. The composition of the vector is such that the ligand EGF can be replaced by any other ligand, if the appropriate coupling conditions are used, retaining the ability of the ligand to bind to the target protein and internalize with it. We propose to replace EGF by a number of other ligands, such PSMA binding ligand (targeting prostate cancer) and Her-2 affibodies. Although only a fraction of women who over-express Her-2 respond to Herceptin, it is likely that they will respond to PolyIC/PP-Her-2 affibody.

2 054 340 €

Start date: 2010-02-01, End date: 2015-01-31

The response to drug therapy varies widely between individuals. Proteins involved in the absorption, distribution, metabolism and excretion of drugs play a central role in determining the concentration of a drug at the target site, and thus drug efficacy and toxicity. Transporters are membrane proteins that mediate the translocation of chemicals into and out of cells using active and passive mechanisms. We have identified a single nucleotide variant in the SLCO1B1 gene encoding the organic anion transporting polypeptide 1B1 (OATP1B1), which severely impairs the hepatic uptake of the cholesterol-lowering drug simvastatin leading to an increased systemic exposure to the drug, and a markedly increased risk of simvastatin-induced muscle toxicity. The effects of this variant differ significantly between statins, forming a rational basis for individualized lipid-lowering therapy. In addition to OATP1B1, also OATP1A2, OATP1B3, and OATP2B1 are known to transport several drugs in vitro (e.g., anticancer, cardiovascular and anti-infective drugs). However, the roles of these transporters in the pharmacokinetics of drugs in vivo in humans are unknown. The aim of this project is to systematically search for genetic variants of SLCO1A2, SLCO1B3 and SLCO2B1, which have functional significance in vivo in humans. This project will enable studies to determine the roles of these transporters in the pharmacokinetics of drugs and in the disposition of endogenous compounds in vivo, with implications for drug development and drug safety. Moreover, functionally significant variants in these genes may be used to personalize drug therapies. Overall, the project can significantly facilitate the development of new drugs and can improve the safe and effective use of drugs already in clinical use, thus increasing the health and well-being of mankind and reducing the overall costs of healthcare and drug development.

1 882 212 €

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

Polyploidization is a widespread phenomenon among plants and is considered a major speciation mechanism. Before becoming evolutionary successful, newly formed polyploids often have to overcome fertility bottlenecks, because mating with partners of lower ploidy causes incompatibilities in the endosperm leading to the formation of mainly non-viable progeny. This reproductive barrier is called the triploid block. Nevertheless, the most frequent route to polyploid formation is probably through unreduced gametes, suggesting that the triploid block can be overcome. Recent work from our laboratory uncovered a genetic pathway leading to unreduced gamete formation at high frequency and revealed that the triploid block is mainly caused by malfunction of Polycomb group (PcG) proteins. PcG proteins are evolutionary conserved proteins, which assemble into multimeric complexes with chromatin-modifying enzymatic activity, implicating epigenetic regulatory mechanisms as an important element of speciation. Here, I propose to unravel the underlying molecular mechanism(s) of the triploid block by identifying the responsible genes causing endosperm failure upon deregulation and their mechanism of regulation in response to interploidy crosses. I also plan to investigate whether genes that contribute to the triploid block are as well responsible for establishing interspecies incompatibilities within the Arabidopsis genus. This project will combine genetics, genomics and epigenomics and will make extensive use of knowledge and tools that we have been established in my laboratory over the recent years, making it likely that the proposed objectives can be achieved. The results of this project will be of interest to a broad scientific community, including biologists with a strong interest in epigenetic mechanisms as well as ecologists interested to understand mechanisms of plant speciation.

1 447 596 €

Start date: 2011-10-01, End date: 2016-09-30