The visualisation of the internal structure of biological organism is essential for the advance of life science. Greater demands are placed upon the field of microscopy to develop techniques that are able to image more complicated structures. One of the most promising techniques that provides, sensitive, chemically selective, high resolution 3-D images of unstained living cells is Coherent anti Stokes Raman scattering (CARS) microscopy. This technique is underused due to its complexity and expense. A main aim of this project is to investigate Photonic Badgap Fibre (PCF) CARS microscopy, a new technique that has the potential to be far simpler and cheaper than alternative methods. In parallel, a non-destructive, rapidly scanning, non-linear microscope will be built to provide, real-time, high resolution images. I also aim demonstrate the use of a cheap, compact femtosecond laser as the laser source for non-linear microscopy rather than current expensive laser systems, which again limits the use of non-linear microscopy to only the best funded laboratories. Finally I intend to incorporate optical tweezing into this work to provide imaging of living molecules within their natural medium (i.e. not placed between a microscope slide and a cover slip). My previous experience consisted of the development and characterisation of ultrashort pulsed lasers, the work I plan to carry out here is applying these lasers for a specific application. I will be working, for the first time, directly with experts in Bio-photonics and Biology. This will enable me to gain vast experience and will be invaluable for my future career development helping me to be propelled into life science.

Eukaryotic cells store their genetic information in the form of chromatin, a complex of DNA packed with structural and regulatory proteins. The functional repeating unit of chromatin is the nucleosome, an octamer of histone proteins wrapped around 150 base pairs of DNA. Chromatin presents a barrier to many proteins that require access to DNA. Nuclear reactions therefore depend on molecular mechanisms that allow regulatory factors to gain access to DNA in chromatin. ATP-dependent chromatin remodeling and covalent modification of histone amino termini, including acetylation, methylation and phosphorylation, play central roles in determining chromatin accessibility 3,4. Although, a wealth of data is available on the function and mechanism of action of single chromatin modifications, what is unclear is how the activities of ATP-dependent and covalent modifiers of chromatin are coordinated. I propose to use the fruit fly and the Drosophila ATP-dependent remodeling factor ISWI as a model system to understand how networks of chromatin modifications function. The genetic and biochemical identification of factors regulating chromatin dynamics, which I propose to study in this research project, will hopefully help me to shed light into the complex network of functional interactions existing between ATP-dependent and covalent modifications of chromatin. I expect that my backgrounds in biochemistry, molecular biology and genetics, developed during the course of my scientific career, will allow me to become an active contributing member of the chromatin field.

Directed evolution is promising technique to improve enzyme properties, but the successful evolution of a novel enzyme activity has not been reported. From an evolutionary point of view, bacterial carbon-halogen bond-cleaving enzymes that convert anthropogenic halogenated hydrocarbons are especially intriguing, as some of them may have only recently emerged in response to environmental pollution. Although the evolutionary origin of most dehalogenases remains elusive, structural characterization of a haloalcohol dehalogenase clearly showed that these enzymes have evolved from at least two different NAD-dependent short-chain dehydrogenases/reductases (SDRs). Whereas their precursors facilitate NAD-dependent REDOX reactions, the dehalogenases catalyze an one-step intramolecular substitution reaction. They have retained the SDR catalytic triad, but contain a variable halide-binding site that replaces the SDR cofactor-binding site. Several important NAD-binding residues are still present in the dehalogenases, suggesting that adaptation of a selected SDR enzyme to a dehalogenase could be within reach of directed evolution experiments. This conclusion inspired me to explore the possibilities of directed evolution to evolve a dehalogenation activity in the NAD-dependent 7alpha-hydroxysteriod dehydrogenase from Escherichia coli. Selection assays will be based on selective growth advantages of transformants on haloalcohol-containing minimal media in the absence of sugars. This research will complement my background in structural biology, and broaden my research expertise with novel experimental approaches to study structure-function relationships in an evolutionary context.

Glutamate is the major excitatory neurotransmitter in the brain and its release can result in rapid synaptic signals as well as long term effects. Glutamate transporters maintain a low extracellular concentration of glutamate by mediating uptake into neurons and glia. The peri-synaptic location of neuronal glutamate transporters also enables them to regulate the spillover of glutamate from the synapse to activate nearby receptors as well as glutamate spill-in from other synapses. Control of these different forms of synaptic signaling means that glutamate transporters may be important in influencing neuronal excitability and have profound effects on long-term changes associated with learning and memory. We aim to investigate how specializations in glutamate transporters allow them to regulate glutamate spillover we will examine the effects of up- and down-regulating expression of different neuronal transporters. We also wish to study the mechanisms behind dynamic insertion and retrieval of transporters at the synapse. Such mechanisms could have bearing on synaptic strength changes thought to contribute to learning and memory. Previously I have investigated how ion channels affect neuronal excitability. In this fellowship I wish to extend my knowledge of other receptors and transporters, expand my existing experience of brain-slice recording and learn molecular biological and imaging techniques. The fellowship will also afford the opportunity to experience a vibrant research environment and build contacts in the wider American research community for future collaborations. Such skills and experience will prove invaluable when I eventually set up my own research lab in the UK.

Auditory brainstem and cortical networks are tuned to natural stimuli (Rieke et al. 1995 Barbour and Wang 2003). The underlying neural mechanisms for this tuning are not known, although it is likely to be combination of intrinsic cell and network properties. Neurons in auditory cortex are dynamically complicated structures which show nonlinear responses to input stimulus. However, most descriptions of neuronal assemblies often greatly reduce single neurons complexity. This proposal outlines a methodology that aims to increase the understanding of how nonlinearities of single neuron dynamics influence neural network coding within the framework of auditory cortex coding of naturalistic stimuli. This will involve an interdisciplinary approach involving novel experimental techniques using in vitro hybrid virtual/biological cell networks. Theses experimental studies will be guided by modeling expansions of stochastic neural network models. My past research has focused on modeling studies of cellular and network neuronal functioning in the electric sense of weakly electric fish. While the auditory and electric sense share features, they are fundamentally quite distinct sensory systems, presumably with different basic rules of operation. In addition, my past training has been entirely computational in nature whereas I will perform physiological experiments during my tenure at NYU. This exposure to both new questions and techniques will offer an increased understanding of sensory neuroscience and provide me with the requisite training to continue to answer neuroscience questions with a greater range of methods than I currently possess.

An important question in cell biology is how complex cellular signaling networks are coordinated to allow the cell to effectively respond to its environment. My goal is to understand how signaling to the actin cytoskeleton is coordinated with signaling to other cellular functions. I have identified a protein complex that acts downstream of Rac in signaling to the actin cytoskeleton. Rac activates the complex by dissociating an inhibitory sub-complex (PIR121, Nap125 and Abi2) from the actin-assembly stimulating sub-complex (WAVE and HSPC300). The co-regulation by Rac of these five proteins may be used to coordinate signaling to the actin cytoskeleton and to other cellular functions. Specifically, PIR121 has been shown to interact with FMR1 (fragile–X mental retardation protein) on synaptosomal vesicles. FMR1 is involved in local translation control of an unknown number of target mRNAs in neurons. In addition, a genetic interaction has been reported between Rac and FMR1. I propose to study the mechanism of coordination between signaling to the actin cytoskeleton and to FMR1-dependent translation control. Moreover, preliminary data indicates that FMR1 activity is enhanced by PIR121 only after dissociation of the WAVE complex by Rac. The PIR121-enhanced activity of FMR1 will be used in an activity-based screen for FMR1 target mRNAs. I also propose to identify additional cellular activities that are potentially co-regulated by Rac by identifying proteins that interact with the inhibitory sub-complex. This work will build on my accomplishments in the actin cytoskeleton field and establish my group as an attractive partner for future collaborations in the cell biology community.

My project will clarify how information from different sensory modalities (vision, touch and muscle sense) are integrated in the brain to produce the sensation of our own body (the ‘body image’). My hypothesis is that brain areas in the posterior parietal cortex contain a ‘multimodal’ representation of the body that is modifiable by sensory experience. To test this hypothesis I will use functional magnetic resonance imaging (fMRI) and transcranial magnetic stimulation (TMS) to register and disturb brain activity when subjects perceive changes in their body imaged during relevant perceptual illusions. The fellowship will allow me to use new techniques and analysis methods to study human brain function using an interdisciplinary approach. I will learn how to use fMRI to study dynamic modulations in the brain signal (‘event-related fMRI’) and how to analyze the spread of neural information between brain centers (mathematical modeling of ‘effective connectivity’). I will also learn to disturb the function of brain areas using repetitive TMS. My project differs conceptually form my previous work partly because the new methods will allow me to test new types of hypotheses, for example how modification of the body image can be caused by dynamic interactions between brain areas. The host laboratory, Wellcome Department of Imaging Neuroscience, is one of the worlds leading centers for human brain research. In this outstanding scientific environment I will be able to broaden my knowledge in neuroscience, learn new cutting edge methods, and get new ideas from interacting with other researchers (8 Professors, and 32 post-docs working together in the same brain imaging laboratory).

Synapses are specialized cell adhesions that are the fundamental functional units of the nervous system. However, the extracellular signals that induce synapse formation in the central nervous system (CNS) are poorly understood. By using highly purified populations of neurons and glia, the Barres Lab has shown that the number of synapses on retinal ganglion cells (RGCs) in culture is enhanced 7-fold by soluble signals released by astrocytes. Recently, the Barres Lab has identified thrombospondin (TSP), a 450kD, astrocyte-secreted protein, as a protein that plays a crucial role in astrocyte-enhanced synaptogenesis. TSP is sufficient to induce structural synapses in vitro and is highly localized to synapses throughout the developing brain. The goal of this proposal is to understand the molecular and cellular mechanisms by which TSP promotes structural synapses in vitro and to determine whether TSP also regulates synaptogenesis in vivo. I will first investigate how TSP increases synapse number. Next I will test a group of potential TSP receptors in order to identify which one mediates TSP action in synaptogenesis. Then I will test the role of TSP and its receptor in vivo using available knockout mice. These studies have the potential to teach us more about how the synaptic structure of the brain is formed during development.

HIV-1 and HIV-2 are closely related viruses that share up to 60% of their genetic sequence and both can cause severe immunodeficiency disease. Nonetheless, most people with HIV-2 infection will die of other cause and the virus remains relatively confined to West Africa whereas 90% of HIV-1 infected people rapidly develop AIDS and the virus has spread throughout the world. Preliminary studies suggest that HIV-2 is not attenuated compared to HIV-1, but that the immune system is more efficient in controlling the replication of HIV-2. Whilst a considerate amount of knowledge about HIV-1 infection has accumulated over the last two decades, much less is known about HIV-2 infection and in particular the reason for its reduced pathogenicity. We hope that gaining a better appreciation of the immune control of HIV-2 infection will help in our understanding of the requirements for protective immunity in HIV infection. My previous research was based on primary human deficiencies with little inference of medecine, whereas this project is aimed at understanding of the cellular immune response to HIV-1 and HIV-2 viruses, working on infected patients with their consent, in close collaboration with clinicians and epidemiologists. We think that medical knowledge can improve our comprehension of immune responses in general and the development of efficient cytotoxic responses in particular.

B-cells are the antibody-producing cells of the immune system. Their development is a complex, stepwise process. While random B-cell receptor gene re-arrangements ensure a diversified antibody repertoire, B-cells recognising self-antigens must be deleted during their development to prevent autoimmunity. This process is initiated by the interaction of the B-cell receptor (BCR) on immature B-cells with stromal cells in the bone marrow, where the recognition of self-antigen transmits a signal that induces a change of specificity (receptor editing) or the death of the B-cell. We aim to investigate how the BCR works in concert with adhesion molecules and other co-receptors during this process. Unique real-time visual systems in combination with the use of cell lines and animal models will allow to relate the dynamic re-organisation of the BCR/adhesion molecules during B-cell/stromal cell interactions to cellular responses on a single-cell level. These studies will lead to a more detailed understanding of the processes involved in B-cell development and in the prevention of autoimmunity. While I will benefit from my expertise in B-cell development, I will shift my focus to the mechanisms of B-cell mediated autoimmunity. Further, I will learn a variety of new techniques. As I am fascinated in the processes involved in conveying an extracellular signal or environmental clue into a cellular response, this work will broaden my insight and expertise in a field where I hope to establish myself as an independent research scientist in the future.

I am interested in genetic factors that mediate cellular growth and body size control. The cyclin-dependent protein kinase complex CycD/Cdk4 regulates growth through a previously unknown mechanism. In a genetic screen, I identified two proteins: mRpL7/L12 and Hif-1 prolyl hydroxylase (Hph), which function, in a common pathway, downstream CycD/Cdk4. mRpL7/L12 is a mitochondrial ribosomal protein. Hph regulates the cellular response to low oxygen (hypoxia). I showed that Hph, through hydroxylation of yet unidentified proteins, has a novel function in growth control. The screen was successful, but not designed to saturate the genome. Thus I propose to continue the screen, on a larger basis, to identify more components of this pathway. In particular, I will be focusing on new, growth-specific, hydroxylation targets of Hph. CycD/Cdk4 stimulate the metabolism, however, our understanding how the metabolism and cellular growth are linked is still limited. In a parallel approach, I propose biochemical assays and two-dimensional gels to better understand the effect of CycD/Cdk4 and Hph on the metabolism. A combination of these assays will allow a rapid characterization of the mutants isolated in the genetic screen, and will give a global view of CycD/Cdk4’s function on the metabolism. The University of Zurich offered me an ‘Oberassistenz’ position, which allows me to perform independent research. This position does not include salary and funding. Next year, I will be applying for a ‘Forderungsprofessur’ (assistant prof.) of the Swiss National Science Foundation. Therefore the CDA would allow me to carefully prepare such a position, and to gain expertise in guiding lab personal.

The tumour suppressor protein p53 induces cell cycle arrest or apoptosis of potentially malignant cells in response to oncogenic stress, preventing the malignant transformation. p53 is mutated in around 50% of human cancers. The mechanism and regulation of p53-induced apoptosis are of great importance, but are still poorly understood. Recently, the protein ASPP2 was shown to specifically stimulate the apoptotic response of p53. The research aim is to elucidate the molecular mechanism and regulation of the p53 – ASPP2 interaction. We will use an interdisciplinary approach combining protein biochemistry and biophysical techniques such as NMR, fluorimetry and calorimetry. First, we will study the kinetics and thermodynamics of the p53 – ASPP2 interaction and use NMR to study the dynamics of these proteins upon binding each other. To elucidate its mechanism of action, we will study the effect of ASPP2 on the DNA-binding of p53 core domain, and how it stimulates the transcriptional activity of p53 despite binding it in its DNA binding site. We will then study the thermodynamics, kinetics, and NMR dynamics of ASPP2 interactions with the apoptosis-related proteins Bcl-2 and NF-kB and explore the possible role of these proteins in ASPP2 regulation at the molecular level. Being a young scientist at the beginning of my independent career, the project is of vital importance for my professional growth. My research interests are the molecular mechanism and dynamics of interactions between macromolecules, and how to restore such interactions when impaired. The project will be the first application of my experimental system for a biological pathway that is crucial for the cell.