To what extent and why do the effects of mutations depend on the genetic background? How common are genetic interactions? Do deleterious mutations act synergistically? Answers to these questions are relevant not only to functional genomics, but also to problems such as the evolution of sex. Owing to the huge number of potential gene combinations, progress in answering these questions is, however, limited by the lack of efficient genome-scale experimental mapping of genetic interactions. To overcome this difficulty, I propose a novel combination of in silico and in vivo studies to screen for synthetic lethal relationships in the yeast metabolic network. My idea is to find candidate interactions by applying flux balance analysis to the genome-scale metabolic model of S. cerevisiae. Then, these predicted interactions will then be verified experimentally. With a large set of interactions in hand I plan to investigate the mechanisms of compensation between enzymes. In particular I shall ask whether the presence of isoenzymes explains the majority of these, or whether compensation mainly arises from interaction between unrelated genes. Additionally I shall ask whether compensating interactions depend on environmental conditions. If so, are they more common under nutrient rich or nutrient poor conditions? The project will utilize my previous experience in metabolic modelling and statistical analysis and allow me to acquire training in experimental genomics in the laboratory of Stephen G. Oliver. The fellowship would enable me to efficiently combine computational and experimental approaches and to perform hypothesis driven functional and evolutionary genomic research.

My aim is to understand mitotic spindle positioning in the one-cell embryo of C. elegans. This is a good system for genetic, molecular biology and imaging experiments. The first division results in two cells of different sizes and is accompanied by two types of spindle movement: posterior displacement and transverse oscillations. Both movements are thought to be driven by motor proteins on the cortex pulling on microtubules that grow out from the spindle. Because oscillations result from particular types of interactions between system components, we believe that they may provide very good clues to the molecular mechanism of spindle positioning. The novel experimental strategy is to develop an image processing system that can track the movement of the mitotic spindle with such high accuracy that the actions of individual molecules can be detected. For example, motor proteins binding to or unbinding from the microtubules are expected to cause discrete changes in the speed of the spindle the resulting fluctuations thus give information about the number of motors and their kinetics. The molecular pathways can be identified using RNAi and laser-cutting can give insight into the mechanics (e.g. whether microtubules are under tension or compression). High precision measurements of the amplitude and frequency of the oscillations, together with their fluctuations, will be used to test and refine non-linear dynamic models of the motion. All the biological part will be new for me as my previous training has been in statistical physics and computer science. Working with biologists in the institute will allow me to make the transition to biophysics which is my professional goal.

G protein-coupled receptor kinases (GRK) provide the rate limiting step for terminating incoming signals at G protein-coupled receptors (GPCR) to protect the cell from overstimulation. GRK2, the most commonly expressed GRK, has been shown to regulate heart function during development as GRK2 knockout (KO) mice die during embryonic development. Since this has been analyzed only preliminary, I want to uncover the molecular mechanism to provide a biochemical explanation for the action of GRK2 ensuring normal cardiac development. The aims of the project are: 1) Generation of a GRK2 knockdown in zebrafish and its histological and biochemical characterization. 2) Determination of the GRK2 structures relevant for the assessed phenotype. 3) Investigation of affected signaling cascades. 4) Transfer of the zebrafish results back to mice. The novelty of the project is based on a combined in vivo approach to explain the biochemical function of a protein of highest relevance in man by using a second model organism instead of extrapolating in vivo relevance from in vitro or cell culture studies. To accomplish this, I will be trained in manipulating and analyzing zebrafish and in techniques commonly used in signal transduction such as site-directed cloning and biochemical assays, which will complement my previous research experience in mice and chicken embryos. Therefore, the HFSP Fellowship will serve my professional growth by enabling me to gain knowledge in the investigation of signal transduction and the handling of a third model organism, giving me the possibility to choose the technique and the organism whichever is best for the question I want to address in my future research.

A fundamental question in sensory neuroscience is how animals process self-generated sensory information in comparison to external stimuli. It has been suggested neural signals feedforward from motor to sensory networks and modulate the response to self-generated stimuli. In this way, animals can prevent self-generated information from desensitising their own sensory pathway and/or from being confused for external stimuli. This concept has been hard to verify, as identification of a modulatory synaptic input requires intracellular recording, which is technically difficult in behaving animals. Almost all studies of neuronal activity in behaving vertebrates have been limited to extracellular recordings. In the last two years, however, improvements in the mechanical stability of the whole-cell patch-clamp configuration has allowed routine intracellular recordings from awake rodents. In this fellowship I will apply this technique to the somatosensory and motor cortex of awake, behaving mice. These experiments will provide fundamental information on the membrane potential of identified groups of neurons in the awake mammalian brain. In combination with behavioural analysis, I will go on to examine the role of feedforward signals in sensory perception. I now wish to transfer my skills from an invertebrate to a vertebrate preparation to gain a broader understanding of the way sensory and motor systems interact have the opportunity to work on a more complex nervous system and learn new neuronal recording techniques. This fellowship would provide me with a perfect opportunity to be part of an exciting field of neuroscience.

Deamination of cytidine in DNA has been found to be involved in both adaptive and innate immune reactions. The activation-induced deaminase (AID) is essential for antibody gene diversification by somatic hypermutation and gene conversion as well as for antibody isotype class switch recombination. The cytidine deaminase APOBEC3G has been shown to inhibit retroviral infection, mutating dC to dU in viral first strand cDNA. These different processes require specific recruitment of the deaminases to their target sequences. The objective of this project is to explore the mechanisms underlying the recruitment of the deaminases to their physiological targets and the structural basis for the observed local sequence preferences of the deamination. The specific aims of this project are: The generation of various chimerical AID/APOBEC3 molecules and their functional analysis A) in vitro and in bacterial mutation assays to explore the molecular basis of the local sequence specificity of the deamination by AID and APOBEC3G. B) in chicken DT40 B cells to identify regions of the AID molecule involved in targeting of deamination to immunoglobulin variable region genes C) in retroviral infection assays to ascertain, using mouse APOBEC3/human APOBEC3G chimeras, the molecular basis by which both mAPOBEC3 and hAPOBEC3G can be incorporated into and act on HIV virions whereas only hAPOBEC3G can act on mouse leukaemia virus (MLV). This functional mapping of the deaminase molecule will be complemented by a screen for binding partners for the deaminases. During this fellowship, I will gain experience in molecular genetics and biochemistry that will complement my expertise in in vivo immunology.

For a few years, chemical genetics have turned out to be an elegant way to suppress the activity of a protein without the irreversibility caused by RNA mutations. The use of small molecules in exploring biological processes has yielded in 2001 to an original strategy based on the natural way for maintaining cellular function, the post-translational regulation of cellular proteins by ubiquitin-dependant proteolysis. PROTACS (PROteolysis TArgeting Chimera MoleculeS) are dimeric molecules establishing an artificial link between an E3 ligase and a protein. Then, polyubiquitination is induced by the E3 ligase and leads to the degradation of the protein. This PROTAC method is a reversible chemical analog of genetic knockout, providing a selective removal of one targeted protein in wild type cells. This original and elegant way to cancel the cellular function of the protein has broad applicability in cancer treatments or as molecular probes for protein function. In this project, the design of a new PROTAC is proposed. The linker will be based on nutlin molecules, new MDM2-p53 interaction inhibitors which bind strongly to MDM2. For this purpose, an efficient and versatile synthesis providing various nutlin analogs will be developed and the best candidates for binding to MDM2 will be incorporated in a PROTAC. Moreover, after validation of this new PROTAC using a preexisting system degrading in vivo androgen receptors, biologically active molecules will be associated to nutlin in different PROTACS to investigate new cellular functions.

Primates, including humans, have the ability to formulate complex plans to achieve far-removed goals. The brain region most closely associated with this is the prefrontal cortex (PFC). The PFC is the most anterior portion of the frontal lobes and it controls the highest-level cognitive processes. It is densely interconnected with the aminergic nuclei of the brainstem, including the dopaminergic and serotonergic systems. Dopamine (DA) and serotonin (5-HT) have an important role in PFC cognitive functions. However, the nature of their actions in the PFC remains controversial. Moreover, the PFC is enriched in serotonin 5-HT1A and 5-HT2A and dopamine D1 receptors. Growing evidence suggests that 5-HT1A antagonists and 5-HT2A and D1 agonists prevent memory impairment and facilitate learning. In contrast, 5-HT1A agonists and 5-HT2A and D1 antagonists have opposite effects. The main goal of the project is to study the role of 5-HT1A, 5-HT2A and D1 receptors in cognitive processes in the monkey. For this we will use a conditional visuomotor learning task that evaluates the ability of the monkey to form and rearrange arbitrary cue-response associations rapidly and examine the neural substrate of the association by performing multiple recordings of PFC neurons. We will analyze the effect of the pharmacological activation of the receptors known to alter cognitive function. The fellowship will allow me to move from a cellular analysis of the PFC in rodents to the study of PFC cognitive processing in awake monkeys and will get me in touch with the most advanced physiological and behavioral methodology and the latest discoverings about the functioning of primate cognitive processes.

Multiple sclerosis (MS) is an inflammatory disorder that causes neurological dysfunction. Several studies in MS and experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, suggest that MS results from the complex interaction of genetic and environmental factors. Studying the genetic bases of MS is therefore necessary to understand its pathology and identify new therapies, but these studies are not easily performed in mammals. Genetic models like worms and flies were useful in identifying the genes involved in innate immunity. However, these animals lack an adaptive immune system and therefore they are not very useful for the study of MS. The zebrafish (Danio rerio), however, is a genetic model that harbors a complete immune system similar to the one in mammals. The aim of this project is to establish a model of EAE in zebrafish (zEAE) to facilitate the identification of genes linked to MS and new therapeutic approaches for its treatment. During my PhD studies I analyzed the immuno-regulation of autoimmune diabetes and arthritis using DNA vaccines, cellular immunology techniques, antigen microarrays and bioinformatic tools. This project will let me acquire an array of techniques used in zebrafish research, enjoying the support of Prof. Weiner’s expertise in MS research and our collaborators’ knowledge of zebrafish biology. The successful completion of this project will allow me to establish an independent research career centered on the study of the complex interactions existing between the immune system, the genome and the environment that result in the development of autoimmune disease.

Ubiquitin E3 ligases are essential enzymes in the cell. They mediate poly-ubiquitination of substrate proteins which targets them for proteasomal degradation. Because of their implication in the regulation of many cellular functions, such as signal transduction or cell cycle progression, their study is of high general interest. SCF-type E3 ligases form an important family of multi-subunit E3s that are organized around a scaffolding cullin protein. Cullins can be modified by the ubiquitin-like protein Nedd8, a process termed neddylation. Interestingly, cycles of neddylation/deneddylation appear to be required for the function of cullin-based E3 ligase however the functional significance of these cycles is unknown. To better understand the function and regulation of cullin neddylation, I will take an interdisciplinary approach using S. cerevisiae as a model system. First, I will perform two independent genetic screens to identify, and subsequently characterize, factors regulating neddylation of Cdc53, a typical yeast cullin. Second, I will biochemically test models for the function of Cdc53 neddylation and characterize its possible consequences on the E3 ligase composition. This will be complemented by live cell imaging experiments to assess the functional dynamics of the E3 ligase and its potential modifications upon Cdc53 neddylation. This project will give me the chance to address fundamental questions about the function of cullin-based E3 ligases. Its interdisciplinarity will broaden my scientific curriculum and provide me with an essential training for my scientific career.

Synaptic vesicles fuse with the plasma membrane to release neurotransmitter. The vesicles then reform some models of reformation predict that direct pinch-off from the plasma membrane generates new synaptic vesicles. An important fraction of the vesicles may use a different reformation route, though: after pinch-off from the plasma membrane the endocytic vesicles fuse with another compartment (early endosome, EE), from which new synaptic vesicles are subsequently generated. I will investigate the processes of EE fusion at the synapse. I will purify EEs from presynaptic terminals and will analyze their ability to fuse together (homotypic fusion) or with EEs from other cells, by use of an in vitro fluorescence assay. I will determine the proteins involved in the fusion event, by competitively inhibiting fusion through addition of soluble purified proteins and/or Fab fragments from antibodies specific for different fusion proteins. I will also introduce these inhibitors into the presynaptic nerve terminal of the rat calyx of Held synapse, to investigate (through electrophysiological tools) the effects of blocking EE fusion in vivo. My previous research relied mainly on fluorescence and electron microscopy of the neuromuscular junction. With the current proposal, I will use biochemical techniques such as synaptosome formation, subcellular fractionation and protein expression and purification. Also, I will have the chance to become skilled at electrophysiological recording. In addition to improving on my technical skills, I will also become familiar with the field of vesicle fusion, which would complement well my current knowledge of synaptic morphology and physiology.

My first goal is to establish the proteinaceous and lipidic “minimal requirements” for membrane fission in-vitro. As fission is a transient event, this study requires an assay that allows video-microscopy. I propose to use Giant Unilammelar Vesicles of various lipid compositions as membrane source, and mix them with proteins involved in fission. This way, I will be able to determine which proteins and lipid composition are absolutely required for fission in vitro. I will also study the sequence of protein and lipid recruitments driving fission. I will test the idea that proteins induce lipid phase separation to fission membranes. Many of the proteins involved in fission can tubulate membranes. My second goal is to measure the forces applied by proteins on the membrane during such processes. Hopefully, in conjunction with a mathematical model, this will help to better understand the mechanism by which the various fission proteins deform the membrane. Once a more comprehensive scheme of how these proteins interact with the membrane is established, I will be able to compare the different models for membrane fission proposed in the literature. My third goal is to follow lipid and protein recruitment during fission in living cells, using time-lapse imaging of clathrin endocytosis. This will enable us to compare the sequence of events driving fission in-vitro to the one driving fission in cells. I will also attempt to determine whether lipid phase separation does occur during fission in live cells. More generally, the in-vitro assay I will establish will be useful for the understanding of the interactions between membranes and the proteins that transiently bind to them.

An important issue in fear memory research is how to reduce fear that has been associated with neutral stimuli. Extinction is demonstrated as a reduction in fear after stimuli that were once associated with an aversive event are no longer coupled with that aversive event. This approach to reducing fear forms the basis of most behavioural therapies in treating anxiety disorders. Approximately 13.1 to 18.7% of the population suffers from anxiety disorders. Advances in the mechanistic understanding underlying extinction holds great promise in directly benefiting patients through improved behavioural and pharmacological treatments. Extinction involves new learning and memory formation rather than erasure of the original memory. The precise molecular mechanisms and brain structures involved are still unclear. I propose to utilize a combination of electrophysiological and molecular biology techniques to test the hypothesis that consolidation of extinction requires the same molecular processes as the consolidation of the original acquisition. Thus far, I have acquired a considerable amount of experience in invertebrate memory research. By studying fear conditioning in a mammalian preparation, questions regarding how structures, such as the amygdala, are involved in the extinction of fear memories can be answered. Dr. Pape's group is the only laboratory that currently combines electrophysiological, behavioural and gene expression analyses of fear memory formation and extinction in mice. By combining electrophysiological and molecular biological techniques while simultaneously examining behaviour I will be able to investigate my research question utilizing multiple approaches.

To determine how GABAergic cells develop in response to sensory experience and influence a vertebrate neuronal circuit during activity I will resort to the retinotectal projection of the Xenopus tadpole. This system allows for direct collection of in vivo images of neuronal structures as animals are stimulated by light (sensory experience), the targeted expression of multiple genes of interest into particular components of the system (bulk/single cell transfections, transgenesis) and the simultaneous analysis of GABAergic cells and their presynaptic (retinal afferents) and postsynaptic (projection tectal cells) counterparts. I thus plan to determine if, like the glutamatergic neurons of the retinotectal projection, GABAergic cells develop in an activity-dependent fashion, considering two plasticity parameters: a) axonal and dendritic arbor structures of GABAergic neurons and b) the formation and maintenance of synaptic inputs and outputs of these cells. I will also determine if synaptic maturation stabilizes the structure of developing GABAergic neurons. Then I will analyze how the formation of synaptic contacts between glutamatergic retinal inputs and GABAergic cells and between these and projection tectal cells affects the structure of the retinotectal circuit. This fellowship will bring my research forward as I move from the in vitro study of molecular mechanisms in individual neurons to the in vivo investigation of communication between different neuronal types and the consequent formation of functional circuits. Professionally, I will enrich my intelectual and methodological porfolio while directly pursuing my research interests on circuitry formation and function

Lysophosphatidic acid (LPA) is an extracellular lipid mediator that signals through G-protein coupled receptors to mediate various cellular responses,including those involved in neuronal development,neurite retraction, and the inhibition of neurite outgrowth. Inhibition or attenuation of LPA signaling in the central nervous system is mediated via the recently identified family of integral membrane proteins known as plasticity-related genes (PRGs). Previously,we have shown that PRG-1 attenuates LPA-induced neurite retraction through its lipid ecto-phosphatase activity,thereby degrading LPA,whereas PRG-3 induces spontaneous neurite outgrowth and promotes filopodia extensions with no detectable ecto-enzymatic activity. Preliminary experiments indicate that PRG-3 induces rapid neurite outgrowth after initial LPA-induced growth cone collapse.Here,we propose a series of novel experiments to characterize the mechanisms of PRG-3 in neurite outgrowth, which are conceptually and technically novel and a challenge from my previous line of research.The focus is on siRNA based knockdown experiments and the characterization of protein interactions with the C-terminal tail of PRG-3. In parallel, we propose to generate transgenic mice overexpressing GFP-tagged PRG-3 under the control of the neuron-specific promoter Thy-1, as this will enable us to analyse neurite outgrowth in vivo and in brain sections in vitro. These transgenic mice will allow us to establish the role of PRG-3 in axon growth during development and regenerative sprouting. We expect that our investigations will yield new insights into LPA signaling in the brain, which will have implications for neurodegenerative diseases.

Initiation of translation is a complex process that involves fine coordination between the ribosome and eukaryotic initiation factors (eIFs). Abnormal levels or mutant forms of eIFs are associated with deregulation of translation and cancer. Structural information is needed to reveal the molecular mechanisms used by these factors to produce an 80S ribosome ready for protein synthesis. I propose to determine the structures of several complexes in the pathway of translation initiation by cryo-electron microscopy. Simpler complexes, such the 40S ribosomal subunit bound to eIF1 and 1A will be pursued first, followed by larger assemblies. The ultimate goal is a 3D model of a 43S complex including eIF1, 1A, 2, 3, 5 and initiator tRNA, possibly with mRNA. To help interpret cryo-EM maps I will concurrently undertake crystallographic studies of the two factors without high-resolution models, eIF3 and 5. Structures of initiation complexes are required to reveal the overall architecture of the translation initiation machinery and the conformational changes that occur during this dynamic process. They will address how initiator tRNA is presented to the 40S ribosomal subunit, and perhaps how the initiator codon of mRNA is scanned for and recognized. This fellowship would enrich my research experience greatly. Going from a large American lab to a more intimate group, from X-ray crystallography to cryo-EM, and switching research fields will broaden my perspective and teach me new tools. This training will prepare me to answer both large-scale and fine structural questions pertaining to the mechanisms of function of macromolecular machines as an independent investigator in an academic lab.

In eukaryotes, co-transcriptional packaging of RNA polymerase II transcripts into messenger ribonucleoprotein particles (mRNPs) is a prerequisite for their proper nuclear export. Formation of mRNPs is monitored by surveillance systems, some of which act already at (or near) transcription sites. One such quality control system ensures that aberrant mRNPs are retained, and degraded, at their sites of transcription, hereby preventing nuclear export and possible contamination of the cell by unwanted products. Retention of aberrant transcripts has been observed in a growing number of yeast mutants and also in mammalian cells. The proposed project aims to elucidate in detail the molecular mechanism underlying transcript retention using the yeast, Saccharomyces cerevisiae, as model system. Two main strategies will be pursued: (i) genetic screens to identify mutants that are deficient in transcript retention, and (ii) biochemical approaches that aim to characterize retained mRNPs and their chromatin environment. The project will allow me to change my research focus and to acquire new skills in yeast genetics, biochemistry and general microscopy. I also intend to use a new application of the ChEC technique that I established during my PhD to isolate specific chromatin regions. This will allow a detailed molecular analysis of chromatin that contains retained mRNPs, and should furthermore be of general interest to chromatin researchers.

Covalent modifications of histones and DNA, as well as histone variants affect chromatin structure and activity. One of the major challenges is to understand how histone modifications and how alterations in the histone composition are translated into changes in gene expression and chromatin structure. The proposal is designed to address: A) Little is known how histone H1 controls the function of chromatin since the field is currently focused on core histones. Elucidating the role of H1 modifications and variants will elevate our understanding of the "epigenetic code" to the next higher level of chromatin organisation. Furthermore we will use DNA accessibility as a marker for chromatin structure and establish a new, high-resolution/genome-wide DNA accessibility assay. We will combine these results with position data for histone modifications, H1 variants or HMGs as a first step to analyse their effect on chromatin structure. B) DNA methylation is one of the longest studied epigenetic modifications. However a DNA demethylase has not yet been identified. We will use embryonic germ cell lines to purify a DNA demethylase with the help of a new assay and we will aim to link DNA demethylation with histone modifications. C) To identify new, yet uncharacterised types of modifications we will purify histones under various mild conditions and identify modifications by MassSpec. This will be the basis for investigating the biological function of these modifications. The proposed projects will expand the scope of my ongoing research and will only be possible with additional funding, which will allow me to establish cutting edge technology and new interdisciplinary collaborations.

The exosome is an evolutionarily conserved complex of 3’-5’-exoribonucleases that plays a key role in RNA metabolism. It functions in RNA degradation (e.g. defective nuclear RNA processing intermediates and cytoplasmic mRNAs) as well as in the accurate 3’-end processing of non-coding RNAs (snRNAs, snoRNAs, rRNAs). The exosome must therefore distinguish between RNAs destined for destruction and those that should be matured to functional RNAs. The mechanisms, which activate the exosome to degrade specific targets while inhibiting the non-specific activity of the component exonucleases, remain poorly understood. To address this question, we propose to study the enzymatic activity of the components of the exosome and its structural organisation in the budding yeast Saccharomyces cerevisiae. We will test how many individual exosome components show exonuclease activity in vitro and whether free proteins in yeast are chaperoned and inactivated by heat shock proteins. We will genetically deplete or mutate individual exosome proteins and then analyse the composition and function of the resulting complexes. In a second set of experiments we will focus on the nuclear exosome component Rrp6p, which appears to have additional functions connected with mRNA quality control. To investigate the link between the exosome and nuclear mRNA surveillance, we will isolate and functionally characterise non-exosomal, Rrp6p-containing complexes. In summary, this project will allow me to combine the advantages of the model organism yeast with my earlier experience in higher eukaryotes thus providing me with a variety of powerful tools to study other aspects of gene expression in my future research.

In RNA interference (RNAi), small RNAs direct a protein-RNA complex to degrade complementary target RNAs. Endogenous siRNAs function via the RNAi pathway to regulate development, titrate gene expression, and direct the formation of heterochromatin [2], and exogenous siRNAs are widely used to inhibit the expression of specific genes in a wide variety of organisms and cultured cells [1]. Yet, our molecular understanding of the RNAi pathway remains rudimentary. In particular, how cells build the RNA-induced silencing complex (RISC), which acts as the effector enzyme complex of RNAi is poorly understood. I propose to use biochemical and biophysical methods to explore the molecular events that lead to the production of the active RISC. Biochemical analysis of RISC assembly will be conducted using an established cell-free system that recapitulates RNAi in vitro. However, to answer outstanding questions regarding the composition and homogeneity of the RISC will require novel methods capable of resolving individual molecules of RISC. I propose to develop single-molecule tools to study RISC composition and function. Further development of this technique may even allow single-complex analysis of the cycle of molecular events that underly RISC production and catalytic function. [1] Y Dorsett and T Tuschl. Nat Rev Drug Discov, 3(4):318-329, 2004 [2] G J Hannon. Nature, 418(6894):244-251, 2002