Polo-like kinase 1 (Plk1) and Aurora A kinases are key regulators of the cell cycle. Their timely activation determines the onset of crucial events in cell division and their overexpression has been linked to many types of cancers. Recent studies have shed light into the mechanism by which Plk1 is activated, showing that Aurora A is the trigger kinase supported by the regulatory protein Bora. The objective of this proposal is to obtain structural information regarding the molecular mechanism by which Bora mediates Plk1 phosphorylation by Aurora A. In particular, the Bora-Aurora A complex will be analysed and due to the flexible nature of Bora, NMR spectroscopy and other biophysical techniques such as isothermal titration calorimetry and surface plasmon resonance will be used. Particularly, in-cell NMR experiments will be attempted to observe Bora conformational changes inside eukaryotic cells. In Prof. Shirakawa´s laboratory, I will be able to use the state of the art apparatus and learn from pioneer researchers in the in-cell NMR field. The combined biophysical data obtained from the experiments proposed will provide the first structural characterization of human Bora and help towards the understanding of centrosome and cell cycle regulation at the molecular level.

The formation of epithelial layers with apical-basal polarity and a central lumen is essential for organ development. Epithelial cells use polarized traffic pathways to deliver proteins to specific regions of the plasma membrane. During development, such pathways are modified. The subject of this proposal is to characterize new proteins required for lumen formation in epithelial morphogenesis, and to further understand their functions and molecular mechanisms.
The first objective of the proposal is the screening for proteins required for lumen formation. We will perform a two-step screening, a genome-wide microarray, and a siRNA screening with a limited library, using the three-dimensional MDCK system to identify candidate proteins involved in lumen formation. Our whole strategy is expected to unveil a new set of proteins of importance in epithelial morphogenesis. Then, we will select a discrete number of the most interesting candidates to perform a functional characterization of these proteins in vitro. We will prioritize the analysis of those candidates of unknown function in epithelial polarity, but associated with membrane exocytosis. In vitro systems, however, can not reconstitute the complexity of the in vivo architecture. To complement the in vitro approach, I will use the zebrafish gut morphogenesis system to address the role of proteins involved in lumen formation and to further characterize the phenotypes observed in vitro.
This proposal is central to human health. Many human diseases, such as cancer, derive from defects in epithelial organization. A molecular understanding of tube morphogenesis could lead to new ways of preventing and treating these conditions.

Two membranes border mitochondria: the outer membrane (OM) and the inner membrane (IM). In contrast to the OM the IM is invaginated into cristae. This morphological characteristic leads to two regions of high membrane curvature: (i) the cristae apex and (ii) the cristae junctions. The aim of the project is to find out which proteins are responsible for this membrane curvature and to understand the molecular characteristics of these proteins that lead-to/facilitate membrane remodelling, and to ask if particular functions of the mitochondria are facilitates by the different degrees of curvature. In case of the cristae apex it was speculated that the ATP synthase is responsible for the curved membrane. We want to show whether the ATP synthase on its own has membrane bending activity and whether this depends on its oligomeric state and whether the synthase can function differently in different curvature environments. In case of the cristae junctions we would like to investigate whether the mitochondrial fusion protein Opa1/Mgm1, which is also important for cristae maintenance, can bend membranes. We are interested whether two different isoforms of Opa1/Mgm1 need to oligomerize for membrane bending. Further we would like to get insights into the structural/function details of Opa1/Mgm1.

The present research proposal is an interdisciplinary study using the tapered microcapillaries developed in the RIKEN lab in Tokyo to irradiate living cells with well‐defined doses of ionizing radiation. Due to the extreme focusing of the beam by the microcapillaries down to less than one cubic μm, irradiation with a spatial resolution high enough to selectively target parts of a living cell becomes possible.
This technique for micro-irradiation is to be used to study the mechanisms of cell damage and cell repair after exposure to ionizing radiation. The effects of nucleus irradiation in mammalian (including human) cells will be compared to effects after irradiation of their cytoplasm, evaluating the relative effectiveness in terms of cell death and bystander effects (i.e. survival rate of cells not directly irradiated). Later on, the extremophile bacterium Deinococcus radiodurans (DR) will be studied to explain the unusually high radiation resistance of this species, where an unambiguous identification of the mechanisms behind its resistance has not been achieved so far.
The micro-irradiation technique at RIKEN has a precision high enough to selectively aim parts of these relatively small bacteria, while other micro-irradiation facilities are limited to much larger cells. This proposal therefore presents the first chance to decide between the different models for the large radiation resistance of DR using micro-irradiation. It is anticipated that this insight will also facilitate the understanding of radiation damage repair in human cells.

The eukaryotic genome is transcribed into abundant long noncoding RNAs (lncRNAs), that the applicant has shown are specifically expressed in precise cellular and subcellular locations. This project aims to test the hypothesis that that a major function of lncRNAs is to bind transcription factors and recruit their action to enhancers throughout the genome. RIP-Seq methodology will be employed to identify the total RNA and DNA sequences associated with three well known transcription factors, thereby to identify the full complement of lncRNAs associated with these transcription factors and provide an unbiased genome-wide map of the enhancer sites bound by them. A comparison of these two data sets will allow determination whether enhancers are transcribed into lncRNAs that then associate with transcription factors that bind to the original enhancers. This will establish the breadth of this regulatory strategy employed throughout the genome and inform subsequent functional characterization of individual lncRNAs. In addition the applicant will receive invaluable post-doctoral training in the international research environment provided at the Broad Institute and will initiate a long-term collaboration between the two leading proponents of lncRNA biology, the Mattick and the Rinn laboratories. The applicant will also be trained in new techniques that include high-throughput sequencing, an increasingly integral feature of genomic research. More broadly, the successful completion of this proposal will constitute a fundamental conceptual advance in our understanding of gene regulation and the importance of lncRNAs transcripts in cell biology.

Functional neuroimaging is an important and widely available approach to studying the awake and performing human brain. Still the fMRI has several crucial limitations in its ability to resolve and interpret how signals vary over time. This proposal is designed to use fMRI to develop new methods to extract more complete and accurate information about the time course of neural signals and brain connectivity.
Several investigators have developed methods to decode information in the fMRI signal by applying multivariate pattern classifier (MPC) techniques. Such analyses successfully decode the spatial pattern of activations in visual cortex in a way that is not possible with the ordinary fMRI. A particularly powerful approach for interpreting neural signals uses the spatial pattern of fMRI signals to estimate the parameters of the neural model. My project is designed to combine neuronal models of cortical activity parameters with MPC techniques and improve the time-resolution of fMRI.
The first phase of the proposed project examines the temporal sequencing of input stimulation from fMRI measurements in order to uncover the brain response over time. The project's second phase expands the analysis for the order of brain region processing in time. This analysis will estimate brain connectivity which is considered to play a major role in the human brain computational capability. To validate the results, I will repeat the time decoding analysis using a high time resolution methodology (e.g, EEG) and use structural brain connectivity measurements (DTI white matter tractography) to analyze potentially connected regions.

Interactions between hosts and their parasites are critical aspects of organismal evolution, yet the simultaneous effects of environmental temperature and host nutrition upon such interactions are rarely explored. We propose to infect caterpillars (Spodoptera spp.) with a virulent microparasitic pathogen (nucleopolyhedrovirus) and measure the effects of this challenge upon survivorship, development, and macronutrient intake and utilization at a range of temperatures from 15ºC to 40ºC. The Human Frontier Short-term Fellowship would allow the merging of my background in ecology and physiology with the insect immunity expertise in Professor Lee’s (host) laboratory. Learning viral challenge techniques, as well as working for the first time with the caterpillar (Spodoptera) model system, will allow me to pursue a career in insect immunoecology.

Autoimmune diseases are believed to involve the breakdown of multiple immunological tolerance pathways which results in overall disequilibrium of the immune balance. In type 1 diabetes, which affects 0.4% of Western populations, autoreactive lymphocytes progressively destroy the pancreatic insulin-producing cells, leading to a loss of glucose homeostasis. To allow better understanding of the tolerance breakdown leading to autoimmunity, animal models that develop a disease with features similar to human diabetes, like the nonobese diabetic (NOD) mouse, have been developed. Until now, the tolerance breakdown occuring in NOD mice has been mainly associated with peripheral defects. Recently, experimental evidence has shown that the autoimmune attack is also caused by central tolerance defects, i.e. failures to eliminate self-reactive thymocytes by negative selection. Although the transcriptional programs mediating this central breakdown were characterized by genomics and genetics, the molecular mechanism linking the NOD T cell receptor to altered nuclear programs is still unknown. Therefore, I propose to perform a molecular dissection of the signaling pathways in differentiating NOD populations. By comparing NOD and B6 signaling networks through focused biochemical analysis of known signaling molecules and large-scale proteome screening, and testing the candidate molecules by transgenic overexpression of modified or dominant-negative forms and by lentivirus-based knock-down, I intend to map the signaling nodes where the genetic variation is disturbing the signaling balance and determine by which mechanism these modifications result in alterations of the NOD transcriptome.

Karyogamy is the process where the nuclei fuse during yeast mating. Nuclear fusion is known to require microtubules (MTs), MT-associated-proteins (MAPs), and MT motors. Many of these proteins are known, but how they interact to bring about karyogamy is not well understood. My aim is to define the molecular mechanisms of nuclear fusion in fission yeast. Fission yeast mitosis is characterized by the nuclei moving apart due to the mitotic MT spindle. Karyogamy is characterized by the nuclei moving together due to the astral MT arrays. In many aspects, karyogamy can be viewed as “reverse mitosis”, and I hypothesize that many of the proteins involved in mitosis will also play similar roles in nuclear fusion. Through high-resolution imaging with a spinning disk confocal microscope, I will test my hypothesis by comparing the dynamics of the wild-type MT cytoskeleton with those of mutant cells where the MTs, MAPs, and/or motors are defective. My work will define mechanisms of nuclear fusion during karyogamy, and may further our understanding of the mechanisms of mitosis.

Hematopoietic stem cells (HSC) have the ability to cure fatal blood diseases due to their unique ability to self-renew and sustain multilineage differentiation for individual's lifetime. However, when challenged during in vitro culture, stress or ageing, HSCs may lose their unique functional properties. As recent studies in the Mikkola lab have shown that the transcription factor Mef2c is an anti-aging regulator in B-cells, and preliminary data suggest that it may also modulate HSC homeostasis, my proposal aims to investigate whether Mef2c acts to protect HSCs from premature aging.
I will first verify whether loss of Mef2c affects the frequency of phenotypic and functional HSCs differentially in young and older mice. I will then perform gene expression analysis on HSCs from Mef2c-deficient and control mice to identify Mef2c dependant pathways, and also CHIP-chip analysis to define genes that are regulated by Mef2c directly.
Understanding how Mef2c protects the longevity of HSCs may have major therapeutic implications for treating blood diseases.

ABSTRACT

State transitions involve a balancing of the light excitation energy between photosystem II (PSII) and photosystem I (PSI) which allows oxygenic photosynthetic organisms to optimize their photosynthetic yield and their metabolic state upon changes in their light environment. This process is governed by the redox state of the plastoquinone pool and involves the cytochrome b6f complex and the phosphorylation of the light-harvesting system (LHCII) and its displacement from PSII to PSI or the dephosphorylation of LHCII and its return from PSI to PSII. A genetic approach has recently identified the Stt7/STN7 thylakoid protein kinase in Chlamydomonas and Arabidopsis as a key component of state transitions and light acclimation. The aim of my proposal is to understand how the activity of Stt7/STN7 is regulated and to identify novel regulatory components of state transitions using a combination of biochemical, genetic and bioinformatic approaches. In particular I wish to characterize a large Stt7/STN7 membrane complex and to establish the interactome of this kinase. Novel regulatory components of state transitions, such as phosphatases and immunophilins, will be identified by a novel genetic screening method using mitochondrial mutants. The expertise in moleculer genetics and photosynthesis in Chlamydomonas and Arabidopsis of the host laboratory and my knowledge in membrane biology of mitochondria will complement each other for this project. I expect at the end of the fellowship to transfer the acquired knowledge and expertise to study the functional crosstalk between organelles and nucleus in alga and green plants as an independent researcher.

This research project will provide fundamental new insights into the mechanisms and functions of neuronal network activity in the basal ganglia, brain areas critical for movement. The main goal is to elucidate how synchronized neuronal oscillations are either advantageous or counterproductive for the operation of these brain circuits during movement. Excessive synchronization of rhythmic neuronal activity in the basal ganglia is a critical functional change accompanying Parkinson’s disease (PD). Studies in unmedicated PD patients show that synchronized oscillations preferentially occur at ‘beta’ frequencies (15-30 Hz), and suggest that, by inappropriately coordinating neuronal activities, exaggerated beta oscillations may play ‘anti-movement’ roles in PD. To answer the key questions of where and how exaggerated beta oscillations emerge, I will exploit a rat model of PD that shows these oscillations. I will conduct large-scale electrophysiological recordings throughout the basal ganglia and partner circuits, thus addressing ‘Where’. By applying pharmacological manipulations, large-scale network recordings and intracellular recordings of single neurons in key circuit components, I will determine the inputs, outputs and intrinsic properties necessary for generating beta oscillations, thus addressing ‘How’. Finally, after defining the synchronized oscillations present during both normal and Parkinsonian movement, I will test whether inducing beta oscillations causes movement problems and whether targeted disruption of beta oscillations restores performance. Thus, I will also lay foundations for developing new therapies that better counteract these pathological rhythms in PD.

Oscillations in neural activity are a prominent feature observed in recordings obtained from hippocampal neurons and extracellular space. These oscillations span multiple frequency ranges, and have been demonstrated to be important for neural coding. Despite this, the responses of neurons to inputs arriving at various oscillatory frequencies are not well understood. Voltage-gated ion channels play crucial roles in modulating neuronal responses to various oscillatory input patterns, with the possibility that each channel contributing to intrinsic neuronal responses to inputs at distinct frequency ranges. Inactivating potassium channels express in high densities in hippocampal pyramidal neurons, and exhibit intraneuronal gradients in their density and undergo activity-dependent plasticity. The overall goal of this proposal is to understand the role and relevance of these two characteristics to temporal coding of neuronal information. The specific hypothesis that will be tested is that the gradients and plasticity in inactivating potassium channels endow the neuron to tune its various dendritic regions to specific properties of inputs in the time-frequency space. This hypothesis will be tested through the development of time-frequency measures that are sensitive to inactivation, and experimentally using them to localize gradients and plasticity in the time-frequency space. A detailed interdisciplinary approach integrating advanced signal processing, biophysically realistic computational modeling, and long-term electrophysiological recordings from the cell body and dendrites of hippocampal pyramidal cells, with a specific timeline for various experimental modules is presented.

Although sleep is universal and critical for survival, its functions are still unclear. Increasing evidence suggests a role for sleep in brain plasticity, but the underlying mechanisms are unclear. Our current understanding is limited by an inherent gap between human behavioral studies and animal physiological studies. I aim to clarify how sleep may support neuronal plasticity by recording multiunit activity in humans during paired associative learning and during subsequent sleep. The project is a multidisciplinary collaboration between the UW sleep center and the UCLA epilepsy surgery program, which is specialized in human intracranial recordings. Subjects will learn associations between pairs of images eliciting selective responses in the recorded neurons. Neural activity from the same neurons will be recorded during subsequent sleep. In the morning, subjects will verbally recall response stimuli upon presentation of trigger stimuli and their performance evaluated. A counterbalanced learning session will be performed at a different time by having subjects learn in the morning and recall in the evening without intervening sleep. The proposed study will allow us to investigate the relation of learning to subsequent sleep, the relation of sleep to subsequent memory, and the information flow between cortex and hippocampus. To help interpret these results, I will perform parallel multiunit recording experiments in rats exposed to novel objects and during sleep. This proposal represents an excellent training opportunity to develop my expertise in sleep research, and will better prepare me to direct my own research to try and understand the function of sleep.

In eukaryotes, translation is initiated by a complex mechanism in which the 40S ribosomal subunit is recruited to the mRNA 5’-end cap structure, recognizes the start codon by a scanning mechanism before association with the 60S ribosomal subunit to form the active 80S ribosome. In an alternative pathway used by viruses such as the human pathogenic hepatitis C virus (HCV), an RNA structure called internal ribosomal entry site (IRES) located in the 5’ untranslated region recruits, properly positions, and activates the ribosome in a cap- and end-independent process which involves only a small subset of the initiation factors (eIFs) needed for canonical translation initiation. The aim of this proposal is to investigate the molecular basis of HCV IRES translation initiation using X-ray crystallography complemented by biochemical methods. Specifically, (i) the crystal structure of the human 80S ribosome bound to the HCV IRES shall be solved; (ii) essential IRES residues and their function in the 80S assembly process will be determined; (iii) the mechanism of IRES dissociation from the ribosome after translation has been initiated will be investigated, and (iv) whether IRES can reassociate with the ribosome after translation initiation thereby possibly enhancing multiple rounds of translation. The goal of the project is to provide new insights into both the function of the eukaryotic ribosome and the IRES-mediated translation initiation pathway. Insight from the proposed research may help designing new anti-viral drugs.

ATP-fuelled molecular motors are responsible for rapid and specific transfer of double-stranded DNA during several fundamental processes, such as cell division, sporulation, bacterial conjugation, and viral DNA transfer. A dramatic example of intercompartmental DNA transfer occurs during sporulation in Bacillus subtilis, in which ~3Mbp of chromosomal DNA are transported across a division septum by the SpoIIIE ATPase. The goal of this project is to elucidate the molecular mechanisms of DNA transfer machines by developing single-molecule and microscopy methods and applying them to the study of DNA translocation by SpoIIIE. We recently showed that SpoIIIE translocates DNA at ~5kbp/s while specifically interacting with highly skewed chromosomal sequences (SRS) that guide its directional motion. In this project, we will develop and use hybrid transverse magnetic tweezers, high-speed atomic force microscopy and a novel assay for monitoring DNA translocation in vivo to understand the molecular mechanism by which SpoIIIE/SRS interactions impart translocation directionality. Central to the understanding of this mechanism is the architecture of the membrane-bound SpoIIIE complex. Novel super-resolution microscopy methods will allow us to directly visualize the architecture and the assembly dynamics of this complex. Our studies showed that SpoIIIE can wirestrip DNA of nucleo-protein complexes and thus reset the cellular epigenetic program. We will use our new technologies to shed light on the molecular mechanism of DNA wirestripping and its biological relevance. The CDA will be key at this early stage in my career by helping me establish my first independent research project.

Helper and regulatory CD4+ T cells are major determinants and modulators of immune responses.
To date, well-defined helper CD4+ T cell subsets are Th1, Th2, Th17 and immunosuppressive regulatory T cells(Treg). These subsets can be characterized by the high expression of “lineage-specific” transcription factors (TFs), namely, T-bet/Runx3, GATA-3/c-Maf, RORgt, and FoxP3, in Th1, Th2, Th17 and Treg, respectively. However, it remains to be determined what signals control the expression of these lineage-specific TFs and other undefined key genes, and which genes are the targets of these TFs during T cell differentiation. In order to understand the dynamics of gene regulation during T cell development, I will combine mathematical modelling with genome-wide analysis of transcriptional regulation by microarray, and molecular and immunological experiments. With help of mathematical modelling, I will try to identify key target genes of the lineage-specific TFs with statistically ranked data. I will also use mathematical models of the “lineage-specific” TF interactions, most of which are in fact expressed by other T cell lineages, to identify the factors that determine T cell fate and assess the models by immunological experiments. Finally, I will predict signaling pathways that control the development of these T cell subsets by mathematical modelling and assess the prediction by molecular and immunological experiments including retroviral gene transduction and RNA interference. This will establish an iterative approach between mathematical modelling and wet experiments in this field.

nimals display remarkable diversity in body plans. However, the origins of this diversity and the mechanisms by which it evolved remain largely unresolved. Recent efforts to understand the evolution of body form have focused on the role of changes in the establishment of body axes and organization early in development arising from differences in gene expression during development. Research into the molecular basis for such changes in gene regulation have largely focused on single morphological traits and target genes from isolated genomic regions, yielding data that is too sparse and scattered to lead to general mechanistic principles. Systematic integrated studies of sequence evolution and the effect of sequence changes on the molecular events that underlie gene regulation are needed to infer general principles governing the evolution of genetic regulatory networks and thus of body shapes. I propose to apply new experimental genomic technologies to study the evolution of gene regulation and gene expression in multiple species from the fruit fly genus Drosophila, placing experimental analysis of gene regulation into an evolutionary framework. Genomes of both closely and distantly related Drosophila species (spanning dozens of million years) have been sequenced, and many genetic and developmental tools are available. This unique combination of developmental and genomic tools on a wide evolutionary range for the first time allows studying the evolution of the regulation of gene expression on a large scale, and may pave the way toward general laws governing body shape evolution.

Gene therapy approaches are based on the replacement of a mutated gene with a correct allele. Homologous recombination promoted by a specific DNA break in the proximity of the gene of interest is the most effective way to precisely insert the therapeutic gene. Homing endonucleases are employed in nature for similar tasks, but their sequence requirements do not necessarily match the target genes for therapy. The focus of my work will be, thus, the design of new homing endonucleases to attain the specificity required for gene therapy. Homing endonucleases will be computationally designed to target genes responsible for severe combined immunodeficiencies (X-SCID and ADA-SCID). The designed endonucleases will be further refined using directed evolution techniques. Therefore, the designed proteins will be of immediate impact as potential therapy. Moreover, the methods developed will represent a general approach for the generation of homing endonucleases with new specificities and for the design of new DNA binding proteins and nucleases with potential applications in the fields of genome engineering and gene therapy.

The maintenance of an undifferentiated state or the commitment to a specific lineage are fundamental transitions in the cell fate that involve nuclear reprogramming and changes in transcription. Alternative splicing affects the majority of mammalian genes and it can greatly expand proteomic complexity and regulate key biological processes. However, its role in cell fate determination during early embryogenesis is largely unknown.
We will use high-throughput analyses of gene expression, including whole genome splicing-sensitive microarray and deep sequencing, to explore changes in alternative splicing during the transition from totipotency to pluripotency in mouse preimplantation development, and from stem cell pluripotency to lineage differentiation in embryoid bodies derived from embryonic stem cells.
In addition, we aim to correlate these transitions with changes in the expression or activity of splicing factors, with the goal of understanding the logic of the regulatory programs behind these changes in posttranscriptional regulation.
Hypotheses derived from the transcriptome data analyses will be tested in vivo by monitoring the effect of downregulation of selected splicing factors on these developmental transitions. In addition, we will employ bichromatic fluorescent reporters that recapitulate specific splicing events to follow changes in splicing regulation in space and time at single-cell resolution during mouse preimplantation development.
The results of this research project should shed light on posttranscriptional mechanisms of genomic regulation, at exon-level resolution, during the acquisition of crucial cell fate decisions at the onset of embryogenesis.