The ability to perceive our environment through interactions with light represent billions of years of cumulative engineering resulting in the complex process of human vision. This required the careful optimization of subtle interactions between chromophores that absorb visible light and their surrounding rhodopsin proteins. Detailed knowledge of these interactions is important for not only understanding vision, but also for the development of novel proteins possessing new light activated functions. Often, the direction and efficiency of photo-induced reactions in proteins are controlled by rapid dynamics occurring as fast as a picosecond, which requires the use of ultrafast techniques to directly explore. This proposal describes a multi-disciplinary approach involving direct feedback between time-resolved measurements and rational protein design to develop new proteins that mimic the photodynamic features of the rhodopsin pigment protein. This allows for a more comprehensive investigation without the inherent limitations to the direct study of rhodopsin. This "bottom-up" approach to reengineering proteins serves as a powerful platform for designing specific protein interactions of interest. Key to the success of this approach is the construction of a state-of-the-art ultrafast laser laboratory capable of performing different time-resolved techniques for exploring transient electronic and vibrational properties. Support from Career Development Award will be highly instrumental in enticing talented international researchers to aid in its construction, which will allow for the samples to dictate which techniques are the most appropriate for their study and not the opposite.

Embryonic stem (ES) cells are defined by pluripotency and their capacity to self-renew. Epigenetic modifications play a crucial part in controlling ES cell identity, and an expanding body of evidence suggests that particularly histone methylation is involved in this process. Histone methyltransferase (HMT) enzymes, that catalyse the addition of methyl groups to histones, and histone demethylases (HDM) that are required for the reverse reaction, carry specific domains that are responsible for catalytic activity. Based on these domains, I have identified 43 potential HMT and 32 HDM enzymes in the mouse genome. The goal of this project is to determine which of the HMT and HDM enzyme in the genome is crucial for ES cell maintenance and differentiation. To this end, lentiviral-mediated expression of siRNA will be used to knock down HMTs and HDMs specifically transcribed in undifferentiated ES cells. If the targeted enzymes are crucial for ES cell maintenance, a knock-down would result in the promotion of differentiation. Moreover, lentiviral transduction to overexpress HMT and HDM genes, which are activated in the course of differentiation, will be employed to identify enzymes that can induce differentiation of ES cells. Furthermore, target genes regulated by the identified enzymes will be determined by genome-wide mapping of their binding sites using ChIP-chip analysis. This project will contribute to our understanding of which role chromatin regulation plays in the maintenance of ES cell identity.

New neurons are continually recruited throughout the adulthood and participate in functionally remodeling the neural network in the brain. One of the most important questions is how the new-born neurons integrate into the pre-exiting circuitry in the adult brain. Recent accumulating evidence supports a role for tonic and phasic GABA induced depolarize and NR1 activation of neural progenitors and new-born neurons in the dentate gyrus in promoting the differentiation and the synaptic integration in the adult brain. However, little is known regarding the role of intrinsic activity of progenitor cells in their incorporation in the adult brain. In this proposal, I plan to utilize retrovirus specific manipulate of new born neurons in the adult mouse brain in vivo, a technique developed by Professor Gage’s lab and sheds light on the study of neural stem cells. I propose to suppress the excitability in the new born neuron within active and mature hippocampal neural network by over-expression inward rectifier K+ channel using retroviral vectors. I plan to examine whether the supression of activity in new born neurons have any effect on their axon and dendrit development, on their circuit formation and competation with surrounded mature neurons. This project contributes to the mechanism of the integration of new born neurons into the preexisting circuitry and strong current interest of scientists in the neural stem cell field. This project will also allow me to investigate a new field, gain insights into new biological concepts and learn new technologies. This fellowship experience will undoubtedly help me to head my own research laboratory in future.

CLC chloride transport proteins are conserved from prokaryotes to eukaryotes and are involved in many physiological functions, such as muscle excitability, hippocampal development, or endocystosis. Defects in CLCs lead to many diseases. The CLC family encompasses both Cl- channels and Cl-/H+ antiporters. Crystal structures of the antiporters have been solved, but there are as yet no structures of the channels. Although the antiporters have been crystallized under a variety of different conditions, none of the structures are significantly different. This is surprising because the process of catalyzing stoichiometric exchange of Cl- for H+ must involve a series of conformational changes. Thus, it is likely that additional conformations exist but that only one of the conformations crystallizes. Additional methods of examining conformational changes are essential. I aim to study conformational changes of the CLC-ec1 antiporter using hydrogen/deuterium exchange (HDX) detected by mass spectrometry. HDX does not require crystallization of the protein and therefore allows mapping of all conformational states. Likewise, I propose to use HDX to study conformational changes associated with gating in the CLC-0 channel. This will yield the first structural data on a full-length CLC channel. Comparison of CLC-0 and CLC-ec1 will reveal to what extent the structure of a eukaryotic channel resembles that of prokaryotic transporter and will allow me to evaluate how conformational changes that lead to Cl-/H+ exchange in transporters are related to gating in channels. These results will help us understand how similar structures have evolved to carry out different physiological functions.

There is a wealth of converging evidence that sleep-dependent mechanisms of neural plasticity lead to the consolidation of learning and memory across a range of animal species. But the way in which it does so remains largely unknown. In the proposed study, we will directly address this issue in Drosophila, an ideal neuroscience model with its conserved mechanisms for sleep and memory. We will address the role of sleep on learning and memory. In addition, we will address how synaptic plasticity influences sleep. Are wake and experience/training comparable in terms of gene regulation and their ability to induce sleep? Finally, we will examine the effect of sleep on the MB a center for learning and memory, specifically assessing changes in gene expression. Taken together, the proposed studies should address at behavioral and molecular levels what is the role of neural plasticity in sleep regulation and in turn, what is the role of sleep in neural plasticity for optimizing cognitive function. This work should not only provide important advances in our understanding of the critical function of sleep, the interactions of sleep and memory, but also might provide some helpful clues for treating sleep and memory disorders.

A remarkable property of living cells is their ability to rapidly generate phenotypically diverse populations. For example, in response to nutrient limitation, individual cells in B. subtilis can undergo a variety of responses, including differentiating into an environmentally-resistant spore, or undergoing competence, a transiently differentiated state in which cells take up extracellular DNA. Using the sporulation initiation and competence circuits in B. subtilis as a simple model system, I intend to study the fundamental problem of how noise (the random fluctuation of cellular components) causes genetically identical cells grown under the same environment to make different decisions about their cell fate. My PhD research focused on understanding the design principles of the Arabidopsis circadian clock genetic network through a combination of modelling and experiment. In this project I will develop a much more quantitative understanding of the sporulation initiation and competence networks, taking advantage of the ease of genetic manipulation and the accurate quantification of gene expression levels possible in B. subtilis. I will use single cell multi-colour time-lapse microscopy to analyze and elucidate the design principles of the sporulation initiation circuit and the competence circuit, as well as the connections between these two developmental pathways. I will use mathematical modelling throughout to analyze the circuits and design experiments. This work will allow me to address fundamental biological questions such as how do cells use noise to create phenotypic diversity? And how does a cell successfully choose one developmental path from a set of options?

The exosome is a macromolecular protein complex of exoribonucleases and RNA binding proteins that plays a vital role in processing and degradation of RNA. The complex is evolutionary conserved from yeast to human as well as in some archaea and its importance is illustrated by the fact that the genetic depletion of any exosome subunit results in a sever growth defect of yeast. Besides influencing gene expression via normal turnover of mRNA, the exosome also participates in various RNA quality control pathways that monitor and swiftly remove aberrant RNA. It has recently been shown that the eukaryotic exosome requires association with additional complexes (TRAMP and SKI complexes) for activation in vivo. So far there are no published structures of eukaryotic exosomes and the molecular mechanism of exosome activation is currently not understood. The project proposed here aims to solve the three dimensional structure of the yeast exosome using cryo electron microscopy (EM). The 3D structure of the eukaryotic exosome will us help to understand the basis of its many function and provide a platform for further functional studies. Additionally, I plan to solve the EM structures of the exosome bound to activating complexes. Such structural data in combination with biochemical and cell biological experiments will provide key insights into the molecular mechanisms of exosome recruitment and activation by the SKI and TRAMP complexes.

Soxb1 genes have been linked with stem cell biology. In particular, SOX2 is clearly associated with several stem cell types from very early stages of development through to the adult mouse. SOX2 has been demonstrated to have special relevance in neural stem cells. In addition, the host lab has also noted the expression of SOX2 in several other locations where stem cells exist. Sox2 (and the other Soxb1 family members) could therefore be considered to a “stem cell gene”, i.e. one required for the establishment and maintenance of a stem cell state. We have therefore adopted a number of approaches to further investigate the role of Sox2 in stem cells, in particular of the nervous system. Specifically, it would be important to identify and study the target genes for Sox2 (and the other Soxb1 proteins) in stem cells function

Although immune cells are well recognized as critical in the defense against pathogens, the contribution of macrophages and other cells of the innate immune system to tumor development is less clear and remains controversial. The physiological functions of macrophages that are present at tumor sites probably depend on the type and stage of progression of the tumor. Therefore, it is important to systematically address the physiological roles of macrophages through the use of in vivo models that allow tight control of both tumor initiation and the presence of macrophages. Here, I will use mouse models (established and available in the host lab) that permit the control of the initiation of lung tumor development through adenovirus-mediated Cre-dependent expression of K-ras and p53 mutants. I will first characterize inflammatory cells infiltration inside the tumors, monitor the expression of cytokines and chemokines in vivo and in vitro, and examine molecular pathways that are activated in these tumors and that may contribute to recruitment of inflammatory cells. Secondly, I will cross these mice with recently established transgenic mice that allow a time-controlled depletion of macrophages. Such an approach should enable to decipher the differential contribution of macrophages during the progression of lung tumors.

The goal of my proposal is to extend MADM system to study the logic of neural circuit organization and formation in the mouse brain. MADM (for Mosaic Analysis with Double Markers) is a recently developed molecular genetic method that can be used to label small populations of cells, to perform lineage analysis, to visualize wiring patterns, and to conditionally knockout genes only in labeled cells. The current MADM system has two major limitations for general phenotypic analysis that I plan to address: (i) MADM-based gene knockouts rely on the availability of a pair of the MADM cassettes between the gene of interest and the centromere. With a novel strategy that utilizes a random integration of MADM-transgene into ES cells, I plan to construct a “MADM mouse library” where all mouse euchromosomes are targeted by MADM cassettes. This library will enable researchers to make conditional mutant cell populations for nearly all genes in the mouse genome, and will be shared worldwide as a useful genetic resource. (ii) Spatial regulation of MADM is currently still limited. I will generate new Cre lines using BAC transgenic mice to provide spatial control of cell labeling and gene disruption. As a model, I will establish a spatially regulated MADM system in higher olfactory neurons using a precursor marker gene in the olfactory brain. I will analyze the spatial organization and conditional mutant phenotypes of olfactory cortical afferents. These experiments will be an essential first step in the broad application of MADM-based molecular genetics to other brain regions for the better understanding of the logic of mammalian neural circuit organization and formation.

Membrane compartmentalization is a rapidly emerging concept to explain the regulation and coordination of a wide variety of biological processes mediated by bacterial plasma membranes (PMs). However, the mechanisms and physiological significance of bacterial PM compartmentalization remain largely unknown. Mycobacteria, which include important pathogens such as Mycobacterium tuberculosis, rely on the PM as a major biosynthetic site of PM and cell wall (CW) components. The highly complex structure of the mycobacterial CW implies that the PM must have a mechanism to regulate and coordinate the numerous biosynthetic pathways involved in PM and CW biogenesis. Indeed, our recent studies on mycobacterial subcellular fractions demonstrated that the mycobacterial PM is compartmentalized into two domains with distinct biosynthetic capacities, implying that membrane compartmentalization is an important mechanism to regulate and coordinate PM and CW biogenesis. The proposed research will investigate the mechanisms of membrane compartmentalization in mycobacteria. First, lipid domains will be visualized microscopically using domain-specific marker proteins, and examined if they are dynamic. Second, the physicochemical properties of these lipid domains that define specific protein targeting will be examined. Third, the targeting signals of marker proteins will be determined. The multilateral approach taken in this proposal will establish the concept of membrane compartmentalization in mycobacteria, and allow the expansion of research pursuing the molecular mechanisms of bacterial PM dynamics. These studies will also lay the groundwork for the development of anti-tuberculosis drugs.

A great diversity of shoot system architecture exists among flowering plants. This diversity resides in ability of the plant to activate axillary buds present on leaves axils which otherwise remain in a dormant stage. In many species, buds outgrowth is inhibited by a signal produced by actively growing shoot apex. This signal, auxin, is nevertheless not acting directly on buds outgrowth. A new signal, graft transmissible, mediates this apical dominance. Branching mutants in Pea, Petunia, Arabidopsis and Rice identified loci essential to this new signal production and perception. The amino acid sequence of the orthologous genes is highly similar despite the drastically different shoot architectures of those species. This high degree of conservation raises the question of how shoot architecture evolved. Were there modifications in this new hormone pathway that are responsible for shoot system divergence? If so, did it occur at the protein level, or did it occur at the transcriptional level (different expression level, pattern and/or responsiveness)? I intent to elucidate those questions by isolating all orthologous genes from a wide range of species (Physcomitrella patens, Poplar, Rice, Medicago, Snapdragon, Pea). I will determine whether they can substitute for the activity of their orthologues and whether their expression is regulated similarly. Furthermore, I will identify the CIS-elements involved in the variation in expression levels, expression pattern or responsiveness to other hormones.

Reinforcement learning models of decision making describe how basal-ganglia (BG) structures learn to evaluate different stimuli in terms of their future reward value. But how does the brain identify which, of all the available stimuli, are the relevant ones to evaluate? We hypothesize that the prefrontal cortex (PFC) constructs representations of the world, identifying and directing attention to stimuli dimensions that are relevant for the task, and which the BG must learn to evaluate. This allows humans and animals flexibly and dynamically to adjust decision-making in real-world tasks. The goal of this project is to investigate this interaction between the PFC and BG through behavioral and neuroimaging experiments using a novel paradigm that combines instructed and uninstructed task switches. By analyzing the data using computational models of the inference processes underlying performance, we aim to tease apart the contribution of different cortical and subcortical areas to this fundamental process. My previous research experience is in theoretical modeling of animal behavior. At this stage in my career, I would like to augment my theoretical capabilities with in-depth experimental skills. Through this project I intend to acquire expertise in cognitive psychological and neuroimaging methods in humans. The host lab provides an invaluable training opportunity both in cognitive psychology, and in functional imaging techniques. These will provide me with the necessary tools with which to test and evaluate this and future computational hypotheses, and with first-hand insight into cognitive processes as they are manifest in human decision-making behavior and in brain activity.