Animals regulate their behaviours by combining physiologically distinct senses such as smell, taste, sound and vision. One good example for this combination is the interaction between visual and smell modalities. Humans presented with the identical odor of isovaleric acid rated but cued with the semantic label of “cheddar cheese” found the odor more pleasant than when the identical odor was cued with the semantic label of “body odor”(4). In the last decade, considerable effort has been expended in understanding the basis for such multi-modal integration in the human and primate brain using functional imaging approaches, but still little is known about the molecular basis. This proposal aims to use a simple and genetically tractable animal to understand how complex stimuli in the nervous system are integrated and interpreted to cause acceptance vs. avoidance behaviour of food stimuli presented with conflicting sensory values. Drosophila melanogaster larvae represent an excellent system for studying the interactions between taste and smell because these animals possess defined set of chemosensory neurons and connections (1). However, little attention has been paid to how conflicting sensory stimuli are evaluated in the larval brain. By using innovative genetic tools to manipulate and observe neuronal function, I will study how chemosensory neurons transmit information to higher brain centers, where conflicting appetitive and aversive sensory information converge, and which molecules are involved in this process. My long-term goal is to understand the basic principles of sensory integration in a simplified system and to discover possible conserved molecular mechanisms.

Delineating the mechanisms of dendrite arbor morphogenesis is crucial for a complete understanding of neuronal development; it is also clinically important as disrupted arbor development is a common feature in mental retardation. Development of the complex neuronal dendrite arbor is a multi-step event governed by both intrinsic genetic programs and extrinsic factors. To date, much effort has been devoted on elucidating how extra-cellular cues regulate neuron development. Conversely, less is understood about the intrinsic neuron class-specific programs that mediate the formation of the diverse dendrite arbor shapes. Recent advances in our understanding of the intrinsic genetic programs of dendrite development have been achieved using the Drosophila melanogaster dendritic arborization sensory (DA) neuron model. DA neurons have highly complex dendritic arbors, which are shaped in a class-specific manner by the combinatorial expression of the transcription factors (TFs), Abrupt, Knot and Cut in different subsets of DA neuron classes. By using genetic manipulation and in vivo imaging techniques in Drosophila I will examine how these TFs modulate cytoskeletal components during arbor elaboration and elucidate their downstream effectors. My goal is to understand the mechanisms by which class-specific alterations in DA dendrite arbor morphogenesis lead to differences in mature dendrite arbor shape. This project marks a significant change in research direction for me, and providing opportunity to develop new skills and learning new research model. This fellowship will enrich my scientific experience and prepare me for future independent research in developmental neurobiology.

Cardiovascular disease is a leading cause of morbidity and mortality in the western world. The low-density lipoprotein receptor (LDLR) is a critical determinant of plasma cholesterol levels and contributes to the pathogenesis of hypercholesterolemia and atherosclerosis. Therefore, the pathways controlling LDLR abundance are an important facet of cholesterol metabolism with major therapeutic implications. During the course of my HFSP long-term fellowship I identified the sterol-responsive gene, LDLR Regulating Gene (LRG), as a novel modulator of the LDLR. Acting as an E3-Ubiquitin ligase, LRG promotes the degradation of the LDLR and diminishes LDL uptake both in vitro and in vivo through a previously unrecognized pathway. Conversely, loss of LRG expression results in increased LDL uptake, possibly enabling the development of therapeutics that specifically target this novel pathway. These findings underpin the hypothesis that LRG has a role in establishing cholesterol homeostasis and atherogenesis. The studies proposed here aim to advance our understanding of the physiological role of LRG and to specifically elucidate:
1) The spatial and temporal sequence of events in the LRG-dependent pathway for degrading the LDLR.
2) The in vivo consequence of gain- or loss-of LRG function on whole-body sterol homeostasis and atherogenesis.
Understanding the mechanisms that govern cholesterol homeostasis is an important aspect of metabolism and is a central goal in my career. The proposed studies constitute a significant step in this direction.

Stem cell trafficking to a targeted tissue location is central to its generative, regenerative and immunological functions. However, this process unfortunately is poorly understood and characterized. Aimed to gain more insight of stem cell trafficking mechanisms, we propose to use chemical approaches to introduce biological species, which potentially contribute to stem cell trafficking, on the stem cell membrane, and to elucidate their individual (or combinational) roles in stem cell rolling, adhesion and migration. The chemical approach is advantageous because of its simplicity, and ability for control over graft density and the distance between grafted biological species and cell surface, and their versatility of co-immobilization, which cannot be readily achieved by the previous methods.Specifically, we will develop a generic chemical approach platform where covalent bonding and streptavidin/biotin interaction is used to introduce cell rolling and adhesion ligands (i.e SLeX and anti-VCAM-1) to mesenchymal stem cell membrane. We will study both the individual and combinational roles of these ligands with respect to cell rolling and adhesion. We will also investigate how the graft density of these ligands and extension from cell surface influence the cell trafficking. Furthermore, the biological consequences (differentiation, viability, and proliferation) of MSC membrane at each chemical modification steps will also be studied. Having the proposed platform established, we will ultimately introduce and study all major cell traffick factors, which will eventually provide us a comprehensive understanding of stem cell trafficking mechanisms.

Virtually all of the world's food, fuel and oxygen supply originate from chloroplasts in photosynthetic organisms. But maintaining harmony between the chloroplast and nuclear genomes is a major challenge for a cell. Therefore, unique intracellular signaling systems have evolved to coordinate transcription among nuclear and organellar genomes. Much progress has been made in clarifying the nucleus-to-plastid signaling, but the retrograde signal is less well understood. Scientists in Cornell have recently mapped and cloned a plastid differentiation mutant, and shown it to be equivalent to Gregor Mendel’s stay-green (sgr) locus. SGR is a chloroplast protein involved in chlorophyll degradation, but its tomato mutant also fails to regulate correct nuclear gene expression. I hypothesize that SGR acts in a novel retrograde signaling pathway, which might use chlorophyll or other pigment breakdown products as signals to trigger nuclear reprogramming during plastid differentiation. To test this, I propose a multi-disciplinary approach that would bring together the fields of biochemistry, bioinformatics, and genetics. In brief, I will identify 1) target genes of the SGR pathway in tomato, and 2) its role in plastid differentiation; then the model plant Arabidopsis will be used to 3) dissect the SGR pathway, 4) identify the signaling molecules and 5) develop mutant screens for additional signal components. The proposed research will uncover the missing retrograde signal(s) regulating plastid differentiation. Given the importance of amyloplasts and elaioplasts in starch and oil production, I anticipate that my results will have relevance beyond the field of plant sciences.

Defining the precise molecular strategies that coordinate patterns of transcriptional responses to specific signals is central for understanding normal development and diseases. Recently, we identified of a novel histone H2A deubiquitinase (2A-DUB) that permits delineation of a strategy for gene activation. 2A-DUB interacts with the histone acetylase PCAF in a coregulatory protein complex, and activates AR-dependent transcription. Compared to other histone modifications, the role of histone ubiquitination remains the least understood. In this proposal, characterization of novel histone deubiquitinase/acetylase complexes will direct an avenue to address the role of histone ubiquitination in transcriptional regulation. In particular, a link of the histone deubiquitinases to androgen receptor could greatly enrich our understanding of androgen signaling in prostate cancer. To achieve these goals, two specific aims are proposed: 1) To functionally characterize the histone deubiquitinase/acetylase (HDUB/HAT) complexes 2A-DUB/PCAF and USP22/hSAGA, and to elucidate their roles in coordination of histone acetylation and ubiquitination in transcriptional regulation; 2) To investigate the roles of 2A-DUB and USP22 and specific histone ubiquitination in AR-dependent transcriptional regulation, and their pathological roles in prostate cancer development. Understanding the role of histone ubiquitination will provide novel conceptual basis for transcriptional regulation in general, and may provide novel approach to therapeutics of hormone-resistant
prostate cancer.

How cellular structures self-assemble is one of the most exciting questions in biology, with potential implications ranging from nanoscience to medicine. In eukaryotes many self-assembly processes are mediated by cytoskeleton proteins such as actin. In rod-shaped prokaryotes the actin-homolog MreB, an essential protein involved in cell shape maintenance, forms a helical pattern along the bacterial axis. This research aims to elucidate the biochemical and biophysical origin of MreB self-assembly by introducing new techniques into this field. Cellular structures in micron-sized organisms are hard to study because of optical diffraction limits. Using the recently developed method of fluorescent photoactivatable localization microscopy, MreB structure will be determined in-vivo with sub-diffraction resolution. Statistical analysis of different cytoskeleton characteristics, such as number of helical turns and helical pitch, will be measured at various steady-state and kinetic conditions. These results will be compared to theories of MreB polymerization and to a new numerical method. To further elucidate the affects of polymerization on membranes' shape, MreB cytoskeleton will be reconstituted in-vitro within lipid vesicles. This reconstituted system will allow measurement of forces applied by MreB cytoskeleton on the cell walls. Liposomes confined within polydimethylsiloxane microchannels will be used to assess how geometry affects cytoskeleton structure. The combination of in-vivo, in-vitro and in-silico approaches will shed new light on the fascinating question of self-assembly in bacteria.

The brain is a highly dynamic system that can change at different time scales, from very fast changes within fraction of a second to long processes of weeks and years. Understanding of brain-behavior coupling and its dynamics is at the heart of modern brain research. It also has practical implications, as we suggest here in relation to sight restoration efforts using sensory substitution devices (SSDs) that convert visual information into auditory input. Blind children might adapt and utilize SSDs better, as their brain is more plastic. Thus, teaching them to use SSDs for detailed artificial vision could prove beneficial. Surprisingly, no one has systematically attempted this before. Here we plan to adapt a method (we developed during my HFSP fellowship Amedi et al, Nature Neuroscience 2007) to teach artificial vision to blind children and adults and study brain dynamics while learning this new crossmodal transformations using safe advanced neuroimaging and electrophysiological methods. Specifically, we will test for the development of specialized functional architecture in the visual cortex (spatial topography, visual streams and object categories) using SSDs. On top of advancing sight restoration and yielding new insights in human-machine interfacing, it will contribute to knowledge of brain plasticity. By comparing blind to sighted controls we will also be able to gain insights on the development and dynamics of multisensory integration. The CDA will help to concretize multimodal approaches, fund demanding longitudinal studies and collaborate with neuroscientists, ophthalmologists (sight restoration using prostheses) and engineers (implementing new SSD algorithms).

Abstract The function of dendritic cells (DC) is to recognize specific pathogens associated molecules (PAMPs) and prime an immune response by presenting the information to dedicated professional immune cells. The networks responsible for DC to process information from recognition of specific pathogenic antigen, to the precise activation, of the DC, promoting its migration into lymph nodes, to activate T helper cells, are unclear. The focus of my post-doctoral research is to elucidate the mechanism controlling the genetic and epigenetic modifications that underlie the specific response of dendritic cells to PAMPs. Specifically, I will address a model whereby the information of a specific PAMP pattern is dynamically stored in modular components of signaling and transcription machinery, that along with changes in chromatin accessibility and microRNAs underlie the fine-tuned change in expression in each target gene leading to the desired cellular function. Utilizing mRNA and microRNA microarrays, along with chip-chip arrays for different chromatin modifications I will establish the dynamics of the signaling and transcriptional events induced by challenging DC with PAMPs. Computational methods will then be used to reconstruct a predictive model of the regulatory networks and chromatin state that underlie the response of DCs. Finally RNAi libraries will allow us to both test our predicted models and improve their function. This project will involve integration of biochemical and molecular proficiency I gained in my Ph.D. along with immunological, genetic and computational approaches I will acquire with the help of Drs Regev and Hacohen at the Broad institute.

It has long been postulated that long lasting changes in synaptic strength, known as synaptic plasticity, is the cellular basis underlying learning and memory. Synaptic strength is determined in major part by the number of AMPA receptors in the postsynaptic membrane. Recent studies have shown that the AMPA receptor auxiliary subunit, stargazin and its binding partner PSD95 play an important role in the trafficking and targeting of AMPA receptors to synapses. Stargazin is the best studied member of transmembrane AMPA receptor regulatory proteins (TARPs), a subfamily of the calcium channel gamma subunit gene family. While it is known that stargazin is phosphorylated in vivo, it is still not well characterized. I propose to define the molecular mechanisms of how different TARPs, together with their phosphorylation status, regulate AMPA receptor maturation, trafficking and function in synaptic plasticity in vivo. I will identify novel interacting proteins and phosphorylation-regulated proteins for each TARP by taking advantage of my previous biochemical skills. Next, I will investigate the functional roles of the TARPs-interacting proteins and the cellular dynamics of TARPs by taking advantage of the host laboratory’s experience in neurobiological and neuroelectrophysiological approaches, which are indispensable for the success of the proposed project. This postdoctoral investigation will allow me to develop my expertise in live imaging, electrophysiology, production of knockout mice and ultimately in behavioral analyses. The knowledge I will gain from these studies will better prepare me to direct my own research to understand the cellular basis of brain function.

The human innate immune system represents an effective first line of defense against pathogens. Therefore, drugs that enhance innate responses might prove extraordinarily useful in the battle against infectious diseases. However, our current understanding of the innate immune system is limited and many of the molecules, mechanisms and cascades that provide resistance against microorganisms remain to be elucidated. Salmonella enterica and enterohaemorrhagic E. coli (EHEC) use secretion systems to inject effector proteins into host cells. These effectors play a central role in pathogenesis. Therefore, it is my hypothesis that many effectors interact directly with components of the host innate immune system and can be used as baits to identify novel molecules involved in host innate responses. Hence, I aim at identifying host cell interaction partners of S. enterica and EHEC effector proteins using a mass spectrometry based quantitative proteomics technique. I plan to characterize further one or a few of the effectors that interact with known or suspected host innate immunity molecules in order to understand how these interactions lead to disease. Hence, this study will provide insight in the pathogenic mechanisms employed by Salmonella and E. coli and will further our understanding of human innate immunity.

Collagen fibrils are the principal source of tensile strength in animal tissues and define the shape and form of tissues in which they occur. Type I collagen is the major component of fibrils in tendon, bone and skin. Collagen is synthesized as Type I procollagen (PC-1) in a precursor that is co-translationally translocated into the endoplasmic reticulum (ER) (1). Most lethal cases of Osteogenesis Imperfecta (OI) are caused by mutations in PC-1 that yield improperly folded PC-1 molecules which are retained in the ER (2). The mechanism by which 300nm long fibril of PC-1 destined for secretion from the cell is transported from the ER through the Golgi to the plasma membrane has long been of challenge to cell biologists (3). It has been proposed from previous work that PC-1 trafficking is not mediated by typical COPII vesicles which are usually 60-70nm in diameter, although PC-1 transport depends on COPII components (4-7). However, recent work done by the Schekman group has provided evidence that Sec23A (COPII component) is involved in collagen transport as evidenced by accumulation of collagen in the ER of fibroblasts from patients having Cranio-lenticulo-sutural dysplasia (8). In an attempt to resolve this issue and get clear insights into this fundamental problem of trafficking of large secretory molecules, I would like to investigate PC-1 transport from the ER using human fibroblasts cells and an in vitro vesicle budding reaction (9). This study will provide a platform to explore the effects of different lethal OI associated PC-1 mutations on its folding and export from the ER and to unravel the mechanism of protein misfolding defects associated with human diseases.

Vertebrae, ribs and most skeletal muscles, derive from somites, which are transient embryonic segments, and mutations affecting somite formation are responsible for human congenital scoliosis. The segmentation of the presomitic mesoderm (PSM) into somites is a unique example of a temporally and spatially repeated embryonic process and the favored hypothesis to explain this process is the “clock and wavefront” model. The “segmentation clock” generates coordinated oscillations of gene expression in the PSM while the “wavefront” determines the site where these oscillations arrest and where the next segment boundary is positioned. The canonical Wnt signaling pathway is thought to be involved in both clock and wavefront. However, direct evidence is lacking because of the effects of this pathway on posterior embryonic outgrowth and early specification of the mesoderm from which somites originate. For this fellowship, I will use inducible transgenic zebrafish lines to transiently modulate Wnt signaling after mesoderm has been specified and before tail outgrowth is affected. Our preliminary results clearly show that this transient disruption of Wnt specifically affects somite patterning. I therefore propose to use a mathematical model side by side with an exhaustive experimental study to elucidate the parameters of the segmentation process that are affected. In addition to molecular markers for both clock and wave front, I will use a new quantitative method to measure the rate of somite formation in the live embryo. This project will thus take advantage of the zebrafish model to investigate the spatial and temporal functions of Wnt signaling in somitogenesis.

The main objective of this project is to study the evolution signaling pathways in yeast in order to understand how genotypic variability impacts on cellular interaction and how these in turn affect phenotypic properties. Quantitative epistatic interactions of S. cerevisiae and S. pombe environmental response pathway components will be determined under different environmental conditions. In parallel to the experimental work, a computational analysis of these pathways will be set-up to determine the extent of sequence and structural changes. This will include (1) determining ortholog/paralogs in currently sequenced yeast genomes to calculate rates of change, sequence divergence and gene gain/loss (3) determining by structural analysis the divergence of binding specificities, and finally (4) reconstructing ancestral states of the pathway components of interest at different evolutionary times. The inferred sequences together with available structural information will permit me to study the mutational pathways that could underlie differences in cellular interactions and phenotypes detected in the experimental analysis. This will allow linking divergence in environmental responses with changes in cellular interactions (genetic interactions, pathways and complexes) and the underlying mutations. Finally, the ancestral network states of diverged pathways will be reconstructed by phylogenetic analysis and recombined into extant species for detailed analysis.