The innate immune system protects us from bacteria, viruses, and cancer. Natural killer (NK) cells are innate immune cells that can detect and destroy cancerous and infected cells. How NK cells do this requires understanding of the molecules they use to sense what is harmless or dangerous, and how these molecules assist in the decision to ignore or kill the target cell. We have identified a receptor-ligand pair (NKR-P1B:Ocil) on NK cells and their targets, respectively, that mediates immune recognition at the molecular level. Because the NKR-P1B receptor is inhibitory, normal cells express the "self" Ocil protein to prevent NK cell attack, while infected and cancerous cells lose Ocil protein ("non-self") and are killed by NK cells. We have already shown that the NKR-P1:Ocil interaction is important in fighting cancer, and we anticipate a novel role for this interaction in infectious disease. I propose to advance our understanding of cancer biology by studying what molecular or genetic signals cause the loss of "self" Ocil protein during formation of tumour cells. I also intend to study the role of this molecular interaction in immunity to viral and bacterial infection, and how pathogens can evade this immune recognition. The HFSP fellowship award facilitated my repatriation to my home country, but I now need further support to continue this important work. An HFSP CDA award would give me the financial and scientific independence, support, and tools that I need to make this work possible. The CDA award would also facilitate my promotion from Lecturer and Principal Investigator to Scientist, so that I may pursue this research full-time without my current teaching duties.

The eukaryotic cell division cycle, including DNA replication and mitosis, is driven by a complex regulatory network. Stochastic elementary steps involved in these processes as well as large morphological transformations of the cell (such as spindle formation, or bud formation in yeast) introduce time variability in the duration of the cycle. How does the cell control the timings and ensure the robustness of the cell cycle oscillator? Many pathways of the cycle are duplicated and over-determined is the role of such redundancy to reduce the temporal fluctuations? The molecular pathways that control the cell cycle are well known, and the emerging challenge in systems biology is to connect the molecular data to the dynamical properties of the whole system. Such dynamical understanding requires study at the single cell level, to eliminate averaging effects encountered in bulk studies. Here, I propose to investigate quantitatively the temporal variability of the division cycle using time-lapse microscopy on monolayer yeast microcolonies. First, by tracking the expression of fluorescent reporters, I will precisely time the different events occurring during the cycle, to determine the noise contribution of each step in the cycle, and check the effect of gene redundancy. Second, using an external trigger signal, I will force the cycle to beat at an imposed frequency, in order to examine the non-linear properties of this essential biological oscillator. This project pools disparate approaches including yeast molecular genetics, cell biology, biophysics, and physics of dynamical systems, and thus constitutes a promising novel cross-disciplinary approach in biology.

T cells play a key role in maintaining immunity against pathogens. T cell progenitors originate in the bone marrow. They then move to the thymus where they undergo education and development which ensure that resulting T cells are able to recognize pathogens and infected cells and tolerate normal healthy cells. These key immunological processes occur in several stages within distinct anatomical compartments of the thymus. To develop normally a T cell progenitor must travel between these compartments in a carefully orchestrated manner. Certain receptors expressed on the surface of thymocytes at different stages of development may play important roles in regulating cell migration. The aim of my postdoctoral project is identify the mechanisms which control thymocyte migration. Using multi-photon microscopy I will be able to track thymocytes in intact thymi. I will use cells, labeled with fluorescent markers, which either lack or have increased expression of particular receptors to identify the roles of these receptors in thymocyte migration. I will also look at how developmental signals influence thymocyte migration. This project will complement my PhD work, where I studied mature T cells at the molecular level looking at the patterns of gene expression in particular subsets. For my postdoctoral project I will examine the development of T cell progenitors at the cellular level using multiphoton imaging of intact thymus. My postdoctoral project will address fundamental issues in immunology and will also extend my knowledge in Developmental and Cell biology and give me an opportunity to learn new cutting-edge techniques.

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.