Recent work on decision making suggests that the brain accumulates information in favour of various alternatives, committing to a decision when one alternative surpasses a critical threshold. Although this explanation is consistent with neural activity preceding rapid eye movements, recent work of mine suggests that two thresholds control orienting eye-head gaze shifts: a higher threshold controlling gaze shift initiation, and a lower threshold controlling head movement initiation. The goal of this project is to reconcile these findings specifically I seek to test the seemingly paradoxical hypothesis of whether a decision to commit to an orienting head movement is made before a decision to commit to a gaze shift. If so, this would suggest that the brain "hedges its bets" by beginning to move the head while still deciding on whether to make a gaze shift. Accordingly, I will record neural activity throughout the oculomotor system while non-human primates participate in a task that requires them to decide rapidly whether to generate an eye-head gaze shift or not. Simultaneously, I will record electromyographic activity from neck muscles in order to sensitively assess the neural signals issued to the head. This project marks an important change in the direction of my professional career as it marks the first time I apply my unique experimental skills to an exciting question in cognitive neuroscience. If my hypothesis is borne out, there are a number of subsequent experiments in other forms of decision making or covert attentional allocation, the outcomes of which could substantially alter our understanding of how the brain decides to commit to alternative courses of action.

The family of EH domain-containing proteins (EHDs) is present in all higher eukaryotes and has an N-terminal nucleotide binding domain (NBD), a central coiled-coil domain and a C-terminal Eps15-homology domain. Cell biological studies indicate that EHDs are involved in endocytosis in the process of membrane budding or pinching of vesicles, but their exact role is completely obscure. To clarify the function of EHDs, various EHD members will be recombinantly expressed in bacteria or in the baculo-virus system and purified. Nucleotide binding and hydrolysis studies will clarify nucleotide specificity and the nucleotide influence on the EHD assembly status. Interactions with proposed binding partners will be followed in the presence of different nucleotides to find out whether EHDs act as molecular switches. Lipid binding studies will establish whether EHDs bind directly or indirectly to membranes. It is planned to solve the X-Ray structure of an EHD member in the presence of various nucleotides to follow nucleotide-dependent conformational changes. Finally, the process of EHD dependent membrane budding or pinching should be reconstituted in vitro and followed using electron-microscopy using synthetic or cellular lipid vesicles as substrate. These experiments will elucidate the role of EHDs in endocytosis and might establish EHDs as model system for nucleotide-dependent membrane budding / pinching processes and may be more tractable than dynamin studies.

Alzheimer’s disease (AD) is a fatal progressive dementia of complex pathology, a prominent feature of which is synaptic deterioration. AD has no cure and its molecular basis is not completely established. Oligomers of the Abeta peptide play a central role in the neuropathology of AD. Evidence for oligomer neurotoxicity has come from studies with various experimental models and synthetic Abeta oligomeric preparations. However, the human relevance of Abeta oligomers is still unclear. This project aims to provide direct evidence for the role of human Abeta oligomers in the neurotoxicity and occurrence of AD, and to investigate the conformation-dependent toxicity of lysozyme and prion oligomers. If Abeta oligomers prove to be part of AD, they could represent ideal drug targets and their elimination might even lead to reversal of memory failure. In addition, verification of AD-dependent oligomers in CSF or plasma could represent a significant opportunity for future clinical diagnostics. All my training so far has been obtained in a protein physical chemistry lab in Brazil. This provided me with the opportunity to learn about protein folding, misfolding and thermodynamics. This fellowship will permit my advanced training in basic and applied research directed at new approaches in the diagnosis and treatment of AD. More importantly, this proposal will significantly broaden my knowledge and skills in Neurosciences. As there is no group working in Neurobiology in the Department where I have a position in Brazil, I am confident that my research experience at Northwestern will be of great importance for the development of my own independent research group upon my return.

I propose to exploit unique features of the zebrafish larva, including its transparency and the accessibility of the brain to experimental manipulation, in addition to its suitability for unbiased genetic screening, for a novel approach to an important neurobiological problem. In particular I will focus my work on the cellular and molecular understanding of the function of the zebrafish visual sistem. In my research project, I propose to mapp of motion-sensitive areas in the zebrafish visual pathways by in vivo calcium imaging and to functional test motion-sensitive areas by GFP-guided laser ablation. In parallel by a genetic approach I will analyze the basis of a particular kind of visual response, the optomotor response, which is specificaly impaired in the zebrafish mutant bladerunner. During my scientific formation, I gained experience in molecular biology, and in several microscopy techniques as well. I used this background to approach classical developmental biology problems, such as neural patterning and control of cell proliferation in organogenesis. Now, by joining the laboratory of Herwig Baier, I will be able to build on these skills to learn, at a molecular and cellular level, about brain function and the neurobiological basis of behavior. This will give me the opportunity to complement my experience in the fields of molecular genetics, optical imaging and functional neuroanatomy. This training will allow me, in the future, to approach problems that represent some of the most exiting in modern biology, not only for their possible contribution to human neurological diseases and psychiatric disorders, but also for their deep philosophical implications.

The signals transduced by Notch receptors influence the differentiation and proliferation of a wide variety of cell types. Notch activation results in the release of its intracellular domain, which then migrates to the nucleus and activates the transcription of target genes. This transcriptional activation is mediated by a ternary complex composed of the intracellular part of Notch and two partner proteins. The goals of this proposal are: (i) to develop a structural model for the Notch transcriptional activation complex, (ii) to determine which residues of Notch and its partner proteins participate in energetic stabilization of the complex, and (iii) to identify small molecules that prevent ternary complex assembly using a high-throughput screen. These studies have broad relevance not only to basic developmental events influenced by Notch signaling, but also to human cancer biology and cancer therapeutics, because inappropriate activation of human Notch1 is implicated in the pathogenesis of human T-cell acute leukemias. Thus, the proposed research will contribute to a deeper understanding of the mechanism of Notch signaling and contribute to efforts in the development of therapeutic agents for cancer treatment. My prior research exposed me to a variety of techniques for the structural characterization of metalloproteins by Nuclear Magnetic Resonance. The proposed research substantially differs from my prior studies, and I feel that biophysical techniques coupled with molecular biology is a powerful approach to the study of structure/function relationships. Such training will complement my scientific background and will be an important step for my career.

The neural processes underlying simple decisions have been explored primarily under conditions in which sensory cues or differences in payoffs indicate the action that maximizes intake of reward. Evidence suggests that signals related to perceptual evidence and reward contingencies are relayed to brain regions involved in the planning of actions and it is here that effector-specific decisions are formed. However, for many decisions - especially those involving competition with a strategic opponent - there is no single 'correct' choice, and instead, a dynamic and stochastic choice strategy is required. The current proposal will test the hypothesis that brain regions involved in the planning of actions are also involved in the formation of stochastic decisions. Monkeys will compete against a computer opponent in the mixed strategy game 'odds-evens' while preparatory activity associated with generating saccades to the two choice targets is recorded from either the superior colliculus or frontal eye fields. Project 1: Correlation between preparatory activity sampled at successive epochs and upcoming choices will provide insight into the time course of developing strategic decisions. Project 2: The extent to which this preparatory activity represents the moment-by-moment bias in the decision process will be assessed by its ability to predict whether a visual probe will trigger a premature saccade. Project 3: A model of the neuronal processes underlying stochastic decisions will be developed from the results obtained in Projects 1 and 2. The validity of this model will be assessed by its ability to perform online 'mind-reading' to defeat the monkey during strategic competition.

RNA interference (RNAi) is a post-transcriptional process where double-stranded RNA (dsRNA) triggers sequence specific degradation of RNA targets thus silencing a gene or family of genes. The RNAi machinery has now also been shown to play a role at the transcriptional level through the modification of higher-order chromatin structure. In plants, fission yeast, Drosophila, the RNAi pathway has been demonstrated to play a role in heterochromatin formation. Most recently, a component of the RNAi pathway has been shown to be essential for heterochromatin formation in vertebrate cells. It is the aim of this fellowship to directly determine whether components of the RNAi pathway play a similar role in mammalian heterochromatin formation. Using a protein tethering approach, epitope tagged RNAi components will be anchored at specific chromosomal loci in the vicinity of a reporter gene and the effect on the reporter gene and chromatin neighbourhood analysed. Most importantly, this will allow specific RNAi/Chromatin silencing complexes to be isolated and characterised. The work is proposed to be carried out under the supervision of Professor Witold Filipowicz at the Friedrich Miescher Institute of Biomedical Research (FMI), Basel, Switzerland. The host institute offers state-of-the-art facilities and provides good multi-disciplinary training and collaboration opportunities thus providing the ideal environment for a successful postdoctoral stay. This fellowship experience will again mean switching fields and countries which is something that I have come to enjoy and now realise has played an essential role in my professional growth.

My proposal focuses on understanding the mechanisms that contribute to self-renewal of stem cells and cancer stem cells using stem cell subsets of the human haematopoietic system in normal and transformed situations. Although this is a fundamental question in stem cell biology and for cancer therapy, the mechanisms governing stem cell self-renewal are only partially understood. I propose to identify the genes involved in self-renewal of normal and cancer stem cells by genetic, genomic and immunological approaches. Moreover, I suggest here to characterize for the first time the target genes of the Notch, Shh and Wnt/b-catenin pathways and to use this information to define the interconnections between these pathways in normal and transformed stem cells. The identification of the key regulatory pathways that promote proliferation of leukemic cells is likely to facilitate the development of new diagnostic tools and therapeutics that target these dysregulated self-renewing processes. My future work differs from my Ph.D studies in that I will be studying processes that contribute to self-renewal and cancer in contrast to questions of tissue rejection. I feel that my choice will be optimal for pursuing an academic career. The change of field will allow me to mature as a scientist, expanding my horizons with regard to biological processes and experimental techniques. The dedication of Dr. Weissman and his excellent group, and the ability to carry out intelligent and sophisticated experiments will undoubtedly provide me the ideal environment for my post-graduate training.

Cells respond to environmental changes by regulating the expression of specific genes. This process involves binding or interaction of regulatory proteins to the genome, resulting in either enhancement or repression of the transcription of genes. We propose to develop a novel method for mapping regulatory proteins to both their cognate DNA binding site and to their interacting protein partners. The method is based on specifically tagging stretched DNA and proteins with luminescent quantum dots, and analyzing them using a combination of AFM and a unique optical imaging technique. The high precision of this technique will enable precise localization of the transcriptional complexes within the genome, and will provide significantly improved data for computational prediction. The success of this technique is an important step towards the understanding of how cellular activity is modulated at the level of transcription. In the last few years I have been developing tools for addressing fundamental problems in nano-science and nano-optics. My desire is to take these tools and study a “real” biological problem. This project gives me the opportunity to be engaged in research that will have a more direct impact on our basic understanding of life with a prospect of improving it. It perfectly bridges my acquired skills and my desired ones, combining familiar technologies like high resolution imaging and AFM with methods from molecular biology and biochemistry like hybridization and protein chemistry. Apart from the hands on experience in these new fields, I will gain knowledge and understanding of biological processes, opening pathways for independent research in nano-bio science

Developmental processes proceeds reliably despite a wide range of variation that may impinge on their underlying molecular mechanisms. These include genetic heterogeneity, environmental variability and the underlying stochasticity of biochemical reactions. My PhD research focused on theoretical analysis of the robustness of developmental patterning networks in Drosophila to changes in gene dosage. In the proposed project I aim at experimental characterization of the effects of stochastic variation on sporulation of Bacillus subtilis. This elaborate process of a resistant spore formation includes many well characterized stages, both on the morphological and the genetic levels. It is used as a simple model for elucidating basic aspects of Developmental processes. A key event during this process is the asymmetric compartmentalization of the bacteria and the subsequent expression of different genes in each compartment. This step happens on a restricted and precise timing despite a large underlying variation in the system's response as revealed by examination of wild-type cells and relevant mutants. Using time-lapse microscopy and image analysis tools I will continuously track single bacterium and observe their sporulation by recording the temporal evolution of morphological markers and fluorescently-tagged molecules. This will enable me to extract statistical behavior on the colony level and analyze temporal correlations between the different components. By comparing the behavior of wild-type and mutant bacteria, I will elucidate the role of developmental checkpoints and cell-state switches on the cells ability to buffer noise during this developmental process.

Shigella invades epithelial cells of the human colon. Internalization of Shigella via macropinocytosis requires the secretion of several effector proteins through a type III apparatus. Following secretion the two major effectors, invasion plasmid antigens B and C (IpaB and IpaC) assemble into a multiprotein complex and integrate into the host cell membrane to allow translocation of other type III substrates into the host cell. Here, the structure and dynamics of the secreted IpaB/C complex will be studied in model or host cell membranes to clarify its role as a translocator and in signaling to host cells. First, I will investigate the topology of the IpaB/C complex in model membranes using genetically tagged fusions of IpaB or IpaC. Atomic force microscopy and electron microscopy will reveal the stoichiometry and structure of the IpaB/C complex. Secondly, I will establish an in vivo assay to study the dynamics of IpaB and IpaC during bacterial entry using tetracysteine tagged Ipas. Thirdly, in vivo fluorescence resonance energy transfer (FRET) and high resolution fluorescence microscopy will be used to analyze interactions between the IpaB/C complex and host targets or components of the type III apparatus, as well as the hierarchy and kinetics of secretion of type III substrates during invasion. With this fellowship, I will develop approaches to reveal the role of the IpaB/C complex during the early events of Shigella infection. This is a dramatic shift from my previous studies on the nuclear pore complex. My new experimental tools can be applied very generally to study bacterial-host interactions, and will be fundamental for me to succeed as independent researcher.