Axonal pruning and synapse elimination reorganize neural circuits in many parts of the developing mammalian nervous system. Much of what is known on this process comes from the study of the neuromuscular junction where axons compete for the occupation of the same synaptic sites on muscle fibers. Muscle fibers, however, are end-organs, so the result of this competition cannot have any downstream effects. The objective of this project is to understand the temporal and spatial relationships between circuit rearrangements that occur during development at different levels of the synaptic relay between brain and salivary end-organ. In order to do accomplish these aims I will use advanced physiological and imaging techniques with de-novo transgenic mice expressing different mixtures of multiple fluorescent proteins in each neuron ("Brainbow" mice) in order to follow these developmental reorganizations over time. This research will provide novel information on the postnatal development of a two level pathway allowing me to test, for the first time, the particular hypothesis that the purpose of synapse elimination is to allow a circuit-wide rearrangement in a neural pathway such that each cell upstream in a polysynaptic pathway drives the output of a limited synchronously active territory in the downstream synaptic target.

The dentate gyrus of the hippocampus is one of the brain regions in which neurons are generated during adulthood. Increasing evidence supports the view that adult newborn granule neurons play an important role in hippocampus-dependent learning and memory. In recent years, it has been reported that immature new neurons approximately one month after their birth may make particularly significant contributions to memory formation. However, at what exact stage the young new neurons become important for memory formation, and the memory processes they mediate, have not been yet determined. Therefore it is necessary to identify the specific functions of young new neurons in memory formation in order to understand the mechanisms of newborn-neuron-based memory processing in the hippocampus. Identifying a specific function of young new neurons in learning and memory requires the establishment of new temporally and spatially controlled ablation techniques. Here we propose to develop a virus-based ablation system capable of specifically ablating young newborn neurons at different times during memory processing. Furthermore, we will evaluate the direct effect on memory formation resulting from virus-based specific elimination of young newborn neurons. Through these analyses, we aim to determine the specific function of young newborn neurons as a component of learning and memory. The achievement of the goals of this project will provide us with new insight into the issue of what kind of memory young newborn neurons are involved in, and give us an idea as to how newborn neurons compute memory information.

White fat in different depots is heterogeneous with respect to its metabolic functions and impact on overall glucose and lipid homeostasis. Accumulation of excess visceral fat (central or “apple-shaped” obesity) is associated with insulin resistance and increased risk of type 2 diabetes and metabolic syndrome, whereas excess of subcutaneous adipose (peripheral or “pear-shaped” obesity) does not have such a risk.
Identifying the pathways that distinguish visceral from subcutaneous fat is essential for finding solutions for the metabolic complications of obesity. Recent work has identified several differentially expressed genes. Among those Glypican-4 (Gpc4) is very interesting in that it is one of the few that is highly correlated with the level of obesity, as measured by body mass index, and the fat distribution as measured by waist-hip ratio.
Gpc4 is a poorly characterised cell surface heparan sulphate proteoglycan, suggested to facilitate or enhance the interaction of growth factors with their receptors. The overall goal of this proposal is to address the role of the differential expression of Gpc4 in subcutaneous vs. visceral fat and its relationship to obesity.
The functional consequences of down- and upregulation of Gpc4 expression will be addressed using a battery of cell biological and biochemical methods. Adipocytes and preadipocytes will be phenotypically and functionally analysed, and Gpc4 interaction partners will be identified using mass spectrometry and characterised to relate them with the observed phenotypes. This project will not only provide insights into Gpc4 function in different fat depots but also unravel its role during weight loss and gain.

Our cells are faced with more than 10.000 DNA lesions per day. Failure to repair such lesions can lead to cell death or the accumulation of genetic aberrations, which could contribute to cancer development. Efficient repair of DNA damage, however, is complicated by the fact that genomic DNA is packaged, through histone and non-histone proteins, into a condensed structure called chromatin. The repair machinery has to circumvent this natural barrier to gain access to damaged DNA and repair DNA lesions properly. We found that the ATP-dependent INO80 chromatin remodeling complex helps to overcome this barrier at sites of DNA damage. INO80 is recruited to chromosomal double-strand breaks (DSBs), where it removes histones to facilitate the accessibility and processing of damaged DNA. Along with INO80 more than 50 different proteins involved in diverse aspects of the DNA damage response are recruited to a DSB. Some of these proteins may interact with the INO80 complex to regulate its activity through post-translational modification of distinct subunits. However, the interplay between INO80 and any of these proteins remains an enigma. Therefore, we propose genetic and biochemical approaches to identify and characterize (I) proteins that interact with INO80 during the DNA damage response and (II) possible post-translational modifications of INO80 subunits. Their role in the DNA damage response will be examined in order to decipher the mechanism that regulates INO80’s activity at chromosomal DSBs. These studies will extend our basic knowledge on the pivotal role that chromatin remodeling complexes play in the signaling, processing and repair of DNA damage.

In eukaryotes, most intracellular membrane fusion reactions are mediated by the formation of a tight core-complex, consisting of complementary SNARE-proteins that are present in both fusing membranes (trans-complex). Of all SNARE-mediated fusion reactions, neuronal exocytosis is best characterized. This is a heterotypic fusion reaction, meaning that the synaptic vesicles and the plasma membrane each contain a different and complementary set of SNAREs. Other intracellular fusion reactions, such as the less well characterized endosomal fusion, are homotypic and both membranes contain a full set of SNAREs. It is unknown what prevents instant formation of a core complex within the same membrane (cis-complex), which would deplete all free SNARE-proteins. Furthermore, it is unclear in which combination active SNAREs need to be distributed between the membranes for effective fusion of endosomes and which regulatory factors may be involved. In this research proposal, it is planned to address these questions using an in vitro approach involving SNAREs reconstituted in liposomes. Using a fluorescence resonance energy transfer (FRET)-based approach, fusion mediated by endosomal SNAREs will be characterized, and a search for regulatory factors will be carried out.

Understanding how cells respond to internal and external forces is important for many biological processes, ranging from cell division to movement and differentiation. A principal determinant of the mechanical properties of living cells is the cell cortex, a shell of cross-linked actin filaments attached to the plasma membrane. This cortical actin network not only provides mechanical strength to the cell; myosin motor proteins create internal stresses within it that can lead to mechanical instabilities, active contractions, and deformations. The aim of this project is to understand the properties of such active, thin actin networks. To this end, we will develop a reconstituted model system that mimics the cell cortex, consisting of a thin actin network linked to a phospholipid monolayer adsorbed to an oil/water interface. By combining advanced microscopy and interfacial rheology, we will systematically study the relation between the structure and composition of this reconstituted cell skin and its mechanical properties. By incorporating myosin motors in the gel layers, we will be able to study, for the first time, active flows and contractions under well-controlled conditions. It is anticipated that these in-vitro studies of mechanics, (in)stability, contraction, and flow in active actin/myosin gel layers will reveal important details on the dynamic behavior of living cells.

An important, but still poorly understood, mechanism underlying our daily social interactions is the ability to anticipate what the other person will do next. Most studies in both humans and monkeys have focused on the neural mechanisms that underlie either (1) the perception of other’s actions or (2) the ability to manipulate and use tools. The project during this short-term fellowship will focus on the question how previously acquired conceptual knowledge is integrated with action-related information to make inferences about the other’s action goals. By repeatedly presenting subjects with pictures of predictable and unpredictable actions and by using a repetition-suppression approach to the fMRI and EEG data, the brain networks that habituate to the predictability of an action will be identified. The outcome of the study will give us new insight in the neural mechanisms underlying the inference of other’s intentions. The present proposal is a strong and logical extension of the work that I did during my PhD-project and directly addresses the brain mechanisms involved in conceptual knowledge for action. The short-term fellowship will give me the opportunity to (1) collaborate with one of the experts in the field (2) master new skills for data-acquisition and analysis, such as fMRI repetition suppression and combining fMRI with EEG measurements, and (3) conduct a project in a lab with state-of-the-art research facilities. This short-term fellowship would greatly expand both my theoretical and practical capacities to proceed as an independent researcher in cognitive neuroscience.

Regulation of endothelial barrier function is essential for vascular homeostasis. Experimental evaluation of vascular permeability currently relies either on in vitro models or in vivo invasive techniques. Both approaches suffer from relevance to this dynamic and complex process. Zebrafish (Danio rerio) is an emerging research model particularly suited for the study of vascular physiology. We propose to generate a vascular leak reporter transgenic zebrafish based on the bimolecular fluorescent complementation technology. In our model, vascular leak in any organ and at any time will be revealed as a fluorescent signal when a reporter plasma protein leaking from vessels meets its cognate receptors inside perivascular tissues. We propose to use this model to analyse the development of vascular semi-permeability in parallel to the development of the vasculature endothelial bed itself. We plan to perform the first forward genetic screen in vertebrates focused on vascular permeability, which we expect to reveal new molecular players in this process. Finally, this model should allow evaluating the impact of any gene product or drug of interest on the vasculature at the organism level. As an example, the study of the function of the apolipoprotein L family members will be initiated and their anticipated role in inflammation-induced permeabilisation of the vascular barrier will be assessed in our model.

Cancer is caused by somatically acquired
changes in DNA sequence. Among these changes, rearrangements of genes through
chromosomal translocations which lead to fusion genes have been observed to be
predominant in human leukaemias, lymphomas and sarcomas but their contribution
to solid tumours remains uncertain. Ovarian cancer is among the most commonly
encountered tumours in women and the patients for this type of cancer normally
experience disease relapse. Accordingly, the identification of gene rearrangements
in this kind of cancer constitutes a good opportunity to ultimately identify
the molecular pathways involved in the ovarian cancer and to find new targets
for the design of new treatments. In the present project, we propose to carry
out genome-wide sequencing analysis using paired-end sequencing in a
Solexa/Illumina platform to identify potential genome rearrangements in ovarian
cancer samples. The newly identified rearrangements, after being correctly
evaluated and filtered, will be subsequently compared to other rearrangements
found in different cancer types being analysed at Sanger Institute. This
comparative analysis will provide us with novel information on the processes of
DNA repair that are involved in ovarian cancer. Finally, the strongest candidates
for being new cancer-causing fusion genes will be functionally analyzed to explore
their role in the developing of ovarian cancer.

Theta frequency synchronization of neuronal activity is a fundamental phenomenon tightly connected to information encoding and memory formation in the mammalian brain. Cholinergic neurons in the medial septum have a central role in the activation of the reciprocally connected septo-hippocampal GABAergic network, the core of the theta generating circuitry. Anti-cholinergic agents abolish some types of theta along with causing severe behavioral impairments. Moreover, cholinergic cells were thought to be part of the MS pacemaker population. However, MS cholinergic neurons have recently been shown to fire in a theta-unrelated fashion. Additionally, cholinergic tone was proved to start increasing only after theta onset. Thus, the role of the cholinergic system in the formation of theta oscillation is still unresolved. We propose that instead of being a permissive modulator of theta, the MS cholinergic system can function as a fast switch contributing to the reset of the GABAergic network at theta onset. The recently introduced optogenetic technique provides an efficient and minimally invasive approach to test our hypothesis. I plan to selectively activate cholinergic neurons by light-induced depolarization while recording the response of theta bursting putative pacemakers in the MS and hippocampal theta. I will employ a combination of techniques already established (juxtacellular recording) as well as novel methods to be implemented (optogenetics, multisite electrode recording) in our laboratory based on the experience obtained during my visit in the host laboratory, the head of which is one of the most respected experts in the field of hippocampus electrophysiology.

The polymerization of actin into filamentous networks provides the driving force for various biological processes involving cell motion such as wound repair, immune response, cancer metastasis and the establishment of neuronal connections. For a cell to migrate, it must first polarize orienting itself along the gradient of chemoatractants and guidance cues. At the leading edge of cells, the actin filaments organize into extended lamellipodial and filopodial networks that span several microns. A large number of techniques including electron microscopy and fluorescence microscopic techniques such as photobleaching recovery analysis and single-molecule observations have been used to monitor the dynamics of structural components of the actin network. Accumulating evidence indicates that the actin network undergoes constant remodeling throughout the lamellipodium. These remodeling processes likely involve actin filament severing into short oligomers, oligomer diffusion, depolymerization of oligomers into monomers, and reassembly by polymerization and filament annealing. However, little is known about the dynamics and the gradient of diffusible actin species in live cells. This project is aimed to elucidate the role of diffusible actin and its regulators in actin reorganization at the leading edge of motile cells. Development of novel image analysis methods will extend single molecule fluorescence microscopy to the study of diffusion and concentration of proteins within live cells. Single-molecule fluorescence observations of formin constructs (FH1-FH2 and FH2 alone that differentially respond to profilin-actin concentrations) will also be used to measure local nucleation and elongation rates of F-actin, and the concentrations of G-actin and profilin-actin. Single-molecule observations of Arp2/3 complex, cofilin and AIP1 will be used to probe actin assembly and disassembly. The results of the analysis of experiments will be used to develop 2D and 3D analytical and computational models involving reaction, diffusion, binding-dissociation, fragmentation and annealing. Using the feedback between experiment and theory, the goal of this work is to reveal the role of concentration gradients and local fluctuations of soluble actin regulators during cell polarization in response to administration of drugs, ligands and physical forces.

The RIG-I and Toll-like receptor (TLR) family are pattern recognition receptors critical for the innate immune response required to combat pathogen infection. The aim of this research proposal is to identify new genes that regulate the activity of these innate immune signalling pathways. The first approach is to determine whether the inhibitor of apoptosis (IAP) proteins, cIAP1 and cIAP2, play a role in modulating innate immune signalling. Both cIAP1/2 regulate components essential for correct TNF superfamily receptor signalling, and these cIAP1/2 target proteins have also been shown to be important for correct RIG-I and/or TLR activity. However, any direct role for cIAP1/2 in modulating innate immunity has yet to be investigated. I will therefore use cIAP1/2 gene knockout mice and cells to determine if these proteins function in TLR and/or RIG-I mediated innate immunity. The second part of this project will investigate the role of the mitochondria in innate immunity. Recent research identified a mitochondrial protein, CARDIF, that is critical for RIG-I signalling. However, why the mitochondrial localization of CARDIF is crucial to its function remains unclear. I will utilize a specific mitochondrial siRNA library to determine potential mitochondrial protein(s) required for the correct function of CARDIF. A second approach, involving the purification of the CARDIF signalling complex and identification of its components by proteomic analysis, will also be employed to isolate novel mitochondrial-CARDIF adaptors. This work therefore aims to elucidate novel immune signalling molecules and examine how innate immunity is regulated.

Salmonellae are Gram-negative bacterial pathogens that are able to infect numerous vertebrates, resulting in a variety of predominantly enteric diseases. A specialized apparatus, termed the type III secretion system, which is highly related to bacterial flagella, is essential to Salmonella pathogenesis and the colonization of host tissues. I propose to concentrate my efforts on the so far little investigated group of accessory membrane proteins of the export apparatus of the secretion systems needle complex. They are vital to the function of type III secretion by mediating substrate protein recognition, translocation and substrate switching. My research will focus on three aspects: to analyze the assembly of the export apparatus with respect to the needle complex, to identify interactions among its components, and to reveal the function of the individual components. I will use blue native gel electrophoresis, in vivo photo-cross-linking and split-GFP techniques to address the assembly and structure of the export apparatus as well as molecular biology and bacterial genetics techniques to investigate its precise function. The study of type III secretion systems, is leading to unique insights into not only the assembly of macromolecular complexes and protein secretion but also mechanisms of symbiosis and pathogenesis.

Cells respond to their environment and to each other by transducing signals across their membranes to elicit transcriptional responses. One mechanism by which information is transduced across membranes involves the cleavage of a transmembrane protein by a membrane-embedded protease within the lipid bilayer. This type of signaling is broadly referred to as Regulated Intramembrane Proteolysis (RIP). Although the number of important signaling pathways that employ RIP continues to grow, the molecular mechanism underlying the regulation of intramembrane proteolysis (the “R” in RIP) remains poorly understood. This proposal is aimed at dissecting one of the first characterized examples of RIP that occurs during spore formation in the bacterium Bacillus subtilis. The ease with which genetic, biochemical and cytological analysis can be carried out in this organism makes this an ideal system to study this important class of signaling pathways.
The guiding hypothesis for this proposal is that the regulation of intramembrane proteolysis is governed by accessibility of the substrate to the caged interior (the catalytic center) of the membrane embedded protease. The molecular dissection of this signaling pathway will provide a more complete picture of how gene expression is properly coordinated during the course of cellular development. In addition, it will serve as a powerful guide for the more complicated mammalian signaling pathways that employ similar logic and similar intramembrane cleaving proteases.

Transcription is a complex process that involves fine coordination between RNA Polymerase (RNAP) and various transcription factors. Being the first step of gene expression it is also the target for a wide variety of regulatory processes. Transcriptional pausing plays a central role and is a conserved mechanism in pro- and eukaryotes. It is involved in the regulation of gene expression and believed to be a prerequisite for termination of transcription in bacteria.
I propose to use X-ray crystallography and cryo-electron microscopy (cryo-EM) to determine the structures of several elongating RNAP complexes trapped in a paused state. Elongating RNAP can be stabilized in a paused state in vitro using well-characterized nucleic acid scaffolds. Transcription factors that are involved in pausing and termination, such as bacterial NusA or NusG can be added to study their mechanism.
This fellowship would enrich my research experience greatly, particularly because I will move from the translation field into transcription. Moving from a European to an American lab will expose me to a different scientific environment. Expanding my experience with X-ray crystallography, learning 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 a principle investigator in an academic lab.

Functional magnetic resonance imaging (fMRI) allows for non-invasive localization of brain activity. In addition to standard activation mapping, effective connectivity analysis allows for statistical testing of comprehensive network models incorporating modulatory factors such as attention or pharmacological treatment. However, applications of this analysis to the ventral brain (e.g., orbitofrontal cortex, amygdala) are rare as acquisition of high-quality data in these regions is hindered by severe signal dropout and geometric distortion effects from intra-voxel dephasing caused by strong field inhomogeneities from nasal cavities. It is therefore the objective of the current application to enable the applicant to work at the Wellcome Trust Centre for Neuroimaging at University College London to obtain training in advanced MR sequence programming, develop a novel approach for minimizing susceptibility-induced signal loss in the ventral brain based on the methods established at the host institution, and establish a new research collaboration with the host institution focussing on improving fMRI data quality in the ventral brain to a level adequate for applying effective connectivity modelling approaches at high magnetic fields. The techniques developed within this fellowship will represent an important step towards extensive connectivity analysis in the ventral brain. Due to the importance of ventral brain areas in emotion and reward processing, as well as a variety of psychiatric and neurological diseases including schizophrenia, depression, phobia, bipolar disorder, personality disorder, ADHD, addiction etc this fellowship will have significant impact on future studies.

The generation of complex body plans during development involves the organization of epithelial tissues into three-dimensional shapes. Germband extension in the Drosophila embryo is a model system for studying epithelial morphogenesis. This process relies on the directional intercalation of cells between their neighbors along one axis, resulting in extension of the tissue along a perpendicular axis. Intercalation occurs via a stereotyped and irreversible sequence of events involving a remodeling of cell-cell junctions. These junctions are maintained by the cell adhesion molecule E-cadherin, but the molecular mechanisms that modulate adhesion during junction remodeling are currently unclear. The proposed research aims to use molecular genetic and live imaging approaches to identify the mechanisms regulating adhesion in intercalating cells. In particular, I will investigate the interaction of E-cadherin with a- and ß-catenin, two essential regulators of its adhesive properties. Live imaging of junctional protein localization in intercalating cells can reveal potential anisotropies in the distribution of adhesive complexes as well as temporal dynamics that could underlie junction remodeling. Moreover, I will develop methods to modulate the cadherin-catenin link in a spatiotemporally controlled manner to assess the role of the cadherin-catenin complex during cell intercalation. Insight from these experiments will be used to characterize the molecular mechanisms by which upstream signaling pathways influence cadherin-based adhesion. The proposed experiments will shed light on how regulated adhesion between cells provides the basis for dynamic processes of tissue morphogenesis.

In mammals, genetically-directed cell labeling technologies have not yet been applied to the morphologic analysis of neurons in the context of CNS disease or treatment. We will make use of the CreER/loxP technology to non-invasively label very small numbers of neurons so that their morphologies and the distribution of pre- or post-synaptic structures can be fully visualized. By developing transgenic mice lines expressing different fluorescent reporters that localize throughout the neuron or that localize to specific subcellular structures (such as synapses), we will be able to define these structures within single neurons in the brain at high resolution. In particular, I plan to express epitope-tagged and GFP-tagged synaptic proteins such as PSD-95, SV2B, Bassoon and SynGAP, together with a membrane tethered red fluorescent protein (tdTomato) so that the rare neuron that has undergone Cre-mediated recombination exhibits puncta of green signal at synapses and a uniform red signal that reveals its overall morphology. We will apply this technology to define, at single cell and subcellular resolution, some of the morphologic changes that accompany the progression of disease in mouse models of Alzheimer’s disease and that accompany drug therapy for depression. The genetically-based neuronal sparse labeling technology that we propose to develop will be a powerful tool to investigate in unprecedented detail a wide variety of developmental, disease-associated, and drug-induced alterations in the structure and connectivity of CNS neurons.

Histone H3 lysine 56 acetylation (H3 K56Ac) is a cell cycle regulated modification involved in the DNA damage response in yeast and human cells. In the yeast Saccharomyces cerevisiae, H3 K56Ac participates in chromatin assembly and is present in all the newly synthesized histone molecules deposited behind replication forks during S-phase. Mutant cells that cannot acetylate H3 K56 are extremely sensitive to a broad spectrum of genotoxic agents that perturb DNA replication, including analogues of chemicals currently used in cancer chemotherapy. However, it is currently unclear how H3 K56Ac protects cells against chemicals that damage DNA replication forks. The project outlined in this proposal is aimed at finding gene deletions that modulate the DNA damage sensitivity of cells lacking H3 K56Ac through a high-throughput genetic screen. These genetic interactions will certainly help to understand the molecular mechanisms that link chromatin assembly and the response to DNA damage during replication. In addition, because chromatin assembly, DNA replication and DNA repair are evolutionarily conserved, the information garnered in yeast may prove crucial to elucidate the function of H3 K56Ac in human cells. Dr Boeke's group is well known for the development of innovative high throughput genetic screens. The opportunity of working in his laboratory to learn these technologies will be very profitable to establish my own research group in the future.

According to the most accepted hypotheses, primitive self-replicating biopolymers were carriers of early genetic information and able to accomplish catalytic functions. Contemporary biopolymers that exhibit these characteristics are however relatively complex molecules, which poses the question of how the first complex sequences could arise in the early earth.
The main goal of this research project is to understand what physico-chemical mechanisms permitted the appearance of such molecules. To achieve this we will focus the project on two basic points. The first one will be the theoretical study of template-directed ligation reactions, which is assumed to be one of the best candidates for physico-chemical processes responsible for the appearance of the first biopolymers. The second research point will be the study of biopolymer sequences from the viewpoint of the theories of information and complexity, which will ultimately lead us to the analysis of molecular fitness landscapes. The expected output of this research is to shed light on the processes by which the first molecules emerged and understand evolutionary processes from a molecular viewpoint.
My background as a theoretical physicist and my experience in the field of complex networks will help me deal with the physics of primitive molecular reactions and analyze the topological properties of the fitness landscapes, respectively. This fellowship will assist me as I want to shift the focus of my research towards biophysics. I look forward to be trained in new biological techniques and make significant contributions to the work which is being done in the research group of Irene Chen.