Michael Levine - US grants

The aim of this research proposal is to assess some of the morphological and neurophysiological correlates of the aging process in the central nervous system. To study these the basal ganglia will be used as a model system. In one set of experiments we will determine the effects of the aging process on the cytoarchitectural and ultrastructural characteristics of basal ganglia neurons in cats. In a second set of experiments the effects of the aging process on the neurophysiological characteristics of basal ganglia neurons in cats will be determined. Wherever possible the morphological and functional alterations that occur in aged animals will be correlated to provide a picture of the neuronal dysfunctions that occur in senescence. In a third set of experiments th anatomical and physiological effects of aging will be related to some of the neurological disturbances that occur.

DESCRIPTION (adapted from application): This is an application to continue his investigations of complex loci of Drosophila to understand the regulation of promoter-enhancer interactions. He made seminal contributions in this area by elucidating how localized patterns of the gene even-skipped (eve) are established along the anteroposterior axis of the precellular Drosophila embryo. The regulation of the segmentation stripes is due to the combined action of broadly distributed activators and narrowly-expressed, short-range repressors. Short range repressors act at distance of less than 100 bp and repress the action of specific activators while long range repression regulates specific enhancers that lie >1000 bp away and are thought to act through repression of general machinery. The present application extends beyond this earlier work focusing on two general goals: 1) The mechanism of action of repressors, both short-range and long-range, and 2) The mechanism by which long-range enhancer-promoter interactions are modulated, in both a positive sense via facilitator proteins and in a negative direction via the use of insulator DNA or competition. There are 3 specific aims: (i) Identify repressors as either short-range or long-range and investigate the mechanism of repression by identifying co-repressors. (ii) Identify proteins required for the enhancer-blocking activity of the specific attenuator Fab-7 and the gypsy insulator. (iii) Investigate the role of promoter competition in the regulation of specific enhancer-promoter interactions in the ANT-C and BX-C gene complexes. The general approach in all of these aims is to use transgenic reporters whose expression is modulated by precisely engineered promoter and enhancers. For example, the initial experiments use a fly strain with a beta-galactosidase reporter transgene. The expression is controlled by enhancers from the eve loci, known as stripe 2 and stripe 3 enhancers since they are responsible for eve expression in these localized region of the precellular embryo. Repressor binding sites positioned close to stripe 2 enhancer and at a distance from stripe 3 can reconstruct the two modes of repression. Repression activity is monitored by the disappearance of blue stripes in the embryo. Other transgenes use Drosophila eye color gene white as the reporter.

The research in this proposal will elucidate mechanisms underlying changes that occur in the functional properties of neurons due to the process of aging. We will focus on a major part of the basal ganglia, the neostriatum, because this ares of the brain is in- volved in pathological conditions associated with aging. We will determine the nature and time-course of the age-related changes in the dopaminergic nigrostriatal connections and their interactions with corticostriatal afferents. In one set of experiments, the effects of the aging process will be determined on the ability of the putative neurotransmitters glutamate, gamma-aminobutyric acid, and dopamine to alter neurophysiological activity of caudate neurons. These experiments will concentrate on demonstrating that dopamine's ability to modulate corticostriatal responses is compromised during aging. The changes in membrane properties of striatal neurons during aging will also be determined. In a second series of experiments immunohistochemical procedures will be used to identify age-related changes in inputs to the striatum from the substantia nigra that contain dopamine and inputs that contain glutamate. The age-related changes of the relationship between dopamine containing synapses and synapses of corticostriatal inputs will be assessed in a final experiment. The studies outlined in this proposal will indicate how neurotransmitter changes during aging are reflected in neuronal function and how they relate to morphological alterations. The information obtained from these studies is important because pathologies in the neostriatum and/or the substantia nigra are involved in age-related neurological disorders exemplified by Parkinson's Disease and Huntington's Disease.

A core of instrumentation will be established to facilitate and promote a variety and ongoing series of projects in molecular biology and molecular genetics. The following new equipment items requested are: a DNA sequencer, and an expanded centralized computer system for DNA/protein sequence analysis. The facility will also include a DNA synthesizer, a fluorimeter, an HPLC, and a high voltage paper electrophoresis apparatus. The facility would be supervised and maintained by a dedicated technician, who would also carry out the DNA syntheses and the DNA sequencing gel runs. Major users of this facility are studying the expression of the dihydrofolate reductase gene in cultured mammalian cells, the role of the enhancer in immunoglobulin gene regulation, the mechanism of RNA splicing and 3' end formation, biogenesis of mitochondria in yeast, transcription termination and antitermination in E. coli, and the characterization of genes controlling cell differentiation in C. elegans. The facility would be available to all 24 laboratories of the Columbia Department of Biological Sciences, and to a limited extent, to undergraduates.

In insects, segmental identity is specified by the homeotic genes, which in Drosophila are found in two separate clusters (ANT-C and BX-C) and in the beetle Tribolium in a single one (HOM-C). These genes contain a conserved DNA sequence, the homeobox, which encodes a 60 amino acid DNA-binding region. Recent work on the mouse has resulted in the identification of four clusters of homeobox containing genes, the Hox genes, which appear to be true homologues of the insect ANT-C plus BX-c, with the same spatial organization along the chromosome and showing the same relative order of expression along the anterior- posterior axis. Similar clusters have been found in other vertebrates, including humans. While the function of the vertebrae genes is mostly unknown, the structural homologies of the Hox and HOM clusters have led to the proposal that an ancestral version of these gene clusters arose before the protostome-deuterostome evolutionary divergence and that it was involved in the specification of positional information during development. Dr. Macagno proposes to characterize the corresponding cluster (LOX-C) of these genes in a more primitive invertebrate, the leech and to compare it to the insect and mammalian clusters in terms of structure, expression and organization, He has already identified several leech homeobox genes which are expressed in spatiotemporal patterns resembling those of vertebrate Hox and insect homeotic genes. %%% This investigation will contribute to our understanding of the evolution and function of a set of genes important in morphogenesis.

Dorsal is a maternal regulatory factor that initiates dorsoventral patterning of the precellular Drosophila embryo. It is a member of the Rel family of transcription factors that is initially distributed throughout the cytoplasm of unfertilized eggs, but shortly after fertilization it translocates into nuclei. This nuclear transport process is regulated by the Toll signaling pathway, which is employed in a variety of processes, including insect immunity and acute/inflammatory responses in mammals. The resulting Dorsal nuclear gradient specifies the embryonic mesoderm, neurogenic ectoderm, and dorsal ectoderm, through the differential regulation of target genes such as snail, sog, and dpp. The snail gene encodes a zinc finger repressor that establishes the boundary between the presumptive mesoderm and neurogenic ectoderm. It is a short-range repressor that must bind within 100 bp of either upstream activators or the core promoter in order to inhibit gene expression. Dpp is a member of the TGF-beta family, and a Dpp activity gradient subdivides the dorsal ectoderm into the amnioserosa and dorsal epidermis. The Dpp gradient depends on sog, which encodes a secreted inhibitory protein that binds Dpp. High levels of Sog are thought to inhibit Dpp signaling, while low levels enhance signaling. The proposed study represents a continuation of our efforts to determine how the Toll-Dorsal signaling pathway controls the dorsoventral patterning of the early Drosophila embryo. The research plan includes three specific aims: (i) determine how Snail functions as a repressor; (ii) identify the components of the Toll signaling pathway that diffuse in precellular embryos to create the Dorsal nuclear gradient; and, (iii) determine how Sog- Dpp interactions create a peak Dpp signaling threshold that specifies the amnioserosa.

Abstract University of Illinois - Chicago - K. DahlenID,u Connection to NSFNET Proposal No. NCR-9317686 The University of Illinois at Chicago (UIC) needs to significantly expand its intra-campus connections to resources available through the NSFNET. At the same time, the University needs to provide a means of integrating Internet resources and networked connectivity into its existing information services. An integral part of this plan is to educate nursing students, educators, researchers, and practitioners to the advantages of campus and Internet resources by promoting access and networked connectivity and mechanisms for improving communication, increasing information access, and for sharing knowledge. The long-term goal is to extend this prototype to the other health professional schools and to the campus as a whole. The main objectives are: to streamline library operations and connectivity; to improve patron service; to develop new forms of collaboration and knowledge-sharing among nursing practitioners in the field; and to test educational strategies to integrate Internet resources into both the curriculum and practice of nursing.

9502957 Levine The proposed project is to add additional hardware infrastructure to provide low latency and high bandwidth connectivity between the vBNS and supercomputers at each supercomputing center. It will also connect network-attached disk arrays to provide very short term storage buffering for running the distributed applications. These added components will integrate the four independent supercomputing centers into a single distributed teraflop computing facility. This integration will provide a testbed for a number of development and development projects in applied computer science and computational sciences. ***

DESCRIPTION (adapted from applicant's abstract) The experiments in this proposal are designed to continue investigations into neuronal processes controlling dopamine modulation of responses mediated by activation of glutamate and GABA receptors in the striatum. The first two aims are a direct continuation of studies begun in the presently funded proposal and will examine the hypothesis that the direction of DA modulation of responses mediated by activation of glutamate receptors is a function of the subtype of glutamate receptor activated and the subtype of DA receptor activated. The third aim is new and will examine the hypothesis that the direction of DA modulation of responses mediated by activation of GABA-A receptors is a function of the DA receptor subtype activated, its site of location on pre- and/or postsynaptic elements and the type of neuron.

The goal of this project is to develop a theory for nonparametric, multidimensional, item-level measurement. The investigator's approach to multidimensional modeling relies on two recent advances in measurement: the submodel theorem and ForScore. The submodel theorem asserts that smooth multidimensional measurement models have equivalent unidimensional submodels. ForScore, a suite of unidimensional model fitting programs, provides means for fitting various unidimensional models that are equivalent to the smooth multidimensional models. The project will result in a set of computer programs for implementing the theory and a demonstration application of the theory in counseling psychology. Latent variable models are used in physical, biological, and social sciences. Multivariate latent variable modeling is less well developed than univariate modeling. If the project is successful, more complex and accurate applications of latent variable models will quickly become possible because researchers will be able to reduce problems involving many variables to univariate problems. For example, consider the analysis of gender differences. Men and women on average respond differently to questions of vocational choices. This research will allow the application of powerful unidimensional differential item functioning measures to be used in the two-dimensional domain of vocational choice. The analysis will increase our understanding of the etiology of gender differences in vocational choice and assist in the design of instruments that will be more useful to women.

DESCRIPTION: This proposal addresses the functional development of glutamate receptors (GluRs) in the neostriatum (NS), concentrating on the N-methyl-d-aspartate (NMDA) receptor. NMDA receptors are considered one of the most important subtypes of GluRs and there is considerable evidence that nervous system development is critically dependent upon NMDA receptor function. Understanding developmental regulation of NMDA receptors is particularly important in the NS where control of motor programs and cognitive abilities are determined. The main driving force behind NS activation and the most important transmitter system in the NS is the Glu-containing system that originates from the cortex. This system makes monosynaptic contacts with all subtypes of NS cells, has a primary role in NS information processing, is implicated in use-dependent plasticity and during early developmental periods may have trophic influences. If Glu inputs and receptors are the prime activators of adult NS cells, the development of GluRs can be expected to have major implications for NS functioning during postnatal maturation. Experiments are designed to examine when GluRs become functional and when Glu-containing synapses make functional contacts on two subpopulations of NS cells, medium- and large-size cells. One hypothesis forms a framework for this proposal. It states that there are two age periods when NMDA receptor function in the NS will be particularly important, an early period [from postnatal days (PNDs) 7-14] as asymmetrical synapses are forming, and a later period (PNDs 20-22) at the end of the peak period of formation of corticostriatal synapses. To test this hypothesis, NMDA receptor development will be compared with that of (-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate (KA) receptors in the two subpopulations of NS neurons. State-of-the-art electrophysiological methods in which cells in slices are visualized before and during whole cell clamp recordings will be used to assess the developmental alterations in GluR agonist-evoked currents. Mechanisms by which NMDA receptor function changes developmentally will be examined. These include development of voltage-dependence, development of receptor binding, development of mRNA and protein expression for GluR subunits, and development of modulation of NMDA receptor function by metabotropic GluRs, dopamine and protein kinases. In combination, complementary information will be integrated to provide a complete picture of the development of NS GluRs. The outcomes will provide information necessary to understand the role of these receptors in NS development and provide clues for generating rational strategies to treat GluR dysfunction during development and in the adult.

The hypothesis to be examined in this project is that loss of nigrostriatal dopamine (DA) neurons in Parkinson's disease (PD) and in animal models of PD1 as well as the pharmacological and surgical treatments of PD alter the functional characteristics of glutamate, Y-amino butyric acid (GABA) and DA receptors in subthalamic nucleus (STN) neurons. The STN has become important for understanding changes in basal ganglia function in PD since it is clear that one outcome in PD is a marked change in the activity of STN neurons. The current model of why STN neuronal activity changes is based on the hypothesis that DA loss in PD leads to a release of the STN from tonic inhibition by the extemal pallidurn. Increased activity of the STN, the only excitatory projection nucleus in this systern1 then provides the major excitatory drive onto basal ganglia outputs. There is a growing consensus that this model of PD is unsatisfactory. Thus, this project is aimed at examining other alternatives. We will use in vifm electrophysiology in STN slices to examine changes in receptor function after unilateral DA depletion and after three treatment paradigms, the classic, chronic L-DOPA treatment, the recent approach of deep brain stimulation of the STN, and a novel approach of implanting GABA-producing cells into the STN. There are four aims that will test our central hypothesis. Aim I will determine if DA depletion alters responses mediated by activation of specific glutamate receptor subtypes and GABA receptors in STN neurons. Aim II will determine if DA depletion alters subsequent DA modulation of responses mediated by activation of specific glutamate receptor subtypes and GABA receptors in STN neurons. Aim III will test the hypothesis that treatment with L-DOPA after DA depletion restores normal responses to the activation of glutamate, GABA/A and DA receptors in the STN. Aim IV will test the hypothesis that implantation of cells that produce excess GABA in STN or deep brain stimulation of STN after DA depletion (two procedures that silence STN neurons) alter abnormal responses induced by activation of glutamate, GABA/A, and DA receptors in either the entopeduncular nucleus (homologue of the internal pallidal segment of primates) and/or the substantia nigra pars reticulata. The results from this project, combined with those from Projects 1 and 3, will provide a more complete understanding of the mechanisms underlying changes in STN neural activity to design new, rational pharmacotherapies for PD that can use the STh as a therapeutic target.

The overall goal of the proposed study is to exploit the extensive lineage maps and simple embryonic development of the Ascidian, Ciona intestinalis, to characterize the regulatory pathway underlying notochord differentiation. Ciona is a simple chordate that possesses a small genome (comparable in size to the genomes of C. elegans and Drosophila). This simplicity suggests that common features of all chordates, such as the notochord, might be specified by "stripped down" pathways that are directly applicable to vertebrate systems. A detailed notochord lineage has been characterized; after just nine embryonic cleavages the fully differentiated notochord contains only forty cells (about 10-12 hours after fertilization). Dr. Levine believes that Ciona is well poised for molecular analyses, particularly the elucidation of developmental pathways. Much of the project's effort will be devoted to the establishment of a blastomere disaggregation assay, whereby the embryonic activities of potentially interesting regulatory proteins are defined within the context of detailed lineages. This assay will involve the microinjection of early embryos with appropriate mRNAs. Individual, defined blastomeres will be cultured after disaggregating staged embryos, thereby permitting an assessment of autonomous versus nonautonomous regulatory interactions. Special efforts will focus on three regulatory proteins that have been implicated in notochord development in other systems, including axial/pintallavis, floating head/Xnot, and brychyury/T.

Area; Charge; Communication; Communities; Computer Programs; Computer software; Computers; Data; Data Banks; Data Bases; Data Storage and Retrieval; Databank, Electronic; Databanks; Database, Electronic; Databases; Development and Research; Devices; Distributed Systems; Electromagnetic, Laser; Equipment; Faculty; Human Resources; Individual; Information Technology; Institution; Internet; Investigators; Lasers; Manpower; Medical center; Microscope; Modeling; Policies; Posters; Posters [Publication Type]; Preparation; Printing; Production; Programs (PT); Programs [Publication Type]; Purpose; R &D; R&D; Radiation, Laser; Research; Research Personnel; Research Resources; Researchers; Resources; Savings; Schools, Medical; Security; Services; Site; Slide; Software; Students; System; System, LOINC Axis 4; Systems, Distributed; Time; Universities; Update; Vendor; WWW; clinical data repository; clinical data warehouse; computer program/software; cost; data acquisition; data repository; data retrieval; data storage; design; designing; digital; laptop; medical schools; member; personnel; posters; programs; relational database; research and development; response; web; world wide web

[unreadable] DESCRIPTION (provided by applicant): This proposal will examine cellular mechanisms underlying the dysfunctions detected in Huntington's disease (HD) using four different murine models. The lethal mutation in HD produces an expanded trinucleotide (GAG) repeat within the protein huntingtin. It causes selective neurodegeneration especially in the striatum and cortex, by an unidentified mechanism. Each of the HD models we will examine exhibits a different phenotype produced by unique transgene constructs or 'knocked-in" GAG repeat lengths. By evaluating multiple models we will be able to examine the dysfunctions in more detail and understand the specificity and sequence of physiological changes common to HD and the models. Based on our preliminary studies, we have uncovered several common cellular deficits in two models. These are enhanced responsiveness of N-methyl-D-aspartate (NMDA) receptors in the striatum associated with increased Ca2+ flux, a marked decrease in K+ conductances and a change in the corticostriatal synaptic response. A third model also displays the enhanced response to NMDA. Some of these changes potentially predispose striatal medium-sized spiny neurons to excitotoxic damage. Using a physiological approach, we will examine four hypotheses concerning the cellular mechanisms of dysfunction in HD: 1) alterations in ionotropic glutamate receptor function and changes in evoked and spontaneous excitatory synaptic inputs to striatal neurons 2) alterations in metabotropic glutamate and dopaminergic receptor modulation of ionotropic glutamate receptor function, 3) alterations in K+ conductances and 4) alterations in Ca2+ conductances. The precise onset of changes will be investigated in relationship to the expression of behavioral deficits by using animals that are presymptomatic or after development of overt motor signs. We will examine striatal and corticostriatal neurons, visualized in the slice preparation or acutely dissociated cells, to characterize basic functions by current- and voltage-clamp analyses. Because HD destroys so many different capabilities - intellectual, physical and emotional - the insights gained from this research elucidating the cellular malfunctions in HD are relevant to understanding other GAG repeat disorders and neurological diseases associated with protein aggregate pathologies like Alzheimer's and Parkinson's disease.

This award is to organize a workshop entitled: "New Paradigms for Developing Peta-scalable Codes" to be held at the Pittsburgh Supercomputer Center on May 3-4, 2003. The two-day workshop will focus on languages and frameworks to facilitate the development of computer programs that will efficiently utilize many thousands of processors. The organizers have identified a number of outstanding speakers who will provide training on modern parallel programming languages, and specialized libraries that are useful in developing scaleable computer codes for massively parallel architectures.

The 3rd EPSRC/NSF/UK/US Workshop will be held in Washington, D.C. April 15 -16, 2004. The meeting will again be organized on the basis of the Memorandum of Understanding (MOU) signed between the National Science Foundation (NSF) and the Engineering and Physical Sciences Research Council (EPSRC( to promote collaboration and co-operating in research activities between the two countries.

The primary object of this 3rd Workshop will be to promote more co-operation and interaction among the computational sciences from the United States and the United Kingdom in the area of High Performance Computing, focusing on materials and biological applications, especially those requiring access to the
large national computing facilities. Goals for the meeting are to articulate current and future needs (for the bio and materials areas) with respect to the following areas at a minimum: current and future uses of existing HPC machines and data rchives; current and future needs for grid based applications and next generation HPC hardware and software needs. The meeting will also discuss areas where the biological and materials sciences could profitable collaborate and how to facilitate the development of US-UK collaborative projects and will discuss impediments to such projects (funding, use of each others facilities, etc.).

The US organizers are Ralph Roskies of the Pittsburgh Supercomputing Center and Michael Klein of the University of Pennsylvania. The UK organizer will be Mark Sansom from Oxford. Representatives of the NSF will also be invited, as will representatives form the UK funding agencies.

[unreadable] DESCRIPTION (provided by applicant): The experiments in this proposal are designed to continue our investigations into cellular electrophysiological processes controlling dopamine (DA) modulation of responses mediated by activation of ionotropic glutamate receptors (iGluRs) in medium-sized spiny neurons of the striatum (MSSNs). The complex interactions between DA and iGluR-mediated neurotransmission within the striatum form the underpinnings of movement sequencing, motivation and reward responses, and psychological normalcy, just to provide a few examples. Imbalances in the interplay of these neurotransmitters have devastating consequences that are apparent in prevalent neurological and neuropsychiatric diseases such as Parkinson's and Huntington's diseases, attention deficit hyperactivity disorder (ADHD), schizophrenia, Tourette's syndrome, and many addictions. We have shown that DA, via D1 receptor activation enhances responses mediated by NMDA receptors while D2 receptor activation attenuates responses mediated by non-NMDA receptors (AMPA/KA). For example, when a D1 agonist was applied and a response was mediated by NMDA receptors, 98% of the time the response was enhanced. When a D2 agonist was applied and a response was mediated by non-NMDA receptors the response was attenuated 100% of the time. Other combinations (D2-DMDA, D1-non-NMDA) were less predictable. We will continue to focus on these interactions as an underlying theme, but will evaluate new areas pertaining to DA modulation. First, we will assess DA-iGluR interactions in a novel mouse model of ADHD that has the DA transporter (DAT) knocked down to 10% of basal levels. This produces a hyperDA state. Our working hypothesis is that DA modulation of iGluR transmission is altered in this genetic model and we have preliminary data to support it. Second, we will further examine mechanisms that control the predictability of DA modulation of GluR responses determining why the D2-NMDA and D1-non-NMDA receptor interactions are less predictable. Our hypothesis is that if factors controlling these interactions can be reduced, the interactions become predictable. We will use a novel mouse model in which enhanced green fluorescent protein is expressed under the control of the promoters for the D1 or D2 DA receptors or the M4 muscarinic acetylcholine receptor. This will allow electrophysiological recording in identified MSSNs that make up the direct or indirect output pathways of the striatum. Third, we will begin to dissect the NMDA receptor in MSSNs to determine how DA modulation is affected when selective subunits or their components (NR2A, NR2A-C-terminal, NR2B) have been removed or blocked pharmacologically. Our working hypothesis is that MSSN subunit composition of the NMDA receptor is an important determinant in predicting the outcome of D1 modulation. Together, these studies will provide important and new information about the physiological uniqueness and fundamental characteristics in ADHD, new information about the factors underlying the direction of DA modulation of iGluR neurotransmission and distinguish the contribution of NMDA receptors to DA modulation. We possess the unique tools and reagents to perform these studies and they will contribute to development of novel drug strategies to intervene in the treatment of ADHD as well as other diseases involving DA-iGluR interactions. [unreadable] [unreadable]

Michael S. Levine, IOS-0745322
Heart Morphogenesis in the Ascidian, Ciona intestinalis

In organisms such as humans, internal organs within the body are evenly distributed throughout the body cavities. However, the "rudiments" of the respective organs arise from nearby locations within the fetus. The orderly arrangement of the organs depends on "directed cell migration," a process whereby the precursors of a specific organ, such as the heart, move from its site of origin to its final location in the adult body. Disruptions in directed cell migration cause many human diseases, including congenital heart defects (cardia bifida). It has been very difficult to study directed cell migration in vertebrate embryos, even relatively simple vertebrates such as fish and frogs, since the progenitors of the heart and other internal organs consist of hundreds or thousands of cells at the time of their migration. During the past few years the Levine lab has developed a simple model organism for studying heart migration, the sea squirt or ascidian, Ciona intestinalis. A variety of recent molecular studies suggest that the sea squirts are the closest living relative of the vertebrates and the same basic mechanisms of cell migration are likely used for heart cell migration in sea squirts and vertebrates. However, it is much easier to study this process in the sea squirt tadpole since it is composed of relatively few cells. At the time of its migration the sea squirt heart rudiment is composed of just four cells. In the proposed work, the Levine lab will identify and characterize the genes required for heart cell migration. The basic mechanisms revealed in this study should apply to heart cell migration in vertebrates, including humans. The Broader Impacts of this work include the elaboration of the fundamental principles of heart development using a powerful, yet simple, model system, generation of resources for the Ciona community, and training of a wide range of researchers, from undergraduates to postdoctoral fellows.

genetics; model

Genetic factors recently have been implicated in rare, familial forms of Parkinson=s disease (PD) and these factors may also function in the more common sporadic form of the disorder. Different genes appear to be involved including alpha-synuclein, parkin, UCH-L1, NR3A2 (NURR1) and DJ-1. Discovery of specific genes involved in the disease is extremely useful in understanding the progression of PD, basic cellular mechanisms, risk factors and provides opportunities to test out new treatment paradigms to halt or delay the progress of the disorder. The general working hypothesis is that mutations in genes involved in PD can be utilized in mouse models to identify early underlying neurotransmission alterations in PD both in the classical nigrostriatal pathway as well as other brain regions critical for the progression of this disorder. The areas studied will be the striatum and the substantia nigra pars compacta [which contains the dopamine (DA) neurons that project to the striatum]. In addition, we hypothesize that areas outside of the striatum and substantia nigra also will display a progression of neuronal dysfunctions that may be linked to PD behaviors. Thus, we also will examine the progression of electrophysiological changes in sensorimotor cortical areas. It is within multiple areas of the brain that physiological changes lead to many of the abnormalities that ultimately cause the symptoms of PD. There are two specific aims: 1) To determine how genetic manipulations known to cause PD in humans when replicated in mice alter the synaptic responses of medium-sized spiny striatal neurons, cortical pyramidal neurons of sensorimotor cortex and their modulation by DA and how the basic electrophysiological properties of these neurons and SN DA neurons are altered; and 2) To determine how knock-out of parkin or over-expressionof alpha-synuclein alters amino acid receptor function and its modulation by activation of DA receptors in medium-sized striatal neurons and cortical pyramidal neurons of sensorimotor cortex. We will examine basic neuronal communication and electrophysiological characteristics and then the changes in the functional properties of glutamate, _- aminobutyric acid (GABA) and DA receptors in the striatum, sensorimotor cortical pyramidal neurons and substantia nigra DA neurons in multiple genetic mouse models. We will use existing mouse models (parkin knock-out and alpha-synuclein over expressing mice) that we have obtained through collaborations as well as the new mouse models generated in the Core and tested in Projects 1 and 2. This project is closely integrated with Project 1 which will examine the progression of behavioral and neuropathological changes in the same mouse models (we will obtain time points for electrophysiological analyses based on data from that project) and Project 2 which will concentrate on the progression of neurochemical changes (we will obtain information on pharmacological approaches from that project). This project also will benefit from information obtained on the association of parkin and alpha-synuclein with components of synaptic vesicles obtained in Project 4. In a similar manner the information we provide will benefit Projects 1, 2, and 4 (by providing specific functional information about synaptic changes) and could provide a bridge for the clinical data obtained in Project 5 by indicating the progression of the types of synaptic changes that occur. Thus, the analyses of these models provides our Center with unique opportunities in a highly interactive, multidisciplinary environment aimed at understanding the progressive mechanisms of cellular dysfunction causing PD. Of primary clinical significance, such analyses can provide novel tools for preclinical testing of neuroprotective strategies.

This award provides participant support costs for the third Kent-Purdue Mini-Symposium on Financial Mathematics, to be held on April 21-22, 2007. The conference will expose participants to an array of cutting-edge research topics in financial mathematics, while promoting the bidirectional nature of the interactions between Mathematics and Finance.

The conference features three distinguished speakers in quantitative finance, who will deliver one-hour talks, as well as five invited 25-minute presentations. A poster session will be held for graduate students and postdocs.

DESCRIPTION (provided by applicant): The fatal mutation in Huntington's disease (HD) leads to an expanded glutamine repeat within the huntingtin protein which causes neuronal dysfunction typically followed by selective neurodegeneration especially within the striatum and cortex. These dysfunctions in neurons and circuits occur during the development of the disease phenotype, well before there is significant cell loss. The experiments in this application are designed to understand the functional changes that occur in specific populations of neurons during the progression of the HD phenotype and to uncover new targets and approaches for therapies. Our working hypothesis is that the most conspicuous cellular dysfunctions leading to pathology in HD result from a combination of cell- autonomous changes and cell-cell interactions. This two-hit hypothesis implies that mutation of the gene in the cell alone may not be sufficient to cause significant dysfunction; other changes have to occur to cause symptoms of the disease, and some of these include altered intercellular synaptic interactions. Previously, we examined changes in the striatum, the cortex and corticostriatal interactions, as the cortical input is one of the two major excitatory inputs to the striatum. However, the excitatory thalamic input to the striatum may be as important as the cortical input in the HD phenotype. It is presently unclear if both thalamostriatal and corticostriatal pathways contribute equally or differentially to alterations in striatal neurons. Aim 1 will use optogenetics to specifically and separately activate striatal glutamatergic inputs to identified subpopulations of striatal neurons and determine their relative contribution to cellular alterations. Medium-sized spiny neurons of the direct and indirect striatal output pathways also display unique, selective and complex alterations as the HD phenotype progresses. These will affect their targets in globus pallidus and substantia nigra. To our knowledge, striatal outputs in HD have not been studied in any detail, especially in mouse models, yet they are extremely important because they determine how the basal ganglia influence the thalamus and cortex. Aim 2 will specifically examine alterations in striatal output target structures while Aim 3 will manipulate striatal output pathwas differentially in an attempt to counter the imbalance of direct and indirect pathways as the disease progresses. Our studies use state-of-the-art optogenetic techniques to specifically activate or inhibit subclasses of neurons as well as genetic techniques to remove expression of the mutant huntingtin gene in subclasses of neurons. Together, the studies will provide the basis for novel and rational treatments for HD by delineating more restricted targets spatially and temporally and will be relevant for understanding other CAG triplet repeat diseases and neurodegenerative disorders.

[unreadable] DESCRIPTION (provided by applicant): The experiments in this proposal are designed to continue our investigations into cellular electrophysiological processes controlling dopamine (DA) modulation of responses mediated by activation of ionotropic glutamate receptors (iGluRs) in medium-sized spiny neurons of the striatum (MSSNs). The complex interactions between DA and iGluR-mediated neurotransmission within the striatum form the underpinnings of movement sequencing, motivation and reward responses, and psychological normalcy, just to provide a few examples. Imbalances in the interplay of these neurotransmitters have devastating consequences that are apparent in prevalent neurological and neuropsychiatric diseases such as Parkinson's and Huntington's diseases, attention deficit hyperactivity disorder (ADHD), schizophrenia, Tourette's syndrome, and many addictions. We have shown that DA, via D1 receptor activation enhances responses mediated by NMDA receptors while D2 receptor activation attenuates responses mediated by non-NMDA receptors (AMPA/KA). For example, when a D1 agonist was applied and a response was mediated by NMDA receptors, 98% of the time the response was enhanced. When a D2 agonist was applied and a response was mediated by non-NMDA receptors the response was attenuated 100% of the time. Other combinations (D2-DMDA, D1-non-NMDA) were less predictable. We will continue to focus on these interactions as an underlying theme, but will evaluate new areas pertaining to DA modulation. First, we will assess DA-iGluR interactions in a novel mouse model of ADHD that has the DA transporter (DAT) knocked down to 10% of basal levels. This produces a hyperDA state. Our working hypothesis is that DA modulation of iGluR transmission is altered in this genetic model and we have preliminary data to support it. Second, we will further examine mechanisms that control the predictability of DA modulation of GluR responses determining why the D2-NMDA and D1-non-NMDA receptor interactions are less predictable. Our hypothesis is that if factors controlling these interactions can be reduced, the interactions become predictable. We will use a novel mouse model in which enhanced green fluorescent protein is expressed under the control of the promoters for the D1 or D2 DA receptors or the M4 muscarinic acetylcholine receptor. This will allow electrophysiological recording in identified MSSNs that make up the direct or indirect output pathways of the striatum. Third, we will begin to dissect the NMDA receptor in MSSNs to determine how DA modulation is affected when selective subunits or their components (NR2A, NR2A-C-terminal, NR2B) have been removed or blocked pharmacologically. Our working hypothesis is that MSSN subunit composition of the NMDA receptor is an important determinant in predicting the outcome of D1 modulation. Together, these studies will provide important and new information about the physiological uniqueness and fundamental characteristics in ADHD, new information about the factors underlying the direction of DA modulation of iGluR neurotransmission and distinguish the contribution of NMDA receptors to DA modulation. We possess the unique tools and reagents to perform these studies and they will contribute to development of novel drug strategies to intervene in the treatment of ADHD as well as other diseases involving DA-iGluR interactions. [unreadable] [unreadable]

The aim of this project is to develop the methodology for estimation and testing of the nonlinear time series models involving a large number of predictor variables. The key idea is to adapt a number of techniques that are used in the nonparametric regression theory and that can be, after suitable justification, transferred to the time series setting. In addition to the above, the decision-theoretic properties of the resulting estimators are established for the first time. This is achieved by establishing asymptotic equivalence results between the nonparametric regression and various nonlinear time series models.

The models studied in this project are very commonly encountered in different areas of application. Many of these are the areas of federal strategic interest. As an example, one can mention forecasting the levels of future flooding and predicting the volume of production in many areas of industry. Another very important area of application is building the models that explain long-term changes in sea surface temperature and, by doing so, help explain and predict the future changes in the global climate. All of the above models often include a lot of predictor variables and this makes the choice of the model needed very difficult in practice. Based on the methods and tests proposed in this project, efficient model selection procedures can be enacted that can greatly help in choosing the right model. After the right model is chosen, high quality forecasts can be obtained that are not only of interest for researchers in science, but that also benefit society as a whole.

1123512
Levine

This Pan-American Advanced Studies Institutes (PASI) award, jointly supported by the NSF and the Department of Energy (DOE), will take place during April 16-25, 2012 at the Universidad de la República and the Institut Pasteur in Montevideo, Uruguay. Organized by Dr. Michael S. Levine and Dr. Ida Chow from the Society for Developmental Biology (SDB) in Bethesda, Maryland, the institute will focus on a systems biology approach to understand the mechanisms of organismal evolution. This PASI is part of a collaborative program between the SDB and the Latin American Society for Developmental Biology (LASDB) to train junior scientists on integrating bioinformatics and systems biology approaches with traditional experimentation to further our understanding of evolutionary processes.

The increased sophistication of computational methods, whole-genome analysis, and high-resolution imaging technologies provide concrete opportunities for cross-disciplinary approaches to elucidate the mechanisms of Darwinian evolution. Using these cutting edge tools and innovative approaches on different model and indigenous organisms, this PASI will contribute to the training of future scientists in the Americas. Participants with different backgrounds?from computer and mathematical sciences to biology and chemistry?who are interested in evolutionary biology will be considered and the most qualified will be selected, with care to maintain diversity on training, research themes and background within the student population. Participants will stay on (April 26-29) to attend the 6th International Meeting of the LASDB, where they will be able to present their own work to a larger international audience.

The Society for Developmental Biology has held annual meetings since its founding in 1939 and they have become the major conference to attend to learn about the latest findings in developmental biology. This three-year award will support three annual meetings: in Montreal, Canada (2012), in Cancun, Mexico (jointly with the 17th International Congress, 2013), and at the University of Washington, Seattle, (2014). The meetings attract an increasingly international audience, especially now that technological advances provide cross-disciplinary approaches to probe previously unresolved mechanisms. They cover questions that have fascinated scientists as well as many lay people for centuries: How does the genome of a fertilized egg specify all the different cell types and organs in the adult? What are the environmental (macro and micro) effects on the development and maintenance of organisms? The meetings are timely, describing major progress toward understanding developmental processes at the genetic, cellular, and systems biology levels. Speakers at the platform (plenary and concurrent) sessions, as well as authors of posters discuss topics from the core of developmental biology and report on exciting and important new advances. The meetings also traditionally provide an important venue to share and discuss unpublished data. Essential training is provided, as students and postdoctoral fellows have ample opportunity to discuss their results with their peers and with senior investigators. SDB meetings also provide a forum to educate practicing scientists and trainees to communicate with non-specialists, especially in regard to issues such as the "debates" appearing in news media and political circles concerning teaching evolution, human cloning, and stem cell research. This is important because developmental biology represents a significant opportunity for understanding evolution and preservation of species, and for contributing to the well being of our planet and its biodiversity.

This project aims to develop theory and methods for estimation of the functional components and weights in the nonparametric multivariate finite mixtures. These mixtures only assume that the components are drawn from some family of multivariate density functions without any parametric specification. The investigators, who are already active in this emerging area of research, adapt a number of estimation methods that are known from finite parametric mixture theory to the nonparametric context. The PI and the co-PI propose a number of practically feasible and fast algorithms that can be used to compute the resulting estimators in practice. Finally, both investigators show how to obtain large-sample asymptotic results for the proposed estimators.

Finite nonparametric mixtures of distributions can provide answers to many practically important questions. As an example, they can be used to help a physician in establishing the definitive diagnosis in case of a complex medical condition with a number of possible diagnoses. An example of such a situation is a patient with a possible heart attack where other differential diagnoses are also possible. Developmental psychology provides another useful example. Indeed, study of cognitive development in children, in particular identification of strategies used by children to accomplish various tasks, can also be modeled easily using these mixtures. This has important implication for developmental psychology, providing answers to many difficult questions faced by child psychologists while helping children mature and develop in an optimal way. The PI and the co-PI propose a number of efficient algorithms to estimate these mixtures and accomplish the practical tasks mentioned above. These algorithms will be publicly available and easy to use as part of the R software package called mixtools.

This project focuses on development of number sense in 7 to 11 year old students through a 3D action game that will train the brain. The project tests a hypothesis that playing a number-sense action game can help children learn beyond the game, making them better overall at number-line sense, precision of numerosity, speed of numerical judgments, and ability to multitask numerical tasks. The innovation is twofold: a game, adapted from first-person shooter games, to train number sense, and a platform that makes it easy to vary aspects of the game (e.g., repetition, speed, number of possibilities) to be able to analyze what is it that is making a difference with respect to learning. Research is identifying the qualities of computer games that will train the brain to be automatic in its judgments and at qualities of experiences that promote such automaticity.

This project aims to promote fundamental mathematical competence in a way that makes the mathematics learning feel effortless and genuinely fun. In particular, the aim is to help children develop number sense, an ability to very quickly make estimates and numerical judgments. Such capability is key to keeping up in mathematics; children without that sense tend to lose interest in mathematics because mathematical computation becomes too frustrating. Training number sense, and, in turn, fostering mathematical performance, should allow more learners to think of themselves as "good at math." Cultivating a child's interest in mathematics from a young age has been identified as key to feeding STEM career paths.

The vehicle for promoting such mathematics learning is a new type of video game with foundations in first-person shooter games. Rather than shooting, players cast spells that require quick numerical estimates. There is reason to believe that such a game will be as engaging as first-person shooter games are. First-person shooter games have been shown to increase attention and executive control in adults, the kinds of cognitive skills known to foster academic achievement. Because engaging video games will reach and be played by a wide range of children; such a game has potential to reach at-risk populations who most need to better develop their number sense and those important skills. The intent is for the game to be used in informal settings, providing a complementary tool for educators to foster mathematics skills. Research that is done in the context of this game will identify the characteristics of game play that promote skill automation, foundational knowledge that is needed to provide a road map for creating child-friendly consumer video games to improve other important aspects of core cognition beyond number sense.

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a genetic autosomal neurodegenerative disorder that is always fatal and for which there are no effective treatments or cures. Patients carrying the mutation display motor dysfunction, cognitive impairment and psychiatric disturbances. Neuropathologically, HD is characterized by neuronal loss in the striatum and cortex and a progressive disconnection between cortex and striatum, interrupting the flow of information from the cortex to the basal ganglia. We have shown that imbalances in synaptic activity in the direct and indirect striatal output pathways differ during early and late stages of the disease and contribute to motor symptoms in two full-length transgenic mouse models of HD. In early stage HD, there is increased glutamate and GABA release onto direct pathway medium-sized spiny neurons (MSNs) while GABA release is increased in the late stage but only onto indirect pathway MSNs. Changes in synaptic activity are associated with increased repetitive behaviors in early stage HD mice and with decreased locomotion in late stage mice. Early stage changes may be mediated by elevated striatal dopamine (DA), because depletion of endogenous DA reduced repetitive behaviors and reversed some of the electrophysiological alterations. In contrast, decreased locomotion in late stage HD might be mediated by decreased DA function. The goal of this application is to employ novel optogenetic approaches, using light stimulation to activate and/or inhibit DA terminals in a mouse model of HD, to better understand the electrophysiological and behavioral dysfunctions. In Aim 1 we will selectively inhibit DA release in the striatum in early stage HD, using optogenetics by expressing halorhodopsin (which inhibits firing when activated by yellow light) in DA neurons. In Aim 2 we will selectively increase DA release in the striatum in late stage HD using optogenetics by expressing channel rhodopsin (which increases firing when activated with blue light). We hypothesize that reducing striatal DA release in early stage HD will restore synaptic activity of MSNs and will have beneficial effects on abnormal repetitive movements. In late stage HD, increasing DA release will restore some of the balance in MSN activity and will alleviate motor symptoms. PUBLIC HEALTH RELEVANCE: In Huntington's disease, abnormal striatal dopamine transmission induces time-dependent alterations in excitatory and inhibitory synaptic transmission that contribute to imbalances in activity of the direct and indirect striatal output pathways leading to motor and cognitive disturbances. In order to modify the differential symptoms in early and late stages of Huntington's disease, this application will alter dopamine release using novel optogenetic approaches to uncover new targets to alleviate symptoms and slow the progression of this devastating genetic disorder.

This award provides funding for Sherlock, a Cray YarcData uRiKA data appliance consisting of a next-generation Cray XMT supercomputer (NG-XMT) running the uRiKA application architecture and augmented by Cray XT5 compute nodes to broaden the range of relevant applications to address the challenges of graph-based data analytics. The system will be deployed at the Pittsburgh Supercomputing Center (PSC). The Cray NG-XMT is a massively multithreaded supercomputer, based on the Cray XT5 infrastructure and specialized for analytics. Its Cray-proprietary Threadstorm 4.0 processors implement multiple, powerful features to support lightweight multithreading, latency hiding, and advanced memory interfaces with AMD HyperTransport-attached SeaStar2 interconnect chips to provide a flat, globally-addressable memory. This will be the first system of its kind to be available to the NSF research community.

DESCRIPTION (provided by applicant): We propose to use the sea squirt, Ciona intestinalis, as a simple chordate model to study key vertebrate developmental processes. Ciona embryos and larvae resemble simplified vertebrate tadpoles with a prominent notochord, dorsal hollow neural tube, and centralized brain. Recent molecular phylogenetic studies indicate that Tunicates (Urochordates) such as Ciona are the closest living relatives of the vertebrates. Ciona possesses a number of exceptional features for elucidating vertebrate developmental processes. The genome is small (~16,000 genes and 160 Mb) and lacks the whole-genome duplication events that bedevil functional genetic analyses in vertebrates. The tadpole is composed of only ~2,000 cells that arise from simple and well-defined lineages, comparable to those seen in C. elegans. It is possible to transform thousands of synchronously developing embryos with desired transgenic DNAs via electroporation. Simply put, the gene networks underlying key vertebrate developmental processes, such as specification of neural crest, are highly conserved in Ciona but not in C. elegans, Drosophila or other invertebrate model systems. The Gans and Northcutt new head theory proposed that most tissues of the vertebrate head represent novel innovations with no homologous counterparts in invertebrates. In vertebrates, these tissues arise from cranial neural crest and placodes, derived from the boundary between the neural tube and anterior neural plate. The focus of this revised proposal is the specification of the Ciona rudimentary cranial placodes and neural crest at the anterior border of the neural tube. We will use a combination of cell- specific labeling methods, cis-regulatory analysis, targeted misexpression assays, gene disruption methods, cell sorting techniques and computational modeling to delineate gene regulatory networks controlling these processes. The research plan includes the following three specific aims: (1) to determine gene regulatory networks governing the specification and invagination of the stomodeum and associated placodes; (2) to determine the gene networks regulating the specification of the putative pituitary; and (3) to characterize the specification of the pigmented otolith and ocellus, and identify Twist target genes responsible for the directed migration of mesenchyme and synthetic ectomesenchyme.

The Pittsburgh Supercomputing Center (PSC) will carry out an accelerated, development pilot project to create, deploy and test software building blocks and hardware implementing functionalities specifically designed to support data-analytic capabilities for data intensive scientific research. Building on the successful Data Supercell (DSC) technology which replaced a conventional tape-based archive with a disk-based system to economically provide the much lower latency and higher bandwidth data success necessary for data-intensive activities, PSC will implement and bring to production quality additional functionalities important to such work. These include improved local performance, additional abilities for remote data access and storage, enhanced data integrity, data tagging and improved manageability. PSC will work with partners in diverse fields of science, initially chosen from biology, astronomy and computer science, who will provide scientific and technology drivers and system validation. The project will leverage current NSF/CI investments in data analytics systems at PSC. Those investments include DSC, Blacklight (an SGI UV1000 with 2×16TB of hardware-enabled cache-coherent shared memory), and Sherlock (a YarcData ?Urika? graph-analytic appliance which also supports a globally accessible shared memory), both very capable for data analytic applications. Their tight coupling to the pilot storage system will allow synergistic development of analytical capabilities with development of increasingly sophisticated mechanisms for data handling. Working with the new, multi-petabyte data store, they will constitute a system specifically optimized for data intensive work as contrasted with conventional HPC systems. Blacklight will be upgraded with more powerful technology, specifically architected to satisfy the more demanding needs of data analytics in years 3,4. When successful, PSC will engage the NSF to consider larger-scale deployment aiming at exascale capacity.

1. Abstract: Nontechnical Description

The Pittsburgh Supercomputing Center (PSC) will provide an innovative and groundbreaking high-performance computing (HPC) and data-analytic system, Bridges, which will integrate advanced memory technologies to empower new communities, bring desktop convenience to HPC, connect to campuses, and intuitively express data-intensive workflows.

To meet the requirements of nontraditional HPC communities, Bridges will emphasize memory, usability, Title and effective data management, leveraging innovative new technologies to transparently benefit applications and lower the barrier to entry.
Three tiers of processing nodes with shared memory ranging from 128GB to 12TB will address an extremely broad range of user needs including interactivity, workflows, long-running jobs, virtualization, and high capacity. Flexible node allocation will enable interactive use for debugging, analytics, and visualization. Bridges will also include a shared flash memory device to accelerate Hadoop and databases.

Bridges will host a variety of popular gateways and portals through which users will easily be able to access its resources. Its many nodes will allow long-running jobs, flexible access to interactive use (for example, for debugging, analytics, and visualization, and access to nodes with more memory. Bridges will host a broad spectrum of application software, and its familiar operating system and programming environment will support high-productivity programming languages and development tools.

Bridges will address data management at all levels. Its shared Project File System, connected to processing nodes by a very capable, appropriately scaled fabric, will provide high-bandwidth, low-latency access to large datasets. Storage on each node will provide local filesystem space that is frequently requested by users and will prevent congestion to the shared filesystem. A set of nodes will be optimized for and dedicated to running databases to support gateways, workflows, and applications. Dedicated web server nodes will enable distributed workflows.

Bridges will introduce powerful new CPUs and GPUs, and a new interconnection fabric to connect them. These new technologies will be supported by extremely broad set of applications, libraries, and easy-to-use programming languages and tools.
Bridges will interoperate with and complement other NSF Advanced Cyberinfrastructure resources and large scientific instruments such as telescopes and high-throughput genome sequencers, and it will provide convenient bridging to campuses.

Bridges will enable important advances for science and society. By supporting pioneers who set examples in fields not traditionally users of HPC, and by lowering the barrier of entry, this project will spur others to recognize the power of the technology and transform their fields, as has happened in traditional HPC fields such as physics and chemistry. The project will engage students in research and systems internships, develop and offer training to novices and experts, extend the impact of the new system to minority schools and EPSCoR states, impact the undergraduate and graduate curriculum at many universities, raise the level of computational awareness at four-year colleges, and support the introduction of computational thinking into high schools.

2. Abstract: Technical Description

The Pittsburgh Supercomputing Center will substantially increase the scientific output of a large community of scientific and engineering researchers who have not traditionally used high-performance computing (HPC) resources. This will be accomplished by the acquisition, deployment, and management of Bridges, a HPC system designed for extreme flexibility, functionality, and usability. Bridges will be supported by operations, user service, and networking staff attuned to the needs of these new user communities, and it will offer a wide range of software appropriate for nontraditional HPC research communities. Users will be able to access Bridges through a variety of popular gateways and portals, and the system will provide development tools for gateway building.

Innovative capabilities to be introduced by Bridges are:

1. Three tiers of processing nodes will offer 128GB, 3TB, and 12TB of hardware-supported, coherent shared memory per node to address an extremely broad range of user needs including interactivity, workflows, long-running jobs, virtualization, and high capacity. The 12TB nodes, featuring a proprietary, high-bandwidth internal communication fabric, will be particularly valuable for genome sequence assembly, graph analytics, and machine learning. Bridges will also include a shared flash memory device to accelerate Hadoop and databases. Flexible node allocation will enable interactive use for debugging, analytics, and visualization.

2. Bridges will provide integrated, full-time relational and NoSQL databases to support metadata, data management and efficient organization, gateways, and workflows. Database nodes will include SSDs for high IOPs and will be allocated through an extension to the XRAC process. Dedicated web server nodes with high-bandwidth connections to the national cyberinfrastructure will enable distributed workflows. The system topology will provide balanced bandwidth for nontraditional HPC workloads and data-intensive computing.

3. Bridges will introduce powerful new CPUs (Intel Haswell and Broadwell), GPUs (NVIDIA GK210 and GP100), the innovative, high-performance Intel Omni Scale Fabric to support increasingly productive development of advanced applications, supported by an extremely broad set of applications, libraries, and easy-to-use programming languages and tools such as OpenACC, parallel MATLAB, Python, and R.

4. A shared Project File System (PFS) will provide high-bandwidth, low-latency access to large datasets. Each node will also provide distributed, high-performance storage to support many emerging applications, intermediate and temporary storage, and reduce congestion on the shared PFS.

Bridges will enable important advances for science and society. By supporting pioneers who set examples in fields not traditionally users of HPC, and by lowering the barrier of entry, this project will spur others to recognize the power of the technology and transform their fields, as has happened in traditional HPC fields such as physics and chemistry. The project will engage students in research and systems internships, develop and offer training to novices and experts, extend the impact of the new system to minority schools and EPSCoR states, impact the undergraduate and graduate curriculum at many universities, raise the level of computational awareness at four-year colleges, and support the introduction of computational thinking into high schools.

? DESCRIPTION (provided by applicant): One of the most fundamental problems in modern biology is to understand dynamic gene activity in time and space in the context of native chromosomes in living cells. The goal of the proposed study is to measure the levels of transcription produced by defined long-range chromosomal interactions in living cells. Traditional live imaging methods lack the spatial resolution to accurately determine the dynamics of gene activity, while bulk assays using fixed material strongly limit investigation of temporal dynamics. Here we propose to overcome these limitations by developing new methods of microscopy and computational analysis. Most of the studies will exploit the unique advantages of the early Drosophila embryo for the development of quantitative live cell imaging methods. Previous studies have identified hundreds of such interactions, and we will sample several of these to provide a titration of varying distances, from tens to hundreds of kilobases, as seen in mammalian systems. There are two specific aims: 1. Develop high-resolution imaging methods and associated computational algorithms for the visualization and quantification of dynamic enhancer-promoter interactions at select endogenous loci in living embryos. 2. Label regulatory regions and associated transcription units of individual genetic loci exhibiting long-range interactions, including trans-homolog associations during transvection at Hox loci, to measure in vivo the effect of chromosome topology on transcriptional activity. We plan to extend this approach to include the visualization of several hundred fluorescent DNA foci in a library of genetically engineered fly lines to establish a general overview of the dynamics of an entire chromosome in a living embryo and its impact on transcription. The successful realization of the proposed studies will greatly augment our current capacity to superimpose whole-genome maps based on fixed tissues onto the dynamic chromosomes of living cells. The resulting technologies will be immediately applied to the visualization of chromosome dynamics in mammalian tissues, particularly multipotent progenitor cells such as mouse hepatoblasts.

Fostering Science, Technology, Engineering and Mathematics Trajectories: Bridging Early Childhood Education Research, Practice, and Policy

Early childhood education is at the forefront of the minds of parents, teachers, policymakers as well as the general public. A strong early childhood foundation is critical for lifelong learning. The National Science Foundation has made a number of early childhood grants in science, technology, engineering and mathematics (STEM) over the years and the knowledge generated from this work has benefitted researchers. Early childhood teachers and administrators, however, have little awareness of this knowledge since there is little research that is translated and disseminated into practice, according to the National Research Council. In addition, policies for both STEM and early childhood education has shifted in the last decade.

The Joan Ganz Cooney Center and the New America Foundation are working together to highlight early childhood STEM education initiatives. Specifically, the PIs will convene stakeholders in STEM and early childhood education to discuss better integration of STEM in the early grades. PIs will begin with a phase of background research to surface critical issues in teaching and learning in early childhood education and STEM. The papers will be used as anchor topics to organize a forum with a broad range of stakeholders including policymakers as well as early childhood researchers and practitioners. A number of reports will be produced including commissioned papers, vision papers, and a forum synthesis report. The synthesis report will be widely disseminated by the Joan Ganz Cooney Center and the New America Foundation.

The Discovery Research K-12 program (DRK-12) seeks to significantly enhance the learning and teaching of science, technology, engineering and mathematics (STEM) by preK-12 students and teachers, through research and development of innovative resources, models and tools (RMTs). Projects in the DRK-12 program build on fundamental research in STEM education and prior research and development efforts that provide theoretical and empirical justification for proposed project.

CORE C: Abstract The need for physiological assessments was generated by UC-TRaN faculty who required electrophysiological studies beyond collaborations. These electrophysiological assessments consisted of experiments performed in brain slices, acutely isolated neurons or in cultures, providing a functional analysis of changes in neurons, local circuits and microcircuits induced primarily by genetic alterations in cellular, mouse or rat models. The first objective of Core C will be to continue these analyses because the need remains and the Core will assist investigators with functional analysis at the cellular, circuit, and systems level using state-of-the-art electrophysiology and optogenetic recording methods in in vitro and in vivo preparations. Recent advances in both genomic and stem cell technologies, particularly hESC and hiPSC, have opened the door to new approaches in IDD research based on human cells. For example, cortical neurons harboring genetic mutations with a given disorder can in principle be readily produced from hiPSC generated from patients. A significant challenge remains to translate this promise into new discoveries about the basis of IDD and therapies. The second objective of this Core will be to facilitate this process by assisting UC-TRaN investigators to produce, propagate, and differentiate hESC and hiPSC into neural and glial cell types of interest to create in vitro models of IDD. Another function of the Core is to work with investigators and molecular screening resources available at UCLA to harness the potential of these in vitro models for drug discovery.

? DESCRIPTION (provided by applicant): The fatal mutation in Huntington's disease (HD) leads to an expanded glutamine repeat within the huntingtin protein which causes neuronal dysfunction typically followed by selective neurodegeneration especially within the striatum and cortex. These dysfunctions in neurons and circuits occur during the development of the disease phenotype, well before there is significant cell loss. Recent studies in animal models have emphasized that synaptic cell-cell interactions play a role in the pathophysiology of this disease. For example, removing mutant huntingtin from the cerebral cortex ameliorates some HD symptoms. The experiments in this application are designed to understand the functional changes that occur in specific populations of cortical neurons during the progression of the HD phenotype and to uncover new targets and approaches for therapies. However, little is known about functional changes in cortical neurons, although these neurons also degenerate in HD. Before motor symptoms become apparent, sensory, cognitive and emotional disturbances occur and these seem to depend on aberrant communication in the cortex that probably involves thalamocortical pathways. These pathways have never been examined in HD. Our overarching hypothesis is that sensory and motor cortical areas are differentially and asynchronously affected during HD progression. We propose that sensory thalamocortical pathways are downregulated early leading to faulty integration and interpretation of sensory signals. In turn, the motor cortex becomes upregulated and disorganized, leading to altered corticostriatal communication and motor symptoms. In this grant proposal we will use state-of-the-art techniques in three different laboratories at UCLA. Aim 1 uses optogenetics and slice electrophysiology to examine mechanistically altered synaptic communication between thalamic sensory and motor nuclei and their corresponding cortical projection areas. Aim 2 uses high-density silicon microprobes to record firing of hundreds of neurons simultaneously in sensory and motor cortical areas as well as thalamic nuclei. Aim 3 uses genetically encoded calcium indicators to visualize neuronal activity in sensory and motor cortical areas. Together, the studies will provide new and important mechanistic insights into the understudied cortical dysfunction and will provide the basis for novel and rational treatments for HD by delineating more restricted targets spatially and temporally. These studies also will be relevant for understanding other CAG triplet repeat diseases and neurodegenerative disorders.

? DESCRIPTION (provided by applicant): During the past 25-30 years the two grants under consideration for merger in the MIRA program (GM46638 and GM34431) have provided numerous insights into the mechanisms underlying the control of gene expression during animal development. Specific highlights include evidence that Hox proteins function as sequence-specific transcription factors, the characterization of the complex eve stripe 2 enhancer, and the elucidation of the affinity threshold model for the differential regulation of gene expression by te Dorsal (NF-kB) gradient. These studies have established the early Drosophila embryo as a premiere system for the study of gene activity in animal development. It is the goal of the proposed MIRA grant to exploit the advent of new imaging technologies to uncover novel mechanisms of gene control. During the past year we have begun to use live imaging methods to visualize the dynamic regulation of gene expression during Drosophila embryogenesis. These studies have led to a number of striking observations that we wish to follow-up during the upcoming funding period, including transcriptional bursts of eve stripe 2 expressions, the non-additive activities of shadow enhancers, transcriptional memory, and allelic communication. We will determine whether transcriptional bursting is due to unstable enhancer-promoter looping interactions, and examine the possibility that the two alleles at a given genetic locus somehow communicate to ensure balanced levels of expression during development. The research plan includes four specific aims: 1) investigate the dynamics by which a single enhancer activates two different target promoters; 2) explore the possibility that temporal precision depends on enhancer switching, whereby enhancers with overlapping activities work in a sequential manner during development; 3) determine whether transcriptional memory depends on specific histone modifications such as methylation; and 4) substantiate our preliminary evidence for allele communication by examining a variety of sensitized transgenes in living embryos.

Summary One of the grand challenges of modern biology is to understand how gene activity is controlled in space and time, in the context of native chromosomes and in individual living cells. The goal of this proposal is to tackle exactly this challenge: we will develop new approaches to measure and manipulate long-range chromosomal interactions and quantify their effects on gene expression, in real-time and in living cells and tissues. By quantitatively mapping the relationship between transcription factor assembly (e.g. formation of biomolecular condensates), chromosome organization and transcription kinetics, our study will define how gene expression is controlled at unprecedented resolution. Transcriptional regulation forms the basis of cellular differentiation during organismal development, and its defects underlie a variety of disease states, from developmental disorders to cancer. Yet current methods are limited: traditional live-imaging lacks the spatial resolution to accurately define chromosome organization at the scale of individual genes, while bulk assays using fixed material are ill-suited for studying temporal dynamics. In addition, membrane-less nuclear condensates, which form through liquid-liquid phase separation, are thought to play key but as-yet-undefined roles in regulating transcription. To address these challenges, we will develop new imaging methods to measure chromosomal distances in living cells and build optogenetic tools to assemble/disassemble chromosome loops and nuclear condensates. We will deploy these tools to examine regulatory interactions at genomic scales characteristic of enhancer? promoter interactions in flies and mammals (from tens to hundreds of kilobases), and study their implications in the context of cell fate specification in the developing Drosophila embryo. The resulting technologies will be applied to analogous transcriptional loci in mouse embryonic stem cells and organoids derived from these cells. Together, the proposed studies will help reveal how robust mechanisms of cell type specification emerge from stochastic processes such as transcriptional bursts, fluctuations in the size and stability of biomolecular condensates, and dynamic instability of chromatin architecture. The overall goal of this project is to establish a quantitative link between chromatin architecture and transcriptional activity, which will ultimately allow us to take control of gene activity by re-engineering the transcriptional programs underlying developmental and disease processes.