ERC FUNDED PROJECTS

Telomeres are heterochromatic nucleoprotein complexes located at the end of linear eukaryotic chromosomes. Contrarily to a longstanding dogma, we have recently demonstrated that mammalian telomeres are transcribed into TElomeric Repeat containing RNA (TERRA) molecules. TERRA transcripts contain telomeric RNA repeats and are produced at least in part by DNA-dependent RNA polymerase II-mediated transcription of telomeric DNA. TERRA molecules form discrete nuclear foci that co-localize with telomeric heterochromatin in both interphase and transcriptionally inactive metaphase cells. This indicates that TERRA is an integral component of telomeres and suggests that TERRA might participate in maintaining proper telomere heterochromatin. We will use a variety of biochemistry, cell biology, molecular biology and microscopy based approaches applied to cultured mammalian cells and to the yeast Schizosaccharomyces pombe, to achieve four distinct major goals: i) We will over-express or deplete TERRA in mammalian cells in order to characterize the molecular details of putative TERRA-associated functions in maintaining normal telomere structure and function; ii) We will locate TERRA promoter regions on different human chromosome ends; iii) We will generate mammalian cellular systems in which to study artificially seeded telomeres that can be transcribed in an inducible fashion; iv) We will identify physiological regulators of TERRA by analyzing it in mammalian cultured cells where the functions of candidate factors are compromised. In parallel, taking advantage of the recent discovery of TERRA also in fission yeast, we will systematically analyze TERRA levels in fission yeast mutants derived from a complete gene knockout collection. The study of TERRA regulation and function at chromosome ends will strongly contribute to our understanding of how telomeres are maintained and will help to clarify the general functions of mammalian non-coding RNAs.

1 602 600 €

Start date: 2009-10-01, End date: 2014-09-30

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

2 467 000 €

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

Comparative genomics revealed that ~5% of the human genome is conserved among mammals. This fraction is likely functional, and could harbor pathogenic mutations. We have shown (Nature 2002, Science 2003) that more than half of the constrained fraction of the genome consists of Conserved Non-Coding sequences (CNCs). Model organisms provided evidence for enhancer activity for a fraction of CNCs; in addition another fraction is part of large non-coding RNAs (lincRNA). However, the function of the majority of CNCs is unknown. Importantly, a few pathogenic mutations in CNCs have been associated with genetic disorders. We propose to i) perform functional analysis of CNCs, and ii) identify the spectrum of pathogenic CNC mutations in recognizable human phenotypes. The aims are: 1. Functional genomic connectivity of CNCs 1a. Use 4C in CNCs in various cell types and determine their physical genomic interactions. 1b. Perform targeted disruption of CNCs in cells and assess the functional outcomes. 2. Pathogenic variation of CNCs 2a. Assess the common variation in CNCs: i) common deletion/insertions in 350 samples by aCGH of all human CNCs; ii) common SNP/small indels using DNA selection and High Throughput Sequencing (HTS) of CNCs in 100 samples. 2b. Identify likely pathogenic mutations in developmental syndromes. Search for i) large deletions and duplications of CNCs using aCGH in 1500 samples with malformation syndromes, 1000 from spontaneous abortions, and 500 with X-linked mental retardation; and ii) point mutations in these samples by targeted HTS. The distinction between pathogenic and non-pathogenic variants is difficult, and we propose approaches to meet the challenge. 3. Genetic control (cis and trans eQTLs) of expression variation of CNC lincRNAs, using 200 samples.

2 353 920 €

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

Cell adhesions play an important role in the organization, growth, maturation, and function of living cells. Interaction of cells with the extracellular matrix (ECM) plays an essential role in a variety of disease states , inflammation, and repair of damaged tissues. At the cellular level, many of the biological responses to external stimuli originate at adhesion loci, such as focal adhesions (FA), which link cells to the ECM . Cell adhesion is mediated by receptor proteins such as cadherins and integrins. The precise molecular composition, dynamics and signalling activity of these adhesion assemblies determine the specificity of adhesion-induced signals and their effects on the cell. However, characterization of the molecular architecture of FAs is highly challenging, and it thus remains unclear how these molecules function together, how they are recruited to the adhesion site, how they are turned over, and how they function in vivo. In this project, I aim to conduct an interdisciplinary study that will provide a quantum step forward in the understanding of the functional organization of FAs. We will analyze, for the first time, the three-dimensional structure of FAs in wild-type cells and in cells deficient in the specific proteins involved in the cell-adhesion machinery. We will study the effect of specific geometries on the functional architecture of focal adhesions in 3D. A combination of state-of-the-art technologies, such cryo-electron tomography of intact cells, gold cluster chemistry for in situ labeling, and modulation of the underlying matrix using micro- and nano-patterned adhesive surfaces, together with correlative light, atomic force and electron microscopy, will provide a hybrid approach for dissecting out the complex process of cell adhesion.In summary, this project addresses the properties of FAs across a wide range of complexities and dimensions, from macroscopic cellular phenomena to the physical nature of these molecular assemblies

1 294 000 €

Start date: 2009-11-01, End date: 2015-10-31

Small RNA-mediated regulation of gene expression is a recent addition to fundamental gene regulatory mechanisms that directly or indirectly affect possibly every gene of a eukaryotic genome. The predominant sources of small RNA in somatic tissues are microRNA genes that encode short dsRNA hairpins of evolutionary conserved sequence. Disorders of metabolism, such as obesity and type 2 diabetes are poorly understood at a molecular level. In this application we propose to explore if miRNA regulatory networks play a role in these diseases. We will employ state of the art methods for identification of small RNAs and their regulated targets and use biochemical, cell and animal model systems to study the detailed molecular mechanisms of metabolic gene regulation by miRNAs. In addition, we will investigate the underlying principles of how RNAs are taken up by cells and develop methods that will improve delivery of miRNA mimetics or inhibitors through cell-specific uptake. The specific aims of this study are: Aim 1: To define the small regulatory miRNA content of liver, muscle and adipose tissue that are associated with abnormal glucose and lipid homeostasis and to dissect the underlying molecular pathways that govern their expression. Aim 2: To characterize the functions of miRNAs in insulin resistance, glucose uptake and production, fatty acid oxidation and lipogenesis. Aim 3: To identify factors and dissect the pathways that regulate RNA uptake by cells and to develop novel pharmacological treatment strategies to manipulate miRNA-expression. Together, this proposal will shed light on the function that miRNA regulatory networks play in metabolism and in the pathophysiology of obesity/type 2 diabetes. In addition, these studies will contribute to the development of new RNA delivery technologies that are urgently needed as experimental tools as well as for novel therapeutic strategies.

2 021 235 €

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

If we are ever to unravel the mysteries of brain function at its most fundamental level, we will need a precise understanding of how its component neurons connect to each other. Furthermore, given the many recent advances in genetic engineering, viral targeting, and immunohistochemical labeling of specific cellular structures, there is a growing need for automated quantitative assessment of neuron morphology and connectivity. Electron microscopes can now provide the nanometer resolution that is needed to image synapses, and therefore connections, while Light Microscopes see at the micrometer resolution required to model the 3D structure of the dendritic network. Since both the arborescence and the connections are integral parts of the brain's wiring diagram, combining these two modalities is critically important. In fact, these microscopes now routinely produce high-resolution imagery in such large quantities that the bottleneck becomes automated processing and interpretation, which is needed for such data to be exploited to its full potential. We will therefore use our Computer Vision expertise to provide not only the necessary tools to process images acquired using a specific modality but also those required to create an integrated representation using all available modalities. This is a radical departure from earlier approaches to applying Computer Vision techniques in this field, which have tended to focus on narrow problems. State-of-the-art methods have not reached the level of reliability and integration that would allow automated processing and interpretation of the massive amounts of data that are required for a true leap of our understanding of how the brain works. In other words, we cannot yet exploit the full potential of our imaging technology and that is what we intend to change.

2 495 982 €

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

How does the assembly of neuronal circuits contribute to the emergence of function controlling dedicated animal behaviors? Finding answers to this question requires a deep understanding of the connectivity map of neuronal circuits controlling a behavior as well as the mechanisms involved in the generation of these specific circuit maps. In the project outlined here, I propose the analysis of the neuronal circuits involved in the generation of motor output, a behavior representing the ultimate output of nearly all nervous system activity. Studying the mouse motor output system will allow the analysis of neuronal circuit connectivity at an exquisite degree of specificity. Owing to the anatomical arrangement of motor neuron pools innervating individual muscles, this system offers the possibility to combine genetic, anatomical and physiological analysis of synaptic specificity with a direct link to a behavioral output. Generation of coordinated motor behavior is functionally linked to the high degree of specificity in presynaptic connections controlling the activation of individual motor neuron pools, yet knowledge on the specificity map of premotor circuits is currently missing. The aim of this research project is to acquire information on the general principles guiding the acquisition, maintenance and developmental plasticity of neuronal connectivity between premotor neurons and functionally defined subpopulations of motor neurons. This project is now possible due to the unique combination of our detailed know-how of the motor system in mice including a variety of genetic animal models, and the application of novel viral circuit tracing technology revealing monosynaptically connected premotor neurons, which we have recently applied successfully to the motor system in mice in vivo. Together, our project will elucidate the anatomical connectome of the motor output system as well as the principles governing the specificity with which motor circuits assemble.

2 499 354 €

Start date: 2010-03-01, End date: 2016-02-29

How sensory processing is occurring into the brain and how to relate behavior to neuronal activities are key questions in modern neuroscience. Understanding the neural codes underlying brain function will be of great importance for future implementation of brain-machine interfaces. This research project proposes to study the cellular and network mechanisms controlling sensory perception. In particular, we would like to precise how sensory stimuli are coded by brain networks and how these representations may be influenced by experience or modulatory brain centers. In order to address these general questions, we propose to study olfaction as model sensory system. The olfactory system is central to the behavior of rodents (animal models that we study), is highly plastic and largely modulated by the neuromodulatory brain centers. We propose to use a combination of genetic, electrophysiological, imaging and behavioral methods to study how odor information is processed in the central nervous system as it moves from the periphery to higher areas of the brain. We showed in the past that sensory information can be contained in dynamic neural ensemble. We propose to show that ensemble dynamics may be the basis of odor coding in the olfactory bulb and to describe the mechanisms underlying cortical coding that would allow us to relate neuronal activity to behavior. In addition, we hope to show the existence of a novel form of plasticity in the olfactory bulb namely ensemble plasticity. We believe that the general questions addressed in the study of these sensory systems go beyond understanding olfactory sensory perception and could potentially be generalized to the function of many brain regions.

1 399 998 €

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

Our goal is to develop fundamentally new architectures for wireless networks that offer the convenience of wireless communication while achieving the performance, predictability and security of wired networks. The wireless channel is inherently a shared medium characterized by limited resources and complex signal interactions between transmitted signals. The question we address is how do we transmit information over wireless and how do we exploit the wireless channel properties to share its resources. Ours is a fundamentally different approach to existing strategies, that builds on new physical and packet layer sharing and cooperation paradigms that we have been working on, to extract the optimal throughput and reliability performance from the wireless medium. These are recent breakthroughs in (i) network coding and (ii) wireless cooperation. Network coding is a new area bringing a novel paradigm for network information flow that enables cooperation at a packet level to optimally share the network resources. Deployment of the first network coding ideas in wireless have already indicated benefits as large as a factor of ten in terms of throughput. Complex signal interactions caused by the inherent broadcast nature of wireless channels, is traditionally viewed as an impediment to be mitigated. Recently it has been demonstrated that one can utilize interference to develop cooperation at the wireless signal level (physical layer) for arbitrary wireless networks. This can give significant capacity advantages over techniques that mitigate interference. Both these ideas can radically affect the way information is communicated, stored and collected, and can revolutionize the design of future wireless networks. In this project we plan to addess several fundamental questions that develop on these themes. We take a complete view of these ideas by not only developing the underlying theory but also through validation on wireless testbeds.

1 771 520 €

Start date: 2009-09-01, End date: 2014-08-31

Oligomers are toxic in an array of protein misfolding and aggregation (PMA) disorders. However, the chain of events from protein aggregation to dysfunction is poorly understood. Prion diseases are marked by accumulation of PrPSc, a misfolded variant of wild-type PrPC. PrPC mediates PrPSc neurotoxicity and counteracts toxic PrPC mutants, indicating that a subversion of normal PrPC function may underlie neurodegeneration, and this may not be limited to prion disease. Here, we propose to explore these newly discovered physiological functions of PrPC in three paradigms. We show that PrPC assembles into a multiprotein complex containing a protease; neurotoxic PrPC mutants generate a smaller complex that is uncleaved. We show that neuronal expression of PrPC is required in trans for long-term myelin maintenance in peripheral nerves. We will therefore investigate the hypothesis that a fragment of PrPC transmits signals crucial for axomyelinic integrity. We show that PrPC physically interacts with both amyloid b and islet amyloid polypeptide and attenuates functional impairment mediated by these peptides. We therefore propose to test whether subversion of normal PrPC function is involved in diverse PMA disorders. We developed an ex vivo model that accurately reproduces major features of prion infections, most notably neurodegeneration. We have identified several unexpected PrPSc-induced cellular stress pathways which may be common to other PMA disorders. Using this model system, we will clarify the role of PrPC in cell survival pathways and determine the requirement for PrPC in the pathology of other PMA disorders. This proposal capitalizes on provocative recent results and, if successful, will provide valuable insights into PMA toxicity that will go far beyond prion diseases.

2 500 000 €

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

Reconstruction of methane flux from lakes: development and application of a new approach

1 554 000 €

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

The hallmark of social insect colonies is reproductive division of labour which is often associated with dramatic morphological and behavioural differences between queens, workers and males. The aim of this proposal is three-fold. First, we will use our recently developed fiducial identification system to investigate the general principles of social organisation and division of labour. The video tracking of workers labelled with markers derived from the augmented reality library ARTag allows us for the first time to distinguish up to 2000 individuals and precisely locate them every 500ms, hence allowing large-scale experiments addressing the question of how the behaviour of individual workers is influenced by the joint effects of environmental factors and social interactions. The second related aim is to investigate how the level of altruism within colonies and the reliability of communication systems are shaped by colony kin structure. Because it is not possible to conduct artificial evolution with social insects we will use a new experimental system consisting of colonies of small mobile robots with simple vision and communication abilities. This system permits to conduct hundreds of generations of experimental evolution in colonies with variable group composition to identify the factors affecting the evolution of altruism and communication. Finally, we will complement these studies with a genetic perspective using a remarkable genetic social polymorphism that we recently discovered in the fire ant Solenopsis invicta. The advent of new ultra high-throughput sequencing techniques will allow us to document the steps involved in the evolution of this genetic social polymorphism and test the suggestion that the chromosome involved in the social polymorphism has the properties of a sex chromosome. This project will be highly interdisciplinary, involving skills in evolutionary biology, the study of animal behaviour, bioinformatics, engineering and molecular biology

2 497 500 €

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

Spatial audio is a field, which investigates technologies to capture and reproduce sound in a way that the spatial properties of it are either preserved or modified depending on application. For example, modern surround sound techniques try to reproduce the sound scene perceived by a human listener in the same way than in the original occasion. The principal investigator (PI) has been able to develop a number of technologies in spatial audio field and to transfer them to the industry. The project would have two work packages, one concentrating on development of technology (WP1) and the other on perceptual studies (WP2). The perceptual studies are assumed to help technology development, and new technologies are assumed to reveal new phenomena in perception. The main issue for WP1 is the development of generic audio format. In future all music records and movie audio tracks are targeted to be in this format, which would be suitable for listening with any loudspeaker setup and also with headphones, always with optimal spatial and timbral quality. The development of the format is based on a technique by the PI, which is extended in this work for enhanced playback over loudspeakers and over headphones. Also, new techniques are developed for sound input from different types of microphones and from existing audio formats. The perceptual issues studied in WP2 would be the functioning of spatial hearing with wide sources and complex sound scenarios, together with computational modeling of brain mechanisms devoted to binaural hearing. The crossmodal effects between vision and auditory systems would also be investigated in the anechoic chamber specially equipped for spatial sound research. As the final task, the perceptual quality of developed generic audio format in different listening scenarios would be evaluated with subjective and objective tests.

1 879 458 €

Start date: 2009-09-01, End date: 2014-08-31

The main objective of this interdisciplinary research project is to elucidate regulatory mechanisms through which the circadian timing system coordinates temporal physiology. This system has a hierarchical architecture, in that a master clock in the brain s suprachiasmatic nucleus synchronizes subsidiary oscillators in nearly all body cells. The establishment of phase coherence is obviously of utmost importance in the coordination of circadian physiology. While recent studies have identified feeding cycles, hormone rhythms, and body temperature oscillations as timing cues for peripheral clocks, the molecular makeup of the involved signalling mechanisms is largely unknown. Using liver and cultured cells as model systems, we will employ two innovative strategies for the elucidation of relevant signalling pathways. (1) STAR-Prom (Synthetic TAndem Repeat-PROmoter display), a technique developed in our laboratory, will hopefully identify most if not all immediate early transcription factors activated in cultured cells by rhythmic blood-borne and temperature-dependent signals. (2) A transgenic mouse model with conditionally active liver clocks will be explored in the genome-wide identification of coding and non-coding transcripts whose rhythmic accumulation is system-driven. The in vivo significance of the components emerging from these approaches will be assessed via RNA interference. Thus, relevant siRNAs will be injected into the tail vein of mice, and their effect on the phase of circadian liver gene expression will be monitored in freely moving mice by using whole body fluorescence imaging. Physiologically important components will serve as entry points for the identification of upstream and downstream constituents in the corresponding signal transduction cascades.

2 360 136 €

Start date: 2010-04-01, End date: 2015-12-31