The Histone H3-variant CENP-A (CID in Drosophila) has been previously demonstrated to be necessary for the kinetochore in a variety of organisms. We have shown that CID overexpression can also nucleate the formation of ectopic centromeres at a subset of CID incorporation sites. I am now interested in determining why some sites are chosen over others and preliminary data suggest that this occurs preferentially near telomeres. Other factors might therefore also contribute to functional kinetochore formation, such as proximity to flanking heterochromatin, other epigenetic marks or underlying DNA sequence. Flies and tissue culture cells with inducible acentric chromosomes will provide additional tools to test for preferential formation of neocentromeres. To address whether CID alone may be sufficient for kinetochore formation, CID will be fused with different DNA binding domains and targeted to specific DNA regions. Such sites will then be tested for acquisition of centromere function. I’m also planning to expand this study into human cells, which would provide a valuable tool in elucidating the well-documented but poorly understood generation of neocentric chromosomes in human patients. The tools and knowledge generated in this project will further help to investigate whether pulse-overexpression of CENP-A can also overcome the inefficiency of centromere formation in artificial chromosomes. The CDA would allow me to particularly support the more novel and innovative directions of the proposed project for which no preliminary data has been obtained yet.

The project aim is to investigate how the unconscious affects overt human behaviors. I will conduct fMRI and EEG in human subjects. They will first be primed by presentation of a variety of stimuli, including subliminal stimuli, followed by behavioral experiments to examine the consequences for overt behavior. Importantly, I will apply an independent component analysis (ICA) approach to extract individual signals from mixed signals of neuroimaging experiments. Using ICA, I will categorize the signal depending on the signal pattern and amplitude, and then relate them to behaviors. Furthermore I will use TMS to examine the functional importance of areas identified with fMRI and then use DWI and probabilistic tractography to examine whether co-activated areas are interconnected. This research field is entirely different from the themes of my previous molecular research work. I will now, however, attempt to investigate whether analytic approaches similar to those used in my previous research, focusing on Ca2+ patterning and cell function, are useful for understanding the factors that determine human behaviors. The focus on the identification of activation patterns is common in both researches. I expect that this project will help me broaden my knowledge and understanding of basic cognitive neuroscience and that it would also provide a good opportunity for training in functional imaging techniques. Importantly, I can learn to understand different points of view and interact with people from different scientific backgrounds. As a result, this project may potentially allow me to liaise between researchers in the fields of molecular biology and cognitive neuroscience.

The neural map embedded within the olfactory system is an internal representation of the external odor world. Odor coding mechanisms and establishment of olfactory sensory maps are best understood for olfactory sensory neurons within the nasal olfactory epithelium and for the olfactory bulb. In contrast, little is known with respect to processing of odorant information encoded in the olfactory bulb to the olfactory cortex and higher brain centers. To address this issue I will apply and develop mouse genetic techniques that allow (I) high resolution tracing of individual relay mitral cell axonal arbors during development with the aim to define the precise topographical image of the olfactory sensory map imprinted in the olfactory cortex and (II) elucidation whether neural activity is involved in the establishment, fine tuning or maintenance of the olfactory odorant map in the olfactory cortex and to which extent this map is innately predetermined. The proposed research will greatly complement my previous training in developmental brain research and represents the most valuable advancement towards the establishment of my independent research program. Directions in my future research have a strong basis on the proposed approaches including elucidation of molecular mechanisms responsible for establishment of precise synaptic networks in higher brain centers during development that serve as scaffolds to allow plastic changes in response to experience culminating in conscious behavior. The insight into these molecular mechanisms might support better characterization of pathology of the diseased brain and development of appropriate therapies.

The ubiquitin-proteasome system orchestrates cellular processes such as cell cycle, DNA repair, transcription, signal transduction, stress response, development, and apoptosis by selectively degrading regulatory proteins or damaged proteins. Hence, defects in this system are linked to severe diseases such as neurodegenerative disorders, inflammation and cancer. Currently, very little is known about the regulation of proteasome activity during the life of a multicellular organism. This proposal aims at establishing how proteasome activity is regulated during an animal’s lifespan and how this activity can be modified by genetic factors or stress cues by using C. elegans as the animal model. A combination of in vivo and in vitro approaches, including a live animal proteasome activity reporter system, are being developed to enable characterization of the proteasome activity at the organismal, tissue and cellular level. Genome-wide RNAi screening will be conducted to identify novel genetic modifiers of the ubiquitin–proteasome system and this approach should open up further research avenues. A deeper understanding of the ubiquitin-proteasome system and the subsequent effects on protein homeostasis could translate into the development of therapeutic options for treatment of proteasome-associated human diseases. As a starting Junior Group Leader at the University of Helsinki in 2006, I am required to obtain all my funding from competitive sources. The CDA would facilitate my transition into an independent investigator with my own research team as well as help me introduce C. elegans as an animal model system to the Finnish research community.

To unravel the molecular and cellular basis of learning and memory is among the most ambitious goals of modern science. Progress in recent years has provided evidence that remodeling of synapses is a fundamental mechanism for information storage and processing in the brain. Synaptic strength is determined in major part by the number of AMPA receptors in the postsynaptic membrane. Recent studies have shown that the AMPA receptor-binding protein GRIP1 plays an important role in the targeting of AMPA receptors to synapses. Although the biological function of GRIP1 remains unclear to date, data obtained during my HFSP Long-Term Fellowship in Morgan Sheng’s Lab at MIT suggest that GRIP is involved in the transport of AMPA receptors and various other synaptic cargo molecules in neurons (Hoogenraad et al., 2005). This proposal describes experiments that address the role of GRIP in synaptic protein trafficking in neurons. I propose to make use of a new (inducible) transport system to functionally characterize the GRIP motor binding domains and to study the regulation of motor binding to synaptic cargo in live hippocampal neurons during development and in response to synaptic activity. The GRIP protein gives me a tool to efficiently relocalize various cargos and study the functional relevance of intracellular targeting of synaptic proteins. This project allows me to extend my knowledge in the mechanisms of synaptic plasticity and to use my new technical skills (e.g. neuron culture, fluorescent time lapse imaging). My training at MIT provides a basis to successfully accomplish the more ambitious goals set out in this proposal.

Cognition and action are realized by integrated functions of the brain, which consist of interconnected structures, such as the cerebral cortex, basal ganglia, cerebellum, and thalamus. Studies have revealed that each structure and its subregions play distinct roles in perceiving sensory signals, linking sensory signals to appropriate movements, and executing movements. By adopting a multidisciplinary approach, I will study the neural mechanisms underlying cognition and action. The methodologies include 1) analyzing neuronal activity recorded from subjects performing a behavioral task, and 2) analyzing the anatomical connections of multiple structures from which the neuronal activity was recorded. As a behavioral model, I will use a motor behavior whereby subjects are required to perceive visual information and then generate motor information based on the visual signal. This paradigm is appropriate because it calls for both sensory recognition and motor planning processes. In the first series of experiments, I will analyze anatomical pathways. In the second series, I will analyze the neuronal activity of the interconnected areas. By combining these two approaches, I will investigate the structural and functional basis of cognition and action, and integrate them.

The understanding of cardiovascular morphogenesis and function is of fundamental interest to developmental biologists and physicians. The proposed cross-disciplinary project combines the techniques of selective plane illumination microscopy, fluorescent proteins, and transgenics to describe and understand the zebrafish heart function in physical terms. The zebrafish is the ideal system for in-vivo cardiovascular studies and a dedicated microscope will be set up to image the zebrafish heart in vivo at high spatial and temporal resolution. I will develop new transgenic lines that express fluorescent proteins in endocardial and myocardial cells. Photoactivatable and photoconvertible fluorescent proteins will be used to highlight individual cells and tissues to investigate their fate and function. The cardiac conduction system will be studied by developing a transgenic line expressing a calcium sensitive fluorophore. A four dimensional physical model of the zebrafish heart will be generated on the basis of the multi-color microscopy data showing function and development of wildtype and mutant hearts. This fellowship will allow me to make the transition from optics to developmental biology. With my background in physics I will now learn about the maintenance and breeding of zebrafish, I will acquire cloning and transgenics techniques and study the developmental principles of cardiovascular morphogenesis. The experience in my new area of research will be very different from my previous studies and will be essential for my professional growth focused on developmental biology. It will help me to establish my own developmental biology laboratory working with zebrafish.

Selective maturation of T cells in the thymus is critical for establishing one of the major limbs of the adaptive immune system. To produce a useful repertoire that lacks most potentially autoreactive clones, immature T cells undergo "positive" and "negative" selection steps. In this proposed study, I plan to investigate how regulation of ion fluxes in response to antigen receptor interaction with self peptide-presenting stromal cells in the thymus contributes to the positive and negative selection of double-positive (CD4+CD8+) immature T cells. To accomplish this specific aim, I will study the in situ behavior and intracellular Ca2+ response of double-positive T cells in freshly prepared thymic slices using two-photon laser scanning microscopy (established in the host laboratory) in combination with electrophysiological and biophysical techniques that I have acquired in my previous research on the physiological regulation of signaling in cells with excitable membranes, such as cardiomyocytes and neurons. The special focus of this proposed project is on the Ca2+ response and on ion channels. I will examine the potential roles of ion channel regulation in fine-tuning the Ca2+ response evoked by T cell receptor stimulation during selection Ca2+ events in the thymus. I believe that such interdisciplinary studies would enable me to make significant contributions in this field.

The specific aim of this proposal is the direct observation of the protein dynamics at the active site in the presence or absence of an intramolecular disulfide bond by use of vibrational echo methods. While the conformational changes of a protein are intimately coupled to its function, the conformational dynamics of a protein are difficult to discern. The Fayer lab at Stanford University specializes in physical and biophysical chemistry and performed the first ultrafast vibrational echo experiments approximately 10 years ago. On the other hand, I have used several molecular biological approaches to investigate the relationship between structure and function of proteins. I will combine physical chemistry and molecular biology to investigate the regulatory role of a disulfide bond. Neuroglobin (Ngb) is induced by neuronal hypoxia and protects neurons from hypoxia, which suggests that this protein may have a role in sensing or responding to neuronal hypoxia. Structural analysis has shown the presence in Ngb of a intramolecular disulfide bond which stresses the protein, and breaking the disulfide bond provides additional structural degrees of freedom of the protein, resulting in a release of O2. To understand the effects of the disulfide bond for the activity of Ngb, I will utilize vibrational echo methods for direct detection of the protein dynamics on time scales not accessible by other methods. The direct observation of the protein dynamics for Ngb in the presence or absence of the intramolecular disulfide bond will provide information on the redox-regulated activity of proteins.

In response to myocardial infarction or long-standing cardiac hypertrophy the myocardium suffers from a loss of cells due to apoptosis and necrosis, which can lead to heart failure. Therapeutic approaches to combating these disease sequelae have typically relied on the prevention of myocyte death or to promote replacement of lost myocytes. With respect to the potential replacement of myocytes, much investigation has centered on defining the molecular signaling pathways that mediate myocyte renewal. However, significant controversy persists as to the prevalence of myocyte self renewal in the heart and its potential physiologic impact. Here we propose a series of genetic studies in the mouse to definitively define the rates of myocyte renewal in the heart with aging and after stress stimulation such as pressure overload (hypertension) or myocardial infarction. We will make double transgenic mice to mark only cardiac myocytes that proliferate using a cyclin B1 promoter fused to the MerCreMer cDNA, which will be crossed with mice containing an LoxP-dependent transgene that Cre can rearrange to give expression of eGFP, marking only renewed cardiac myocytes. The MerCreMer cDNA permits labeling of cells only after tamoxifen injection, so that the experiment can be temporally controlled. This detection system would be coupled with genetic manipulation of signaling pathways in the heart that are likely to participate in the renewal process. For example, the sponsor's laboratory has generated cardiac-specific transgenic mice that overexpress the conducting subunit for the T-type calcium channel. Much evidence suggests that re-emergence of T-type calcium current is required for proliferation of smooth muscle cells, and many other cell types. In response to injury the smallest myocytes in the heart re-express some T-current, suggesting a potential role in renewal (T-current is normally only seen during cardiac development as the myocytes proliferate). Other pathways will also be investigated. Thus, we propose an in depth analysis of the manner in which myocyte self-renewal may be regulated in the heart and its ultimate physiologic significance. This area of investigation is substantially different than anything I have performed previously, as are all the proposed techniques.

The expansion of optical telecommunications has coincided with a heightened recognition of the benefit of light-based tools for biomedical sciences. The opportunity to utilize the tremendous advances in fiber optic devices and miniature components for basic and applied biology is therefore at hand. My research goal is to leverage my expertise in telecom photonics for the development of new optical imaging technologies for life science. In this program, I propose to develop fiber-optic endoscopic microscopy that can visualize internal organs of a living small animal minimally invasively through a small surgical opening in the skin. With this new instrument, biologists will be able to study the behavior cells, their interactions with internal organs in situ, and the efficacy of therapeutics with high resolutions at the cellular level. Furthermore, the minimally invasive procedure will allow the study to be performed over an extended period and repeatedly at multiple time points in the same animal without having to sacrifice it each time. This program will leverage the laser scanning intravital microscopy technologies developed in host institute, combined with my expertise in specialty optical fibers for high power pulse delivery, fiber lasers for low-cost compact multiphoton microscopy, and micro optics for the development of low-aberration endoscopes. Upon successful completion of the development, I intend to participate actively in the application of the instrument to biological researches. I like to take this opportunity to challenge myself in a highly multidisciplinary environment and broaden my skills to become an independent academic investigator in biomedical optics.

Genomic stability and DNA repair have been suggested to affect mammalian aging. In yeast, Sir2 maintains this genomic stability and longevity by its enzymatic function of NAD dependent histone deacetylase. This protein has seven homologs in mammals and is conserved throughout evolution. However, the functions of these proteins are largely unknown and by deciphering their function, network of protein in which they are involved and the mechanisms in which they are involved will lead to better understanding of genomic stability, cellular senescence and aging in mammals. By using in-vitro biochemical assays and in-vivo mice models I would like to determine the functions of these SIRT proteins and dissect the molecular mechanisms in which these SIRT proteins are involved. I Would like to concentrate on analyzing SIRT7 knock out mice to elucidate the function of SIRT7 and its involvement in genomic stability, cellular senescence and mammalian aging. These studies have great implications towards understanding malignancies related to aging and tumorigenesis.

Epigenetic modifications impart additional layers of heritable information upon the DNA code, regulating the underlying genetic information. Cytosine DNA methylation, a major epigenetic modification in higher eukaryotes, is involved in the regulation of gene expression, but the full extent and mechanisms of control are unknown. I propose to use Arabidopsis as a model system to identify the genome-wide extent to which DNA methylation regulates gene expression, and identify genes/genetic elements that control this regulation. Whole-genome tiling microarrays will be used to detect genome-wide sites of DNA methylation and transcriptional activity between wild-type and methylation-deficient mutant plants, identifying the genes transcriptionally regulated by DNA methylation. Furthermore, genes differentially regulated by DNA methylation between 19 resequenced Arabidopsis strains (ecotypes) will be identified. By forward-genetic mapping, genetic elements responsible for the differential methylation will be identified, thus elucidating molecular mechanisms controlling the regulation of gene expression by DNA methylation. My previous research used biochemical/molecular biological techniques to investigate plant mitochondrial biogenesis. Through a significant change in my research direction, this project will use forward genetic techniques, large-scale genomic analyses, functional genomics, and bioinformatics to investigate mechanisms of epigenetic regulation. This fellowship will give me the opportunity to broaden my research and training, and will provide me with experience crucial for achieving my goal of becoming an independent researcher in the field of epigenetic regulation.

Hematopoietic stem cells (HSC) have been extensively studied and manipulated, however very little is known about their interaction with their microenvironment in the bone marrow. Direct observation of the HSC niche has revealed a more complex picture than previously believed, with HSC residing in close proximity not only of the bone endosteum but also the stromal capillaries. I intend to combine advanced imaging techniques to visualize all marrow compartments in vivo at the same time and investigate the relationship between endosteal and vascular niches. Moreover, as Notch and Wnt pathways have been linked to HSC self-renewal, I will use a cell-specific gene targeting approach to delete Jagged1 and beta-catenin in the osteoblasts and analyze their role within the niche. These studies will bring together two areas of investigation developed in Dr. Scadden’s laboratory, namely models of stem cell/niche communication and high resolution in vivo microscopy, to gain a unique perspective on the dynamic interactions between HSC and their specialized microenvironment in physiological and altered conditions. After investigating lineage commitment of epidermal stem cell progeny during my PhD, this fellowship will give me the opportunity to broaden my experimental skills and address a different question in stem cell biology by focusing directly on HSC regulation. My long-term goal is to understand how similar signaling pathways cooperate in the maintenance of different self-renewing tissues and I expect that the expertise acquired during my PhD and with Dr. Scadden will put me in a particularly favorable position for the establishment of my own research group in the future.

Embryonic stem cells (ESCs) can differentiate into any tissue of the adult organism, including blood. The goals of the proposed project are (i) to determine whether morphogens that induce blood cell fate from embryonic mesoderm will direct ESCs to differentiate into hematopoietic stem cells (HSCs), (ii) to define the genetic pathways induced by morphogen signaling in ESCs, and (iii) to test if morphogens act as true instructive signals during hematopoietic differentiation. In order to activate morphogen signaling in differentiating ESCs, I will either directly supplement growth media with Indian Hedgehog (IHH), Bone morphogenetic protein (BMP4), and Wnt3a proteins, or express these factors from an inducible transgene. I will quantify the extent of HSC differentiation using known parameters of hematopoiesis. By RT-PCR and Chromatin Immunoprecipitation, I will test whether blood cell commitment induced by morphogen signaling occurs via the direct activation of the cdx and hox master regulatory genes of hematopoiesis. Finally, I will isolate cells not fated to form blood and determine the potential of morphogens to re-direct these cells toward the hematopoietic lineage. Previously, I used Drosophila as a model to study the epigenetic inheritance of gene expression states, which is required for the maintenance of cell identities. With this fellowship, I will investigate the role of morphogens in cell fate decisions in ESCs. Thus, I will be able to study a fundamental developmental process in one of the most dynamic fields at the interface between basic biology and applied research, which will be an excellent preparation for my long-term goal to establish my own laboratory.