The present proposal focus on the role of the BTAF1 protein in transcription regulation. DNA promoter recognition and subsequent assembly of transcription complexes depend on TBP (TATA-binding protein) containing complexes. Among them B-TFIID is unique in containing only one TBP interacting protein, BTAF1 and in exhibiting ATPase activity which regulates BTAF1-TBP interaction. Little is known about the regulation of B-TFIID/BTAF1 activity. The proposal aims i) to identify new interactors of BTAF1 and to study effects of them on BTAF1 function both in transcription and in ATPase assays and ii) to visualize conformational changes within B-TFIID. To understand more precisely the chromatin remodelling function of BTAF1 in this process, emphasis will be made on the Myb partners of BTAF1, transcriptional activators involved in some cancers. The laboratory of Marc Timmers will act as my host and is located at the University Medical Centre of Utrecht in the Netherlands. This laboratory is recognized as expert concerning the regulation of transcription. With my background in structural biology and more specifically in NMR spectroscopy of membrane and soluble proteins acquired up to the end of my Ph.D., this project essentially based on cellular biology, biochemistry of DNA-protein interactions and electron microscopy represents a new promising challenge and will be useful for carrying on my research career.

The phylogenetic position of Xenoturbella has long remained obscure, and although they were recently reported as deuterostomes, further studies are essential to confirm their phylogenetic position. By revealing their life cycle and gathering the traits showing the evolutionary history of the animal, I aim to elucidate the phylogenetic position of the animal. If they are confirmed to be deuterostomes, new knowledge of deuterostome evolution will be obtained through this project. The early history of deuterostomes, to which we belong, remains unclear, but Xenoturbella may represent the simple body plan of the last common ancestor of the deuterostomes. Research on their morphology, gene expression patterns, and embryology should reveal important features of the early evolution of deuterostomes and the body plan of their common ancestor. The first important task will be examining the reproductive cycle of Xenoturbella. The breeding season will be determined followed by induction of spawning and/or embryo release. Developmental stages will be reared to adults. Examples of each stage will be fixed and analyzed by several morphological methods. In addition, expression patterns of developmental regulatory genes will be investigated to better understand the structure of Xenoturbella. Molecular studies will be a new experience to me. As molecular techniques become essential to most areas of biology, it is vital for me to acquire them early in my career. I will also learn ecological methods and establish a long-term culture system for Xenoturbella. Long-term culture of live specimens will enable observation of natural spawning, mating behavior, fission, and regeneration.

The entorhinal cortex (EC) acts as a gateway for information flow to and from the hippocampus and neocortex, and plays a crucial role in various memory-dependent tasks. It has been postulated that principal neurons in layer II (LII) of the EC mediate a comparison of hippocampal input and output signals, making the EC part of a novelty detection network. While hippocampal inputs reaching LII EC neurons are from the neocortex, hippocampal outputs reach them through intra-entorhinal projections from layer V. The objectives of this proposal are to test if principal neurons in LII act as novelty detectors and to study the ionic mechanisms underlying such function. I propose to achieve these goals by using a combination of experimental and theoretical techniques. First, I plan to use electrophysiological and imaging techniques to experimentally characterize dendritic information processing. Specifically, I propose to (i) study spike timing-dependent plasticity (STDP) of intra-entorhinal and cortical projections onto LII EC neurons (ii) assess the roles of dendritic ion channels in novelty detection by presenting LII EC neurons with paired inputs from these two pathways and using blockers for various ion channels and (iii) analyze the possibility that STDP of either pathway also causes plasticity of dendritic ion channels. Then, I will build morphologically realistic models of LII EC neurons taking into account their intrinsic properties and the outcomes of the above experiments. Finally, feeding these model cells with paired inputs with varied synaptic strengths and timing, I plan to test the relevance of these experimental findings to novelty detection at the systems level.

The cytoskeleton is the physical backbone that maintains the architecture and integrity of eurkaryotic cells and especially, of CNS neurons, featured by complex dendritic arborization and long axon that can reach one meter in Human. In early life, a destabilization of this highly ordered dynamic structure triggers neurodevelopmental disorders while in the adult CNS, cytoskeletal alterations promote neurodegenerative disorders. The interconnection of three networks- microfilaments (MFs), intermediate filaments (IFs) and microtubules (MTs)- builds the cytoskeleton. Enriched in CNS neurons, MTs serve as tracks for intracellular transport through molecular motors to deliver neurotrophic factors, organelles, and vesicles to appropriate cellular compartments. With MFs and IFs, MTs modulate the shape of the neuron in accordance to its developmental stages, maturation, energetic needs and functions. Thus, regulation of MTs dynamics and stability by Microtubule associated proteins (MAPs) impacts directly on the behavior of the neuron. Protein 600 (p600) encodes for a polypeptide of 5184 amino acids (GenBank Accession Number: NP_065816) with unknown functions in the CNS. The objective of the present research project is to define the roles of p600 in the CNS. Based on its homology with proteins linked to cytoskeleton-mediated transport, p600 may constitute a novel cytoskeleton-associated protein and, specifically, a novel MAP that couples the development of the CNS to its maturation and demise. Along with my interest for studying the cytoskeleton, this project will eventually permit me to build career and develop independent research.

The striatum is a brain region involved in motor coordination and cognitive function. Several degenerative disorders are associated with striatal dysfunction, including Parkinson’s and early stage Alzheimer’s diseases. Symptoms appear to be caused by an imbalance of synaptic transmission, in particular of the slow modulatory synaptic signals provided by dopamine (DA) and acetylcholine (ACh) to the main striatal cell type, the medium spiny neuron (MSN). The effect of DA and ACh on synaptic processing in the striatum is unclear. The proposed research addresses the controversial question of whether DA and ACh regulate postsynaptic channels in MSNs, specifically: a) do DA and ACh regulate postsynaptic glutamate receptors? b) do DA and ACh regulate the calcium influx evoked in spines by action potentials in MSNs? and c) what is the amplitude, timecourse and spatial extent of modulation following synaptically released neuromodulator? Post-synaptic channels directly in the spine will be examined using optical methods that allow rapid local application of neurotransmitter, thus avoiding confounds from DA and ACh modulation of glutamate release. Optical methods also permit the identification of modulatory synapses in cell pairs, which would otherwise be difficult because these synapses are not associated with the fast currents usually used to indicate the presence of a connection. This project would add experience with optical experiments to my skills in electrophysiology and mathematical modelling. In addition, I would like to extend my training in synaptic biophysics to more complex processes including synaptic integration and neuromodulation.

Growth and development of a plant requires precise control and co-ordination mechanisms of carbon assimilation, transport and storage. In spite of the fundamental importance of these processes –almost all of the organic carbon on earth is derived from them– crucial aspects remain poorly understood. Progress in understanding carbon metabolism, transport and signalling is severely hampered by a lack of knowledge of sugar concentrations inside living cells in intact plants. The proposed project aims to overcome these obstacles: During the first phase of the fellowship I will design and create a sucrose nanosensor and use it together with existing nanosensors to study glucose, maltose and sucrose levels in living cells under different environmental and genetic conditions. In the second phase I will apply these techniques in combination with functional genomics techniques to study mechanisms controlling carbon partitioning. This approach will lead to deeper understanding of how carbon availability controls growth and development of the whole plant. The proposed research differs from my previous experience in its interdisciplinary nature. I will be using latest techniques in photometrics, microscopy, computational design of proteins, computer modelling and functional genomics to answer key problems in biology. I will be supervised by an expert in transport physiology and nutrient sensing and collaborate with a photometrics specialist to ensure that I will acquire expertise in both fields. Most importantly the project is expected to generate information that will provide the basis of major new research programs, on which an independent research career can be based.

The development of multicellular organisms occurs in four dimensions, the three axes of space and a fourth axis of time. While a great deal is known about the fundamental mechanisms of spatial pattern formation, much less is known about temporal patterning during development. The objective of my project is to unravel the molecular mechanisms underlying developmental timing. To achieve the goal, I plan to reveal the molecular circuits functioning with the hunchback-like-1 (hbl-1) transcription factor in C. elegans. hbl-1 has unique features because it is a target of temporal microRNAs and its orthologues are essential for regulation of developmental timing in C. elegans and Drosophila. I propose to: (1) screen for mutations that suppress hbl-1 and clone the modifying genes, (2) perform a DNA microarray analysis to identify downstream targets of hbl-1, and (3) functionally characterize these genes at molecular level. Since hbl-1 is evolutionally conserved in structure and function, this project will provide a solid basis for understanding evolutionally conserved mechanisms of developmental timing. The research field (developmental timing, transcription factor, microRNA), the methodology (genetic modifier screening, genome-wide analysis of gene expression) and the model organism (C. elegans) are all different from my previous work (cell biology of actin cytoskeleton and insect molecular endocrinology). I have not experienced these techniques, and they are important for understanding complex developmental events in this post-genomic era. This experience will be very useful in the future when I establish my own research program and educate students as an independent investigator.

My past studies mainly focused upon chemical reactions catalyzed by proteins in vitro. In order to analyze the reaction mechanisms of some enzymes, I have been trained in X-ray crystallography, the synthesis/assays of chemical compounds, and protein expression/purification. I have succeeded in analyzing the reaction mechanisms of macrophomate synthase (natural Diels-Alderase) and ACC deaminase by mutagenesis from structural information combined with chemical methods. Expertise in enzyme chemistry will make important contributions to studies of cellular function. I am going to focus on the biochemical and structural studies of protein-protein interactions and multi-component assemblies of scaffold proteins in a signal-transduction pathway in the host laboratory. Scaffold proteins play a central role in integrating the input and output of signaling systems and are increasingly the focus of cancer research. I think that my expertise in chemistry, crystallography and NMR spectroscopy is a good starting point to explain protein-protein interactions. One of the most efficient methods to identify and evaluate protein-protein interactions is using NMR spectroscopy prior to X-ray analysis. In parallel with crystallographic studies I will carry out HSQC measurements NMR spectroscopy will also be a useful tool in judging whether the recombinant proteins are folded correctly and suitable for crystallization. Biological techniques that will be available in the host laboratory range from tissue culture (and cell based assay employing confocal microscopy) to in silico drug design.

Tiny RNAs have been recently recognized as a novel mechanism to regulate gene expression in animals and plants. MicroRNAs (miRNAs) are processed from larger precursors that can form fold-back structures. Mature miRNAs recognize partially complementing sequences in target mRNAs and guide them to cleavage or to translational arrest. Although it is clear now that miRNAs are a widespread tool to control a great number of genes, many aspects of this regulatory process remain to be elucidated. This project will address some of them using Arabidopsis thaliana as a model organism. In order to study the following points we will focus on four different miRNAs, each one harboring specific characteristics in order to search for general answers about miRNA mechanisms. (1) MiRNAs precursors are processed by Dicer-like ribonucleases, however the specific cues that determine the position of the miRNA along the larger fold-back structure are unknown. In vitro biochemical systems, together with genetic screens and manipulation of candidate sequences will be integrated in order to study miRNA biogenesis. (2) MiRNAs with similar sequences can have very different in vivo specificity. A combination of in vitro and in vivo studies will be used to determine the structural basis for these effects. (3) The concepts of phylogenetic footprinting and shadowing, so far used mostly for identifying transcription factor binding sites will be used to investigate constraints in miRNA sequence evolution. The information obtained will be used synergistically with the previous aims.

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.