It has been postulated that the integration of adult-born neurons into pre-existing circuitry significantly contributes to the brain plasticity such as the formation of new memories. However, the role of adult-born neurons has not been investigated with intact neural circuitry in behaving animals, due to the lack of appropriate techniques to modulate the activity of adult-born neurons specifically. Here I propose an optogenetic method using microbial light-sensitive proteins Channelrhodopsin-2 (ChR2) and Natronomonas pharaonis halorhodopsin (NpHR) to manipulate the activity of adult-born neuron. Using double-floxed Cre-recombinase-dependent adeno-associated virus (AVV) system, combined with transgenic mice expressing Cre recombinase under the control of the adult neural stem cell or the transit amplifying specific promoter, ChR2 or NpHP proteins were introduced to the adult-born neurons and could drive or suppress of the action potentials by photo-stimulation. This approach can overcome several weaknesses of existing methodologies, and allows the functional analysis of newborn neurons within intact neural circuits. Cell biological, electrophysiological, and behavioral phenotypes after the activation or the inhibition of newborn neurons will be analyzed at different time window during the maturation and the integration of newborn neurons. The role of adult-born neuron containing neural circuits for learning and memory processes will be investigated in depth and be newly interpreted by this method. In addition, this work will give me important exposure to new techniques and broaden my insights for my future career as an independent academic investigator.

Cholesterol levels are regulated through a mechanism of feedback on the transcription of genes involved in cholesterol synthesis. Sterol Regulatory Element Binding Protein (SREBP) is cleaved by specific proteases in the Golgi, and the released fragment serves to promote transcription of the enzymes involved in cholesterol synthesis, as well as of the low density lipoproteins (LDL) receptor which uptakes LDL-bound cholesterol from the blood. SREBP is transported from the ER to the Golgi via incorporation into vesicles formed by coat proteins of the COPII family. A SREBP-cleavage-activating protein (Scap) is used as binding mediator. When cholesterol levels are high transport of Scap/SREBP is inhibited. Scap binds to the COPII protein Sec24 which is required to capture Scap/SREBP into transport vesicles that bud from the ER. The laboratory of David Ginsburg at the University of Michigan has found that that a mouse strain deficient in Sec24A, one of four paralogs of this gene in mammals, are normal but have unusually low levels of cholesterol, possibly indicating a role for Sec24A in Scap/SREBP transport. Endogenous production of cholesterol in mammals might hence be dependent on a specific paralog of a COPII member, raising the questions of how specificity of interaction is achieved at the molecular level. The experimental plan will start with the optimization of a Scap/SREBP expression system. Subsequently, specific interaction of Scap with Sec24A will be investigated by using in vitro COPII budding reactions. The site on Sec24 responsible for the interaction with Scap will be identified by testing the binding of the Scap COPII sorting signal peptide to Sec24A mutant

Glycogen functions as an energy reserve in many species, and its main purpose is to fulfill sudden requirements for glucose. The rate-limiting enzyme during glycogen synthesis is glycogen synthase (GS) that catalyses the transfer of glucose units from UDP-glucose to a growing alpha-1,4 linked glucose chain. Initially, a glucose chain of 6-8 residues is assembled on glycogenin (GN) by a process of self-glycosylation. GN forms a stable complex with GS, which continues to add glucose units to the extent that megadalton glycogen particles are formed. GS is regulated by dephosphorylation and allosterically by glucose-6-phosphate (G6P), both of which contribute to maintenance of whole-body glucose homeostasis. Despite major advances in the last 50 years in understanding glycogen synthesis and GS regulation, structural information for this key enzyme is unavailable. Here I lay out plans for shedding light on the process of glycogenesis and GS regulation, by solving and studying the structure of GS in complex with GN and the allosteric activator G6P. I will build on recently obtained preliminary data, including soluble protein complexes of GS/GN in both active and inactive forms. Structure determination of the GS/GN complex will generate a range of further biochemical hypotheses that will be tested by mutagenesis, pulldown and activity assays, with the aim of understanding how GS and GN cooperate to regulate glycogenesis, how GS is inactivated by phosphorylation and how GS is activated allosterically by G6P. Elucidation of these key regulatory mechanisms will provide new insights in understanding, further studying and possibly curing metabolic disorders such as type 2 diabetes.

Friedreich’s ataxia is a neurodegenerative disease caused by deficiency of frataxin, an essential protein involved in iron metabolism. The depletion of frataxin increases mitochondrial iron levels and decreases the activity of Fe-S proteins. It has been suggested that the frataxin acts as iron chaperon. Nevertheless, recent data shows that bacterial frataxin could act as iron sensor. In order to clarify this point, I will study the interactions between the human frataxin orthologue with the others proteins involved in the Fe-S cluster assembly (Nfs1/Isu/ferrodoxin) by using NMR. In addition, I will do the assignment of Isu which will be an excellent training. The molecular docking between the proteins will be also studied by fluorescence and ITC. The obtained results will be analyzed when I come back to my origin group and completed quantifying in kinetically terms the mechanism of action of frataxin.

Three main reasons have prompted me to seek a stage abroad. The recently purchased 600 MHz NMR-spectrometer in the University of Balearic Islands has encourage me to do this stage which should enhance my knowledge (limited) about this technique. A stage abroad is crucial in my career, not only because it would allow me to be able to improve my position (non-permanent) but to determine my future goals. The development of this project represents collaboration between our group and the Dr. Pastore’s group.

Our group is interested in study how the protein structure/function is modified in the protein glycation. NMR studies should be very useful for this purpose. The knowledge acquired will be important when I come back and allow me to reinforce the use of the new NMR.

To determine how a virus functions, knowledge is needed about the molecular arrangement of its proteins and their interactions with the host cell over the course of the ‘life’ cycle of the virus. The goal of our project is to gain such a time-resolved structural view of the infection process at the molecular level. We propose an innovative multi-disciplinary approach that integrates data from experimental and computational methods, generated at different stages of infection. The method will be applied to one of the largest and most complex viruses, HSV-1 (thousands of proteins of ~100 distinct kinds). We will determine: i) the virions shape and architecture in vivo (cryo electron tomography); ii) the shape of stable subcomplexes (cryo electron microscopy); iii) protein interactions and stoichiometry (proteomic techniques); and iv) the structure or shape of proteins and their relative positions within subcomplexes (bioinformatics). A probabilistic analysis of structural models, consistent with all data, will reveal the localizations of all proteins in each tomogram and their direct interactions. The resulting structures will represent a major advance in our understanding of the molecular and mechanistic details of HSV-1 pathogenesis, a crucial step for identifying drug targets. Our novel approach will be applicable to many viral systems and will open the door for similar studies on other pathogens.

Birdsong is a model system for exploring sensorimotor plasticity in a well defined neural circuit critical for learning and memory. Male zebra finches learn song during development, resulting in a stereotyped adult song that, in normal conditions, does not subsequently change. Altered auditory feedback experiments have shown that song learning and adult maintenance is an active process, but little is known about the neural mechanisms of auditory feedback evaluation. I propose to design an electronic system that will emulate the low-order dynamics of the avian vocal organ and tract in order to generate realistic synthetic sounds similar to the real song. The input will be a physiological signal recorded from the periphery (air sac pressure), so that the bird will be able to control the device and synthetically sing through it. This biomimetic device will address limitations in prior studies that presented unrealistic altered auditory feedback. I will test the hypothesis that a bird exposed to realistic modifications of auditory feedback will rapidly modify its song. I will coordinate these behavioral studies with single neuron recordings during singing. Lesion studies have shown that the basal ganglia-forebrain circuit is involved in song plasticity. In this work I will test the hypothesis that during exposure to biologically realistic altered auditory feedback, the activity of the LMAN forebrain nucleus will be modified in order to send correction signals to the motor pathway. The ultimate goal of these studies is to elaborate the neural code for sensory-motor integration during vocal behavior.

Centrosomes are the dominant microtubule organizers in animal cells. In many cell types, they are tethered to the cell’s nucleus. This promotes the assembly of a mitotic spindle in mitosis and is crucial for cellular functions in many tissues. How centrosomes are attached to the nuclear envelope, and how this is regulated in the cell cycle is not completely understood. I have found that the Polo kinase, a master regulator of cell division, promotes centrosome attachment to nuclei in Drosophila syncytial embryos. When Polo activity is reduced, a single centrosome detaches from nuclei while the other stays attached. The reason for this asymmetry is unknown. Polo consists in a kinase domain and a Polo-Box Domain that mediates protein interactions. I will use chemical inhibition of individual domains, coupled to imaging in embryos to dissect the function of Polo in centrosome attachment. Using an asymmetric centriolar marker, I will also examine the contributions of centriole age and of tension at centrosomes, to the detachment of single centrosomes. Several lines of evidence suggest that the proteins mediating centrosome attachment are different in the fly embryo from those already implicated in this process in other systems. The fly offers the possibility to elucidate this new pathway. I will use a proteomic, genetic and functional approach to dissect the molecular network connecting centrosomes to the nuclear envelope in embryos. I will also combine bioinformatics and biochemistry to identify the targets of the Polo kinase in this network. This work will use a muti-disciplinary, collaborative approach, and will advance our understanding of a basic problem of cell biology.

Plants, either directly or as fodder for livestock, feed the world and are increasingly important sources of fuel and fiber. Unfortunately, approximately half of the global crop yield each year is lost as a result of adverse environmental conditions. Drought is the abiotic stress with the greatest impact on crop yield worldwide and is expected to be exacerbated by global climate change. Plant physiology is affected by a number of other stressors including salinity, heavy metal ions, and reactive oxygen species. During times of stress, osmolytes and potassium ions accumulate to high concentrations inside of plants. These species are known to generally affect the folding of RNA, which often alters gene expression. Our hypothesis is that plants use certain RNA and DNA switches as a way to sense stressors and their indicators, which subsequently alters gene expression. Several plants have been fully sequenced, including the model plant species Arabidopsis thaliana, which allows us to apply a full complement of bioinformatic, biochemical, biophysical, and biological approaches. Our overall approach includes discovery and characterization of RNA/DNA switches in plants, including new and novel classes of stress-responsive switches, using a combination of biologically and chemically motivated searches driven by bioinformatics and experiments. We will then characterize the structures and physical and chemical properties of these RNA/DNA elements both in vitro and in planta. Techniques to be applied include enzymatic and chemical structure mapping of the nucleic acid, mutagenesis, thermodynamics, single molecule studies, small angle X-ray scattering (SAXS), fluorescence resonance energy transfer (FRET), and computation modeling. In addition, transgenic approaches will be used to assess the impact of RNA/DNA folding events on plant drought tolerance. Feasibility of this research plan is supported by initial bioinformatics searches and biochemical studies. These studies, if successful, will elucidate key mechanisms for how plants respond to stress at the cellular and molecular levels, and will likely help define mechanisms for gene regulation in response to stress in other eukaryotes.

Folding of epithelial sheets is one of the most ancient mechanisms leading to the formation of three-dimensional structures in developing tissues. In a highly simplified picture, epithelial morphogenesis can be separated into two steps. First, inductive signals establish two-dimensional patterns of gene expression across the epithelia. At the next step, these patterns are converted into spatial patterns of force generation and mechanical properties of cells, thus controlling the folding of a sheet into a target morphology. Previous studies of epithelial morphogenesis focused on single genes and small networks, but a systems-level model of morphogenesis in any given experimental context is yet to be developed. In developing such a picture, the key questions are related to the number and identities of involved genes, diversity and dynamics of their expression patterns, mechanisms of pattern formation, and connection between patterning and morphogenesis. We will investigate these questions in the context of the formation of the Drosophila eggshell, an established genetic model of epithelial morphogenesis.

This will be the first time that genetics, systems biology, cell biology, nonlinear dynamics, and continuum mechanics will be brought together to study patterning and morphogenesis in a system highly amenable to genetic manipulations. We will establish a two-dimensional atlas for the expression of dozens of genes involved in eggshell morphogenesis and formulate quantitative models for the formation of these patterns by signaling pathways. Based on live imaging and genetic experiments, we will formulate the hypotheses regarding the connection between patterns of gene expression and the mechanical properties of patterned epithelia. We will explore these hypotheses computationally, using a continuum mechanics approach, and experimentally, using live imaging. The result of this integrative approach will be the first experimentally validated systems-level model for two-dimensional epithelial patterning and resulting folding into a target three-dimensional morphology.

It is largely unknown how specific connections are established among the hundreds of cell types of the vertebrate CNS. Previous work has implicated cell adhesion molecules of the immunoglobulin, cadherin, and other families as mediators of this process. We have formed a collaborative network to explore a new gene family encoding leucine-rich repeat transmembrane (Lrrtm) cell-surface receptors. Individual members of the Lrrtm family are expressed in mutually exclusive patterns in the retina and tectum of zebrafish. The specific aims are 1) to establish a molecular interaction network for the Lrrtm family using biochemistry and biophysics (G. Wright), 2) to characterize the expression patterns of Lrrtm receptor/ligand pairs in the visual system of zebrafish using two-color fluorescent RNA in situ hybridzations and monoclonal antibodies (Wright & Baier), 3) to test if Lrrtms are necessary and/or sufficient for synaptic specificity by creating loss-of-function mutants, taking advantage of the Wellcome Trust Sanger Institute mutation resource, and by generating transgenic lines able to drive expression of Lrrtms and their genetically engineered variants in subsets of neurons (Baier & Wright), and finally 4) to identify the exact developmental step at which Lrrtms act (Trauner, Baier, & Wright). For this last experiment, we will genetically engineer an Lrrtm that can be masked with a photolabile, chemical compound and “unmasked” with ultraviolet light. We will remove the mask at defined time points before, during, and after synapse formation to determine if Lrrtms are required for neurite guidance, cell-cell recognition, synapse formation, and/or synapse consolidation. Together, these experiments will reveal the function of a novel candidate gene family in neuronal connectivity and will devise a broadly applicable paradigm for the study of other signalling systems in the developing nervous system and beyond.

Zinc is an essential cofactor for numerous proteins, where it serves both structural and catalytic roles. The proper functioning of living cells depends on the acquisition of this metal while, at the same time, an overabundance of zinc can be toxic. The uptake mechanisms and efflux pumps employed by cells to maintain the intracellular concentration of zinc have been previously characterized. On the other hand, little if anything is known regarding zinc trafficking within the cell. An extensive comparative-genomic analysis has led my future supervisor to propose that the ubiquitous family of COG0523 is involved in zinc trafficking. My post-doctoral research will aim in enhancing the understanding of zinc trafficking within the cell by surveying the role played by members of COG0523. First I will use the SEED database and comparative genomics to identify members of COG0523 which are involved in zinc trafficking and their specific targets. Utilizing genetic experiments, mass spectrometry, and Isothermal Titration Calorimetry, I will confirm the role of each members of COG0523 previously identified in zinc trafficking and associate them to their specific targets. I will use these data to build a complete phylogenetic tree of COG0523 where the function of each subfamily is determined. By using bioinformatics, I will identify homologs of COG0523 which are involved in zinc trafficking in eukaryotic cells. Finally this work will lead to a better understanding of the “metallome”. This project will involve integration of biochemical and molecular skills I gained during my Ph.D. along with bioinformatics, and genetic I will acquire with the help of Dr Valérie de Crécy-Lagard.

Respiratory syncytial virus (RSV), a member of the Paramyxovidae family, is one of the most important viral causes of lower respiratory tract disease in infants. Late steps in RSV life cycle include assembly on the cell membrane and virus release, however, the host machinery and the specific protein interactions during budding of RSV are unknown. The matrix (M) protein of the virus was previously shown to play a key role in RSV release, and it is currently the main candidate for recruiting the cellular machinery used by the virus for budding. My goal is to develop a career as an independent researcher, ultimately establishing my own lab focused on investigating the contribution of viral proteins and cellular factors in RSV budding. My main interest now is to learn the basic assays for studying RSV and to determine the feasibility of investigating RSV release. Prof. David Jans group at Monash University, Australia, is a world leader in molecular mechanisms underlying RSV pathogenesis. The main interest of the lab is the role of RSV M protein in virus assembly process, making this lab a good candidate as my host. During my stay in the Jans lab, my project will focus on the interaction between M and the microtubule (MT) network and its importance in RSV budding. Through working in the proposed host lab, I will acquire experience in specific techniques relevant to RSV research, get preliminary results and establish important collaborations with leading labs in RSV field. Mastering various methods will enable me to establish them in my own lab in the near future in order to investigate RSV budding in relevant physiological systems.

As a result of adaptation to different pathogenic environments, one can foresee that different primate species have probably developed different immunological responses against pathogenic infections. Nonetheless, to date, the extent to which natural selection has shaped the immune response to infection in humans and non-human primates remains largely unknown. To address this issue, I propose to identify immunological pathways that have been remodelled in specific primate lineages, by combining cutting-edge genomics with computational analyses and tools from molecular evolution. Specifically, I intend to evaluate in humans, chimpanzees, and rhesus macaques the time-course expression profile of monocytes after stimulation with three synthetic ligands that mimic infection with bacteria and viruses. This research project represents the first genome-wide exploration of differences in the regulation of the innate immune system in response to infection across closely-related primate species. From the analyses I expect to identify genes, and more broadly pathways, which activation after stimulus is shared between the three species, as well as those which activation is restricted to specific primate lineages. Further, by means of different functional assays, I expect to identify the “key genes” that orchestrate species-specific immune responses following infection. Ultimately, this project should help to better elucidate the biological and evolutionary parameters accounting for human-specific responses to infection and eventually to better elucidate why chimpanzees appear to be less susceptible to a number of infectious diseases such as AIDS, malaria, or hepatitis B.

Every day humans and animals are exposed to harmful micro-organisms, yet do not become ill. If you think about it, this is remarkable. We generally consider the absence of disease as not very exciting, and often research in infection biology is done with the aim to cure a disease or to prevent disease by development of specific vaccines. We propose to study the efficient systems that become involved to protect the host at the most vulnerable sites of the body. Mechanical barriers, innate immune receptors, and the adaptive immune system play a vital role in protecting the host from pathogenic invaders that attack the skin, lungs or gastro-intestinal tract. However, before the protection of the innate immune receptors kicks in, another “line of defence” seems to provide immediate protection. This protective system consists of peptides and proteins (“Innate Immune Effector Proteins”, IIEP), that are either soluble or loosely attached to mucus and other extracellular structures, and that exert direct effects (aggregation, killing) on a variety of pathogens ranging from viruses to fungi. Among these fascinating molecules, which appear to have largely physical mechanisms of action, are small antimicrobial peptides (AMPs) as well as large “pattern recognition” proteins such as the collectins. Our aim is to observe (for the first time) and subsequently analyze and understand the first encounters of pathogens with host proteins in the ARENA, a microfluidic model system that will mimic the interface between host and outside world. Our model system will mimic the air-water interface of mammalian lungs, and will contain bioluminescent bacteria that will encounter the lung collectins SP-A and SP-D plus the defensin hBD-1 and the cathelicidin LL-37. The ARENA chip device will permit carefully controlled on-chip dilutions of protein mixtures, bacteria (and host cells) by merging a stream carrying bacteria with a stream of IIEP to form a dynamic interface. Time lapse video microscopy is used to track aggregation and death of bacteria by fluorescent markers. Multiple tests per chip under multiple conditions can be run simultaneously.

Hypotheses: 1. The assembly state of collectins determines the propensity to quickly immobilize invading pathogens. 2. Concerted actions of collectins with AMPs kill bacteria effectively and protect the airways from biofilm formation.

A challenge in elucidating the signaling networks that control the organization of mammalian cells into tissues lies in the complexity of the whole organism, which limits analysis at molecular resolution. We propose to bridge this gap in scale between cell signaling networks and collective cell behaviour during tissue morphogenesis, by studying in experimental models of increasing complexity EphB2/ephrinB1-controlled cell positioning during vasculogenesis. Specifically, we will achieve this goal by integrated use of:

- novel phosphoproteomics strategies for unbiased elucidation of phosphotyrosine (PY)/

protein tyrosine phosphatase (PTP)-signaling networks

- novel strategies for analyzing PTPs, and high-resolution imaging of biosensor-tagged

Eph/ephrin signaling nodes during vasculogenesis.

Functional phosphoproteomics of metabolic (SILAC) labeled Eph+ or ephrin+ cells, and identification of ROS-oxidised PTP’s, will survey PTP-controlled PY signaling during Eph/ephrin-facilitated cell navigation, while distinguishing between distinct cell populations. Genetically encoded optical sensors for Eph/ephrin and PTP signaling activities will allow recording the correlation between bidirectional Eph/ephrin signaling activity within cells and the assembly of tissue during vasculogenesis. The underlying experimental models, with a common concept of confronting Eph-expressing with ephrin-expressing cells, will increase in complexity from simple 2-D co-culture and in-vitro capillary tube formation, to ‘confrontation’ cultures between tumour- and ES cell- spheroids to trigger in-vitro neoangiogenesis. Finally, modification of selected endogenous proteins in ES cells with fluorescent tags and biosensors by homologous recombination will allow assessment of emerging endogenous signalling clusters during ES cell differentiation.

Overall, these strategies will elucidate the molecular concepts underlying self-organized tissue assembly and will provide the experimental platform and knowledge base for our long-term goal - to assess at molecular resolution, but in the context of a whole animal, a signaling circuit controlling tissue patterning. Eph RTKs, as one of the principal signaling systems that control developmental cell navigation through feed-back controlled bi-directional signaling circuits, provide an ideal paradigm and experimental model system for this strategy.

In recent years molecular neuroscience has provided us with an arsenal of novel techniques and genetic tools to monitor brain function on the scale of neuronal populations. Here I propose to use one of these tools, a genetically expressed calcium sensor, to follow neuronal population representations of sounds in the auditory cortex of mice while they learn to associate a particular sound with a fear response. It is known that experience can change neuronal activity in this area, but the role of this plasticity is still elusive. Our goal is to characterize these changes on a network scale and test the hypothesis whether they lead to the establishment of special memory processes, such as the so-called auto-associative memories. Auto-associative memory is phenomenon observed in some theoretical neural networks, which has been long hypothesized to underlie the complex memorization processes occurring in animal and human brains. It has however received till now little experimental confirmation. My project will combine in vivo two-photon imaging of large number of neurons with elaborate analysis of the population response to challenge this hypothesis. I will also take advantage of the high stability of the genetically expressed sensor to follow consistently well defined neural populations over weeks or months. This will allow me to investigate the long term stability of sound representations in parallel to their learned modifications, thereby getting not only some insights on how memories are formed but also are maintained at cortical level.

DNA-templated events are initiated by binding of regulatory proteins to the genomic sequence. In mammals, this interaction occurs in the context of a highly condensed nucleo-protein complex called chromatin. Thus, cellular mechanisms are required to alter chromatin accessibility and facilitate transcriptional regulation essential for differentiation and development. In vitro studies on simple nucleosomal template have shown that ATP-dependent remodeling complexes are able to mobilize histones, thereby destabilizing chromatin structure. However, little is known about the mechanism of action on more complex higher-order chromatin, which is their substrate in vivo. I propose to develop a method to study the order of biochemical events initiated by remodeling complexes and other chromatin regulators during transcriptional reprogramming of mouse ES cells into differentiated neurons. Using small, membrane-permeable molecules to induce protein proximity, I will target remodeling enzymes to a genetic locus in the context of native chromatin and define the subsequent biochemical changes at high temporal resolution. This approach will enable me to address at which degree of chromatin compaction remodelers act and test the effects of histone modifications on their activity. Moreover, this assay will be used to screen all known chromatin regulators for their ability to reactivate pluripotency genes in differentiated cells and thus could provide a tool to identify drug targets for proliferation control.
The proposed project will allow me to explore a new direction of chromatin biology and will expose me to innovative technologies facilitating my own independent research in the future.

Stem cells are emerging as one of the fundamental bases of tissue biology. They allow most mature tissues to be replenished by new cells throughout life. If the regenerative capacity of mature cells is impaired, liver progenitor cells (LPCs), known as oval cells (OCs) in rodents, are activated and expand into the liver parenchyma, generating hepatocytes and cholangiocytes to restore liver mass. The mechanisms that control expansion of the LPC compartment remain elusive and studies delineating this function will be critical to understanding liver regeneration and tumorigenesis.
We have recently shown that deletion of the neurofibromatosis type 2 (Nf2) tumor suppressor gene in the mouse liver yields a dramatic expansion of OCs and the eventual development of both hepatocellular- and cholangio-carcinomas indicating that the Nf2-encoded protein, Merlin, is critical for liver stem/progenitor cell homeostasis. Merlin is a member of the Ezrin, Radixin, Moesin family of membrane associated proteins that are thought to organize membrane domains that mediate signals between the cell and its microenvironment. The mechanism by which Merlin suppresses tumor development is not clear, but evidence from our lab and others supports a key role for Merlin in associating with and regulating certain membrane receptors upon cell:cell contact.The goal of this proposal is to understand the biological and molecular mechanisms of how the liver stem cell niche is established and maintained. We will use both in vitro and in vivo models to determine how Merlin controls the activation, expansion and differentiation of LPCs and how disruption of these mechanisms contributes to liver tumor development.

Noncoding RNAs are being recognized as key molecules in important cellular processes, such as differentiation of adult stem cells. A new class of long noncoding RNA, transcribed from intergenic regions, lincRNA, will be studied systematically for their involvement in the differentiation of human primary keratinocytes. In a project involving substantial collaboration within the host institute, we aim at establishing biological importance and main characteristics of lincRNA by studying their expression, biogenesis and interaction partners. First, on a genome-wide level, we will analyze the abundance, relative expression level and sequence of individual lincRNA using DNA microarrays and massive parallel sequencing of the human keratinocyte transcriptome before and after terminal differentiation. Functionally important lincRNA will then be studied with respect to their genomic origin, promoter architecture, transcribing polymerase, potential processing and subcellular localization, suggesting how the expression of lincRNA is integrated in cellular regulation pathways. Finally, a biochemical purification procedure for the purification and identification of lincRNA-interacting molecules will be developed using the MS2-interaction technology. Components of lincRNA-protein complexes will be functionally tested for their involvement in differentiation using knockdown and overexpression experiments, giving insight into how lincRNA co-govern cellular decisions.

Since protein stability is imperative for a proper function, the physical properties of essential globular proteins dictate the fitness of an organism that carries them. Most globular proteins, however, possess thermodynamic margins within which their stability can be affected by mutations (either destabilizing or stabilizing) without causing an immediate effect on organismal fitness. Although these ‘neutral’ mutations can become deleterious or advantageous when combined with subsequent generational mutations, their impact on the rate and course of protein evolution remains unexplored. The goal of this project is to elucidate by a comprehensive approach, spanning the basic biophysical, molecular level, and organismal fitness level, how apparently neutral amino acid changes in essential proteins can influence the dynamics of protein evolution. To this end, the thermodynamic response of a group of E. coli’s essential proteins to a set of computationally predicted and experimentally verified neutral mutations will be measured against the fitness of competing populations in a real-time evolution experiment. Whole genomes of representative clones will be sequenced, and genomic changes responsible for buffering the stability effects of neutral mutations in evolving populations will be identified. Possible structural, functional and regulatory mechanisms that underlie these genetic changes will be tested computationally and experimentally at the single protein, functional pathway/network, and organism levels. Finally, a mathematical model that relates the thermodynamic response of proteins to neutral mutations with the rate of molecular evolution will be developed.