The centrosome is the main microtubule organising center in animal cells, and it plays an important part in organising many aspects of cell behaviour like cell cycle progression, the organisation of the mitoc spindle and cell division. The centrosome consists of a pair of centrioles surrounded by an amorphous pericentriolar material (PCM) that is the site of microtubule nucleation. During interphase, the centrioles organise a small amount of PCM and nucleate a small number of microtubules. As cells enter mitosis, however, centrosomes “mature” and there is a dramatic recruitment of PCM around the centrioles, and the centrosomes organise many more microtubules. How this centrosome maturation occurs is unclear, but several structural proteins (?-tubulin, ninein and cenexin) and regulatory proteins (Aurora-A and Polo) have been implicated in this process in flies, worms, and vertebrates. In this proposal, I describe a visual genome-wide RNA interference (RNAi) screen designed to identify genes involved in centrosome maturation in Drosophila. Using facilities specifically designed for such genome-wide screens, I plan to assess the affect of individually knocking down all known genes in Drosophila on the recruitment of several PCM components to the centrioles during mitosis. This will enable us to identify new PCM components and will reveal the factors responsible for PCM recruitment in mitosis. Drosophila cell lines have proven to be for very useful for genome-wide screening, and, as centrosome maturation is a conserved feature of cell division in all animal cells, any new proteins we identify are likely to have functional homologues in humans.

DNAzymes are short sequences of single-stranded DNA that act as catalysts when folded in the presence of a cofactor. The most promising DNAzyme, which catalyzes chain scission of mRNA at the start codon, has spawned an active area of clinical research. To date, all DNAzymes have been discovered in the laboratory by directed evolution. Current directed evolution techniques limit DNAzymes to approximately 20 bases, and changing a single base can reduce the catalytic rate by several orders of magnitude. Thus, relating a particular DNAzyme’s sequence to its structure (and fluctuations around that structure) is crucial to understanding their biochemistry, and will aid in the first-principles discovery of new, more powerful enzymes for biotechnology and clinical applications. The specific aim of this proposal is to correlate insights from gained from statistical physics with available biochemical rate data in order to determine the structural features that give rise to stable, highly active DNAzymes. The principle technique to be used is a new Monte Carlo method for simulating mesoscale nucleic acid dynamics on nanosecond time scales. The work is interdisciplinary in that it connects a complex biological function at the molecular scale (the catalytic activity of DNAzymes) to a fundamental physical process (the dynamics of self-interacting, flexible macromolecules). In the context of broader research interests and professional development, the proposed work is the theoretical precursor to a strongly interdisciplinary approach to the design of DNAzymes that will eventually combine theoretical modeling, biochemical assays and fluorescence correlation spectroscopy.

Cell replacement therapy for Parkinson´s disease (PD) is based on the idea that implanted dopamine neurons may be able to substitute for the lost nigrostriatal neurons. How can we generate dopamine neuron in vitro? What are the cues controlling their differentiation? The present project is focused on the identification and characterization of neural stem and progenitor cells, isolated either from the developing CNS with special emphasis on the isolation and expansion of dopamine neuron progenitors for transplantation in animal models of PD. In particular, we are interested in studying the generation and molecular specification of neurons from immature progenitors. By identifying key regulators of a specific neuronal cell fate, we believe that it will be possible to manipulate, or direct, the differentiation of immature progenitors towards a specific neuronal fate such dopaminergic neurons. Most of the work will be performed on mouse cells, which are easier to work with and a wider range of techniques and research tools are available for research on such cells. Important parallel research is performed on human neural and embryonic stem cells, where the results obtained on mouse cells are applied to the more problematic and difficult human cell material. I will dedicate myself to a new and promising field in neuroscience in an exciting intellectual environment. Previously trained in fundamental developmental neurobiology, I would like to focus my career on the therapy of brain disorders. A HFSP fellowship would allow me to broaden my scientific training and establish an independent line of research that will form the basis of my future laboratory.

Photosynthetic activity underlies all life on Earth. Photosynthetic organisms living in different environments have developed different strategies to adapt their photosynthetic apparatus to changing light conditions and to optimize photosynthetic yield. The photosynthetic complexes share fundamental structures in different organisms, but differ in composition, ratio, and dynamic control of the photosynthetic reactions. Current evidence indicates that marine organisms express the highest diversity of all, due to the substantial differences in the light field of marine environments compared to terrestrial environments. My working hypothesis is that selective pressure in the ocean has favoured organisms capable of exploiting the information deriving from light quality and quantity to prepare adaptive responses of the photosynthetic apparatus to changing irradiances. Successful marine algae like diatoms should display this capability. Thus, the novel identification of red/far-red absorbing photoreceptors in addition to those expected for blue light in the recently sequenced genomes of two diatom species, provide the basis to test this hypothesis and to explore the relevance of photoreceptors in structuring and dynamically modifying the photosynthetic apparatus and in regulating its function in marine phototrophs. The CDA represents an important support to independently develop this novel research. Combining molecular and ecophysiological approaches, the project will make me the trait d’union between different laboratories within SZN, and will allow to answer fundamental questions regarding the ecology and evolution of phytoplankton.

Protein complexes play important roles in many cellular processes. Determining the structural aspects of protein interactions will thus further our understanding of these processes, and enhance the potential for intervention in pathologies. Although the structures of many single-chain proteins are known, structures of complexes are rare (of the known biological interactions, where the two protein structures have been solved, only 5% were solved as a complex). My primary research objective is to develop methods for efficient and accurate sampling of docked conformations, including the exploration of backbone flexibility. The improved methods will be used to study biomedically important processes, such as the oligomerization of the capsid protein of HIV. This oligomerization, which involves backbone remodeling, is a crucial step in viral maturation and a long-standing target of therapeutic intervention. I also propose to artificially redesign the sequences of a pair of proteins that do not naturally bind so that they will bind. This result will serve as proof-of-concept for the rational development of protein agonists and antagonists of other proteins. The proposed research involves different methods and concepts from the prediction of transmembrane protein structures which I studied in my doctoral work. Importantly, the protein-design aspects involve experimental work with which I have no prior familiarity. The Baker laboratory has many years of experience in all of the aspects that are new to me. Hence, this fellowship will provide me with the necessary experience for setting up a laboratory for joint computational-experimental study and design of protein interactions.

A combination of different methods is required to unravel the architecture of those macromolecules that are difficult to determine by a single high-resolution method. Small angle X-ray scattering (SAXS) of proteins and complexes in solution can result in structural insights into proteins and complexes in the size range of 30-300 kDa, in particular those that can adopt different functional conformational states. I propose to exploit SAXS for the modeling of large proteins and complexes in combination with other types of information, such as homology-derived restraints and statistical potentials, using the program MODELLER, a widely used program for comparative protein structure modeling. Importantly, I will quantify the ambiguity and accuracy of the resulting models. The method will then be applied to characterize the conformational states of Hsp90, an essential eukaryotic chaperone, throughout the ATP-cycle based on SAXS data and atomic structures of its domains and homologs. Finally, I will explore the theory of electron scattering of vitrified solutions and test it experimentally. This approach may have substantial practical advantages over SAXS because it should require a much smaller volume of less concentrated sample. While my previous research focused on structure at a low-resolution level of ~1 nm, the proposed modeling project will extend my knowledge to atomic representations. Moreover, I will enrich my repertoire of biophysical methods for structural characterization. Both experiences will be indispensable for my future research in the structural characterization of macromolecular assemblies using biophysical methods, in particular by cryo-EM, SAXS, and modeling.

Phagocytosis by specialized phagocytes is a host defense mechanism against pathogens. It is imperative for the cell to efficiently recognize phagosomal content to mediate its further processing for presentation by Class II MHC molecules. A comprehensive understanding of how a cell identifies the substance of a phagosome is still lacking. Signaling molecules attached and clustered in membrane microdomains, such as lipid rafts or the relatively little investigated tetraspanin-webs might well mediate this detection of content. Initial experiments in the Ploegh lab tracked the RFP-tagged version of the tetraspanin CD63 and showed that it is selectively recruited to phagosomes containing C. neoformans and not to phagosomes harboring an equal-sized polystyrene bead. I will investigate if this is a general phenomenon and identify lipid-modified proteins that mediate this effect using azide-tagging of lipids in cells in combination with proteomic analysis of the labeled components. The identified molecules will be tracked with state of the art microscopy as well as knock-down and knock-out technology to elucidate new pathways for phagosomal content recognition. This area of research and the methods are new to me, but a logical continuation of my professional development. Being a chemist by training, I have investigated enzyme kinetics and protein folding using mostly biophysical and biochemical techniques. I am now looking to extend my expertise to the fields of cell biology and immunology in order to be able to combine these approaches in later stages of my career.

In mammals, X-chromosome inactivation is a mechanism that ensures equivalent levels of X linked-gene expression between male and female, by silencing randomly one of the two X chromosomes in female cells. The process is established early in development and is initiated by a unique locus, which produces a large non-coding RNA, Xist. The Xist transcript is involved in cis-silencing by coating the entire inactive X chromosome. This induces chromatin changes that include histone modifications and DNA methylation. It has been proposed that LINE repetitive elements, which are particularly enriched on the X chromosome relative to autosomes, could act as “booster” elements to promote efficient inactivation, either by enhancing initial spreading of the inactivation signal, or by facilitating long term maintenance of gene silencing. Recent observations from the host laboratory, showing a correlation between methylation rates of CpG islands of X linked-genes and LINE density, suggest a possible model for the role of LINE elements in maintenance of X inactivation. The project described here aims at directly testing this model by genetically modifying LINE environments and assessing the consequences for gene silencing and CpG methylation. This work will provide further insights in understanding mechanisms of repeat-mediated gene silencing in mammalian genomes and will be a complementary study from my previous research based on the impact of transposons on plant genomes.

The biogenesis and co-transcriptional processing of eukaryotic and viral messenger RNA (mRNA) is regulated in part by direct recruitment of processing factors to the large subunit and C-terminal domain (CTD) of RNA polymerase II (RNAPII). The CTD recruits factors involved in pre-mRNA splicing, 5’- and 3’-processing, pathways essential for efficient mRNA export, stability, and translation. The CTD is comprised of tandem repeats of the heptad consensus sequence YSPTSPS wherein Ser2 and Ser5 are phosphorylated and dephosphorylated in coordination with the transcriptional cycle. The 5’ mRNA cap is the first co-transcriptional modification to occur on the nascent mRNA chain and cap formation occurs through the coordinated activities of three enzymes including RNA triphosphatase, RNA guanylyltransferase and RNA methyltransferase. Recruitment of these enzymes to RNA polymerase II (RNAPII) occurs via direct recognition of the phosphorylated RNAPII CTD. Interestingly, RNA methyltransferase maintains interactions with the phosphorylated CTD beyond early elongation when the CTD is presumably phosphorylated on both Ser2 and Ser5. After each round of transcription, RNAPII is recycled through dephosphorylation of the CTD by phosphatases such as Fcp1. This proposal will focus on utilizing macromolecular crystallography, genetics, and biochemistry to elaborate the mechanistic characterization of these enzymes and their interactions with RNAPII CTD. Consistent with my long-term goal, this study will provide molecular insights into how cellular factors are coordinated and perhaps coupled with transcription to facilitate co-transcriptional processing of mRNA.

During the last decades lung cancer has become the leading cause of cancer deaths in the world. Inactivation of tumor suppressor genes are the events underlying lung tumorigenesis, mainly genetic changes of p16INK4a and p14ARF. Our group has recently unveiled that aberrant expression of Cdc6 is oncogenic by directly repressing the INK4/ARF locus through the RD (4). In the present project, we propose to dissect the silencing mechanism on the INK4/ARF locus in lung cancer as working model. At the molecular level, I will analyze how ectopic Cdc6 and RNAi-RD result in RDINK4/ARF-dependent transcriptional repression, and, ensuing downregulation of the INK4/ARF locus. Moreover, I plan to characterize both the Cdc6 high levels found in NSCLC and endogenous RNAi-RD as internal regulation of the locus using murine models for lung tumorigenesis. In addition, we have a cohort of NSCLC to study whether the silencing of p16INK4/p14ARF by Cdc6 and RNAi-RD drive or contribute to the genomic alterations of p16INK4/p14AF associated to different transformations stages. These valuable tools will allow not only to understand the basis lung tumor biology but also for the development and validation of new tumor intervention strategies as well as for the identification of markers as Cdc6 or endogenous RNAi-RD for early diagnosis. I believe that the proposed studies on analysis of INK4/ARF locus in malignancy represent a cohesive approach to reach my professional goal as an independent researcher. My experience in molecular biology along with the support and help of Dr. Serrano, known as one of the leading researchers in the field, are vital for the successful execution of this ambitious project.

We have recently enabled photoswitching of a kainate receptor-channel by conjugating it to a tethered ligand. Ligand binding is controlled by a photoisomerizable azobenzene group in the tether, which presents the ligand to its binding site under ultraviolet light, thus opening the ion channel. Visible light removes the ligand from the binding site and results in channel closing. This project aims to extend this approach and develop new photoswitchable glutamate-gated receptors (AMPAR, NMDAR and mGluR). They are multimeric proteins with multiple ligand binding sites, and their operational mechanics are not well understood. Enabling to photoswitch these receptors will open new opportunities to study their gating mechanisms, by allowing to remotely control ligand binding in only one of the receptor subunits, and to distinguish between the events of ligand binding and channel opening. In addition, photoswitchable receptors make it possible to rapidly and reversibly depolarize cells without using invasive electrodes. Fast photoswitching in neurons will allow to generate photo-excitatory post synaptic potentials. In combination with the spatial selectivity of laser scanning microscopes, novel experiments can be designed to study the dynamics of cellular signaling at the intracellular and multicellular levels. The development of these tools is an important step towards the long-term goal of combining remote sensors and actuators of cell activity, in order to rationally interrogate complex cellular systems.

Determining the mechanisms underlying growth and differentiation in mammalian epidermis and its appendages will be important for understanding how this process goes awry in human skin disorders, including cancers. The Wnt signaling pathway is required to instruct skin stem cells (SCs) to make a hair follicle (rather than epidermis). Wnt signaling stabilizes beta-catenin, which in skin SCs acts as a transcriptional co-activator for the DNA binding protein, Tcf3. In the absence of beta-catenin,Tcf3 represses genes. I now propose to 1) elucidate Tcf3’s role in mouse skin SCs, by conditional ablation in vivo and in cultured keratinocytes 2) identify essential direct Tcf3 target genes by microarray,promoter analyses, chromatin immunoprecipitations and other molecular approaches, and 3) explore the functional significance of key Tcf3 targets. My studies should reveal how activated SCs acquire epidermal (Tcf3--) versus early hair follicle (Tcf3++) fates. They should expose whether Tcf3 target genes can be activated by shutting off Tcf3 (epidermal fate) as well as by Wnt-stabilization of beta-catenin (follicle fate) and if so, whether the same Tcf3 target genes are involved in both mechanisms. They should also provide insights as to whether Tcf3/Wnts govern SC activation, SC maintenance/quiescence or epidermal/follicle fate decisions. By means of this proposal, I will not only be able to address these important questions in stem cell biology, but also strengthen my knowledge and background in this field, and acquire the skills and intellectual thinking I will need for a successful academic career in scientific research.

The aim of this project is to investigate the role of 3’-phosphoinositides (PIs) in cancer development using the fruit fly Drosophila melanogaster. Drosophila is a powerful model to study cancer biology, because it mimics most aspects of human cancer development, including uncontrolled cell division, growth and invasion. One of the four 3’-PIs has been strongly correlated to increased cell growth and division and development of human cancer, but its role in metastasis is not known. The correlation between carcinogenesis and the remaining three 3’-PIs has not yet been explored. I will analyse how increase or decrease in levels of the four 3’-PIs affects receptor signalling and the steps of tumour development. This is easily done in Drosophila during larval development, because the larval tissue is transparent and affected cells can be labelled and traced by confocal microscopy. In this way, I will analyse the effects of defective PI metabolism on cell division, growth and metastasis. The genes that affect cancer development will be studied in more detail to determine the underlying molecular mechanisms and their possible involvement in human cancer will be validated in collaboration with geneticists. In this project I will learn the Drosophila model system, tumour biology and confocal microscopy to study cancer development in vivo, whereas I in my previous research have analysed detailed molecular mechanisms using biochemistry and cultured cells. Thus, I will broaden my knowledge significantly. In summary, this project has a great potential to identify novel genes involved in cancer development and will be of great value for my further development as a scientist.

Neurodegenerative diseases are characterized by progressive loss of neurons and synapses. Deregulation of Ca+2 is suggested to be involved in early stages of the disease and trigger its pathology. Ca+2 has been implicated in synaptic plasticity, therefore, the neurodegenerative process may initiate at synapses. Synaptic Ca+2 levels are regulated by voltage-gated Ca+2 channels and glutamate receptors, both implicated in neurodegeneration. Cdk5, a serine/threonine kinase, phosphorylates CASK, a protein that binds N-type voltage-gated Ca+2 channel and alter its activity. Cdk5 also regulates the function of NMDA receptors. We suggest an hypothesis whereby dysregulated cdk5 alters the activity of NMDA receptors and voltage gated Ca+2 channels and by thus modify synaptic Ca+2 levels, leading to synaptic dysfunction and eventually neurodegeneration. We will examine the role of cdk5 in regulating its synaptic substrates, maintaining synaptic Ca+2 homeostasis, and employ mice with altered cdk5 activity to determine if dysregulated cdk5 can induce changes in synaptic Ca+2 levels and in different aspects of synaptic function early before any observed signs of the disease. This proposal will provide new insight on early changes that occur in synapses and on the molecular process which leads to neurodegeneration. This fellowship will provide me the opportunity to conduct research in an area that differs from my previous research experience. I have never worked on synapses, Ca+2 signaling or neurodegeneration. I will be exposed to a variety of ideas and techniques, which will help me to establish my own laboratory and investigate molecular processes that lead to neurodegeneration.