The goal of this project is to investigate the potential use of the human embryonic stem cells(hESC)-derived cerebellar progenitors as a therapeutic strategy in rat model of cerebellar lesions. Recently we developed the first protocol for generation the cerebellar cells from hESC (Erceg et al., 2009, submitted). These cells could be useful in the treatment of different types of cerebellar disorders. The next step will be to evaluate the in vivo function. The proposed project will be to transplant generated cells in a rat model of cerebellar lesion to asses whether these cells are capable to restore already lost motor function in cerebellar lesions. The experiments and short-term stay will be performed in the Laboratory of Prof. Lura Petrosini, Dept.of Psychology, University "Sapienza" di Roma, Italy. In her Lab we will learn and perform the surgical lesions in the rat. This research project requires cerebellar lesions in adult rats. These lesions may involve the surgical ablation of a hemicerebellum with or without the ablation of the deep cerebellar nuclei of the same side. They may involve the surgical ablation of an entire hemicerebellum, or of a cerebellar hemisphere or of the vermian region. After the lesion the animals will be tested for their motor symptomatology. Testing for cognitive symptomatology will performed when no change in the motor symptomatology is observed (about three weeks after the surgical lesion). Animals will submitted to behavioral testing only if they exhibit stable motor symptomatology consistent with a cerebellar lesion.

Brain function depends on the precise assembly of neuronal networks during development, however the mechanisms underlying this process are poorly understood. During spinal cord development, inputs to motor neurons are established sequentially, starting with local interneuron circuits, followed by peripheral sensory input, and ending with the assembly of connections from the motor cortex that are responsible for accurate voluntary movement. I hypothesize that during development each step in the sequence of circuit connectivity influences later events. Specifically, I propose to address whether functional proprioceptive connectivity is required for later development of cortical input to spinal motor neurons. I will first characterize the time course with which corticospinal projections and synapses develop in mice, with a special focus on direct monosynaptic connections from motor cortex to spinal motor neurons. I will anterogradely label corticospinal projections using transgenic mice that express GFP only in the forebrain and I will retrogradely label motor neuron monosynaptic partners by a modified rabies virus expressing GFP or RFP that is able to jump just one synapse. I will also study the topographic distribution of cortical motor neurons projecting to specific motor neuron pools. Secondly, I will determine when established connections become functional combining viral expression of channel rhodopsin in corticospinal projections with intracellular and/or voltage dye recordings of motor neurons. Finally, I will compare the obtained wild-type results with the ones from transgenic mice in which proprioceptive connectivity to motor neurons is ablated.

The question of the origin of life is one of the most fascinating in science. It is commonly believed that, prior to our form of life, there was on Earth another form of life based on RNA, the so-called “RNA world”. Research is ongoing to build a laboratory system that resembles the RNA world in order to study how it might operate. Such a system requires RNA enzymes that have the ability to self-replicate and that have sufficient functional complexity to evolve in a Darwinian manner. The Joyce group has pursued a strategy to create such a system, based on the R3C ligase ribozyme, which they developed previously using in vitro evolution. A complex network is being constructed in which many different RNA enzymes cross-replicate, in the absence of proteins or any other biological entity. Competition and the possibility of occasional mutations should give the system the features of Darwinian evolution. The aim of the proposed research is to develop a kinetic model that could describe such a system. The model will be validated based on comparison with experimental results. Computer simulations will be performed, with the aim of helping to design new experiments that provide a deeper understanding of the system, operating in the context of serial transfer experiments performed within a microfluidic device. The model will describe the time-dependent behavior of the system under various conditions, thereby providing an informative picture of the self-sustained Darwinian evolution of RNA.

Myosin molecules are proteins that function as motors inside cells. These molecular motors play crucial, dynamic roles in most cellular processes, including contraction, movement, and shape change. A variety of diseases owe their origins to defects in myosins, like DFNA22 syndrome, which is characterized by deafness. Myosins transport other biomolecules by hauling them along the actin cytoskeleton. This transport consumes energy at the ‘head’ domain of the motor, while its ‘tail’ domain is traditionally considered more structural and mainly required for dimerisation, since it is rather rigid. Until recently, coiled coils in mechano-sensory proteins like myosins were thought to be relatively straight and geometrically ideal. Detailed investigations of the implications of structural deviations from a strong coiled coil motif on biological function of myosins are just emerging. For instance, myosin VI in both Drosophila and humans contains a peculiar distribution of charged residues in the tail that Jim Spudich’s group has shown, by biophysical approaches, to be a ~70-residue long stable, fairly rigid single α-helix, not the coiled coil that was predicted earlier. Ramanathan Sowdhamini’s lab has developed computer algorithms for the analysis of coiled coil interfaces through energy calculations. Jim Spudich’s lab has a long-standing expertise in biophysical and cellular experiments on myosins. Henrik Flyvbjerg’s lab leads efforts to properly analyze stochastic processes and data in biophysics, such as those manifested in molecular motor studies and in motility assays. The three groups together will make a unique team by uniting their complementary tools of bioinformatics, biophysical studies, and mathematical modeling to obtain important structural and functional understandings of the relatively ignored, but vital, tail domains of the myosin family of molecular motors.

The knowledge of the structures of biological macromolecules is invaluable to elucidate the mechanistic details of their function. Complexes that are associated to lipid membranes carry out many important cellular functions, but their structures are largely elusive to date because these types of macromolecules are extremely difficult to assess by established experimental techniques, such as X-ray crystallography, single-particle cryo-EM, and NMR spectroscopy. I propose to unravel the structures of membrane-associated complexes using cryoelectron tomography (CET) and subsequent modeling. CET is currently the only generally applicable three-dimensional imaging method to retrieve the density of a given complex in its close-to-native state, and the technique is therefore uniquely suited to provide density maps of membrane-associated complexes. Subsequent modeling allows determining those structures, which are compatible with the densities, but also all other available information on the complex such as biochemical interactions as determined by proteomics studies and atomic structures of constituents. The developed methodology will be applied to the machinery that governs endoplasmic reticulum mediated decay of proteins (ERAD). Removal of erroneous proteins from the endoplasmic reticulum and subsequent degradation via ERAD is essential for cellular function. Malfunctions of protein degradation have been implicated in diseases such as Alzheimer and Parkison’s disease. The structure of the ERAD machinery is almost completely uncharted and even low-resolution structures will greatly elevate our rudimentary understanding of ERAD.

The process of myelination, in which myelin sheaths are formed around axons, represents one of the most spectacular processes in biology. Myelination of axons is required for the efficient transmission of nerve impulses and maintainance of axonal integrity in the central nervous system. As well as being of intrinsic biological interest, this process is relevant to our understanding of the regenerative process of remyelination following myelin diseases such as multiple sclerosis. Critical to this process is the mechanism by which a population of neural stem/precursor cells called oligodendrocyte precursor cells (OPCs) differentiates into myelin-forming oligodendrocytes, cells that migrate to and wrap their processes around axons to form the multiple layers of compacted membrane that constitute myelin. Thus far, only the biochemical stimulation of OPC differentiation has been studied. However, OPCs are also subjected to various kinds of mechanical strain during the processes of cell migration, axon engagement, and neuron growth, and it remains entirely unknown how such physical stimuli may regulate or contribute to OPC differentiation. In this project we seek to clarify the importance and modes of mechanical stimuli that impact OPC differentiation, by integrating international expertise in OPC biology, novel biophysical experiments, and material mechanics. Using well-established protocols for growing, differentiating, and characterizing OPCs in vitro used in the Franklin lab, we will systematically consider effects of global, local, and receptor-mediated strain on OPC morphology, molecular markers, and myelination potential. These strains will be applied using optical, scanning-nanoprobe, and substrate-based cell deformation techniques being developed by Guck and Van Vliet, in order to identify the key modes and magnitudes of mechanical stimuli that contribute to OPC differentiation. The conversion of effective mechanical stimulus to intracellular biochemical signaling pathways will then be identified by RNA profiling (including microarray profiling and post-hoc bioinformatic pathway analyis tools) and validated by ICC and Western blotting. In summary, the proposed research will develop and apply innovative experimental approaches to improve our understanding of the process and stimulation of axon myelination, and to identify potential new targets for treating demyelinating diseases. More generally, these systematic studies on a key precursor cell type will serve as a model to clarify the general mechanisms involved in mechanotransduction in the maintenance and differentiation of stem and precursor cells.

Not only are animals able to record the time of day when some behaviourally relevant event has happened, but also they take advantage of this knowledge in their subsequent behaviour. The main goal of the project is to understand how flies are aware of time; and the basic idea is making use of learning and memory to uncover this ability. An initial focused approach consists of evaluating whether memory processes are modulated by the circadian clock. Training in Dr. Dubnau’s laboratory, at Cold Spring Harbor Laboratory (CSHL) is vital for the success of this project since it will provide access to the memory paradigm, provide hands-on experience to manipulate and interpret the behavioral data, and whenever possible, answer several questions regarding a potential circadian modulation of memory, with emphasis on possible mechanisms. In doing so, new issues will certainly take shape which might end up requiring modifications in the original setup to be able to study time awareness at home. The request for a Short-Term Fellowship is based on the fact that it would enable me to spend some time overseas, which in itself will open new venues in my career: not only it will be instrumental in successfully carrying out my post-doctoral project, but also it will allow the setup of this new behavioral paradigm in my home institution; besides, my lab would benefit immensely from the opportunity to spread the research in circadian modulation of memory and, hopefully, time awareness. Training at a high profile institution such as CSHL will most likely impact my career as a young independent researcher. Moreover, it will give a powerful know-how to bring back home and share with others.

Replicative DNA helicases are enzymes that unwind DNA during DNA replication. MCM2-7 is a hexameric protein complex present in all eukaryotes that is composed of six nonidentical, but highly related AAA+ ATPase proteins. There is a wide agreement that the MCM2-7 complex functions as a replicative DNA helicase in replication. So far, it is still a mystery how MCM2-7 complex unwinds DNA on chromosome. Most well-studied replicative DNA helicases function by encircling and translocating along single-stranded DNA (ssDNA). However, various lines of indirect evidence implicate that the MCM2-7 complex may encircle and translocate along double-stranded DNA (dsDNA). In this proposal, we expect to determine whether the MCM2-7 complex translocates along ssDNA or dsDNA during the DNA unwinding. To distinguish these two fundamentally different mechanisms, we will use ChIP to examine the effects of specific DNA lesions on MCM2-7 translocation. Meanwhile, by performing DNA footprinting on a helicase stalled at a specific location, we will independently investigate the way in which MCM2-7 binds to DNA. The proposed study will not only answer one of the biggest open questions in the replication field, but also be instrumental to understand the roles of the many proteins that are required to recruit and activate MCM2-7 during replication. Moreover, the knowledge gained from this study will also provide key insight into the architecture of the replisome.

Calcineurin (Cn) is a serine/threonine protein phosphatase that plays pivotal roles in many physiological processes. Most prominently, calcium-activated Cn dephosphorylates multiple residues within the regulatory domain of the nuclear factors of activated T cells (NFATs). Effective phosphate removal by Cn requires its docking to NFAT. There are still many open questions regarding the biological and structural-related aspects of this mechanism such as what is the mechanisms by which phosphorylated and dephosphorylated NFAT interact with Cn? and how dephosphorylation exposes a nuclear localization sequence?
This plan proposes to structurally characterize the Cn-NFAT association using NMR techniques. This determination of three-dimensional structure for the NFAT regulatory domain before and after the dephosphorylation process will have important implications for understanding the T-cell activation and fundamental signaling pathway mechanisms.

Homologous recombination (HR), albeit being an important mechanism for the repair of DNA double-strand breaks, and thus the maintenance of genome stability, has to be tightly regulated, since unscheduled or excessive HR can lead to gross chromosomal rearrangements characteristic of cancer cells. In yeast, the Srs2 helicase is known to suppress aberrant recombination events, but functional homologues in higher eukaryotes have remained elusive for a long time. Recently, the Boulton lab has identified SPAR1 in a C. elegans screen as a functionally analogous protein to Srs2. SPAR1 is conserved from C. elegans to humans and exhibits many of the genetic and biochemical hallmarks of yeast Srs2.
The aim of this project is to investigate the mechanisms of how HR is restrained in higher eukaryotes. To do so, a two-pronged approach will be employed: The first approach comprises the detailed biochemical characterisation of SPAR1. To this end, I will design an in vitro assay to elucidate a potential role of SPAR1 in synthesis-dependent strand annealing (SDSA). In addition, I will set out to purify and characterise SPAR1 containing complexes from human cells. Novel interacting partners of SPAR1 will be characterised for their role in regulating recombination and their influence on SPAR1 activity. In a parallel approach, I will perform a GFP reporter-based genetic screen in C. elegans to identify spar-1 like genes, which will be subsequently tested for their anti-recombinogenic function in a human cell culture system.
Integration of the results from both approaches can be expected to add considerably to our understanding of how and by which factors homologous recombination is regulated.

Toll-like receptors (TLR) signaling cascades recognize pathogenic components and initiate the innate immune response, which is largely mediated through gene expression changes. Variation in TLR signaling underlies differences in immune homeostasis and susceptibility to infectious and autoimmune diseases. It is unclear, however, how genetic variation between individuals leads to inflammatory diversity and whether variation in gene expression mediates the phenotypic differences. Here, I propose to elucidate the regulatory mechanisms underlying the influence of genetic polymorphism on gene expression variability in inflammatory responses. I will develop new algorithms for simultaneous mapping of multiple gene expression traits and for direct identification of their underlying regulatory mechanisms. The scalable algorithm I will design will be applicable for complex mammalian systems, will provide concrete mechanistic hypotheses, and will be made available to the biomedical community. I will apply this algorithm to genome-wide gene expression and phenotype measurements in dendritic cells from segregating mouse populations exposed to different combinations of TLR ligands. The resulting hypotheses will be then tested in lab, identifying the molecular mechanisms that underlie heritable variation in innate immunity and inflammation. I hope that my work will help to understand individual variation in immune disorders and will constitute an important step toward personalized medicine.

In this project, we will investigate the biological function of the dengue virus helicase. Dengue virus is an important human pathogen that poses a serious health problem around the world. Using reverse genetics, infectious clones, and in vitro assays, we are investigating the interaction of the helicase with the viral RNA replication machinery. Here, we propose to initiate a collaboration to investigate mechanistic aspects of the unwinding activity of this viral helicase. I will learn a technology, developed in the host laboratory, to analyze the RNA unwinding process by single molecule methods. To this end, I will be trained to use optical tweezers. Then, we will design appropriate RNA substrates to determine thermodynamic and kinetic parameters of the unwinding process. The combination of the virology studies that I am carrying on in my home laboratory, together with the mechanistic studies that I propose in this project, will help us to define the biological role of the dengue virus helicase. Importantly, this short term fellowship will be the starting point of a new international collaboration that will allow me, as a young investigator, to have access to new technology and to learn novel biophysical approaches to address a relevant biological question.

Hepatitis C virus (HCV) affects 120 million individuals world-wide and causes serious liver disease in up to 20% of infected patients. To date, the molecular events leading to HCV infection and to HCV-induced liver pathology remain largely elusive.
HCV enters the liver via sinusoidal blood vessels and infects hepatocytes from the basolateral side. HCV entry into hepatocytes requires the tight junction protein claudin-1. In the healthy liver, claudin-1 mediates intimate contacts of adjacent hepatocytes and forms the physical barrier between basolateral and apical compartments. It is unknown whether and how HCV gains access to tight junctions. Since claudin-1 is essential for hepatocyte function, we hypothesize that claudin-1 engagement by HCV contributes to liver pathology. Therefore, I will first investigate how HCV uses claudin-1 for entry and second how this affects the integrity of polarized primary hepatocyte cultures.
To visualize how HCV engages tight junctions, I will establish labeling techniques for HCV and apply single particle tracking methods. Live-cell imaging of HCV and its host receptors will provide a detailed spatio-temporal view of HCV entry. Moreover, I will clarify which cellular trafficking pathways are exploited by HCV using inhibitors and gene silencing.
Engagement of the cell-cell adhesion molecule claudin-1 likely alters liver integrity. I will investigate tight-junction function of HCV-infected primary polarized hepatocytes using permeability assays and electrophysiology. This together with the above imaging techniques will help understand how HCV exploits cellular adhesion molecules for entry and what impact this has on liver pathology.

Dendritic cells (DCs) are the most efficient antigen presenting cells of the immune system, due to their capacity to present peptides derived from endocytosed exogenous antigens on MHC class I molecules. This process is called cross-presentation, resulting in the activation of specific CD8+ T cells (cross-priming).
My proposed work will elucidate functional and molecular mechanisms for efficient cross-priming of viral antigens in vivo and in vitro.
I will establish new experimental systems to assess cross-priming efficiency of viral antigens delivered by autophagic or apoptotic cells used as exogenous antigen sources.
In particular, I will characterize the mechanisms through which autophagic cells internalized by DCs elicit more efficient cross-priming compared to apoptotic cells.
I will evaluate whether antigen processing within the autophagic cell prior to phagocytosis by DCs is required for efficient cross-priming.
By using knock out mice, I will investigate whether specific signaling pathways involving TLR-7, TLR-9 and RIG-I are engaged upon autophagic cell internalization by DCs.
Finally, I propose siRNA screening approaches targeting autophagic cells to identify key molecules potentially responsible for their efficient antigen cross-presentation by DCs.
My proposed work will establish more physiological conditions to characterize the cross-presentation pathway in vivo. Not only this project will give a substantial contribution in the field of immunology, but it will also have important implications for immunotherapeutic approaches.

The brain represents sensory information by complex patterns of electrical activity in groups of neurons. Deciphering this neuronal population code is one of the fundamental challenges in neuroscience. To study this, the neural network of the retina is superbly suited. It combines good experimental accessibility with a well-defined role as the first stage of visual processing and displays intriguing multi-neuronal activity patterns, such as synchronization and other forms of correlated activity. I therefore intend to study how multi-neuronal activity patterns in the retina contribute to representing the information that the brain receives from the eye. In particular, I will ask which types of visual stimuli are best represented by different kinds of multi-neuronal activity patterns. I will begin with synchronous activity of neuron pairs and subsequently include larger groups of neurons and non-synchronous activity patterns. The features of the identified stimuli will provide insight into the role of these patterns for information transmission. My approach will be to design experiments that actively search for suitable stimuli while monitoring the neuronal activity with multi-electrode arrays. An essential ingredient will be the development of closed-loop experiments. Based on automated analysis of the recorded data, this will allow implementation of appropriate search algorithms for systematic adjustments of the applied visual stimuli during the experiment. For this multi-disciplinary endeavor, the support and recognition provided by the CDA will function as an ideal framework to assemble a strong international research team from a diversity of scientific backgrounds.

Epidermis is a multi-layered epithelium composed of hair follicles (HF), interfollicular epidermis (IFE), and sebaceous glands (SG). Its patterning is initiated during embryogenesis by signals coming from the underlying dermis, and is later maintained in adult skin by different populations of stem cells residing in HF bulge, IFE and SG. Patterning changes according to the body region. Several signalling pathways have been implicated in HF spacing and IFE specification in back skin, but the mechanisms responsible for pattern variations remain unknown. Mouse tail represents a good model to study this question because of its highly stereotyped arrangement of HF into triplets, facilitating hair counting, and its different populations of interfollicular epidermal cells, identifiable using markers. The aim of this proposal is to identify the factors regulating HF and IFE patterning using mouse tail as an experimental model. I will analyze interactions between different signalling pathways for their ability to modify HF and IFE patterning. To this end, I will implant beads soaked in molecules that regulate signalling pathways in wild-type and transgenic mouse tail epidermis both in vitro and in vivo. I will test the role of the dermis in controlling tail patterning by changing epidermis orientation relative to dermis, before and after establishment of patterning. Finally, I will investigate the role of the identified pathways in adult tails during induction of ectopic HF. These experiments will enlighten mechanisms regulating embryonic and adult skin patterning and contribute in understanding both development and stem cell regulation in skin.

While animals may be able to attribute knowledge, ignorance and motivation to other individuals (a human ability termed 'Theory of Mind'), direct evidence for such attributions by non-humans has proved very difficult to attain. In order to overcome the difficulty, inherent to non-verbal methods, of revealing what animals expect other individuals to do, I will develop and use a behavioural paradigm to enable obtaining such data. I will first train hand-raised Western scrub-jays (Aphelocoma californica), which readily cache food items under laboratory conditions, to perform an 'expectation test'. Storers will be rewarded when they perform specific behaviours according to their predictions regarding the location in which an observer jay is likely to look for the cached food. Then, I will use this test to reveal whether jays can attribute a false-belief to a conspecific which has observed them caching in one spot, but not subsequently re-caching the item in another spot, while remembering the correct present location themselves. Extending this paradigm to other ecologically-relevant scenarios, I will test whether jays keep track of the knowledge-state of many individuals simultaneously, whether they attribute knowledge based on auditory information to others and whether they act to deceitfully decrease the knowledge of potential cache pilferers. This study may provide a novel and much needed paradigm for the field of animal cognition, advancing our understanding of whether and how animals represent the minds of other individuals in their social environment.

Deregulation of the epidermal growth factor receptor (EGFR) has been identified as a common component of multiple cancer types including head and neck, ovarian, gastric, breast, and colorectal cancers. Over the past two decades, we have learned much about how signals are conveyed from EGF receptor at the membrane to its targets in the cytoplasm or nucleus. However, most traditional methods, have adopted a reductionist approach, focusing on one or a few molecules of interest. Thus, while the traditional methods have yielded abundant information about individual proteins in EGFR signalling, they have been less successful at providing an integrated understanding of biological systems. Here, we will develop a technology that enables the high-throughput, system-wide, and quantitative characterization of an intracellular signalling network, under different perturbation conditions and as a function of time. Our analysis of the mechanics of the EGFR signalling network will provide more thorough characterization as well as opportunities for the development of targeted novel cancer therapeutics. Further, dissecting the EGFR network at systems level should serve as a benchmark to evaluate future efforts in data-driven model discovery. Ultimately, it is our hope that this combination of high-throughput data collection and probabilistic modeling will provide a general route to the rational design of combination therapies.

The mammalian hippocampus is crucially involved in spatial navigation. In this context, hippocampal excitatory principal cells are referred to as place cells, if they distinctly enhance their firing frequency when the animal enters a specific region in the environment, the place field. Hippocampal interneurons are also known to show place-modulated firing activity, although less pronounced and influenced by additional contextual factors. Yet, the dynamic interplay of principal cell and interneuron firing and the topography of neuronal activity patterns underlying behavioral states are little understood. In this fellowship I therefore propose to identify the principal rules of local hippocampal ensemble activity in freely exploring rodents. The major aims are to (1) determine the proportion of place cells to non-spatially selective neurons, (2) characterize the fine-scale topography of place-specific firing in both principal cells and interneurons, and (3) analyze the dynamic interplay between principal cells and interneurons in hippocampal ensembles. To do so I will follow a progression of concerted approaches: Two-photon microscopic imaging of hippocampal neurons in vivo will first be established in anesthetized animals. Transgenic mice expressing GFP in interneurons will then be used to differentiate activity patterns in principal cells and interneurons, and ensemble activity of hippocampal neurons will fiber-optically be monitored in exploring rodents. Extensive data analysis will finally describe the local topography of principal cell and interneuron activity and unravel the balance between local excitation and inhibition in freely behaving animals.

Animals and their organs become patterned as
they grow. Several signaling pathways have been shown to regulate patterning
(e.g. Wingless/Wnt, Hedgehog, Fibroblast Growth Factor, Notch pathways) or
growth (e.g. Hippo and Insulin Signaling pathways) aspects of development. One
of the most intriguing and challenging questions in developmental biology today
is how these processes are coordinated in time and space. Decapentaplegic
(Dpp), a Drosophila member of the transforming growth factor-ß
(TGF-ß) family,
serves as a paradigm to answer this question because it has the ability to
regulate both patterning and growth.

My first aim
focuses on the logic of transcriptional regulation by Smad proteins, the
nuclear effectors of the TGF-ß pathways. The Drosophila Smad proteins, Mad and Medea, lead to activation of some target genes while
inhibiting others. The putative cofactors required for Dpp-dependent activation
have not been identified thus far. Here, I will test
the hypothesis that T-box containing proteins, Dorsocross 1, 2, and 3, fulfil
this function.

In the second
part, I aim to monitor Dpp signaling dynamics in vivo. Fusing Dpp
responsive elements to cDNA constructs that encode highly unstable versions of
GFP or RFP will allow us to detect small and transient fluctuations in Dpp
response. These experiments aspire to achieve a detailed spatio-temporal
analysis of the dynamics of Dpp signaling in growing tissues. Such in vivo
studies will allow putting forward or excluding certain models of how Dpp
exerts its multiple functions.