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.

During the transition from S phase to mitosis, epithelial cells displace their nuclei which subsequently migrate from the basal membrane to the apical surface of the neuroepithelium, a phenomenon termed interkinetic nuclear migration (INM). The nucleus moves from the basal zone to the apical surface in G1 before it replicates its DNA in S-phase. It then returns to the basal zone in G2 and undergoes mitosis. The cellular mechanisms that underlie INM are elusive. So far nuclear positioning has been mainly studied in unicellular organisms. However, applying the knowledge coming from these studies to investigate nuclear positioning during INM in a multicellular organism will establish new nuclear positioning concepts in higher eukaryotes. It will also help to gain insight into related processes like cell migration. I want to study mechanics that trigger INM on a molecular level in the retinal neuroepithelium of the zebrafish embryo. 4D-microscopy will be used to follow nuclei, microtubules and centrosomes as well as actin filaments in the developing embryo. Molecular details of the microtubule-nucleus interactions will be investigated in detail as nuclear positioning depends on microtubules in many organisms. Anchor and motor proteins will be studied using genetic methods mainly morpholino approaches. Knocking down motor proteins as well as molecules that could link microtubules to the actin cytoskeleton and determining their influence on INM will give us insights into the molecular composition of this process. The outcome of these studies will lead to a general understanding of INM mechanics and will open doors to a better understanding of how it is linked to cell cycle events.

Splicing is a fundamental gene regulation process that removes the non-coding regions and joins the coding regions of pre-mRNA to produce proper mature mRNA for protein translation. Most of the eukaryotic genes display alternative splicing patterns that allow the production of multiple proteins from the same pre-mRNA, significantly enriching the complexity of the proteome. Even though the chemistry of the splicing reaction is known, comparatively little is known about the splicing code, namely how splice sites are chosen and splicing decisions are made by the cellular splicing machinery. There is evidence that in mammals constitutive and alternative splicing events are regulated by trans-acting proteins that are recruited by cis-acting elements along the RNA sequence such as exonic and intronic splicing enhancers as well as exonic and intronic splicing silencers. Although substantial effort has been recently put into the identification and characterization of cis-acting elements that regulate splicing, a detailed and comprehensive study of trans-acting proteins recruited by the intronic/exonic splicing enhancers/silencers in mammalian cells remains to be explored. Therefore, this project aims to establish functional relationships between the known cis-acting elements and trans-acting factors, study the biological function of the splicing isoforms of the trans-acting factors and discover novel trans-acting factors by an RNA interference medium-throughput screen. Since many genetic diseases arise from mutations in cis-acting elements, a comprehensive library of the associated trans-acting factors might point to novel therapeutic targets.

Pre-mRNA splicing, the removal of introns from mRNA precursors, is indispensable for the expression of most genes in eukaryotes. Splicing involves two transesterification steps within the spliceosomal complex, an elaborate molecular ensemble formed by the ordered interaction of four small nuclear ribonucleoproteins (snRNPs), U1, U2, U5, and U4/U6, and numerous splicing factors with the pre-mRNA. To date, six kinds of proteins are identified that are specific to U5 snRNPs Snu40p, Dib1p, Prp28p, Snu114p, Brr2p, and Prp8p in yeast. Notably, Prp8p (280 kDa) is highly conserved among species and essential for nuclear pre-mRNA splicing in vitro and in vivo. Prp8p can be crosslinked not only to loop1 of U5 snRNA which tethers the 5’ and 3’ exon during splicing reaction, but also to 5’ splice site (5’SS), branchpoint, and 3’ splice site (3’SS) of the pre-mRNA, perhaps acting as a cofactor in RNA catalysis. In addition, Prp8p binds strongly with the ATP-dependent RNA unwindase, Brr2p, and, EF-2 homologous GTPase / putative translocation inducer, Snu114p in yeast. Moreover, it is interesting that specific mutations near the C-terminus of Prp8p cause the retina disorder, Retinitis Pigmentosa. With this fellowship, I will do a dramatic shift from my previous studies first, I will purify this ternary complex by S.cerevisiae expression system and tri-(U4/U6)•U5 snRNP by tandem affinity purification (TAP) method, then I will determine the structure of Prp8p/Brr2p/Snu114p ternary complex by X-ray crystallography and the envelope of the tri-(U4/U6)•U5 complex by cryo-electron microscopy, which will provide important insights into the catalytic core of the spliceosome.

The molecular mechanisms regulating cell fate and pattern formation during plant development belong to one of the most fundamental questions in developmental biology. Stomata, specialized epidermal structures regulating gas exchange between plant tissues and the atmosphere, constitute an excellent model to study these universal questions. To understand the complete circuit for stomatal development and patterning, we will need to identify genes that positively and negatively regulate multiple divisions and signaling events. Our present knowledge about positive regulators is poor. In this project I will clone a newly identified positive regulator of stomatal development. By performing detailed molecular and functional characterization of the gene I will identify at which stages it is expressed and required and will elucidate genetic interactions between it and known regulators of stomata development. As a complementary approach, I will explore cellular targets of the stomatal development network. Particularly, I want to investigate the dynamic organization of the cytoskeleton, organelle behavior and cell polarity organization during stomata development. By taking advantage of new stomatal-lineage-specific enhancer elements and the phenotypes created by overexpression of key stomatal regulators, I will be able to resolve cellular behavior in a never before seen way. This project is an integrated approach to study genetic and cellular mechanisms regulating stomata development in the model system Arabidopsis thaliana. The output of this work will provide valuable information about molecular and cellular mechanisms regulating cell differentiation and specification in plants.

The synapse is a specialized structure involved in the transfer of information in the brain. Thus, any regulation of the synapse will modulate the quality of the information that is communicated within the brain. Recently, it has been shown that astrocytes take an active role in regulating synaptic function. It is now widely accepted that the synapse comprises three components, not only pre- and postsynaptic neurons, but also processes sent by astrocytes. Astrocytes extend numerous processes contacting many types of synapses where synapse-glia interactions occur and where the information processing is performed. Although some of the general principles of the contribution of astrocytes to synaptic transmission are now becoming established, it is still unknown how a single astrocyte handles the functioning of the different synapses enwrapped by its processes. Thus, it is crucial to understand how individual astrocytic processes talk to their associated synapses and how the activation of individual processes occurs in relation to synaptic signaling. Using state-of-the-art cellular imaging techniques and synaptic recordings, activity of individual astrocytic processes will be monitored to study synaptic cross-talk between astrocytes and synapses. I will test whether activity in individual astrocytic processes is related to synaptic activity, if the glial processes are independently activated and whether they are specialized for one type of synapse. Finally, the ability of the same astrocyte to release different neuromodulators will be tested. This project should further our understanding of the role of glial cells in the regulation of the transfer of information in the brain.