The proposed project aims to delineate the mechanisms by which the salvador/warts/hippo (sav/wts/hpo) pathway restricts organ size during development and disease. When activity of the sav/wts/hpo pathway is reduced, cells grow and proliferate excessively and are resistant to programmed cell death. Defining the mechanisms by which this pathway functions is important, as several components of this pathway have been implicated in the genesis of human cancer. This study will employ a multi-faceted approach to investigate the mechanisms by which the sav/wts/hpo pathway restricts cell growth and number. We will confirm whether candidate downstream targets of the sav/wts/hpo pathway identified by microarray analysis, are genuine transcriptional targets of this pathway. We will then use Drosophila genetics and cell biology to examine whether these novel target genes regulate cell growth downstream of this pathway. Additionally, we will use bioinformatics to identify gene regulatory elements that are used by the sav/wts/hpo pathway to control the expression of known sav/wts/hpo target genes, as well as target genes that have been newly-identified in this study. The CDA is critical for the establishment of my independent research program and will allow me to assemble a team to perform the experiments outlined here. Ultimately I aim to establish a laboratory that makes important insights into the mechanisms that control cell growth and proliferation, and hence, organ size, during development. These studies should have implications for diseases such as cancer, which arise in part due to excessive cell growth and proliferation and insensitivity to programmed cell death.

In order to understand the evolution of genes and their function it is important to identify and quantify the evolutionary forces at work. To this end, neutral substitution rates of genes are estimated, which has so far mainly been done by estimating substitution rates at synonymous sites, i.e. at codon sites at that nucleotide changes do not change the encoded amino acid. However, there is accumulating evidence that synonymous sites might evolve under constraints also in primates. So far it was not possible to approach this question in a quantitative manner, because appropriate models that take into account the different mutation rates in the hypermutable dinucleotide CG (CpG) are missing. During this fellowship we intend to develop a likelihood method to quantify selection on synonymous and on non-synonymous sites at the same time. We will design this method to also accommodate CpGs as mutational hotspots. The neutral substitution rate will be inferred using intronic or flanking sequences. In addition we will estimate the strength of selection at synonymous sites by contrasting between-species divergence with human diversity data. These measures of constraint on synonymous sites will help to unravel the nature of constraints on synonymous sites and also improve the inference of selection at non-synonymous sites. Hence, I will supplement my skills in data analysis with mathematical and statistical knowledge. In the dawn of the post-genomic era with all the diverse large-scale functional datasets, sound statistical skills will be indispensable for successful research.

The overall objective of this proposal is to address key issues in transcriptional control in the yeast Schizosaccharomyces pombe. My focus is on a multiprotein complex, named SAGA, originally discovered in the yeast Saccharomyces cerevisiae, which controls transcription initiation by RNA polymerase II. SAGA is conserved throughout eukaryotes. The proposed experiments are to identify SAGA and its functions in the distantly related fission yeast S. pombe, in which chromatin organization and transcriptional regulation are remarkably different from that in S. cerevisiae and directly relevant to higher eukaryotes. This proposal contains two sets of experiments to address this issue. The first set addresses the roles of SAGA in key cellular processes, such as cell-cell separation and establishment of a specific cell mating identity. In the second set, a thorough biochemical analysis of SAGA in S. pombe will provide a necessary characterization of the complex in this model system. These experiments may help elucidate conserved as well as yet-unidentified specific mechanisms of transcription regulation among eukaryotes. During my Ph.D., I became fascinated by how tissue-specific gene expression is controlled by specific transcriptional mechanisms in mammals. As these mechanisms remain poorly understood, I decided to gain skills into fundamental transcription mechanisms using yeast and eventually to translate this knowledge back into mammals. My research background consists mainly of work in mice and this postdoctoral fellowship will give me the opportunity to learn how to answer new, but related biological questions, using a different model system.

The hepatitis C virus (HCV) core protein plays an important role in virus-host cell interactions. Importantly, transgenic mice expressing core accumulate intracellular lipids, a condition called steatosis, and develop hepatocellular carcinoma. The group of M. Ott has recently shown that a fraction of core localizes to the mitochondrial outer membrane via a C-terminal targeting motif. The goal of this fellowship is to study the role of mitochondrial core protein in HCV pathogenesis. We will generate core proteins mutant in the mitochondrial localization motif and and study their effect on apoptosis and lipid droplet formation in vitro. We will also introduce these mutations into transgenic mice who conditionally express core and study development of steatosis and hepatocellular carcinoma in vivo. Special focus lies on a potential physical and functional interaction of core with the recently identified sorting protein PACS-2 that integrates ER-mitochondria communication and regulates lipid transfer and apoptosis. My previous research was performed in yeast: I described an altruistic aspect of cell death during aging and characterized new cell death pathways and executors of apoptosis in yeast. This fellowship will allow me to move into the new field of molecular virology. I will acquire new expertise by working with primary mammalian cells, virally infected cells, and transgenic mice. This will importantly extend and complement my current training focussed on yeast cell biology and genetics. In the future, I would like to become an independent researcher who continues to investigate molecular mechanisms of diseases with direct implications for diagnosis and treatment.

The innate immune system uses a wide variety of microbial recognition receptors to identify potential pathogens. The main players in innate immunity are phagocytes such as neutrophils, macrophages, and dendritic cells (DC). All these cell types can express on the cell membrane Dectin-1, a C-type lectin-like receptor that recognizes different b-glucans and can mediate phagocytosis of intact yeasts, which contain b-glucans on their cell walls. In an attempt to explore the signaling pathways triggered by this receptor, the host laboratory has observed the specific involvement of Syk in Dectin-1 mediated signaling for synthesis of IL-10 and IL-2 in DCs, and the association of this kinase to the cytoplasmic tail of Dectin through the phosphorylated membrane proximal tyrosine. Dectin-1 also collaborates with other receptors such as TLR2 present on DCs to generate different responses. The purpose of this project is to determine whether ligand engagement can affect the conformation and/or oligomerization state of Dectin-1 and define the signaling pathways that get activated in response to different Dectin-1 ligands and to co-engagement of Dectin-1 with TLR2. Results from this study will lead to a better understanding of how different innate receptors expressed on DCs participate in the transmission of information to generate an adaptive immune response. The work in innate immune response represents a complete change in the field of work at the intellectual and technical level, but, at the same time, I can take advantage of my background for development of this project. This training will be very important to learn to develop a complete project by myself in the near future.

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.